Additional annotation of the pig transcriptome using integrated Iso-seq and Illumina RNA-seq analysis

USDA-ARS?s Scientific Manuscript database

Alternative splicing is a well-known phenomenon that dramatically increases eukaryotic transcriptome diversity. The extent of mRNA isoform diversity among porcine tissues was assessed using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq) and Illumina short read sequencing ...

Integrated Analysis of Dysregulated ncRNA and mRNA Expression Profiles in Humans Exposed to Carbon Nanotubes

PubMed Central

Shvedova, Anna A.; Yanamala, Naveena; Kisin, Elena R.; Khailullin, Timur O.; Birch, M. Eileen; Fatkhutdinova, Liliya M.

2016-01-01

Background As the application of carbon nanotubes (CNT) in consumer products continues to rise, studies have expanded to determine the associated risks of exposure on human and environmental health. In particular, several lines of evidence indicate that exposure to multi-walled carbon nanotubes (MWCNT) could pose a carcinogenic risk similar to asbestos fibers. However, to date the potential markers of MWCNT exposure are not yet explored in humans. Methods In the present study, global mRNA and ncRNA expression profiles in the blood of exposed workers, having direct contact with MWCNT aerosol for at least 6 months (n = 8), were compared with expression profiles of non-exposed (n = 7) workers (e.g., professional and/or technical staff) from the same manufacturing facility. Results Significant changes in the ncRNA and mRNA expression profiles were observed between exposed and non-exposed worker groups. An integrative analysis of ncRNA-mRNA correlations was performed to identify target genes, functional relationships, and regulatory networks in MWCNT-exposed workers. The coordinated changes in ncRNA and mRNA expression profiles revealed a set of miRNAs and their target genes with roles in cell cycle regulation/progression/control, apoptosis and proliferation. Further, the identified pathways and signaling networks also revealed MWCNT potential to trigger pulmonary and cardiovascular effects as well as carcinogenic outcomes in humans, similar to those previously described in rodents exposed to MWCNTs. Conclusion This study is the first to investigate aberrant changes in mRNA and ncRNA expression profiles in the blood of humans exposed to MWCNT. The significant changes in several miRNAs and mRNAs expression as well as their regulatory networks are important for getting molecular insights into the MWCNT-induced toxicity and pathogenesis in humans. Further large-scale prospective studies are necessary to validate the potential applicability of such changes in mRNAs and miRNAs as prognostic markers of MWCNT exposures in humans. PMID:26930275

Structural and Functional Analysis of an mRNP Complex That Mediates the High Stability of Human β-Globin mRNA

PubMed Central

Yu, Jia; Russell, J. Eric

2001-01-01

Human globins are encoded by mRNAs exhibiting high stabilities in transcriptionally silenced erythrocyte progenitors. Unlike α-globin mRNA, whose stability is enhanced by assembly of a specific messenger RNP (mRNP) α complex on its 3′ untranslated region (UTR), neither the structure(s) nor the mechanism(s) that effects the high-level stability of human β-globin mRNA has been identified. The present work describes an mRNP complex assembling on the 3′ UTR of the β-globin mRNA that exhibits many of the properties of the stability-enhancing α complex. The β-globin mRNP complex is shown to contain one or more factors homologous to αCP, a 39-kDa RNA-binding protein that is integral to α-complex assembly. Sequence analysis implicates a specific 14-nucleotide pyrimidine-rich track within its 3′ UTR as the site of β-globin mRNP assembly. The importance of this track to mRNA stability is subsequently verified in vivo using mice expressing human β-globin transgenes that contain informative mutations in this region. In combination, the in vitro and in vivo analyses indicate that the high stabilities of the α- and β-globin mRNAs are maintained through related mRNP complexes that may share a common regulatory pathway. PMID:11486027

Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA.

PubMed

Kim, Bo-Eun; Choi, Soon Won; Shin, Ji-Hee; Kim, Jae-Jun; Kang, Insung; Lee, Byung-Chul; Lee, Jin Young; Kook, Myoung Geun; Kang, Kyung-Sun

2018-01-01

Neural stem cells (NSCs) are a prominent cell source for understanding neural pathogenesis and for developing therapeutic applications to treat neurodegenerative disease because of their regenerative capacity and multipotency. Recently, a variety of cellular reprogramming technologies have been developed to facilitate in vitro generation of NSCs, called induced NSCs (iNSCs). However, the genetic safety aspects of established virus-based reprogramming methods have been considered, and non-integrating reprogramming methods have been developed. Reprogramming with in vitro transcribed (IVT) mRNA is one of the genetically safe reprogramming methods because exogenous mRNA temporally exists in the cell and is not integrated into the chromosome. Here, we successfully generated expandable iNSCs from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via transfection with IVT mRNA encoding SOX2 (SOX2 mRNA) with properly optimized conditions. We confirmed that generated human UCB-MSC-derived iNSCs (UM-iNSCs) possess characteristics of NSCs, including multipotency and self-renewal capacity. Additionally, we transfected human dermal fibroblasts (HDFs) with SOX2 mRNA. Compared with human embryonic stem cell-derived NSCs, HDFs transfected with SOX2 mRNA exhibited neural reprogramming with similar morphologies and NSC-enriched mRNA levels, but they showed limited proliferation ability. Our results demonstrated that human UCB-MSCs can be used for direct reprogramming into NSCs through transfection with IVT mRNA encoding a single factor, which provides an integration-free reprogramming tool for future therapeutic application.

Predicting the Dynamics of Protein Abundance

PubMed Central

Mehdi, Ahmed M.; Patrick, Ralph; Bailey, Timothy L.; Bodén, Mikael

2014-01-01

Protein synthesis is finely regulated across all organisms, from bacteria to humans, and its integrity underpins many important processes. Emerging evidence suggests that the dynamic range of protein abundance is greater than that observed at the transcript level. Technological breakthroughs now mean that sequencing-based measurement of mRNA levels is routine, but protocols for measuring protein abundance remain both complex and expensive. This paper introduces a Bayesian network that integrates transcriptomic and proteomic data to predict protein abundance and to model the effects of its determinants. We aim to use this model to follow a molecular response over time, from condition-specific data, in order to understand adaptation during processes such as the cell cycle. With microarray data now available for many conditions, the general utility of a protein abundance predictor is broad. Whereas most quantitative proteomics studies have focused on higher organisms, we developed a predictive model of protein abundance for both Saccharomyces cerevisiae and Schizosaccharomyces pombe to explore the latitude at the protein level. Our predictor primarily relies on mRNA level, mRNA–protein interaction, mRNA folding energy and half-life, and tRNA adaptation. The combination of key features, allowing for the low certainty and uneven coverage of experimental observations, gives comparatively minor but robust prediction accuracy. The model substantially improved the analysis of protein regulation during the cell cycle: predicted protein abundance identified twice as many cell-cycle-associated proteins as experimental mRNA levels. Predicted protein abundance was more dynamic than observed mRNA expression, agreeing with experimental protein abundance from a human cell line. We illustrate how the same model can be used to predict the folding energy of mRNA when protein abundance is available, lending credence to the emerging view that mRNA folding affects translation efficiency. The software and data used in this research are available at http://bioinf.scmb.uq.edu.au/proteinabundance/. PMID:24532840

Predicting the dynamics of protein abundance.

PubMed

Mehdi, Ahmed M; Patrick, Ralph; Bailey, Timothy L; Bodén, Mikael

2014-05-01

Protein synthesis is finely regulated across all organisms, from bacteria to humans, and its integrity underpins many important processes. Emerging evidence suggests that the dynamic range of protein abundance is greater than that observed at the transcript level. Technological breakthroughs now mean that sequencing-based measurement of mRNA levels is routine, but protocols for measuring protein abundance remain both complex and expensive. This paper introduces a Bayesian network that integrates transcriptomic and proteomic data to predict protein abundance and to model the effects of its determinants. We aim to use this model to follow a molecular response over time, from condition-specific data, in order to understand adaptation during processes such as the cell cycle. With microarray data now available for many conditions, the general utility of a protein abundance predictor is broad. Whereas most quantitative proteomics studies have focused on higher organisms, we developed a predictive model of protein abundance for both Saccharomyces cerevisiae and Schizosaccharomyces pombe to explore the latitude at the protein level. Our predictor primarily relies on mRNA level, mRNA-protein interaction, mRNA folding energy and half-life, and tRNA adaptation. The combination of key features, allowing for the low certainty and uneven coverage of experimental observations, gives comparatively minor but robust prediction accuracy. The model substantially improved the analysis of protein regulation during the cell cycle: predicted protein abundance identified twice as many cell-cycle-associated proteins as experimental mRNA levels. Predicted protein abundance was more dynamic than observed mRNA expression, agreeing with experimental protein abundance from a human cell line. We illustrate how the same model can be used to predict the folding energy of mRNA when protein abundance is available, lending credence to the emerging view that mRNA folding affects translation efficiency. The software and data used in this research are available at http://bioinf.scmb.uq.edu.au/proteinabundance/.

Integrated Analysis of Long Noncoding RNA and mRNA Expression Profile in Advanced Laryngeal Squamous Cell Carcinoma.

PubMed

Feng, Ling; Wang, Ru; Lian, Meng; Ma, Hongzhi; He, Ning; Liu, Honggang; Wang, Haizhou; Fang, Jugao

2016-01-01

Long non-coding RNA (lncRNA) plays an important role in tumorigenesis. However, the expression pattern and function of lncRNAs in laryngeal squamous cell carcinoma (LSCC) are still unclear. To investigate the aberrantly expressed lncRNAs and mRNAs in advanced LSCC, we screened lncRNA and mRNA expression profiles in 9 pairs of primary Stage IVA LSCC tissues and adjacent non-neoplastic tissues by lncRNA and mRNA integrated microarrays. Gene Ontology and pathway analysis were performed to find out the significant function and pathway of the differentially expressed mRNAs, gene-gene functional interaction network and ceRNA network were constructed to select core mRNAs, and lncRNA-mRNA expression correlation network was built to identify the interactions between lncRNA and mRNA. qRT-PCR was performed to further validate the expressions of selected lncRNAs and mRNAs in advanced LSCC. We found 1459 differentially expressed lncRNAs and 2381 differentially expressed mRNAs, including 846 up-regulated lncRNAs and 613 down-regulated lncRNAs, 1542 up-regulated mRNAs and 839 down-regulated mRNAs. The mRNAs ITGB1, HIF1A, and DDIT4 were selected as core mRNAs, which are mainly involved in biological processes, such as matrix organization, cell cycle, adhesion, and metabolic pathway. LncRNA-mRNA expression correlation network showed LncRNA NR_027340, MIR31HG were positively correlated with ITGB1, HIF1A respectively. LncRNA SOX2-OT was negatively correlated with DDIT4. qRT-PCR further validated the expression of these lncRNAs and mRNAs. The work provides convincing evidence that the identified lncRNAs and mRNAs are potential biomarkers in advanced LSCC for further future studies.

Effects of simulated microgravity on microRNA and mRNA expression profile of rat soleus

NASA Astrophysics Data System (ADS)

Xu, Hongjie; Wu, Feng; Cao, Hongqing; Kan, Guanghan; Zhang, Hongyu; Yeung, Ella W.; Shang, Peng; Dai, Zhongquan; Li, Yinghui

2015-02-01

Spaceflight induces muscle atrophy but mechanism is not well understood. Here, we quantified microRNAs (miRNAs) and mRNA shifts of rat soleus in response to microgravity. MiRNAs and mRNA microarray of soleus after tail suspension (TS) for 7 and 14 days were performed followed by target gene and function annotation analysis and qRT-PCR. Relative muscle mass lost by 37.0% in TS-7 but less than 10% in the following three weeks. TS altered 23 miRNAs and 1313 mRNAs with at least 2-fold. QRT-PCR confirmed some of these changes. MiR-214, miR-486-5p and miR-221 continuously decreased. MiR-674 and Let-7e decreased only in TS-7, while miR-320b and miR-187 decreased only in TS-14. But there was no alteration of miR-320 and miR-206 in both time point. For mRNA detection, actn3 (5.1-fold and 13.8-fold) and myh4 (38-fold and 51.6-fold) increased abundantly and a3galt2 decreased. Predicted targeted genes (whyz, ywhaz and SFRP2) of altered miRNAs decreased. GO terms and cellular pathway of these alteration showed enrichment in regulation of muscle metabolism. Integration analysis of the miRNA and mRNA expression profiles confirmed that eleven genes were differently regulated by four miRNAs. This is the first study that showed expression pattern and synergistical regulation of miRNA and mRNA in rat soleus of TS for up to 14 days.

Proteogenomic characterization of human colon and rectal cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Bing; Wang, Jing; Wang, Xiaojing

2014-09-18

We analyzed proteomes of colon and rectal tumors previously characterized by the Cancer Genome Atlas (TCGA) and performed integrated proteogenomic analyses. Protein sequence variants encoded by somatic genomic variations displayed reduced expression compared to protein variants encoded by germline variations. mRNA transcript abundance did not reliably predict protein expression differences between tumors. Proteomics identified five protein expression subtypes, two of which were associated with the TCGA "MSI/CIMP" transcriptional subtype, but had distinct mutation and methylation patterns and associated with different clinical outcomes. Although CNAs showed strong cis- and trans-effects on mRNA expression, relatively few of these extend to the proteinmore » level. Thus, proteomics data enabled prioritization of candidate driver genes. Our analyses identified HNF4A, a novel candidate driver gene in tumors with chromosome 20q amplifications. Integrated proteogenomic analysis provides functional context to interpret genomic abnormalities and affords novel insights into cancer biology.« less

A comparison of non-integrating reprogramming methods

PubMed Central

Schlaeger, Thorsten M; Daheron, Laurence; Brickler, Thomas R; Entwisle, Samuel; Chan, Karrie; Cianci, Amelia; DeVine, Alexander; Ettenger, Andrew; Fitzgerald, Kelly; Godfrey, Michelle; Gupta, Dipti; McPherson, Jade; Malwadkar, Prerana; Gupta, Manav; Bell, Blair; Doi, Akiko; Jung, Namyoung; Li, Xin; Lynes, Maureen S; Brookes, Emily; Cherry, Anne B C; Demirbas, Didem; Tsankov, Alexander M; Zon, Leonard I; Rubin, Lee L; Feinberg, Andrew P; Meissner, Alexander; Cowan, Chad A; Daley, George Q

2015-01-01

Human induced pluripotent stem cells (hiPSCs1–3) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV)4, episomal (Epi)5 and mRNA transfection mRNA6 methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation. PMID:25437882

Identification of novel point mutations in splicing sites integrating whole-exome and RNA-seq data in myeloproliferative diseases.

PubMed

Spinelli, Roberta; Pirola, Alessandra; Redaelli, Sara; Sharma, Nitesh; Raman, Hima; Valletta, Simona; Magistroni, Vera; Piazza, Rocco; Gambacorti-Passerini, Carlo

2013-11-01

Point mutations in intronic regions near mRNA splice junctions can affect the splicing process. To identify novel splicing variants from exome sequencing data, we developed a bioinformatics splice-site prediction procedure to analyze next-generation sequencing (NGS) data (SpliceFinder). SpliceFinder integrates two functional annotation tools for NGS, ANNOVAR and MutationTaster and two canonical splice site prediction programs for single mutation analysis, SSPNN and NetGene2. By SpliceFinder, we identified somatic mutations affecting RNA splicing in a colon cancer sample, in eight atypical chronic myeloid leukemia (aCML), and eight CML patients. A novel homozygous splicing mutation was found in APC (NM_000038.4:c.1312+5G>A) and six heterozygous in GNAQ (NM_002072.2:c.735+1C>T), ABCC 3 (NM_003786.3:c.1783-1G>A), KLHDC 1 (NM_172193.1:c.568-2A>G), HOOK 1 (NM_015888.4:c.1662-1G>A), SMAD 9 (NM_001127217.2:c.1004-1C>T), and DNAH 9 (NM_001372.3:c.10242+5G>A). Integrating whole-exome and RNA sequencing in aCML and CML, we assessed the phenotypic effect of mutations on mRNA splicing for GNAQ, ABCC 3, HOOK 1. In ABCC 3 and HOOK 1, RNA-Seq showed the presence of aberrant transcripts with activation of a cryptic splice site or intron retention, validated by the reverse transcription-polymerase chain reaction (RT-PCR) in the case of HOOK 1. In GNAQ, RNA-Seq showed 22% of wild-type transcript and 78% of mRNA skipping exon 5, resulting in a 4-6 frameshift fusion confirmed by RT-PCR. The pipeline can be useful to identify intronic variants affecting RNA sequence by complementing conventional exome analysis.

Towards systems genetic analyses in barley: Integration of phenotypic, expression and genotype data into GeneNetwork

PubMed Central

Druka, Arnis; Druka, Ilze; Centeno, Arthur G; Li, Hongqiang; Sun, Zhaohui; Thomas, William TB; Bonar, Nicola; Steffenson, Brian J; Ullrich, Steven E; Kleinhofs, Andris; Wise, Roger P; Close, Timothy J; Potokina, Elena; Luo, Zewei; Wagner, Carola; Schweizer, Günther F; Marshall, David F; Kearsey, Michael J; Williams, Robert W; Waugh, Robbie

2008-01-01

Background A typical genetical genomics experiment results in four separate data sets; genotype, gene expression, higher-order phenotypic data and metadata that describe the protocols, processing and the array platform. Used in concert, these data sets provide the opportunity to perform genetic analysis at a systems level. Their predictive power is largely determined by the gene expression dataset where tens of millions of data points can be generated using currently available mRNA profiling technologies. Such large, multidimensional data sets often have value beyond that extracted during their initial analysis and interpretation, particularly if conducted on widely distributed reference genetic materials. Besides quality and scale, access to the data is of primary importance as accessibility potentially allows the extraction of considerable added value from the same primary dataset by the wider research community. Although the number of genetical genomics experiments in different plant species is rapidly increasing, none to date has been presented in a form that allows quick and efficient on-line testing for possible associations between genes, loci and traits of interest by an entire research community. Description Using a reference population of 150 recombinant doubled haploid barley lines we generated novel phenotypic, mRNA abundance and SNP-based genotyping data sets, added them to a considerable volume of legacy trait data and entered them into the GeneNetwork . GeneNetwork is a unified on-line analytical environment that enables the user to test genetic hypotheses about how component traits, such as mRNA abundance, may interact to condition more complex biological phenotypes (higher-order traits). Here we describe these barley data sets and demonstrate some of the functionalities GeneNetwork provides as an easily accessible and integrated analytical environment for exploring them. Conclusion By integrating barley genotypic, phenotypic and mRNA abundance data sets directly within GeneNetwork's analytical environment we provide simple web access to the data for the research community. In this environment, a combination of correlation analysis and linkage mapping provides the potential to identify and substantiate gene targets for saturation mapping and positional cloning. By integrating datasets from an unsequenced crop plant (barley) in a database that has been designed for an animal model species (mouse) with a well established genome sequence, we prove the importance of the concept and practice of modular development and interoperability of software engineering for biological data sets. PMID:19017390

Combined analysis of mRNA and miRNA identifies dehydration and salinity responsive key molecular players in citrus roots.

PubMed

Xie, Rangjin; Zhang, Jin; Ma, Yanyan; Pan, Xiaoting; Dong, Cuicui; Pang, Shaoping; He, Shaolan; Deng, Lie; Yi, Shilai; Zheng, Yongqiang; Lv, Qiang

2017-02-06

Citrus is one of the most economically important fruit crops around world. Drought and salinity stresses adversely affected its productivity and fruit quality. However, the genetic regulatory networks and signaling pathways involved in drought and salinity remain to be elucidated. With RNA-seq and sRNA-seq, an integrative analysis of miRNA and mRNA expression profiling and their regulatory networks were conducted using citrus roots subjected to dehydration and salt treatment. Differentially expressed (DE) mRNA and miRNA profiles were obtained according to fold change analysis and the relationships between miRNAs and target mRNAs were found to be coherent and incoherent in the regulatory networks. GO enrichment analysis revealed that some crucial biological processes related to signal transduction (e.g. 'MAPK cascade'), hormone-mediated signaling pathways (e.g. abscisic acid- activated signaling pathway'), reactive oxygen species (ROS) metabolic process (e.g. 'hydrogen peroxide catabolic process') and transcription factors (e.g., 'MYB, ZFP and bZIP') were involved in dehydration and/or salt treatment. The molecular players in response to dehydration and salt treatment were partially overlapping. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis further confirmed the results from RNA-seq and sRNA-seq analysis. This study provides new insights into the molecular mechanisms how citrus roots respond to dehydration and salt treatment.

Combined analysis of mRNA and miRNA identifies dehydration and salinity responsive key molecular players in citrus roots

PubMed Central

Xie, Rangjin; Zhang, Jin; Ma, Yanyan; Pan, Xiaoting; Dong, Cuicui; Pang, Shaoping; He, Shaolan; Deng, Lie; Yi, Shilai; Zheng, Yongqiang; Lv, Qiang

2017-01-01

Citrus is one of the most economically important fruit crops around world. Drought and salinity stresses adversely affected its productivity and fruit quality. However, the genetic regulatory networks and signaling pathways involved in drought and salinity remain to be elucidated. With RNA-seq and sRNA-seq, an integrative analysis of miRNA and mRNA expression profiling and their regulatory networks were conducted using citrus roots subjected to dehydration and salt treatment. Differentially expressed (DE) mRNA and miRNA profiles were obtained according to fold change analysis and the relationships between miRNAs and target mRNAs were found to be coherent and incoherent in the regulatory networks. GO enrichment analysis revealed that some crucial biological processes related to signal transduction (e.g. ‘MAPK cascade’), hormone-mediated signaling pathways (e.g. abscisic acid- activated signaling pathway’), reactive oxygen species (ROS) metabolic process (e.g. ‘hydrogen peroxide catabolic process’) and transcription factors (e.g., ‘MYB, ZFP and bZIP’) were involved in dehydration and/or salt treatment. The molecular players in response to dehydration and salt treatment were partially overlapping. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis further confirmed the results from RNA-seq and sRNA-seq analysis. This study provides new insights into the molecular mechanisms how citrus roots respond to dehydration and salt treatment. PMID:28165059

Kinetic analysis of the effects of target structure on siRNA efficiency

NASA Astrophysics Data System (ADS)

Chen, Jiawen; Zhang, Wenbing

2012-12-01

RNAi efficiency for target cleavage and protein expression is related to the target structure. Considering the RNA-induced silencing complex (RISC) as a multiple turnover enzyme, we investigated the effect of target mRNA structure on siRNA efficiency with kinetic analysis. The 4-step model was used to study the target cleavage kinetic process: hybridization nucleation at an accessible target site, RISC-mRNA hybrid elongation along with mRNA target structure melting, target cleavage, and enzyme reactivation. At this model, the terms accounting for the target accessibility, stability, and the seed and the nucleation site effects are all included. The results are in good agreement with that of experiments which show different arguments about the structure effects on siRNA efficiency. It shows that the siRNA efficiency is influenced by the integrated factors of target's accessibility, stability, and the seed effects. To study the off-target effects, a simple model of one siRNA binding to two mRNA targets was designed. By using this model, the possibility for diminishing the off-target effects by the concentration of siRNA was discussed.

Integrative mouse and human mRNA studies using WGCNA nominates novel candidate genes involved in the pathogenesis of major depressive disorder.

PubMed

Malki, Karim; Tosto, Maria Grazia; Jumabhoy, Irfan; Lourdusamy, Anbarasu; Sluyter, Frans; Craig, Ian; Uher, Rudolf; McGuffin, Peter; Schalkwyk, Leonard C

2013-12-01

This study aims to identify novel genes associated with major depressive disorder and pharmacological treatment response using animal and human mRNA studies. Weighted gene coexpression network analysis was used to uncover genes associated with stress factors in mice and to inform mRNA probe set selection in a post-mortem study of depression. A total of 171 genes were found to be differentially regulated in response to both early and late stress protocols in a mouse study. Ten human genes, orthologous to mouse genes differentially expressed by stress, were also found to be dysregulated in depressed cases in a human post-mortem brain study from the Stanley Foundation Brain Collection. Several novel genes associated with depression were uncovered, including NOVA1 and USP9X. Moreover, we found further evidence in support of hippocampal neurogenesis and peripheral inflammation in major depressive disorder.

RNA-Seq Reveals an Integrated Immune Response in Nucleated Erythrocytes

PubMed Central

Morera, Davinia; Roher, Nerea; Ribas, Laia; Balasch, Joan Carles; Doñate, Carmen; Callol, Agnes; Boltaña, Sebastian; Roberts, Steven; Goetz, Giles; Goetz, Frederick W.; MacKenzie, Simon A.

2011-01-01

Background Throughout the primary literature and within textbooks, the erythrocyte has been tacitly accepted to have maintained a unique physiological role; namely gas transport and exchange. In non-mammalian vertebrates, nucleated erythrocytes are present in circulation throughout the life cycle and a fragmented series of observations in mammals support a potential role in non-respiratory biological processes. We hypothesised that nucleated erythrocytes could actively participate via ligand-induced transcriptional re-programming in the immune response. Methodology/Principal Findings Nucleated erythrocytes from both fish and birds express and regulate specific pattern recognition receptor (PRR) mRNAs and, thus, are capable of specific pathogen associated molecular pattern (PAMP) detection that is central to the innate immune response. In vitro challenge with diverse PAMPs led to de novo specific mRNA synthesis of both receptors and response factors including interferon-alpha (IFNα) that exhibit a stimulus-specific polysomal shift supporting active translation. RNA-Seq analysis of the PAMP (Poly (I∶C), polyinosinic∶polycytidylic acid)-erythrocyte response uncovered diverse cohorts of differentially expressed mRNA transcripts related to multiple physiological systems including the endocrine, reproductive and immune. Moreover, erythrocyte-derived conditioned mediums induced a type-1 interferon response in macrophages thus supporting an integrative role for the erythrocytes in the immune response. Conclusions/Significance We demonstrate that nucleated erythrocytes in non-mammalian vertebrates spanning significant phylogenetic distance participate in the immune response. RNA-Seq studies highlight a mRNA repertoire that suggests a previously unrecognized integrative role for the erythrocytes in other physiological systems. PMID:22046430

Transient Expression of an LEDGF/p75 Chimera Retargets Lentivector Integration and Functionally Rescues in a Model for X-CGD

PubMed Central

Vets, Sofie; De Rijck, Jan; Brendel, Christian; Grez, Manuel; Bushman, Frederic; Debyser, Zeger; Gijsbers, Rik

2013-01-01

Retrovirus-based vectors are commonly used as delivery vehicles to correct genetic diseases because of their ability to integrate new sequences stably. However, adverse events in which vector integration activates proto-oncogenes, leading to clonal expansion and leukemogenesis hamper their application. The host cell-encoded lens epithelium-derived growth factor (LEDGF/p75) binds lentiviral integrase and targets integration to active transcription units. We demonstrated earlier that replacing the LEDGF/p75 chromatin interaction domain with an alternative DNA-binding protein could retarget integration. Here, we show that transient expression of the chimeric protein using mRNA electroporation efficiently redirects lentiviral vector (LV) integration in wild-type (WT) cells. We then employed this technology in a model for X-linked chronic granulomatous disease (X-CGD) using myelomonocytic PLB-985 gp91−/− cells. Following electroporation with mRNA encoding the LEDGF-chimera, the cells were treated with a therapeutic lentivector encoding gp91phox. Integration site analysis revealed retargeted integration away from genes and towards heterochromatin-binding protein 1β (CBX1)-binding sites, in regions enriched in marks associated with gene silencing. Nevertheless, gp91phox expression was stable for at least 6 months after electroporation and NADPH-oxidase activity was restored to normal levels as determined by superoxide production. Together, these data provide proof-of-principle that transient expression of engineered LEDGF-chimera can retarget lentivector integration and rescues the disease phenotype in a cell model, opening perspectives for safer gene therapy. PMID:23462964

rCAD: A Novel Database Schema for the Comparative Analysis of RNA.

PubMed

Ozer, Stuart; Doshi, Kishore J; Xu, Weijia; Gutell, Robin R

2011-12-31

Beyond its direct involvement in protein synthesis with mRNA, tRNA, and rRNA, RNA is now being appreciated for its significance in the overall metabolism and regulation of the cell. Comparative analysis has been very effective in the identification and characterization of RNA molecules, including the accurate prediction of their secondary structure. We are developing an integrative scalable data management and analysis system, the RNA Comparative Analysis Database (rCAD), implemented with SQL Server to support RNA comparative analysis. The platformagnostic database schema of rCAD captures the essential relationships between the different dimensions of information for RNA comparative analysis datasets. The rCAD implementation enables a variety of comparative analysis manipulations with multiple integrated data dimensions for advanced RNA comparative analysis workflows. In this paper, we describe details of the rCAD schema design and illustrate its usefulness with two usage scenarios.

rCAD: A Novel Database Schema for the Comparative Analysis of RNA

PubMed Central

Ozer, Stuart; Doshi, Kishore J.; Xu, Weijia; Gutell, Robin R.

2013-01-01

Beyond its direct involvement in protein synthesis with mRNA, tRNA, and rRNA, RNA is now being appreciated for its significance in the overall metabolism and regulation of the cell. Comparative analysis has been very effective in the identification and characterization of RNA molecules, including the accurate prediction of their secondary structure. We are developing an integrative scalable data management and analysis system, the RNA Comparative Analysis Database (rCAD), implemented with SQL Server to support RNA comparative analysis. The platformagnostic database schema of rCAD captures the essential relationships between the different dimensions of information for RNA comparative analysis datasets. The rCAD implementation enables a variety of comparative analysis manipulations with multiple integrated data dimensions for advanced RNA comparative analysis workflows. In this paper, we describe details of the rCAD schema design and illustrate its usefulness with two usage scenarios. PMID:24772454

Expression of angiotensin II receptors in the caprine ovary and improvement of follicular viability in vitro.

PubMed

Bruno, J B; Lima-Verde, I B; Celestino, J J H; Lima, L F; Matos, M H T; Faustino, L R; Donato, M A M; Peixoto, C A; Campello, C C; Silva, J R V; Figueiredo, J R

2016-08-01

This study aimed to evaluate mRNA levels of angiotensin II (ANG II) receptors (AGTR1 and AGTR2) in caprine follicles and to investigate the influence of ANG II on the viability and in vitro growth of preantral follicles. Real-time polymerase chain reaction (PCR) was used to quantify AGTR1 and AGTR2 mRNA levels in the different follicular stages. For culture, caprine ovaries were collected, cut into 13 fragments and then either directly fixed for histological and ultrastructural analysis (fresh control) or placed in culture for 1 or 7 days in α-minumum essential medium plus (α-MEM+) with 0, 1, 5, 10, 50 or 100 ng/ml ANG II. Then, the fragments were destined to morphological, viability and ultrastructural analysis. The results showed that primordial follicles had higher levels of AGTR1 and AGTR2 mRNA than secondary follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA than their respective cumulus-oocyte complex (COCs). After 7 days of culture, ANG II (10 or 50 ng/ml) maintained the percentages of normal follicles compared with α-MEM+. Fluorescence and ultrastructural microscopy confirmed follicular integrity in ANG II (10 ng/ml). In conclusion, a high expression of AGTR1 and AGTR2 is observed in primordial follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA. Finally, 10 ng/ml ANG II maintained the viability of caprine preantral follicles after in vitro culture.

Genomic analysis suggests that mRNA destabilization by the microprocessor is specialized for the auto-regulation of Dgcr8.

PubMed

Shenoy, Archana; Blelloch, Robert

2009-09-11

The Microprocessor, containing the RNA binding protein Dgcr8 and RNase III enzyme Drosha, is responsible for processing primary microRNAs to precursor microRNAs. The Microprocessor regulates its own levels by cleaving hairpins in the 5'UTR and coding region of the Dgcr8 mRNA, thereby destabilizing the mature transcript. To determine whether the Microprocessor has a broader role in directly regulating other coding mRNA levels, we integrated results from expression profiling and ultra high-throughput deep sequencing of small RNAs. Expression analysis of mRNAs in wild-type, Dgcr8 knockout, and Dicer knockout mouse embryonic stem (ES) cells uncovered mRNAs that were specifically upregulated in the Dgcr8 null background. A number of these transcripts had evolutionarily conserved predicted hairpin targets for the Microprocessor. However, analysis of deep sequencing data of 18 to 200nt small RNAs in mouse ES, HeLa, and HepG2 indicates that exonic sequence reads that map in a pattern consistent with Microprocessor activity are unique to Dgcr8. We conclude that the Microprocessor's role in directly destabilizing coding mRNAs is likely specifically targeted to Dgcr8 itself, suggesting a specialized cellular mechanism for gene auto-regulation.

LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes

PubMed Central

Singh, Parmit Kumar; Plumb, Matthew R.; Ferris, Andrea L.; Iben, James R.; Wu, Xiaolin; Fadel, Hind J.; Luke, Brian T.; Esnault, Caroline; Poeschla, Eric M.; Hughes, Stephen H.; Kvaratskhelia, Mamuka; Levin, Henry L.

2015-01-01

The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced. PMID:26545813

Identification of novel point mutations in splicing sites integrating whole-exome and RNA-seq data in myeloproliferative diseases

PubMed Central

Spinelli, Roberta; Pirola, Alessandra; Redaelli, Sara; Sharma, Nitesh; Raman, Hima; Valletta, Simona; Magistroni, Vera; Piazza, Rocco; Gambacorti-Passerini, Carlo

2013-01-01

Point mutations in intronic regions near mRNA splice junctions can affect the splicing process. To identify novel splicing variants from exome sequencing data, we developed a bioinformatics splice-site prediction procedure to analyze next-generation sequencing (NGS) data (SpliceFinder). SpliceFinder integrates two functional annotation tools for NGS, ANNOVAR and MutationTaster and two canonical splice site prediction programs for single mutation analysis, SSPNN and NetGene2. By SpliceFinder, we identified somatic mutations affecting RNA splicing in a colon cancer sample, in eight atypical chronic myeloid leukemia (aCML), and eight CML patients. A novel homozygous splicing mutation was found in APC (NM_000038.4:c.1312+5G>A) and six heterozygous in GNAQ (NM_002072.2:c.735+1C>T), ABCC3 (NM_003786.3:c.1783-1G>A), KLHDC1 (NM_172193.1:c.568-2A>G), HOOK1 (NM_015888.4:c.1662-1G>A), SMAD9 (NM_001127217.2:c.1004-1C>T), and DNAH9 (NM_001372.3:c.10242+5G>A). Integrating whole-exome and RNA sequencing in aCML and CML, we assessed the phenotypic effect of mutations on mRNA splicing for GNAQ, ABCC3, HOOK1. In ABCC3 and HOOK1, RNA-Seq showed the presence of aberrant transcripts with activation of a cryptic splice site or intron retention, validated by the reverse transcription-polymerase chain reaction (RT-PCR) in the case of HOOK1. In GNAQ, RNA-Seq showed 22% of wild-type transcript and 78% of mRNA skipping exon 5, resulting in a 4–6 frameshift fusion confirmed by RT-PCR. The pipeline can be useful to identify intronic variants affecting RNA sequence by complementing conventional exome analysis. PMID:24498620

Complex and extensive post-transcriptional regulation revealed by integrative proteomic and transcriptomic analysis of metabolite stress response in Clostridium acetobutylicum.

PubMed

Venkataramanan, Keerthi P; Min, Lie; Hou, Shuyu; Jones, Shawn W; Ralston, Matthew T; Lee, Kelvin H; Papoutsakis, E Terry

2015-01-01

Clostridium acetobutylicum is a model organism for both clostridial biology and solvent production. The organism is exposed to its own toxic metabolites butyrate and butanol, which trigger an adaptive stress response. Integrative analysis of proteomic and RNAseq data may provide novel insights into post-transcriptional regulation. The identified iTRAQ-based quantitative stress proteome is made up of 616 proteins with a 15 % genome coverage. The differentially expressed proteome correlated poorly with the corresponding differential RNAseq transcriptome. Up to 31 % of the differentially expressed proteins under stress displayed patterns opposite to those of the transcriptome, thus suggesting significant post-transcriptional regulation. The differential proteome of the translation machinery suggests that cells employ a different subset of ribosomal proteins under stress. Several highly upregulated proteins but with low mRNA levels possessed mRNAs with long 5'UTRs and strong RBS scores, thus supporting the argument that regulatory elements on the long 5'UTRs control their translation. For example, the oxidative stress response rubrerythrin was upregulated only at the protein level up to 40-fold without significant mRNA changes. We also identified many leaderless transcripts, several displaying different transcriptional start sites, thus suggesting mRNA-trimming mechanisms under stress. Downregulation of Rho and partner proteins pointed to changes in transcriptional elongation and termination under stress. The integrative proteomic-transcriptomic analysis demonstrated complex expression patterns of a large fraction of the proteome. Such patterns could not have been detected with one or the other omic analyses. Our analysis proposes the involvement of specific molecular mechanisms of post-transcriptional regulation to explain the observed complex stress response.

Spatial analysis and high resolution mapping of the human whole-brain transcriptome for integrative analysis in neuroimaging.

PubMed

Gryglewski, Gregor; Seiger, René; James, Gregory Miles; Godbersen, Godber Mathis; Komorowski, Arkadiusz; Unterholzner, Jakob; Michenthaler, Paul; Hahn, Andreas; Wadsak, Wolfgang; Mitterhauser, Markus; Kasper, Siegfried; Lanzenberger, Rupert

2018-08-01

The quantification of big pools of diverse molecules provides important insights on brain function, but is often restricted to a limited number of observations, which impairs integration with other modalities. To resolve this issue, a method allowing for the prediction of mRNA expression in the entire brain based on microarray data provided in the Allen Human Brain Atlas was developed. Microarray data of 3702 samples from 6 brain donors was registered to MNI and cortical surface space using FreeSurfer. For each of 18,686 genes, spatial dependence of transcription was assessed using variogram modelling. Variogram models were employed in Gaussian process regression to calculate best linear unbiased predictions for gene expression at all locations represented in well-established imaging atlases for cortex, subcortical structures and cerebellum. For validation, predicted whole-brain transcription of the HTR1A gene was correlated with [carbonyl- 11 C]WAY-100635 positron emission tomography data collected from 30 healthy subjects. Prediction results showed minimal bias ranging within ±0.016 (cortical surface), ±0.12 (subcortical regions) and ±0.14 (cerebellum) in units of log2 expression intensity for all genes. Across genes, the correlation of predicted and observed mRNA expression in leave-one-out cross-validation correlated with the strength of spatial dependence (cortical surface: r = 0.91, subcortical regions: r = 0.85, cerebellum: r = 0.84). 816 out of 18,686 genes exhibited a high spatial dependence accounting for more than 50% of variance in the difference of gene expression on the cortical surface. In subcortical regions and cerebellum, different sets of genes were implicated by high spatially structured variability. For the serotonin 1A receptor, correlation between PET binding potentials and predicted comprehensive mRNA expression was markedly higher (Spearman ρ = 0.72 for cortical surface, ρ = 0.84 for subcortical regions) than correlation of PET and discrete samples only (ρ = 0.55 and ρ = 0.63, respectively). Prediction of mRNA expression in the entire human brain allows for intuitive visualization of gene transcription and seamless integration in multimodal analysis without bias arising from non-uniform distribution of available samples. Extension of this methodology promises to facilitate translation of omics research and enable investigation of human brain function at a systems level. Copyright © 2018 Elsevier Inc. All rights reserved.

Texture Analysis of Poly-Adenylated mRNA Staining Following Global Brain Ischemia and Reperfusion

PubMed Central

Szymanski, Jeffrey J.; Jamison, Jill T.; DeGracia, Donald J.

2011-01-01

Texture analysis provides a means to quantify complex changes in microscope images. We previously showed that cytoplasmic poly-adenylated mRNAs form mRNA granules in post-ischemic neurons and that these granules correlated with protein synthesis inhibition and hence cell death. Here we utilized the texture analysis software MaZda to quantify mRNA granules in photomicrographs of the pyramidal cell layer of rat hippocampal region CA3 around 1 hour of reperfusion after 10 min of normothermic global cerebral ischemia. At 1 hour reperfusion, we observed variations in the texture of mRNA granules amongst samples that were readily quantified by texture analysis. Individual sample variation was consistent with the interpretation that animal-to-animal variations in mRNA granules reflected the time-course of mRNA granule formation. We also used texture analysis to quantify the effect of cycloheximide, given either before or after brain ischemia, on mRNA granules. If administered before ischemia, cycloheximide inhibited mRNA granule formation, but if administered after ischemia did not prevent mRNA granulation, indicating mRNA granule formation is dependent on dissociation of polysomes. We conclude that texture analysis is an effective means for quantifying the complex morphological changes induced in neurons by brain ischemia and reperfusion. PMID:21477879

Transient Expression of an LEDGF/p75 Chimera Retargets Lentivector Integration and Functionally Rescues in a Model for X-CGD.

PubMed

Vets, Sofie; De Rijck, Jan; Brendel, Christian; Grez, Manuel; Bushman, Frederic; Debyser, Zeger; Gijsbers, Rik

2013-03-05

Retrovirus-based vectors are commonly used as delivery vehicles to correct genetic diseases because of their ability to integrate new sequences stably. However, adverse events in which vector integration activates proto-oncogenes, leading to clonal expansion and leukemogenesis hamper their application. The host cell-encoded lens epithelium-derived growth factor (LEDGF/p75) binds lentiviral integrase and targets integration to active transcription units. We demonstrated earlier that replacing the LEDGF/p75 chromatin interaction domain with an alternative DNA-binding protein could retarget integration. Here, we show that transient expression of the chimeric protein using mRNA electroporation efficiently redirects lentiviral vector (LV) integration in wild-type (WT) cells. We then employed this technology in a model for X-linked chronic granulomatous disease (X-CGD) using myelomonocytic PLB-985 gp91(-/-) cells. Following electroporation with mRNA encoding the LEDGF-chimera, the cells were treated with a therapeutic lentivector encoding gp91(phox). Integration site analysis revealed retargeted integration away from genes and towards heterochromatin-binding protein 1β (CBX1)-binding sites, in regions enriched in marks associated with gene silencing. Nevertheless, gp91(phox) expression was stable for at least 6 months after electroporation and NADPH-oxidase activity was restored to normal levels as determined by superoxide production. Together, these data provide proof-of-principle that transient expression of engineered LEDGF-chimera can retarget lentivector integration and rescues the disease phenotype in a cell model, opening perspectives for safer gene therapy.Molecular Therapy - Nucleic Acids (2013) 2, e77; doi:10.1038/mtna.2013.4; published online 5 March 2013.

Genome-wide DNA methylation profiling integrated with gene expression profiling identifies PAX9 as a novel prognostic marker in chronic lymphocytic leukemia.

PubMed

Rani, Lata; Mathur, Nitin; Gupta, Ritu; Gogia, Ajay; Kaur, Gurvinder; Dhanjal, Jaspreet Kaur; Sundar, Durai; Kumar, Lalit; Sharma, Atul

2017-01-01

In chronic lymphocytic leukemia (CLL), epigenomic and genomic studies have expanded the existing knowledge about the disease biology and led to the identification of potential biomarkers relevant for implementation of personalized medicine. In this study, an attempt has been made to examine and integrate the global DNA methylation changes with gene expression profile and their impact on clinical outcome in early stage CLL patients. The integration of DNA methylation profile ( n  = 14) with the gene expression profile ( n  = 21) revealed 142 genes as hypermethylated-downregulated and; 62 genes as hypomethylated-upregulated in early stage CLL patients compared to CD19+ B-cells from healthy individuals. The mRNA expression levels of 17 genes identified to be differentially methylated and/or differentially expressed was further examined in early stage CLL patients ( n  = 93) by quantitative real time PCR (RQ-PCR). Significant differences were observed in the mRNA expression of MEIS1 , PMEPA1 , SOX7 , SPRY1 , CDK6 , TBX2 , and SPRY2 genes in CLL cells as compared to B-cells from healthy individuals. The analysis in the IGHV mutation based categories (Unmutated = 39, Mutated = 54) revealed significantly higher mRNA expression of CRY1 and PAX9 genes in the IGHV unmutated subgroup ( p  < 0.001). The relative risk of treatment initiation was significantly higher among patients with high expression of CRY1 (RR = 1.91, p  = 0.005) or PAX9 (RR = 1.87, p  = 0.001). High expression of CRY1 (HR: 3.53, p  < 0.001) or PAX9 (HR: 3.14, p  < 0.001) gene was significantly associated with shorter time to first treatment. The high expression of PAX9 gene (HR: 3.29, 95% CI 1.172-9.272, p  = 0.016) was also predictive of shorter overall survival in CLL. The DNA methylation changes associated with mRNA expression of CRY1 and PAX9 genes allow risk stratification of early stage CLL patients. This comprehensive analysis supports the concept that the epigenetic changes along with the altered expression of genes have the potential to predict clinical outcome in early stage CLL patients.

Biomaterials for mRNA Delivery

PubMed Central

Islam, Mohammad Ariful; Reesor, Emma K. G.; Xu, Yingjie; Zope, Harshal R.; Zetter, Bruce R.; Shi, Jinjun

2015-01-01

Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice. PMID:26280625

Stool metatranscriptomics: A technical guideline for mRNA stabilisation and isolation.

PubMed

Reck, Michael; Tomasch, Jürgen; Deng, Zhiluo; Jarek, Michael; Husemann, Peter; Wagner-Döbler, Irene

2015-07-04

The complex microbiome of the gut has an enormous impact on human health. Analysis of the transcriptional activity of microorganisms through mRNA sequencing (metatranscriptomics) opens a completely new window into their activity in vivo, but it is highly challenging due to numerous technical and bioinformatical obstacles. Here we present an optimized pipeline for extraction of high quality mRNA from stool samples. Comparison of three commercially available RNA extraction kits with the method of Zoetendal revealed that the Powermicrobiome Kit (MoBio) performed best with respect to RNA yield and purity. Next, the influence of the stabilization reagent during sample storage for up to 15 days was studied. RIN analysis and qRT-PCR of spiked-in and indigenous genes revealed that RNA Later preserved mRNA integrity most efficiently, while samples conserved in RNA Protect showed substantial mRNA decay. Using the optimized pipeline developed here, recovery rates for spiked-in E.coli cells expressing fluorescing proteins were 8.7-9.7% for SuperfolderGFP and 14.7-17.8% for mCherry. The mRNA of stabilized stool samples as well as of snap-frozen controls was sequenced with Illumina Hiseq, yielding on average 74 million reads per sample. PCoA analysis, taxonomic classification using Kraken and functional classification using bwa showed that the transcriptomes of samples conserved in RNA Later were unchanged for up to 6 days even at room temperature, while RNA Protect was inefficient for storage durations exceeding 24 h. However, our data indicate that RNA Later introduces a bias which is then maintained throughout storage, while RNA Protect conserved samples are initially more similar to the snap frozen controls. RNA Later conserved samples had a reduced abundance of e.g. Prevotellaceae transcripts and were depleted for e.g. COG category "Carbohydrate transport and metabolism". Since the overall similarity between all stool transcriptional profiles studied here was >0.92, these differences are unlikely to affect global comparisons, but should be taken into account when rare but critically important members of the stool microbiome are being studied.

Integrated mRNA and miRNA transcriptome reveal a cross-talk between developing response and hormone signaling for the seed kernels of Siberian apricot

PubMed Central

Niu, Jun; Wang, Jia; An, Jiyong; Liu, Lili; Lin, Zixin; Wang, Rui; Wang, Libing; Ma, Chao; Shi, Lingling; Lin, Shanzhi

2016-01-01

Recently, our transcriptomic analysis has identified some functional genes responsible for oil biosynthesis in developing SASK, yet miRNA-mediated regulation for SASK development and oil accumulation is poorly understood. Here, 3 representative periods of 10, 30 and 60 DAF were selected for sRNA sequencing based on the dynamic patterns of growth tendency and oil content of developing SASK. By miRNA transcriptomic analysis, we characterized 296 known and 44 novel miRNAs in developing SASK, among which 36 known and 6 novel miRNAs respond specifically to developing SASK. Importantly, we performed an integrated analysis of mRNA and miRNA transcriptome as well as qRT-PCR detection to identify some key miRNAs and their targets (miR156-SPL, miR160-ARF18, miR164-NAC1, miR171h-SCL6, miR172-AP2, miR395-AUX22B, miR530-P2C37, miR393h-TIR1/AFB2 and psi-miRn5-SnRK2A) potentially involved in developing response and hormone signaling of SASK. Our results provide new insights into the important regulatory function of cross-talk between development response and hormone signaling for SASK oil accumulation. PMID:27762296

Integrated mRNA and miRNA transcriptome reveal a cross-talk between developing response and hormone signaling for the seed kernels of Siberian apricot.

PubMed

Niu, Jun; Wang, Jia; An, Jiyong; Liu, Lili; Lin, Zixin; Wang, Rui; Wang, Libing; Ma, Chao; Shi, Lingling; Lin, Shanzhi

2016-10-20

Recently, our transcriptomic analysis has identified some functional genes responsible for oil biosynthesis in developing SASK, yet miRNA-mediated regulation for SASK development and oil accumulation is poorly understood. Here, 3 representative periods of 10, 30 and 60 DAF were selected for sRNA sequencing based on the dynamic patterns of growth tendency and oil content of developing SASK. By miRNA transcriptomic analysis, we characterized 296 known and 44 novel miRNAs in developing SASK, among which 36 known and 6 novel miRNAs respond specifically to developing SASK. Importantly, we performed an integrated analysis of mRNA and miRNA transcriptome as well as qRT-PCR detection to identify some key miRNAs and their targets (miR156-SPL, miR160-ARF18, miR164-NAC1, miR171h-SCL6, miR172-AP2, miR395-AUX22B, miR530-P2C37, miR393h-TIR1/AFB2 and psi-miRn5-SnRK2A) potentially involved in developing response and hormone signaling of SASK. Our results provide new insights into the important regulatory function of cross-talk between development response and hormone signaling for SASK oil accumulation.

In silico analysis of stomach lineage specific gene set expression pattern in gastric cancer.

PubMed

Pandi, Narayanan Sathiya; Suganya, Sivagurunathan; Rajendran, Suriliyandi

2013-10-04

Stomach lineage specific gene products act as a protective barrier in the normal stomach and their expression maintains the normal physiological processes, cellular integrity and morphology of the gastric wall. However, the regulation of stomach lineage specific genes in gastric cancer (GC) is far less clear. In the present study, we sought to investigate the role and regulation of stomach lineage specific gene set (SLSGS) in GC. SLSGS was identified by comparing the mRNA expression profiles of normal stomach tissue with other organ tissue. The obtained SLSGS was found to be under expressed in gastric tumors. Functional annotation analysis revealed that the SLSGS was enriched for digestive function and gastric epithelial maintenance. Employing a single sample prediction method across GC mRNA expression profiles identified the under expression of SLSGS in proliferative type and invasive type gastric tumors compared to the metabolic type gastric tumors. Integrative pathway activation prediction analysis revealed a close association between estrogen-α signaling and SLSGS expression pattern in GC. Elevated expression of SLSGS in GC is associated with an overall increase in the survival of GC patients. In conclusion, our results highlight that estrogen mediated regulation of SLSGS in gastric tumor is a molecular predictor of metabolic type GC and prognostic factor in GC. Copyright © 2013 Elsevier Inc. All rights reserved.

Computational, Integrative, and Comparative Methods for the Elucidation of Genetic Coexpression Networks

DOE PAGES

Baldwin, Nicole E.; Chesler, Elissa J.; Kirov, Stefan; ...

2005-01-01

Gene expression microarray data can be used for the assembly of genetic coexpression network graphs. Using mRNA samples obtained from recombinant inbred Mus musculus strains, it is possible to integrate allelic variation with molecular and higher-order phenotypes. The depth of quantitative genetic analysis of microarray data can be vastly enhanced utilizing this mouse resource in combination with powerful computational algorithms, platforms, and data repositories. The resulting network graphs transect many levels of biological scale. This approach is illustrated with the extraction of cliques of putatively co-regulated genes and their annotation using gene ontology analysis and cis -regulatory element discovery. Themore » causal basis for co-regulation is detected through the use of quantitative trait locus mapping.« less

Stress integration after acute and chronic predator stress: differential activation of central stress circuitry and sensitization of the hypothalamo-pituitary-adrenocortical axis.

PubMed

Figueiredo, Helmer F; Bodie, Bryan L; Tauchi, Miyuki; Dolgas, C Mark; Herman, James P

2003-12-01

Predator exposure is a naturalistic stressor of high ethological relevance. In the current study, our group examined central and peripheral integration of stress responses in rats after acute or repeated exposure to a natural predator (cat). Acute cat exposure rapidly induced hypothalamo-pituitary-adrenocortical (HPA) axis activation and paraventricular nucleus (PVN) CRH mRNA production. Repeated daily cat exposure (7 and 14 d) also up-regulated PVN mRNA CRH expression, but did not result in frank adrenocortical hyperactivity. Unlike other chronic homotypic stress regimens, repeated cat exposure facilitated corticosterone secretion after the 6th or 13th day of exposure. Notably, ACTH secretion and central amygdaloid nucleus CRH mRNA expression were enhanced in animals that were preexposed to the holding chamber relative to chamber-naive rats, suggesting that contextual cues can sensitize subsequent responses to a fearful stimulus. Analysis of c-fos activation was then used to identify brain circuits activated by acute predator stress. Cat exposure elicited a pattern of central c-fos activation that differed substantially from that after either restraint or hypoxia. Predator-specific c-fos mRNA induction was observed in several brain regions comprising the hypothetical brain defense circuit (bed nucleus of the stria terminalis, medial region of the ventromedial nucleus, and dorsal premammillary nucleus). Surprisingly, acute cat exposure did not induce c-fos expression in the PVN. In summary, the data indicate that 1) predation stress invokes a unique stress circuitry that promotes homotypic sensitization of the HPA axis, and 2) familiarization of animals to testing environments can prime central stress pathways to respond robustly to novel threats.

RNA Flow Cytometry Using the Branched DNA Technique.

PubMed

Soh, Kah Teong; Wallace, Paul K

2018-01-01

The systematic modulation of mRNA and proteins governs the complicated and intermingled biological functions of our cells. Traditionally, transcriptomic technologies such as DNA microarray and RNA-Seq have been used to identify, characterize, and profile gene expression data. These are, however, considered bulk methods as they are unable to measure gene expression at the single-cell level, unless the cells are pre-sorted. Branched DNA is a flow cytometry-based detection platform that has been developed recently to measure mRNA at the single-cell level. Originally adapted from microscopy, the current system has been modified to achieve compatibility with the detection of surface and intracellular antigens using monoclonal antibodies conjugated to fluorochromes, thus permitting simultaneous detection of mRNAs and proteins. The Branched DNA method offers a variety of advantages when compared to traditional or standard methods used for the quantification of mRNA, such as (a) the detection of specific mRNA on a per cell basis, (b) an alternate detection tool when the measurement of a protein is technically infeasible (i.e., no quality antibody exists) or the epitope is not assessable, and (c) correlate the analysis of mRNA with protein. Compared to earlier attempts at measuring nucleic acid by flow cytometry, the hybridization temperature applied in the Branched DNA assay is much lower, thus preserving the integrity of cellular structures for further characterization. It also has greatly increased specificity and sensitivity. Here, we provide detailed instruction for performing the Branched DNA method using it in a model system to correlate the expression of CD8 mRNA and CD8 protein by flow cytometry.

Integrated Lung and Tracheal mRNA-Seq and miRNA-Seq Analysis of Dogs with an Avian-Like H5N1 Canine Influenza Virus Infection

PubMed Central

Fu, Cheng; Luo, Jie; Ye, Shaotang; Yuan, Ziguo; Li, Shoujun

2018-01-01

Avian-like H5N1 canine influenza virus (CIV) causes severe respiratory infections in dogs. However, the mechanism underlying H5N1 CIV infection in dogs is unknown. The present study aimed to identify differentially expressed miRNAs and mRNAs in the lungs and trachea in H5N1 CIV-infected dogs through a next-generation sequencing-based method. Eighteen 40-day-old beagles were inoculated intranasally with CIV, A/canine/01/Guangdong/2013 (H5N1) at a tissue culture infectious dose 50 (TCID50) of 106, and lung and tracheal tissues were harvested at 3 and 7 d post-inoculation. The tissues were processed for miRNA and mRNA analysis. By means of miRNA-gene expression integrative negative analysis, we found miRNA–mRNA pairs. Lung and trachea tissues showed 138 and 135 negative miRNA–mRNA pairs, respectively. One hundred and twenty negative miRNA–mRNA pairs were found between the different tissues. In particular, pathways including the influenza A pathway, chemokine signaling pathways, and the PI3K-Akt signaling pathway were significantly enriched in all groups in responses to virus infection. Furthermore, dysregulation of miRNA and mRNA expression was observed in the respiratory tract of H5N1 CIV-infected dogs and notably, TLR4 (miR-146), NF-κB (miR-34c) and CCL5 (miR-335), CCL10 (miR-8908-5p), and GNGT2 (miR-122) were found to play important roles in regulating pathways that resist virus infection. To our knowledge, the present study is the first to analyze miRNA and mRNA expression in H5N1 CIV-infected dogs; furthermore, the present findings provide insights into the molecular mechanisms underlying influenza virus infection. PMID:29556219

Nuclear Export of Messenger RNA

PubMed Central

Katahira, Jun

2015-01-01

Transport of messenger RNA (mRNA) from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP) particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex. PMID:25836925

Characterization of X Chromosome Inactivation Using Integrated Analysis of Whole-Exome and mRNA Sequencing

PubMed Central

Szelinger, Szabolcs; Malenica, Ivana; Corneveaux, Jason J.; Siniard, Ashley L.; Kurdoglu, Ahmet A.; Ramsey, Keri M.; Schrauwen, Isabelle; Trent, Jeffrey M.; Narayanan, Vinodh; Huentelman, Matthew J.; Craig, David W.

2014-01-01

In females, X chromosome inactivation (XCI) is an epigenetic, gene dosage compensatory mechanism by inactivation of one copy of X in cells. Random XCI of one of the parental chromosomes results in an approximately equal proportion of cells expressing alleles from either the maternally or paternally inherited active X, and is defined by the XCI ratio. Skewed XCI ratio is suggestive of non-random inactivation, which can play an important role in X-linked genetic conditions. Current methods rely on indirect, semi-quantitative DNA methylation-based assay to estimate XCI ratio. Here we report a direct approach to estimate XCI ratio by integrated, family-trio based whole-exome and mRNA sequencing using phase-by-transmission of alleles coupled with allele-specific expression analysis. We applied this method to in silico data and to a clinical patient with mild cognitive impairment but no clear diagnosis or understanding molecular mechanism underlying the phenotype. Simulation showed that phased and unphased heterozygous allele expression can be used to estimate XCI ratio. Segregation analysis of the patient's exome uncovered a de novo, interstitial, 1.7 Mb deletion on Xp22.31 that originated on the paternally inherited X and previously been associated with heterogeneous, neurological phenotype. Phased, allelic expression data suggested an 83∶20 moderately skewed XCI that favored the expression of the maternally inherited, cytogenetically normal X and suggested that the deleterious affect of the de novo event on the paternal copy may be offset by skewed XCI that favors expression of the wild-type X. This study shows the utility of integrated sequencing approach in XCI ratio estimation. PMID:25503791

Posttranscriptional regulation of albumin gene expression by branched-chain amino acids in rats with acute liver injury.

PubMed

Kuwahata, Masashi; Kuramoto, Yasuko; Tomoe, Yuka; Sugata, Emi; Segawa, Hiroko; Ito, Mikiko; Oka, Tatsuzo; Miyamoto, Ken-Ichi

2004-12-24

We previously demonstrated that the integration of albumin mRNA into functional polysomes was regulated by the supply of branched-chain amino acids (BCAA) in the liver of galactosamine-treated rats. To study the mechanism of this regulation, we investigated interaction between rat liver proteins and albumin transcripts. When albumin transcript was incubated with ribosome salt wash (RSW) extracts prepared from liver, a specific RNA-protein complex (p65) formed. Competition experiments showed that a pyrimidine-rich sequence in the coding region of albumin mRNA was required for the formation of p65. The level of p65 was increased in the RSW extracts prepared from liver of galactosamine-treated rats infused with a standard amino acid formula, compared with a BCAA-enriched amino acid formula. The protein in p65 appears to be polypyrimidine tract-binding protein (PTB), because the formation of p65 was reduced in the RSW extracts pre-incubated with anti-PTB antibody. In cell-free translation analysis, immunodepletion of PTB from rabbit reticulocyte lysate caused an increase in albumin translation. These results suggest that binding of PTB to albumin mRNA suppresses its translation. A supply of BCAA may interfere with this binding and improve the translation efficiency of albumin mRNA in injured liver.

A Systems Biology Approach to Investigating Sex Differences in Cardiac Hypertrophy.

PubMed

Harrington, Josephine; Fillmore, Natasha; Gao, Shouguo; Yang, Yanqin; Zhang, Xue; Liu, Poching; Stoehr, Andrea; Chen, Ye; Springer, Danielle; Zhu, Jun; Wang, Xujing; Murphy, Elizabeth

2017-08-19

Heart failure preceded by hypertrophy is a leading cause of death, and sex differences in hypertrophy are well known, although the basis for these sex differences is poorly understood. This study used a systems biology approach to investigate mechanisms underlying sex differences in cardiac hypertrophy. Male and female mice were treated for 2 and 3 weeks with angiotensin II to induce hypertrophy. Sex differences in cardiac hypertrophy were apparent after 3 weeks of treatment. RNA sequencing was performed on hearts, and sex differences in mRNA expression at baseline and following hypertrophy were observed, as well as within-sex differences between baseline and hypertrophy. Sex differences in mRNA were substantial at baseline and reduced somewhat with hypertrophy, as the mRNA differences induced by hypertrophy tended to overwhelm the sex differences. We performed an integrative analysis to identify mRNA networks that were differentially regulated in the 2 sexes by hypertrophy and obtained a network centered on PPARα (peroxisome proliferator-activated receptor α). Mouse experiments further showed that acute inhibition of PPARα blocked sex differences in the development of hypertrophy. The data in this study suggest that PPARα is involved in the sex-dimorphic regulation of cardiac hypertrophy. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Uniform, optimal signal processing of mapped deep-sequencing data.

PubMed

Kumar, Vibhor; Muratani, Masafumi; Rayan, Nirmala Arul; Kraus, Petra; Lufkin, Thomas; Ng, Huck Hui; Prabhakar, Shyam

2013-07-01

Despite their apparent diversity, many problems in the analysis of high-throughput sequencing data are merely special cases of two general problems, signal detection and signal estimation. Here we adapt formally optimal solutions from signal processing theory to analyze signals of DNA sequence reads mapped to a genome. We describe DFilter, a detection algorithm that identifies regulatory features in ChIP-seq, DNase-seq and FAIRE-seq data more accurately than assay-specific algorithms. We also describe EFilter, an estimation algorithm that accurately predicts mRNA levels from as few as 1-2 histone profiles (R ∼0.9). Notably, the presence of regulatory motifs in promoters correlates more with histone modifications than with mRNA levels, suggesting that histone profiles are more predictive of cis-regulatory mechanisms. We show by applying DFilter and EFilter to embryonic forebrain ChIP-seq data that regulatory protein identification and functional annotation are feasible despite tissue heterogeneity. The mathematical formalism underlying our tools facilitates integrative analysis of data from virtually any sequencing-based functional profile.

Integrating Data Clustering and Visualization for the Analysis of 3D Gene Expression Data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Data Analysis and Visualization; nternational Research Training Group ``Visualization of Large and Unstructured Data Sets,'' University of Kaiserslautern, Germany; Computational Research Division, Lawrence Berkeley National Laboratory, One Cyclotron Road, Berkeley, CA 94720, USA

2008-05-12

The recent development of methods for extracting precise measurements of spatial gene expression patterns from three-dimensional (3D) image data opens the way for new analyses of the complex gene regulatory networks controlling animal development. We present an integrated visualization and analysis framework that supports user-guided data clustering to aid exploration of these new complex datasets. The interplay of data visualization and clustering-based data classification leads to improved visualization and enables a more detailed analysis than previously possible. We discuss (i) integration of data clustering and visualization into one framework; (ii) application of data clustering to 3D gene expression data; (iii)more » evaluation of the number of clusters k in the context of 3D gene expression clustering; and (iv) improvement of overall analysis quality via dedicated post-processing of clustering results based on visualization. We discuss the use of this framework to objectively define spatial pattern boundaries and temporal profiles of genes and to analyze how mRNA patterns are controlled by their regulatory transcription factors.« less

The novel regulatory ncRNA, NfiS, optimizes nitrogen fixation via base pairing with the nitrogenase gene nifK mRNA in Pseudomonas stutzeri A1501

PubMed Central

Zhan, Yuhua; Yan, Yongliang; Deng, Zhiping; Chen, Ming; Lu, Wei; Lu, Chao; Shang, Liguo; Yang, Zhimin; Zhang, Wei; Wang, Wei; Li, Yun; Ke, Qi; Lu, Jiasi; Xu, Yuquan; Zhang, Liwen; Xie, Zhihong; Cheng, Qi; Elmerich, Claudine; Lin, Min

2016-01-01

Unlike most Pseudomonas, the root-associated bacterium Pseudomonas stutzeri A1501 fixes nitrogen after the horizontal acquisition of a nitrogen-fixing (nif) island. A genome-wide search for small noncoding RNAs (ncRNAs) in P. stutzeri A1501 identified the novel P. stutzeri-specific ncRNA NfiS in the core genome, whose synthesis was significantly induced under nitrogen fixation or sorbitol stress conditions. The expression of NfiS was RNA chaperone Hfq-dependent and activated by the sigma factor RpoN/global nitrogen activator NtrC/nif-specific activator NifA regulatory cascade. The nfiS-deficient mutant displayed reduced nitrogenase activity, as well as increased sensitivity to multiple stresses, such as osmotic and oxidative stresses. Secondary structure prediction and complementation studies confirmed that a stem-loop structure was essential for NfiS to regulate the nitrogenase gene nifK mRNA synthesis and thus nitrogenase activity. Microscale thermophoresis and physiological analysis showed that NfiS directly pairs with nifK mRNA and ultimately enhances nitrogenase activity by increasing the translation efficiency and the half-life of nifK mRNA. Our data also suggest structural and functional divergence of NfiS evolution in diazotrophic and nondiazotrophic backgrounds. It is proposed that NfiS was recruited by nifK mRNA as a novel regulator to integrate the horizontally acquired nif island into host global networks. PMID:27407147

mRNA and methylation profiling of radioresistant esophageal cancer cells: the involvement of Sall2 in acquired aggressive phenotypes

PubMed Central

Luo, Judong; Wang, Wenjie; Tang, Yiting; Zhou, Dandan; Gao, Yi; Zhang, Qi; Zhou, Xifa; Zhu, Hui; Xing, Ligang; Yu, Jinming

2017-01-01

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest malignancies worldwide. Radiotherapy plays a critical role in the curative management of inoperable ESCC patients. However, radioresistance restricts the efficacy of radiotherapy for ESCC patients. The molecules involved in radioresistance remain largely unknown, and new approaches to sensitize cells to irradiation are in demand. Technical advances in analysis of mRNA and methylation have enabled the exploration of the etiology of diseases and have the potential to broaden our understanding of the molecular pathways of ESCC radioresistance. In this study, we constructed radioresistant TE-1 and Eca-109 cell lines (TE-1/R and Eca-109/R, respectively). The radioresistant cells showed an increased migration ability but reduced apoptosis and cisplatin sensitivity compared with their parent cells. mRNA and methylation profiling by microarray revealed 1192 preferentially expressed mRNAs and 8841 aberrantly methylated regions between TE-1/R and TE-1 cells. By integrating the mRNA and methylation profiles, we related the decreased expression of transcription factor Sall2 with a corresponding increase in its methylation in TE-1/R cells, indicating its involvement in radioresistance. Upregulation of Sall2 decreased the growth and migration advantage of radioresistant ESCC cells. Taken together, our present findings illustrate the mRNA and DNA methylation changes during the radioresistance of ESCC and the important role of Sall2 in esophageal cancer malignancy. PMID:28367244

Messenger RNA-based therapeutics for the treatment of apoptosis-associated diseases.

PubMed

Matsui, Akitsugu; Uchida, Satoshi; Ishii, Takehiko; Itaka, Keiji; Kataoka, Kazunori

2015-10-28

Gene therapy is a promising approach for treating diseases that are closely associated with excessive apoptosis, because the gene can effectively and sustainably introduce anti-apoptotic factors into cells. However, DNA delivery poses the risk of random genomic integration, leading to overexpression of the delivered gene and cancer development. Messenger RNA (mRNA) can evade integration events in target cells. We examined the use of mRNA-based therapeutics for introducing anti-apoptotic factors by using a mouse model of fulminant hepatitis. For introducing mRNA into the liver, a synthesised polymer-based carrier of polyplex nanomicelles was used for hydrodynamic intravenous injection. Using GFP as a reporter, we demonstrate that mRNA delivery induced efficient protein expression in almost 100% of liver cells, while plasmid DNA (pDNA) delivery provided a smaller percentage of GFP-positive cells. Analyses using Cy5-labelled mRNA and pDNA revealed that efficient expression by mRNA was attributed to a simple intracellular mechanism, without the need for nuclear entry. Consistent with this observation, Bcl-2 mRNA was more effective on reducing apoptosis in the liver of mice with fulminant hepatitis than Bcl-2 pDNA. Therefore, mRNA-based therapeutics combined with an effective delivery system such as polyplex nanomicelles is a promising treatment for intractable diseases associated with excessive apoptosis.

Coordinated Pulses of mRNA and of Protein Translation or Degradation Produce EGF-Induced Protein Bursts.

PubMed

Golan-Lavi, Roni; Giacomelli, Chiara; Fuks, Garold; Zeisel, Amit; Sonntag, Johanna; Sinha, Sanchari; Köstler, Wolfgang; Wiemann, Stefan; Korf, Ulrike; Yarden, Yosef; Domany, Eytan

2017-03-28

Protein responses to extracellular cues are governed by gene transcription, mRNA degradation and translation, and protein degradation. In order to understand how these time-dependent processes cooperate to generate dynamic responses, we analyzed the response of human mammary cells to the epidermal growth factor (EGF). Integrating time-dependent transcript and protein data into a mathematical model, we inferred for several proteins their pre-and post-stimulus translation and degradation coefficients and found that they exhibit complex, time-dependent variation. Specifically, we identified strategies of protein production and degradation acting in concert to generate rapid, transient protein bursts in response to EGF. Remarkably, for some proteins, for which the response necessitates rapidly decreased abundance, cells exhibit a transient increase in the corresponding degradation coefficient. Our model and analysis allow inference of the kinetics of mRNA translation and protein degradation, without perturbing cells, and open a way to understanding the fundamental processes governing time-dependent protein abundance profiles. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

eQTL networks unveil enriched mRNA master integrators downstream of complex disease-associated SNPs.

PubMed

Li, Haiquan; Pouladi, Nima; Achour, Ikbel; Gardeux, Vincent; Li, Jianrong; Li, Qike; Zhang, Hao Helen; Martinez, Fernando D; 'Skip' Garcia, Joe G N; Lussier, Yves A

2015-12-01

The causal and interplay mechanisms of Single Nucleotide Polymorphisms (SNPs) associated with complex diseases (complex disease SNPs) investigated in genome-wide association studies (GWAS) at the transcriptional level (mRNA) are poorly understood despite recent advancements such as discoveries reported in the Encyclopedia of DNA Elements (ENCODE) and Genotype-Tissue Expression (GTex). Protein interaction network analyses have successfully improved our understanding of both single gene diseases (Mendelian diseases) and complex diseases. Whether the mRNAs downstream of complex disease genes are central or peripheral in the genetic information flow relating DNA to mRNA remains unclear and may be disease-specific. Using expression Quantitative Trait Loci (eQTL) that provide DNA to mRNA associations and network centrality metrics, we hypothesize that we can unveil the systems properties of information flow between SNPs and the transcriptomes of complex diseases. We compare different conditions such as naïve SNP assignments and stringent linkage disequilibrium (LD) free assignments for transcripts to remove confounders from LD. Additionally, we compare the results from eQTL networks between lymphoblastoid cell lines and liver tissue. Empirical permutation resampling (p<0.001) and theoretic Mann-Whitney U test (p<10(-30)) statistics indicate that mRNAs corresponding to complex disease SNPs via eQTL associations are likely to be regulated by a larger number of SNPs than expected. We name this novel property mRNA hubness in eQTL networks, and further term mRNAs with high hubness as master integrators. mRNA master integrators receive and coordinate the perturbation signals from large numbers of polymorphisms and respond to the personal genetic architecture integratively. This genetic signal integration contrasts with the mechanism underlying some Mendelian diseases, where a genetic polymorphism affecting a single protein hub produces a divergent signal that affects a large number of downstream proteins. Indeed, we verify that this property is independent of the hubness in protein networks for which these mRNAs are transcribed. Our findings provide novel insights into the pleiotropy of mRNAs targeted by complex disease polymorphisms and the architecture of the information flow between the genetic polymorphisms and transcriptomes of complex diseases. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers.

PubMed

Domínguez-Sánchez, María S; Sáez, Carmen; Japón, Miguel A; Aguilera, Andrés; Luna, Rosa

2011-02-17

One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples. The mRNA levels were measured by quantitative real-time PCR and hybridization of a tumor tissue cDNA array; and the protein levels and distribution by immunostaining of a custom tissue array containing a set of paraffin-embedded samples of different tumor and normal tissues followed by statistical analysis. We show that the expression of two mRNP factors, THOC1 and ALY are altered in several tumor tissues. THOC1 mRNA and protein levels are up-regulated in ovarian and lung tumors and down-regulated in those of testis and skin, whereas ALY is altered in a wide variety of tumors. In contrast to THOC1, ALY protein is highly detected in normal proliferative cells, but poorly in high-grade cancers. These results suggest a differential connection between tumorogenesis and the expression levels of human THO and ALY. This study opens the possibility of defining mRNP biogenesis factors as putative players in cell proliferation that could contribute to tumor development.

Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles.

PubMed

Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

2014-01-01

Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection.

Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles

PubMed Central

Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

2014-01-01

Objectives: Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. Methods: MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. Results: A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. Conclusions: We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection. PMID:25664019

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Jing; Ma, Zihao; Carr, Steven A.

Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this “guilt-by-association” (GBA) approach. Recent advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC).more » Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies. Molecular & Cellular Proteomics 16: 10.1074/mcp.M116.060301, 121–134, 2017.« less

Tools to covisualize and coanalyze proteomic data with genomes and transcriptomes: validation of genes and alternative mRNA splicing.

PubMed

Pang, Chi Nam Ignatius; Tay, Aidan P; Aya, Carlos; Twine, Natalie A; Harkness, Linda; Hart-Smith, Gene; Chia, Samantha Z; Chen, Zhiliang; Deshpande, Nandan P; Kaakoush, Nadeem O; Mitchell, Hazel M; Kassem, Moustapha; Wilkins, Marc R

2014-01-03

Direct links between proteomic and genomic/transcriptomic data are not frequently made, partly because of lack of appropriate bioinformatics tools. To help address this, we have developed the PG Nexus pipeline. The PG Nexus allows users to covisualize peptides in the context of genomes or genomic contigs, along with RNA-seq reads. This is done in the Integrated Genome Viewer (IGV). A Results Analyzer reports the precise base position where LC-MS/MS-derived peptides cover genes or gene isoforms, on the chromosomes or contigs where this occurs. In prokaryotes, the PG Nexus pipeline facilitates the validation of genes, where annotation or gene prediction is available, or the discovery of genes using a "virtual protein"-based unbiased approach. We illustrate this with a comprehensive proteogenomics analysis of two strains of Campylobacter concisus . For higher eukaryotes, the PG Nexus facilitates gene validation and supports the identification of mRNA splice junction boundaries and splice variants that are protein-coding. This is illustrated with an analysis of splice junctions covered by human phosphopeptides, and other examples of relevance to the Chromosome-Centric Human Proteome Project. The PG Nexus is open-source and available from https://github.com/IntersectAustralia/ap11_Samifier. It has been integrated into Galaxy and made available in the Galaxy tool shed.

BIAS: Bioinformatics Integrated Application Software.

PubMed

Finak, G; Godin, N; Hallett, M; Pepin, F; Rajabi, Z; Srivastava, V; Tang, Z

2005-04-15

We introduce a development platform especially tailored to Bioinformatics research and software development. BIAS (Bioinformatics Integrated Application Software) provides the tools necessary for carrying out integrative Bioinformatics research requiring multiple datasets and analysis tools. It follows an object-relational strategy for providing persistent objects, allows third-party tools to be easily incorporated within the system and supports standards and data-exchange protocols common to Bioinformatics. BIAS is an OpenSource project and is freely available to all interested users at http://www.mcb.mcgill.ca/~bias/. This website also contains a paper containing a more detailed description of BIAS and a sample implementation of a Bayesian network approach for the simultaneous prediction of gene regulation events and of mRNA expression from combinations of gene regulation events. hallett@mcb.mcgill.ca.

An Integrated Analysis of MicroRNA and mRNA Expression Profiles to Identify RNA Expression Signatures in Lambskin Hair Follicles in Hu Sheep

PubMed Central

Lv, Xiaoyang; Sun, Wei; Yin, Jinfeng; Ni, Rong; Su, Rui; Wang, Qingzeng; Gao, Wen; Bao, Jianjun; Yu, Jiarui; Wang, Lihong; Chen, Ling

2016-01-01

Wave patterns in lambskin hair follicles are an important factor determining the quality of sheep’s wool. Hair follicles in lambskin from Hu sheep, a breed unique to China, have 3 types of waves, designated as large, medium, and small. The quality of wool from small wave follicles is excellent, while the quality of large waves is considered poor. Because no molecular and biological studies on hair follicles of these sheep have been conducted to date, the molecular mechanisms underlying the formation of different wave patterns is currently unknown. The aim of this article was to screen the candidate microRNAs (miRNA) and genes for the development of hair follicles in Hu sheep. Two-day-old Hu lambs were selected from full-sib individuals that showed large, medium, and small waves. Integrated analysis of microRNA and mRNA expression profiles employed high-throughout sequencing technology. Approximately 13, 24, and 18 differentially expressed miRNAs were found between small and large waves, small and medium waves, and medium and large waves, respectively. A total of 54, 190, and 81 differentially expressed genes were found between small and large waves, small and medium waves, and medium and large waves, respectively, by RNA sequencing (RNA-seq) analysis. Differentially expressed genes were classified using gene ontology and pathway analyses. They were found to be mainly involved in cell differentiation, proliferation, apoptosis, growth, immune response, and ion transport, and were associated with MAPK and the Notch signaling pathway. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of differentially-expressed miRNA and genes were consistent with sequencing results. Integrated analysis of miRNA and mRNA expression indicated that, compared to small waves, large waves included 4 downregulated miRNAs that had regulatory effects on 8 upregulated genes and 3 upregulated miRNAs, which in turn influenced 13 downregulated genes. Compared to small waves, medium waves included 13 downregulated miRNAs that had regulatory effects on 64 upregulated genes and 4 upregulated miRNAs, which in turn had regulatory effects on 22 downregulated genes. Compared to medium waves, large waves consisted of 13 upregulated miRNAs that had regulatory effects on 48 downregulated genes. These differentially expressed miRNAs and genes may play a significant role in forming different patterns, and provide evidence for the molecular mechanisms underlying the formation of hair follicles of varying patterns. PMID:27404636

Identification of microRNAs with regulatory potential using a matched microRNA-mRNA time-course data.

PubMed

Jayaswal, Vivek; Lutherborrow, Mark; Ma, David D F; Hwa Yang, Yee

2009-05-01

Over the past decade, a class of small RNA molecules called microRNAs (miRNAs) has been shown to regulate gene expression at the post-transcription stage. While early work focused on the identification of miRNAs using a combination of experimental and computational techniques, subsequent studies have focused on identification of miRNA-target mRNA pairs as each miRNA can have hundreds of mRNA targets. The experimental validation of some miRNAs as oncogenic has provided further motivation for research in this area. In this article we propose an odds-ratio (OR) statistic for identification of regulatory miRNAs. It is based on integrative analysis of matched miRNA and mRNA time-course microarray data. The OR-statistic was used for (i) identification of miRNAs with regulatory potential, (ii) identification of miRNA-target mRNA pairs and (iii) identification of time lags between changes in miRNA expression and those of its target mRNAs. We applied the OR-statistic to a cancer data set and identified a small set of miRNAs that were negatively correlated to mRNAs. A literature survey revealed that some of the miRNAs that were predicted to be regulatory, were indeed oncogenic or tumor suppressors. Finally, some of the predicted miRNA targets have been shown to be experimentally valid.

Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

PubMed

Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

2018-01-01

DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1 ) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P < 0.05 and more than 5.96% genes presented very strong correlation (R T4  > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers

PubMed Central

2011-01-01

Background One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples. Methods The mRNA levels were measured by quantitative real-time PCR and hybridization of a tumor tissue cDNA array; and the protein levels and distribution by immunostaining of a custom tissue array containing a set of paraffin-embedded samples of different tumor and normal tissues followed by statistical analysis. Results We show that the expression of two mRNP factors, THOC1 and ALY are altered in several tumor tissues. THOC1 mRNA and protein levels are up-regulated in ovarian and lung tumors and down-regulated in those of testis and skin, whereas ALY is altered in a wide variety of tumors. In contrast to THOC1, ALY protein is highly detected in normal proliferative cells, but poorly in high-grade cancers. Conclusions These results suggest a differential connection between tumorogenesis and the expression levels of human THO and ALY. This study opens the possibility of defining mRNP biogenesis factors as putative players in cell proliferation that could contribute to tumor development. PMID:21329510

VDR regulation of microRNA differs across prostate cell models suggesting extremely flexible control of transcription.

PubMed

Singh, Prashant K; Long, Mark D; Battaglia, Sebastiano; Hu, Qiang; Liu, Song; Sucheston-Campbell, Lara E; Campbell, Moray J

2015-01-01

The Vitamin D Receptor (VDR) is a member of the nuclear receptor superfamily and is of therapeutic interest in cancer and other settings. Regulation of microRNA (miRNA) by the VDR appears to be important to mediate its actions, for example, to control cell growth. To identify if and to what extent VDR-regulated miRNA patterns change in prostate cancer progression, we undertook miRNA microarray analyses in 7 cell models representing non-malignant and malignant prostate cells (RWPE-1, RWPE-2, HPr1, HPr1AR, LNCaP, LNCaP-C4-2, and PC-3). To focus on primary VDR regulatory events, we undertook expression analyses after 30 minutes treatment with 1α,25(OH)2D3. Across all models, 111 miRNAs were significantly modulated by 1α,25(OH)2D3 treatment. Of these, only 5 miRNAs were modulated in more than one cell model, and of these, only 3 miRNAs were modulated in the same direction. The patterns of miRNA regulation, and the networks they targeted, significantly distinguished the different cell types. Integration of 1α,25(OH)2D3-regulated miRNAs with published VDR ChIP-seq data showed significant enrichment of VDR peaks in flanking regions of miRNAs. Furthermore, mRNA and miRNA expression analyses in non-malignant RWPE-1 cells revealed patterns of miRNA and mRNA co-regulation; specifically, 13 significant reciprocal patterns were identified and these patterns were also observed in TCGA prostate cancer data. Lastly, motif search analysis revealed differential motif enrichment within VDR peaks flanking mRNA compared to miRNA genes. Together, this study revealed that miRNAs are rapidly regulated in a highly cell-type specific manner, and are significantly co-integrated with mRNA regulation.

Processed pseudogenes of human endogenous retroviruses generated by LINEs: their integration, stability, and distribution.

PubMed

Pavlícek, Adam; Paces, Jan; Elleder, Daniel; Hejnar, Jirí

2002-03-01

We report here the presence of numerous processed pseudogenes derived from the W family of endogenous retroviruses in the human genome. These pseudogenes are structurally colinear with the retroviral mRNA followed by a poly(A) tail. Our analysis of insertion sites of HERV-W processed pseudogenes shows a strong preference for the insertion motif of long interspersed nuclear element (LINE) retrotransposons. The genomic distribution, stability during evolution, and frequent truncations at the 5' end resemble those of the pseudogenes generated by LINEs. We therefore suggest that HERV-W processed pseudogenes arose by multiple and independent LINE-mediated retrotransposition of retroviral mRNA. These data document that the majority of HERV-W copies are actually nontranscribed promoterless pseudogenes. The current search for HERV-Ws associated with several human diseases should concentrate on a small subset of transcriptionally competent elements.

Amyloid protein-mediated differential DNA methylation status regulates gene expression in Alzheimer's disease model cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sung, Hye Youn; Choi, Eun Nam; Ahn Jo, Sangmee

2011-11-04

Highlights: Black-Right-Pointing-Pointer Genome-wide DNA methylation pattern in Alzheimer's disease model cell line. Black-Right-Pointing-Pointer Integrated analysis of CpG methylation and mRNA expression profiles. Black-Right-Pointing-Pointer Identify three Swedish mutant target genes; CTIF, NXT2 and DDR2 gene. Black-Right-Pointing-Pointer The effect of Swedish mutation on alteration of DNA methylation and gene expression. -- Abstract: The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer's disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterationsmore » in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2 Prime -deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the -435, -295, and -271 CpG sites of CTIF, and at the -505 to -341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at -432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory mechanism may contribute to the pathogenesis of AD.« less

Molecular cloning and expression profile analysis of porcine TCAP gene.

PubMed

Cheng, Hunjun; Xu, Xuewen; Zhao, Shuhong; Liu, Bang; Yu, Mei; Fan, Bin

2010-03-01

The gradually discovered sarcomeric proteins play important roles for structural integrity and signal transduction of sarcomere during myofibril genesis. TCAP (also described as telethonin, T-cap), one of the sarcomeric protein genes, is regulated developmentally. In this study, we reported the molecular characteristics of porcine TCAP gene. A 979 bp TCAP cDNA nucleotide sequence was obtained in pig and the deduced amino acid sequence had 92 and 91% identity to those of human and mouse homologous genes, respectively. One SNP was discovered and the allele frequency analysis showed that G allele frequency was low among 221 unrelated pigs from seven breeds. The tissue distribution patterns revealed that TCAP mRNA was expressed abundantly in skeletal and heart muscle tissue. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) results displayed TCAP mRNA was up-regulated in both Tongcheng and Landrace pigs during prenatal skeletal muscle development stages. This study suggested that TCAP gene might be a prospective candidate gene affecting muscle mass and meat quality traits in the pig, and also implicated the possible significance of TCAP on sarcomere assembly.

Smoking-related microRNAs and mRNAs in human peripheral blood mononuclear cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Ming-Wei

Teenager smoking is of great importance in public health. Functional roles of microRNAs have been documented in smoke-induced gene expression changes, but comprehensive mechanisms of microRNA-mRNA regulation and benefits remained poorly understood. We conducted the Teenager Smoking Reduction Trial (TSRT) to investigate the causal association between active smoking reduction and whole-genome microRNA and mRNA expression changes in human peripheral blood mononuclear cells (PBMC). A total of 12 teenagers with a substantial reduction in smoke quantity and a decrease in urine cotinine/creatinine ratio were enrolled in genomic analyses. In Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA), differentially expressedmore » genes altered by smoke reduction were mainly associated with glucocorticoid receptor signaling pathway. The integrative analysis of microRNA and mRNA found eleven differentially expressed microRNAs negatively correlated with predicted target genes. CD83 molecule regulated by miR-4498 in human PBMC, was critical for the canonical pathway of communication between innate and adaptive immune cells. Our data demonstrated that microRNAs could regulate immune responses in human PBMC after habitual smokers quit smoking and support the potential translational value of microRNAs in regulating disease-relevant gene expression caused by tobacco smoke. - Highlights: • We conducted a smoke reduction trial program and investigated the causal relationship between smoke and gene regulation. • MicroRNA and mRNA expression changes were examined in human PBMC. • MicroRNAs are important in regulating disease-causal genes after tobacco smoke reduction.« less

Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation.

PubMed

Tao, Wenjing; Sun, Lina; Shi, Hongjuan; Cheng, Yunying; Jiang, Dongneng; Fu, Beide; Conte, Matthew A; Gammerdinger, William J; Kocher, Thomas D; Wang, Deshou

2016-05-04

MicroRNAs (miRNAs) represent a second regulatory network that has important effects on gene expression and protein translation during biological process. However, the possible role of miRNAs in the early stages of fish sex differentiation is not well understood. In this study, we carried an integrated analysis of miRNA and mRNA expression profiles to explore their possibly regulatory patterns at the critical stage of sex differentiation in tilapia. We identified 279 pre-miRNA genes in tilapia genome, which were highly conserved in other fish species. Based on small RNA library sequencing, we identified 635 mature miRNAs in tilapia gonads, in which 62 and 49 miRNAs showed higher expression in XX and XY gonads, respectively. The predicted targets of these sex-biased miRNAs (e.g., miR-9, miR-21, miR-30a, miR-96, miR-200b, miR-212 and miR-7977) included genes encoding key enzymes in steroidogenic pathways (Cyp11a1, Hsd3b, Cyp19a1a, Hsd11b) and key molecules involved in vertebrate sex differentiation (Foxl2, Amh, Star1, Sf1, Dmrt1, and Gsdf). These genes also showed sex-biased expression in tilapia gonads at 5 dah. Some miRNAs (e.g., miR-96 and miR-737) targeted multiple genes involved in steroid synthesis, suggesting a complex miRNA regulatory network during early sex differentiation in this fish. The sequence and expression patterns of most miRNAs in tilapia are conserved in fishes, indicating the basic functions of vertebrate miRNAs might share a common evolutionary origin. This comprehensive analysis of miRNA and mRNA at the early stage of molecular sex differentiation in tilapia XX and XY gonads lead to the discovery of differentially expressed miRNAs and their putative targets, which will facilitate studies of the regulatory network of molecular sex determination and differentiation in fishes.

Serum-based six-miRNA signature as a potential marker for EC diagnosis: Comparison with TCGA miRNAseq dataset and identification of miRNA-mRNA target pairs by integrated analysis of TCGA miRNAseq and RNAseq datasets.

PubMed

Sharma, Priyanka; Saraya, Anoop; Sharma, Rinu

2018-01-30

To evaluate the diagnostic potential of a six microRNAs (miRNAs) panel consisting of miR-21, miR-144, miR-107, miR-342, miR-93 and miR-152 for esophageal cancer (EC) detection. The expression of miRNAs was analyzed in EC sera samples using quantitative real-time PCR. Risk score analysis was performed and linear regression models were then fitted to generate the six-miRNA panel. In addition, we made an effort to identify significantly dysregulated miRNAs and mRNAs in EC using the Cancer Genome Atlas (TCGA) miRNAseq and RNAseq datasets, respectively. Further, we identified significantly correlated miRNA-mRNA target pairs by integrating TCGA EC miRNAseq dataset with RNAseq dataset. The panel of circulating miRNAs showed enhanced sensitivity (87.5%) and specificity (90.48%) in terms of discriminating EC patients from normal subjects (area under the curve [AUC] = 0.968). Pathway enrichment analysis for potential targets of six miRNAs revealed 48 significant (P < 0.05) pathways, viz. pathways in cancer, mRNA surveillance, MAPK, Wnt, mTOR signaling, and so on. The expression data for mRNAs and miRNAs, downloaded from TCGA database, lead to identification of 2309 differentially expressed genes and 189 miRNAs. Gene ontology and pathway enrichment analysis showed that cell-cycle processes were most significantly enriched for differentially expressed mRNA. Integrated analysis of TCGA miRNAseq and RNAseq datasets resulted in identification of 53 063 significantly and negatively correlated miRNA-mRNA pairs. In summary, a novel and highly sensitive signature of serum miRNAs was identified for EC detection. Moreover, this is the first report identifying miRNA-mRNA target pairs from EC TCGA dataset, thus providing a comprehensive resource for understanding the interactions existing between miRNA and their target mRNAs in EC. © 2018 John Wiley & Sons Australia, Ltd.

Integrated molecular portrait of non-small cell lung cancers

PubMed Central

2013-01-01

Background Non-small cell lung cancer (NSCLC), a leading cause of cancer deaths, represents a heterogeneous group of neoplasms, mostly comprising squamous cell carcinoma (SCC), adenocarcinoma (AC) and large-cell carcinoma (LCC). The objectives of this study were to utilize integrated genomic data including copy-number alteration, mRNA, microRNA expression and candidate-gene full sequencing data to characterize the molecular distinctions between AC and SCC. Methods Comparative genomic hybridization followed by mutational analysis, gene expression and miRNA microarray profiling were performed on 123 paired tumor and non-tumor tissue samples from patients with NSCLC. Results At DNA, mRNA and miRNA levels we could identify molecular markers that discriminated significantly between the various histopathological entities of NSCLC. We identified 34 genomic clusters using aCGH data; several genes exhibited a different profile of aberrations between AC and SCC, including PIK3CA, SOX2, THPO, TP63, PDGFB genes. Gene expression profiling analysis identified SPP1, CTHRC1and GREM1 as potential biomarkers for early diagnosis of the cancer, and SPINK1 and BMP7 to distinguish between AC and SCC in small biopsies or in blood samples. Using integrated genomics approach we found in recurrently altered regions a list of three potential driver genes, MRPS22, NDRG1 and RNF7, which were consistently over-expressed in amplified regions, had wide-spread correlation with an average of ~800 genes throughout the genome and highly associated with histological types. Using a network enrichment analysis, the targets of these potential drivers were seen to be involved in DNA replication, cell cycle, mismatch repair, p53 signalling pathway and other lung cancer related signalling pathways, and many immunological pathways. Furthermore, we also identified one potential driver miRNA hsa-miR-944. Conclusions Integrated molecular characterization of AC and SCC helped identify clinically relevant markers and potential drivers, which are recurrent and stable changes at DNA level that have functional implications at RNA level and have strong association with histological subtypes. PMID:24299561

Emphysema induced by elastase alters the mRNA relative levels from DNA repair genes in acute lung injury in response to sepsis induced by lipopolysaccharide administration in Wistar rats.

PubMed

Sergio, Luiz Philippe S; Lucinda, Leda M F; Reboredo, Maycon M; de Paoli, Flavia; Fonseca, Lídia M C; Pinheiro, Bruno V; Mencalha, Andre L; Fonseca, Adenilson S

2018-03-01

Purpose/Aim of the study: Patients suffering from chronic obstructive pulmonary disease (COPD) in association with acute respiratory distress syndrome (ARDS) present oxidative stress in lung cells, with production of free radicals and DNA lesions in pulmonary and adjacent cells. Once the DNA molecule is damaged, a set of enzymatic mechanisms are trigged to preserve genetic code integrity and cellular homeostasis. These enzymatic mechanisms include the base and the nucleotide excision repair pathways, as well as telomere regulation. Thus, the aim of this work was to evaluate the mRNA levels from APEX1, ERCC2, TP53, and TRF2 genes in lung tissue from Wistar rats affected by acute lung injury in response to sepsis and emphysema. Adult male Wistar rats were randomized into 4 groups (n = 6, for each group): control, emphysema, sepsis, and emphysema with sepsis. Pulmonary emphysema was induced by intratracheal instillation of elastase (12 IU/animal) and sepsis induced by intraperitoneal Escherichia coli lipopolysaccharide (LPS) injection (10 mg/kg). Lungs were removed, and samples were withdrawn for histological analysis and total RNA extraction, cDNA synthesis, and mRNA level evaluation by real time quantitative polymerase chain reaction. Data show acute lung injury by LPS and emphysema by elastase and that APEX1, ERCC2, TP53, and TRF2 mRNA levels are increased significantly (p < 0.01) in emphysema with sepsis group. Our results suggest that alteration in mRNA levels from DNA repair and genomic stability could be part of cell response to acute lung injury in response to emphysema and sepsis.

Correlation analysis of the mRNA and miRNA expression profiles in the nascent synthetic allotetraploid Raphanobrassica

PubMed Central

Ye, Bingyuan; Wang, Ruihua; Wang, Jianbo

2016-01-01

Raphanobrassica is an allopolyploid species derived from inter-generic hybridization that combines the R genome from R. sativus and the C genome from B. oleracea var. alboglabra. In the present study, we used a high-throughput sequencing method to identify the mRNA and miRNA profiles in Raphanobrassica and its parents. A total of 33,561 mRNAs and 283 miRNAs were detected, 9,209 mRNAs and 134 miRNAs were differentially expressed respectively, 7,633 mRNAs and 39 miRNAs showed ELD expression, 5,219 mRNAs and 57 miRNAs were non-additively expressed in Raphanobrassica. Remarkably, differentially expressed genes (DEGs) were up-regulated and maternal bias was detected in Raphanobrassica. In addition, a miRNA-mRNA interaction network was constructed based on reverse regulated miRNA-mRNAs, which included 75 miRNAs and 178 mRNAs, 31 miRNAs were non-additively expressed target by 13 miRNAs. The related target genes were significantly enriched in the GO term ‘metabolic processes’. Non-additive related target genes regulation is involved in a range of biological pathways, like providing a driving force for variation and adaption in this allopolyploid. The integrative analysis of mRNA and miRNA profiling provides more information to elucidate gene expression mechanism and may supply a comprehensive and corresponding method to study genetic and transcription variation of allopolyploid. PMID:27874043

Buffer-dependent regulation of aquaporin-1 expression and function in human peritoneal mesothelial cells.

PubMed

Zhai, Yihui; Bloch, Jacek; Hömme, Meike; Schaefer, Julia; Hackert, Thilo; Philippin, Bärbel; Schwenger, Vedat; Schaefer, Franz; Schmitt, Claus P

2012-07-01

Biocompatible peritoneal dialysis fluids (PDF) are buffered with lactate and/or bicarbonate. We hypothesized that the reduced toxicity of the biocompatible solutions might unmask specific effects of the buffer type on mesothelial cell functions. Human peritoneal mesothelial cells (HPMC) were incubated with bicarbonate (B-)PDF or lactate-buffered (L-)PDF followed by messenger RNA (mRNA) and protein analysis. Gene silencing was achieved using small interfering RNA (siRNA), functional studies using Transwell culture systems, and monolayer wound-healing assays. Incubation with B-PDF increased HPMC migration in the Transwell and monolayer wound-healing assay to 245 ± 99 and 137 ± 11% compared with L-PDF. Gene silencing showed this effect to be entirely dependent on the expression of aquaporin-1 (AQP-1) and independent of AQP-3. Exposure of HPMC to B-PDF increased AQP-1 mRNA and protein abundance to 209  ± 80 and 197  ±  60% of medium control; the effect was pH dependent. L-PDF reduced AQP-1 mRNA. Addition of bicarbonate to L-PDF increased AQP-1 abundance by threefold; mRNA half-life remained unchanged. Immunocytochemistry confirmed opposite changes of AQP-1 cell-membrane abundance with B-PDF and L-PDF. Peritoneal mesothelial AQP-1 abundance and migration capacity is regulated by pH and buffer agents used in PD solutions. In vivo studies are required to delineate the impact with respect to long-term peritoneal membrane integrity and function.

Determination of the Absolute Number of Cytokine mRNA Molecules within Individual Activated Human T Cells

NASA Technical Reports Server (NTRS)

Karr, Laurel J.; Marshall, Gwen; Hockett, Richard D.; Bucy, R. Pat; Curreri, Peter A. (Technical Monitor)

2002-01-01

A primary function of activated T cells is the expression and subsequent secretion of cytokines, which orchestrate the differentiation of other lymphocytes, modulate antigen presenting cell activity, and alter vascular endothelium to mediate an immune response. Since many features of immune regulation probably result from modest alterations of endogenous rates of multiple interacting processes, quantitative analysis of the frequency and specific activity of individual T cells is critically important. Using a coordinated set of quantitative methods, the absolute number of molecules of several key cytokine mRNA species in individual T cells has been determined. The frequency of human blood T cells activated in vitro by mitogens and recall protein antigens was determined by intracellular cytokine protein staining, in situ hybridization for cytokine mRNA, and by limiting dilution analysis for cytokine mRNA+ cells. The absolute number of mRNA molecules was simultaneously determined in both homogenates of the entire population of cells and in individual cells obtained by limiting dilution, using a quantitative, competitive RT-PCR assay. The absolute numbers of mRNA molecules in a population of cells divided by the frequency of individual positive cells, yielded essentially the same number of mRNA molecules per cell as direct analysis of individual cells by limiting dilution analysis. Mean numbers of mRNA per positive cell from both mitogen and antigen activated T cells, using these stimulation conditions, were 6000 for IL-2, 6300 for IFN-gamma, and 1600 for IL-4.

Interdependence between transcription and mRNP processing and export, and its impact on genetic stability.

PubMed

Luna, Rosa; Jimeno, Sonia; Marín, Mercedes; Huertas, Pablo; García-Rubio, María; Aguilera, Andrés

2005-06-10

The conserved eukaryotic THO-TREX complex acts at the interface between transcription and mRNA export and affects transcription-associated recombination. To investigate the interdependence of nuclear mRNA processes and their impact on genomic integrity, we analyzed transcript accumulation and recombination of 40 selected mutants covering representative steps of the biogenesis and export of the messenger ribonucleoprotein particle (mRNP). None of the mutants analyzed shared the strong transcript-accumulation defect and hyperrecombination of THO mutants. Nevertheless, mutants in 3' end cleavage/polyadenylation, nuclear exosome, and mRNA export showed a weak but significant effect on recombination and transcript accumulation. Mutants of the nuclear exosome (rrp6) and 3' end processing factors (rna14 and rna15) showed inefficient transcription elongation and genetic interactions with THO. The results suggest a tight interdependence among mRNP biogenesis steps and transcription and an unexpected effect of the nuclear exosome and the cleavage/polyadenylation factors on transcription elongation and genetic integrity.

Magnesium Induced Nucleophile Activation in the Guanylyltransferase mRNA Capping Enzyme

PubMed Central

Swift, Robert V.; Ong, Chau D.; Amaro, Rommie E.

2012-01-01

The messenger RNA guanylyltransferase, or mRNA capping enzyme, co-transcriptionally caps the 5′-end of nascent mRNA with GMP during the second in a set of three enzymatic reactions that result in the formation of an N7-methyl guanosine cap during mRNA maturation. The mRNA capping enzyme is characterized, in part, by a conserved lysine nucleophile that attacks the alpha-phosphorous atom of GTP, forming a lysine-GMP intermediate. Experiments have firmly established that magnesium is required for efficient intermediate formation, but have provided little insight into the requirement’s molecular origins. Using empirical and thermodynamic integration pKa estimates, along with conventional MD simulations, we show that magnesium binding likely activates the lysine nucleophile by increasing its acidity and by biasing the deprotonated nucleophile into conformations conducive to intermediate formation. These results provide additional functional understanding of an important enzyme in the mRNA transcript life cycle and allow functional analogies to be drawn that affect our understanding of the metal dependence of related superfamily members. PMID:23205906

In silico analysis of stomach lineage specific gene set expression pattern in gastric cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pandi, Narayanan Sathiya, E-mail: sathiyapandi@gmail.com; Suganya, Sivagurunathan; Rajendran, Suriliyandi

Highlights: •Identified stomach lineage specific gene set (SLSGS) was found to be under expressed in gastric tumors. •Elevated expression of SLSGS in gastric tumor is a molecular predictor of metabolic type gastric cancer. •In silico pathway scanning identified estrogen-α signaling is a putative regulator of SLSGS in gastric cancer. •Elevated expression of SLSGS in GC is associated with an overall increase in the survival of GC patients. -- Abstract: Stomach lineage specific gene products act as a protective barrier in the normal stomach and their expression maintains the normal physiological processes, cellular integrity and morphology of the gastric wall. However,more » the regulation of stomach lineage specific genes in gastric cancer (GC) is far less clear. In the present study, we sought to investigate the role and regulation of stomach lineage specific gene set (SLSGS) in GC. SLSGS was identified by comparing the mRNA expression profiles of normal stomach tissue with other organ tissue. The obtained SLSGS was found to be under expressed in gastric tumors. Functional annotation analysis revealed that the SLSGS was enriched for digestive function and gastric epithelial maintenance. Employing a single sample prediction method across GC mRNA expression profiles identified the under expression of SLSGS in proliferative type and invasive type gastric tumors compared to the metabolic type gastric tumors. Integrative pathway activation prediction analysis revealed a close association between estrogen-α signaling and SLSGS expression pattern in GC. Elevated expression of SLSGS in GC is associated with an overall increase in the survival of GC patients. In conclusion, our results highlight that estrogen mediated regulation of SLSGS in gastric tumor is a molecular predictor of metabolic type GC and prognostic factor in GC.« less

Impact of gestational chronodisruption on fetal cardiac genomics.

PubMed

Galdames, Hugo A; Torres-Farfan, Claudia; Spichiger, Carlos; Mendez, Natalia; Abarzua-Catalan, Lorena; Alonso-Vazquez, Pamela; Richter, Hans G

2014-01-01

We recently reported that gestational chronodisruption induces fetal growth restriction and marked effects on fetal adrenal physiology. Here, whole-transcriptome profiling was used to test whether gestational chronodisruption modifies gene expression in the fetal heart, potentially altering cardiac development. At day 10 of gestation (E10), pregnant rats were randomized in two groups: constant light (LL) and control 12 h light/12 h dark photoperiod (LD). RNA isolated from E18 heart was subjected to microarray analysis (Affymetrix platform for 28,000 genes). Integrated transcriptional changes were assessed by gene ontology and pathway analysis. Significant differential expression was found for 383 transcripts in LL relative to LD fetal heart (280 up-regulated and 103 down-regulated); with 42 of them displaying a 1.5-fold or greater change in gene expression. Deregulated markers of cardiovascular disease accounted for alteration of diverse gene networks in LL fetal heart, including local steroidogenesis and vascular calcification, as well as cardiac hypertrophy, stenosis and necrosis/cell death. DNA integrity was also overrepresented, including a 2.1-fold increase of Hmga1 mRNA, which encodes for a profuse architectural transcription factor. microRNA analysis revealed up-regulation of miRNAs 218-1 and 501 and concurrent down-regulation of their validated target genes. In addition, persistent down-regulation of Kcnip2 mRNA and hypertrophy of the left ventricle were found in the heart from 90 days-old offspring from LL mothers. The dysregulation of a relevant fraction of the fetal cardiac transcriptome, together with the diversity and complexity of the gene networks altered by gestational chronodisruption, suggest enduring molecular changes which may shape the hypertrophy observed in the left ventricle of adult LL offspring. © 2013.

Fox-2 protein regulates the alternative splicing of scleroderma-associated lysyl hydroxylase 2 messenger RNA.

PubMed

Seth, Puneet; Yeowell, Heather N

2010-04-01

Scleroderma (systemic sclerosis [SSc]) is a complex connective tissue disorder characterized by hardening and thickening of the skin. One hallmark of scleroderma is excessive accumulation of collagen accompanied by increased levels of pyridinoline collagen crosslinks derived from hydroxylysine residues in the collagen telopeptide domains. Lysyl hydroxylase 2 (LH2), an important alternatively spliced enzyme in collagen biosynthesis, acts as a collagen telopeptide hydroxylase. Changes in the pattern of LH2 alternative splicing, favoring increased inclusion of the alternatively spliced LH2 exon 13A, thereby increasing the levels of the long transcript of LH2 (LH2[long]), are linked to scleroderma disease. This study was undertaken to examine the role played by RNA binding protein Fox-2 in regulating exon 13A inclusion, which leads to the generation of scleroderma-associated LH2(long) messenger RNA (mRNA). Phylogenetic sequence analysis of introns flanking exon 13A was performed. A tetracycline-inducible system in T-Rex 293 cells was used to induce Fox-2 protein, and endogenous LH2(long) mRNA was determined by reverse transcriptase-polymerase chain reaction. An LH2 minigene was designed, validated, and used in Fox-2 overexpression and mutagenesis experiments. Knockdown of Fox-2 was performed in mouse embryonic fibroblasts and in fibroblasts from SSc patients. Overexpression of Fox-2 enhanced the inclusion of exon 13A and increased the generation of LH2(long) mRNA, whereas knockdown of Fox-2 decreased LH2(long) transcripts. Mutational analysis of an LH2 minigene demonstrated that 2 of the 4 Fox binding motifs flanking LH2 exon 13A are required for inclusion of exon 13A. In early passage fibroblasts derived from patients with scleroderma, the knockdown of Fox-2 protein significantly decreased the endogenous levels of LH2(long) mRNA. Our findings indicate that Fox-2 plays an integral role in the regulation of LH2 splicing. Knockdown of Fox-2 and other methods to decrease the levels of fibrosis-associated LH2(long) mRNA in primary scleroderma cells may suggest a novel approach to strategies directed against scleroderma.

Lymphotoxin-beta receptor blockade reduces CXCL13 in lacrimal glands and improves corneal integrity in the NOD model of Sjögren's syndrome

PubMed Central

2011-01-01

Introduction In Sjögren's syndrome, keratoconjunctivitis sicca (dry eye) is associated with infiltration of lacrimal glands by leukocytes and consequent losses of tear-fluid production and the integrity of the ocular surface. We investigated the effect of blockade of the lymphotoxin-beta receptor (LTBR) pathway on lacrimal-gland pathology in the NOD mouse model of Sjögren's syndrome. Methods Male NOD mice were treated for up to ten weeks with an antagonist, LTBR-Ig, or control mouse antibody MOPC-21. Extra-orbital lacrimal glands were analyzed by immunohistochemistry for high endothelial venules (HEV), by Affymetrix gene-array analysis and real-time PCR for differential gene expression, and by ELISA for CXCL13 protein. Leukocytes from lacrimal glands were analyzed by flow-cytometry. Tear-fluid secretion-rates were measured and the integrity of the ocular surface was scored using slit-lamp microscopy and fluorescein isothiocyanate (FITC) staining. The chemokine CXCL13 was measured by ELISA in sera from Sjögren's syndrome patients (n = 27) and healthy controls (n = 30). Statistical analysis was by the two-tailed, unpaired T-test, or the Mann-Whitney-test for ocular integrity scores. Results LTBR blockade for eight weeks reduced B-cell accumulation (approximately 5-fold), eliminated HEV in lacrimal glands, and reduced the entry rate of lymphocytes into lacrimal glands. Affymetrix-chip analysis revealed numerous changes in mRNA expression due to LTBR blockade, including reduction of homeostatic chemokine expression. The reduction of CXCL13, CCL21, CCL19 mRNA and the HEV-associated gene GLYCAM-1 was confirmed by PCR analysis. CXCL13 protein increased with disease progression in lacrimal-gland homogenates, but after LTBR blockade for 8 weeks, CXCL13 was reduced approximately 6-fold to 8.4 pg/mg (+/- 2.7) from 51 pg/mg (+/-5.3) in lacrimal glands of 16 week old control mice. Mice given LTBR blockade exhibited an approximately two-fold greater tear-fluid secretion than control mice (P = 0.001), and had a significantly improved ocular surface integrity score (P = 0.005). The mean CXCL13 concentration in sera from Sjögren's patients (n = 27) was 170 pg/ml, compared to 92.0 pg/ml for sera from (n = 30) healthy controls (P = 0.01). Conclusions Blockade of LTBR pathways may have therapeutic potential for treatment of Sjögren's syndrome. PMID:22044682

Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells

PubMed Central

Lee, Jinwoo; Tong, Tiegang; Duan, Haichuan; Foong, Yee Hoon; Musaitif, Ibrahim; Yamazaki, Takeshi; Jefcoate, Colin

2016-01-01

The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb StAR mRNA molecules via dual hybridization at the 3′- and 5′-ends and reveals an unexpectedly high frequency of 1:1 pairing with mitochondria marked by the matrix StAR protein. This pairing may be central to translation-coupled cholesterol transfer. Altogether, our results show that adrenal cells exhibit high-efficiency StAR activity that needs to integrate rapid cholesterol transfer with homeostasis and pulsatile hormonal stimulation. StAR NBD, the extended 3.5-kb mRNA, SIK1, and Tis11b play important roles. PMID:27531991

Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells.

PubMed

Lee, Jinwoo; Tong, Tiegang; Duan, Haichuan; Foong, Yee Hoon; Musaitif, Ibrahim; Yamazaki, Takeshi; Jefcoate, Colin

2016-01-01

The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb StAR mRNA molecules via dual hybridization at the 3'- and 5'-ends and reveals an unexpectedly high frequency of 1:1 pairing with mitochondria marked by the matrix StAR protein. This pairing may be central to translation-coupled cholesterol transfer. Altogether, our results show that adrenal cells exhibit high-efficiency StAR activity that needs to integrate rapid cholesterol transfer with homeostasis and pulsatile hormonal stimulation. StAR NBD, the extended 3.5-kb mRNA, SIK1, and Tis11b play important roles.

Integrated RNA-Seq and sRNA-Seq Analysis Identifies Chilling and Freezing Responsive Key Molecular Players and Pathways in Tea Plant (Camellia sinensis)

PubMed Central

Zheng, Chao; Zhao, Lei; Wang, Yu; Shen, Jiazhi; Zhang, Yinfei; Jia, Sisi; Li, Yusheng; Ding, Zhaotang

2015-01-01

Tea [Camellia sinensis (L) O. Kuntze, Theaceae] is one of the most popular non-alcoholic beverages worldwide. Cold stress is one of the most severe abiotic stresses that limit tea plants’ growth, survival and geographical distribution. However, the genetic regulatory network and signaling pathways involved in cold stress responses in tea plants remain unearthed. Using RNA-Seq, DGE and sRNA-Seq technologies, we performed an integrative analysis of miRNA and mRNA expression profiling and their regulatory network of tea plants under chilling (4℃) and freezing (-5℃) stress. Differentially expressed (DE) miRNA and mRNA profiles were obtained based on fold change analysis, miRNAs and target mRNAs were found to show both coherent and incoherent relationships in the regulatory network. Furthermore, we compared several key pathways (e.g., ‘Photosynthesis’), GO terms (e.g., ‘response to karrikin’) and transcriptional factors (TFs, e.g., DREB1b/CBF1) which were identified as involved in the early chilling and/or freezing response of tea plants. Intriguingly, we found that karrikins, a new group of plant growth regulators, and β-primeverosidase (BPR), a key enzyme functionally relevant with the formation of tea aroma might play an important role in both early chilling and freezing response of tea plants. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis further confirmed the results from RNA-Seq and sRNA-Seq analysis. This is the first study to simultaneously profile the expression patterns of both miRNAs and mRNAs on a genome-wide scale to elucidate the molecular mechanisms of early responses of tea plants to cold stress. In addition to gaining a deeper insight into the cold resistant characteristics of tea plants, we provide a good case study to analyse mRNA/miRNA expression and profiling of non-model plant species using next-generation sequencing technology. PMID:25901577

Integrated RNA-Seq and sRNA-Seq Analysis Identifies Chilling and Freezing Responsive Key Molecular Players and Pathways in Tea Plant (Camellia sinensis).

PubMed

Zheng, Chao; Zhao, Lei; Wang, Yu; Shen, Jiazhi; Zhang, Yinfei; Jia, Sisi; Li, Yusheng; Ding, Zhaotang

2015-01-01

Tea [Camellia sinensis (L) O. Kuntze, Theaceae] is one of the most popular non-alcoholic beverages worldwide. Cold stress is one of the most severe abiotic stresses that limit tea plants' growth, survival and geographical distribution. However, the genetic regulatory network and signaling pathways involved in cold stress responses in tea plants remain unearthed. Using RNA-Seq, DGE and sRNA-Seq technologies, we performed an integrative analysis of miRNA and mRNA expression profiling and their regulatory network of tea plants under chilling (4℃) and freezing (-5℃) stress. Differentially expressed (DE) miRNA and mRNA profiles were obtained based on fold change analysis, miRNAs and target mRNAs were found to show both coherent and incoherent relationships in the regulatory network. Furthermore, we compared several key pathways (e.g., 'Photosynthesis'), GO terms (e.g., 'response to karrikin') and transcriptional factors (TFs, e.g., DREB1b/CBF1) which were identified as involved in the early chilling and/or freezing response of tea plants. Intriguingly, we found that karrikins, a new group of plant growth regulators, and β-primeverosidase (BPR), a key enzyme functionally relevant with the formation of tea aroma might play an important role in both early chilling and freezing response of tea plants. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis further confirmed the results from RNA-Seq and sRNA-Seq analysis. This is the first study to simultaneously profile the expression patterns of both miRNAs and mRNAs on a genome-wide scale to elucidate the molecular mechanisms of early responses of tea plants to cold stress. In addition to gaining a deeper insight into the cold resistant characteristics of tea plants, we provide a good case study to analyse mRNA/miRNA expression and profiling of non-model plant species using next-generation sequencing technology.

Integrated mRNA and microRNA transcriptome analyses reveal regulation of thermal acclimation in Gymnocypris przewalskii: A case study in Tibetan Schizothoracine fish

PubMed Central

Tian, Fei; Zhao, Kai

2017-01-01

Environmental acclimation is important episode in wildlife occupation of the high-altitude Tibetan Plateau (TP). Transcriptome-wide studies on thermal acclimation mechanism in fish species are rarely revealed in Tibetan Plateau fish at high altitude. Thus, we used mRNA and miRNA transcriptome sequencing to investigate regulation of thermal acclimation in larval Tibetan naked carp, Gymnocypris przewalskii. We first remodeled the regulation network of mRNA and miRNA in thermal acclimation, and then identified differential expression of miRNAs and target mRNAs enriched in metabolic and digestive pathways. Interestingly, we identified two candidate genes contributed to normal skeletal development. The altered expression of these gene groups could potentially be associated with the developmental issues of deformity and induced larval death. Our results have three important implications: first, these findings provide strong evidences to support our hypothesis that G. przewalskii possess ability to build heat-tolerance against the controversial issue. Second, this study shows that transcriptional and post-transcriptional regulations are extensively involved in thermal acclimation. Third, the integrated mRNA and microRNA transcriptome analyses provide a large number of valuable genetic resources for future studies on environmental stress response in G. przewalskii and as a case study in Tibetan Schizothoracine fish. PMID:29045433

University of California San Francisco (UCSF-2): Integrative Genomic Approaches in Neuroblastoma (NBL) | Office of Cancer Genomics

Cancer.gov

The CTD2 Center at University of California San Francisco (UCSF-2) used an integrative genomics approach to reveal unidentified mRNA splicing patterns in neuroblastoma. Read the abstract Experimental Approaches Read the detailed Experimental Approaches

The response of claudin-like transmembrane septate junction proteins to altered environmental ion levels in the larval mosquito Aedes aegypti.

PubMed

Jonusaite, Sima; Kelly, Scott P; Donini, Andrew

2016-07-01

Septate junctions (SJs) occlude the paracellular pathway and function as paracellular diffusion barriers within invertebrate epithelia. However, integral components of SJs and their contribution to barrier properties have received considerably less attention than those of vertebrate occluding junctions. In arthropods, SJ proteins have only been identified in Drosophila and among these are three integral claudin-like proteins, Megatrachea (Mega), Sinuous (Sinu) and Kune-kune (Kune), as well as a receptor-like transmembrane SJ protein known as Neurexin IV (Nrx IV). In this study, mega, sinu, kune and nrx IV are identified and characterized in aquatic larvae of the mosquito Aedes aegypti and a role for these proteins in ionoregulatory homeostasis is considered. Transcripts encoding Mega, Sinu, Kune and Nrx IV were found in iono/osmoregulatory tissues such as the midgut, Malpighian tubules, hindgut and anal papillae, but abundance was greater in the hindgut and anal papillae. Using immunohistochemical and western blot analysis it was found that Kune localized to the regions of intercellular contact between epithelial cells of the rectum and posterior midgut and in the apical membrane domain of the syncytial epithelium of anal papillae. To investigate a potential role for integral SJ proteins in larval A. aegypti iono/osmoregulation, abundance was examined in animals reared in freshwater or brackish water (30 % seawater). In iono/osmoregulatory epithelia, larvae exhibited tissue-specific alterations in mega mRNA and Kune protein abundance, but not sinu or nrx IV mRNA. These studies provide a first look at the potential contribution of integral SJ components to iono/osmoregulatory homeostasis in an aquatic invertebrate.

Complexities and sequence similarities of mRNA populations of cholinergic (NS20-Y) and adrenergic (N1E-115) murine neuroblastoma cell lines.

PubMed

Strauss, W L

1990-07-01

The clonal murine neuroblastoma cell lines NS20-Y and N1E-115 have been proposed as models for examining the commitment of neural crest cells to either the cholinergic or adrenergic phenotype, respectively. The validity of this model depends in part on the extent to which these two cell lines have diverged as a result of their transformed, rather than neuronal properties. In order to quantitate differences in gene expression between NS20-Y and N1E-115 cells, the mRNA complexity of each cell type was determined. An analysis of the kinetics of hybridization of NS20-Y cell mRNA with cDNA prepared from NS20-Y cell mRNA demonstrated the presence of approximately 11,700 mRNA species assuming an average length of 1900 nucleotides. A similar analysis using mRNA isolated from N1E-115 cells and cDNA prepared from N1E-115 cell mRNA demonstrated that the adrenergic cell line expressed approximately 11,600 mRNA species. The species of mRNA expressed by each cell line were resolved into high, intermediate, and low abundance populations. In order to determine whether mRNAs were expressed by the cholinergic, but not by the adrenergic cell line, NS20-Y cDNA was hybridized to an excess of N1E-115 cell mRNA. An analysis of the solution hybridization kinetics from this procedure demonstrated that the two cell lines do not differ significantly in the nucleotide complexity of their mRNA populations. The extensive similarity between the two mRNA populations suggests that only a small number of genes are expressed differentially between the two cell lines and supports their use as models for the differentiation of cholinergic and adrenergic neurons.

Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone

PubMed Central

Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

2016-01-01

Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

Analysis of the 3’ untranslated regions of α-tubulin and S-crystallin mRNA and the identification of CPEB in dark- and light-adapted octopus retinas

PubMed Central

Kelly, Shannan; Yamamoto, Hideki

2008-01-01

Purpose We previously reported the differential expression and translation of mRNA and protein in dark- and light-adapted octopus retinas, which may result from cytoplasmic polyadenylation element (CPE)–dependent mRNA masking and unmasking. Here we investigate the presence of CPEs in α-tubulin and S-crystallin mRNA and report the identification of cytoplasmic polyadenylation element binding protein (CPEB) in light- and dark-adapted octopus retinas. Methods 3’-RACE and sequencing were used to isolate and analyze the 3’-UTRs of α-tubulin and S-crystallin mRNA. Total retinal protein isolated from light- and dark-adapted octopus retinas was subjected to western blot analysis followed by CPEB antibody detection, PEP-171 inhibition of CPEB, and dephosphorylation of CPEB. Results The following CPE-like sequence was detected in the 3’-UTR of isolated long S-crystallin mRNA variants: UUUAACA. No CPE or CPE-like sequences were detected in the 3’-UTRs of α-tubulin mRNA or of the short S-crystallin mRNA variants. Western blot analysis detected CPEB as two putative bands migrating between 60-80 kDa, while a third band migrated below 30 kDa in dark- and light-adapted retinas. Conclusions The detection of CPEB and the identification of the putative CPE-like sequences in the S-crystallin 3’-UTR suggest that CPEB may be involved in the activation of masked S-crystallin mRNA, but not in the regulation of α-tubulin mRNA, resulting in increased S-crystallin protein synthesis in dark-adapted octopus retinas. PMID:18682811

Intercellular communication in the immune system: differential expression of connexin40 and 43, and perturbation of gap junction channel functions in peripheral blood and tonsil human lymphocyte subpopulations

PubMed Central

Oviedo‐orta, E; Hoy, T; Evans, W H

2000-01-01

The distribution and function of connexins (integral membrane proteins assembled into gap junction intercellular communication channels) were studied in human lymphocyte subpopulations. The expression of mRNA encoding connexins in peripheral blood and tonsil‐derived T, B and natural killer (NK) lymphocytes was examined. Connexin43 (Cx43) mRNA was expressed in peripheral blood and tonsil lymphocytes, but Cx40 mRNA expression was confined to tonsil‐derived T and B lymphocytes; Cx26, Cx32, Cx37 and Cx45 were not detected by reverse transcription–polymerase chain reaction (RT–PCR). Western blot analysis also demonstrated the presence of Cx40 and Cx43 proteins in T and B lymphocytes in a manner coincidental to the mRNA detection. Stimulation in vitro of T and B lymphocytes with phytohaemagglutinin (PHA) and lipopolysaccharide (LPS), respectively, increased Cx40 and Cx43 protein expression. Flow cytometric analysis, using antibodies to extracellular loop amino acid sequences of connexins, confirmed the surface expression of connexins in all lymphocyte subpopulations. Assembly of connexins into gap junctions providing direct intercellular channels linking attached lymphocytes was demonstrated by using a dye transfer technique. The exchange of dye between lymphocytes was inhibited by a connexin extracellular loop mimetic peptide and α‐glycyrrhetinic acid, two reagents that restrict intercellular communication across gap junctions. Dye coupling occurred between homologous and heterologous co‐cultures of T and B lymphocytes, and was not influenced by their stimulation with PHA and LPS. The connexin mimetic peptide caused a significant decrease in the in vitro synthesis of immunoglobulin M (IgM) by T‐ and B‐lymphocyte co‐cultured populations in the presence or absence of stimulation by PHA. The results identify connexins as important cell surface components that modulate immune processes. PMID:10792506

Increased expression of transforming growth factor beta s after acute oedematous pancreatitis in rats suggests a role in pancreatic repair.

PubMed Central

Riesle, E; Friess, H; Zhao, L; Wagner, M; Uhl, W; Baczako, K; Gold, L I; Korc, M; Büchler, M W

1997-01-01

BACKGROUND: Transforming growth factor beta isoforms (TGF beta s) belong to a family of multifunctional regulators of cellular growth and differentiation. They are mitogenic and chemotactic for fibroblasts and are potent stimulators of extracellular matrix production (collagen) and deposition. Upregulation of TGF beta transcription has been reported for several in vivo systems during repair after injury. AIMS: To study the expression of the three mammalian isoforms of TGF beta (TGF beta 1-3) and their relation to collagen expression as a marker for fibroblast response in acute oedematous pancreatitis in rats. METHODS: Using northern blot analysis and immunohistochemistry, the expression and localisation of TGF beta isoforms, collagen, and amylase were analysed during the course of acute oedematous pancreatitis in rats, experimentally induced by intravenous caerulein infusion. RESULTS: Induction of acute pancreatitis resulted in a biphasic peak pattern of expression of TGF beta 1, beta 2, and beta 3 mRNA, with a pronounced increase from day 1 to day 3 (sixfold, 2.5-fold, fivefold, respectively) and again from day 5 to day 7 (three-fold, 2.3-fold, 3.5-fold, respectively). The temporal changes in TGF beta mRNA identically paralleled the expression in collagen mRNA. In contrast, amylase mRNA expression, used as a general indicator of acinar cell integrity, was slightly decreased after induction of acute pancreatitis. Immunohistochemical analysis of pancreatitis tissue showed that increased expression of TGF beta s was mainly present in the pancreatic acinar and ductal cells; this was evident within one day after pancreatitis induction. CONCLUSION: Overexpression of TGF beta s after induction of acute pancreatitis suggests a role for these proteins in pancreatic repair and remodelling. The increased levels of TGF beta s may help suppress immune activation, and may contribute to the increase in the extracellular matrix including collagen and to the repair of the pancreatic parenchyma. Images PMID:9155579

Downregulation of aquaporin 9 decreases catabolic factor expression through nuclear factor‑κB signaling ins chondrocytes.

PubMed

Takeuchi, Kazuhiro; Hayashi, Shinya; Matumoto, Tomoyuki; Hashimoto, Shingo; Takayama, Koji; Chinzei, Nobuaki; Kihara, Shinsuke; Haneda, Masahiko; Kirizuki, Shinsuke; Kuroda, Yuichi; Tsubosaka, Masanori; Nishida, Kotaro; Kuroda, Ryosuke

2018-06-13

Aquaporins (AQPs) are small integral membrane proteins that are essential for water transport across membranes. AQP9, one of the 13 mammalian AQPs (including AQP0 to 12), has been reported to be highly expressed in hydrarthrosis and synovitis patients. Given that several studies have identified signal transduction as an additional function of AQPs, it is hypothesized that AQP9 may modulate inflammatory signal transduction in chondrocytes. Therefore, the present study used a model of interleukin (IL)‑1β‑induced inflammation to determine the mechanisms associated with AQP9 functions in chondrocytes. Osteoarthritis (OA) and normal cartilage samples were subjected to immunohistological analysis. In addition, matrix metalloproteinase (MMP)3, MMP13 and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS‑5) mRNA and protein analysis was conducted in normal human articular chondrocytes from the knee (NHAC‑Kn) stimulated with IL‑1β by reverse transcription‑polymerase chain reaction (RT‑qPCR) and western blotting, respectively. AQP9 knockdown was also performed by transfection of AQP9‑specific small interfering RNA using Lipofectamine. AQP1, 3, 7, 9 and 11 mRNA expression levels were detected in OA human chondrocytes and in IL‑1β‑treated normal human chondrocytes. The levels of AQP9, MMP‑3, MMP‑13 and ADAMTS‑5 mRNA were increased by treatment with 10 ng/ml IL‑1β in a time‑dependent manner, while knockdown of AQP9 expression significantly decreased the mRNA levels of the MMP3, MMP13 and ADAMTS‑5 genes, as well as the phosphorylation of IκB kinase (IKK). Treatment with a specific IKK inhibitor also significantly decreased the expression levels of MMP‑3, MMP‑13 and ADAMTS‑5 in response to IL‑1β stimulation. Furthermore, immunohistochemical analysis demonstrated that AQP9 and inflammatory markers were highly expressed in OA cartilage. In addition, the downregulation of AQP9 in cultured chondrocytes decreased the catabolic gene expression in response to IL‑1β stimulation through nuclear factor‑κB signaling. Therefore, AQP9 may be a promising target for the treatment of OA.

Detection of carcinoembryonic antigen messenger RNA in blood using quantitative real-time reverse transcriptase-polymerase chain reaction to predict recurrence of gastric adenocarcinoma

PubMed Central

2010-01-01

Background The existence of circulating tumor cells (CTCs) in peripheral blood as an indicator of tumor recurrence has not been clearly established, particularly for gastric cancer patients. We conducted a retrospective analysis of the relationship between CTCs in peripheral blood at initial diagnosis and clinicopathologic findings in patients with gastric carcinoma. Methods Blood samples were obtained from 123 gastric carcinoma patients at initial diagnosis. mRNA was extracted and amplified for carcinoembryonic antigen (CEA) mRNA detection using real-time RT-PCR. Periodic 3-month follow-up examinations included serum CEA measurements and imaging. Results The minimum threshold for corrected CEA mRNA score [(CEA mRNA/GAPDH mRNA) × 106] was set at 100. Forty-five of 123 patients (36.6%) were positive for CEA mRNA expression. CEA mRNA expression significantly correlated with T stage and postoperative recurrence status (P = 0.001). Recurrent disease was found in 44 of 123 cases (35.8%), and 25 of these (56.8%) were positive for CEA mRNA. Of these patients, CEA mRNA was more sensitive than serum CEA in indicating recurrence. Three-year disease-free survival of patients positive for CEA mRNA was significantly poorer than of patients negative for CEA mRNA (P < 0.001). Only histological grade and CEA mRNA positivity were independent factors for disease-free survival using multivariate analysis. Conclusions CEA mRNA copy number in peripheral blood at initial diagnosis was significantly associated with disease recurrence in gastric adenocarcinoma patients. Real-time RT-PCR detection of CEA mRNA levels at initial diagnosis appears to be a promising predictor for disease recurrence in gastric adenocarcinoma patients. PMID:21040522

Detection of carcinoembryonic antigen messenger RNA in blood using quantitative real-time reverse transcriptase-polymerase chain reaction to predict recurrence of gastric adenocarcinoma.

PubMed

Qiu, Miao-Zhen; Li, Zhuang-Hua; Zhou, Zhi-Wei; Li, Yu-Hong; Wang, Zhi-Qiang; Wang, Feng-Hua; Huang, Peng; Aziz, Fahad; Wang, Dao-Yuan; Xu, Rui-Hua

2010-10-31

The existence of circulating tumor cells (CTCs) in peripheral blood as an indicator of tumor recurrence has not been clearly established, particularly for gastric cancer patients. We conducted a retrospective analysis of the relationship between CTCs in peripheral blood at initial diagnosis and clinicopathologic findings in patients with gastric carcinoma. Blood samples were obtained from 123 gastric carcinoma patients at initial diagnosis. mRNA was extracted and amplified for carcinoembryonic antigen (CEA) mRNA detection using real-time RT-PCR. Periodic 3-month follow-up examinations included serum CEA measurements and imaging. The minimum threshold for corrected CEA mRNA score [(CEA mRNA/GAPDH mRNA) × 106] was set at 100. Forty-five of 123 patients (36.6%) were positive for CEA mRNA expression. CEA mRNA expression significantly correlated with T stage and postoperative recurrence status (P = 0.001). Recurrent disease was found in 44 of 123 cases (35.8%), and 25 of these (56.8%) were positive for CEA mRNA. Of these patients, CEA mRNA was more sensitive than serum CEA in indicating recurrence. Three-year disease-free survival of patients positive for CEA mRNA was significantly poorer than of patients negative for CEA mRNA (P < 0.001). Only histological grade and CEA mRNA positivity were independent factors for disease-free survival using multivariate analysis. CEA mRNA copy number in peripheral blood at initial diagnosis was significantly associated with disease recurrence in gastric adenocarcinoma patients. Real-time RT-PCR detection of CEA mRNA levels at initial diagnosis appears to be a promising predictor for disease recurrence in gastric adenocarcinoma patients.

Integrative omics analysis. A study based on Plasmodium falciparum mRNA and protein data.

PubMed

Tomescu, Oana A; Mattanovich, Diethard; Thallinger, Gerhard G

2014-01-01

Technological improvements have shifted the focus from data generation to data analysis. The availability of large amounts of data from transcriptomics, protemics and metabolomics experiments raise new questions concerning suitable integrative analysis methods. We compare three integrative analysis techniques (co-inertia analysis, generalized singular value decomposition and integrative biclustering) by applying them to gene and protein abundance data from the six life cycle stages of Plasmodium falciparum. Co-inertia analysis is an analysis method used to visualize and explore gene and protein data. The generalized singular value decomposition has shown its potential in the analysis of two transcriptome data sets. Integrative Biclustering applies biclustering to gene and protein data. Using CIA, we visualize the six life cycle stages of Plasmodium falciparum, as well as GO terms in a 2D plane and interpret the spatial configuration. With GSVD, we decompose the transcriptomic and proteomic data sets into matrices with biologically meaningful interpretations and explore the processes captured by the data sets. IBC identifies groups of genes, proteins, GO Terms and life cycle stages of Plasmodium falciparum. We show method-specific results as well as a network view of the life cycle stages based on the results common to all three methods. Additionally, by combining the results of the three methods, we create a three-fold validated network of life cycle stage specific GO terms: Sporozoites are associated with transcription and transport; merozoites with entry into host cell as well as biosynthetic and metabolic processes; rings with oxidation-reduction processes; trophozoites with glycolysis and energy production; schizonts with antigenic variation and immune response; gametocyctes with DNA packaging and mitochondrial transport. Furthermore, the network connectivity underlines the separation of the intraerythrocytic cycle from the gametocyte and sporozoite stages. Using integrative analysis techniques, we can integrate knowledge from different levels and obtain a wider view of the system under study. The overlap between method-specific and common results is considerable, even if the basic mathematical assumptions are very different. The three-fold validated network of life cycle stage characteristics of Plasmodium falciparum could identify a large amount of the known associations from literature in only one study.

Integrative omics analysis. A study based on Plasmodium falciparum mRNA and protein data

PubMed Central

2014-01-01

Background Technological improvements have shifted the focus from data generation to data analysis. The availability of large amounts of data from transcriptomics, protemics and metabolomics experiments raise new questions concerning suitable integrative analysis methods. We compare three integrative analysis techniques (co-inertia analysis, generalized singular value decomposition and integrative biclustering) by applying them to gene and protein abundance data from the six life cycle stages of Plasmodium falciparum. Co-inertia analysis is an analysis method used to visualize and explore gene and protein data. The generalized singular value decomposition has shown its potential in the analysis of two transcriptome data sets. Integrative Biclustering applies biclustering to gene and protein data. Results Using CIA, we visualize the six life cycle stages of Plasmodium falciparum, as well as GO terms in a 2D plane and interpret the spatial configuration. With GSVD, we decompose the transcriptomic and proteomic data sets into matrices with biologically meaningful interpretations and explore the processes captured by the data sets. IBC identifies groups of genes, proteins, GO Terms and life cycle stages of Plasmodium falciparum. We show method-specific results as well as a network view of the life cycle stages based on the results common to all three methods. Additionally, by combining the results of the three methods, we create a three-fold validated network of life cycle stage specific GO terms: Sporozoites are associated with transcription and transport; merozoites with entry into host cell as well as biosynthetic and metabolic processes; rings with oxidation-reduction processes; trophozoites with glycolysis and energy production; schizonts with antigenic variation and immune response; gametocyctes with DNA packaging and mitochondrial transport. Furthermore, the network connectivity underlines the separation of the intraerythrocytic cycle from the gametocyte and sporozoite stages. Conclusion Using integrative analysis techniques, we can integrate knowledge from different levels and obtain a wider view of the system under study. The overlap between method-specific and common results is considerable, even if the basic mathematical assumptions are very different. The three-fold validated network of life cycle stage characteristics of Plasmodium falciparum could identify a large amount of the known associations from literature in only one study. PMID:25033389

Contributions of testosterone and territory ownership to sexually-motivated behaviors and mRNA expression in the medial preoptic area of male European starlings

PubMed Central

Spool, Jeremy A.; Stevenson, Sharon A.; Angyal, Caroline S.; Riters, Lauren V.

2016-01-01

Animals integrate social information with their internal endocrine state to control the timing of behavior, but how these signals are integrated in the brain is not understood. The medial preoptic area (mPOA) may play an integrative role in the control of courtship behavior, as it receives projections from multiple sensory systems, and is central to the hormonal control of courtship behavior across vertebrates. Additionally, data from many species implicate opioid and dopaminergic systems in the mPOA in the control of male courtship behavior. We used European starlings to test the hypothesis that testosterone (T) and social status (in the form of territory possession) interact to control the timing of courtship behavior by modulating steroid hormone-, opioid- and dopaminergic-related gene expression in the mPOA. We found that only males given both T and a nesting territory produced high rates of courtship behavior in response to a female. T treatment altered patterns of gene expression in the mPOA by increasing androgen receptor, aromatase, mu-opioid receptor and preproenkephalin mRNA and decreasing tyrosine hydroxylase mRNA expression. Territory possession did not alter mRNA expression in the mPOA, despite the finding that only birds with both T and a nesting territory produced courtship behavior. We propose that T prepares the mPOA to respond to the presence of a female with high rates of courtship song by altering gene expression, but that activity in the mPOA is under a continuous (i.e. tonic) inhibition until a male starling obtains a nesting territory. PMID:27633459

Contributions of testosterone and territory ownership to sexually-motivated behaviors and mRNA expression in the medial preoptic area of male European starlings.

PubMed

Spool, Jeremy A; Stevenson, Sharon A; Angyal, Caroline S; Riters, Lauren V

2016-11-01

Animals integrate social information with their internal endocrine state to control the timing of behavior, but how these signals are integrated in the brain is not understood. The medial preoptic area (mPOA) may play an integrative role in the control of courtship behavior, as it receives projections from multiple sensory systems, and is central to the hormonal control of courtship behavior across vertebrates. Additionally, data from many species implicate opioid and dopaminergic systems in the mPOA in the control of male courtship behavior. We used European starlings to test the hypothesis that testosterone (T) and social status (in the form of territory possession) interact to control the timing of courtship behavior by modulating steroid hormone-, opioid- and dopaminergic-related gene expression in the mPOA. We found that only males given both T and a nesting territory produced high rates of courtship behavior in response to a female. T treatment altered patterns of gene expression in the mPOA by increasing androgen receptor, aromatase, mu-opioid receptor and preproenkephalin mRNA and decreasing tyrosine hydroxylase mRNA expression. Territory possession did not alter mRNA expression in the mPOA, despite the finding that only birds with both T and a nesting territory produced courtship behavior. We propose that T prepares the mPOA to respond to the presence of a female with high rates of courtship song by altering gene expression, but that activity in the mPOA is under a continuous (i.e. tonic) inhibition until a male starling obtains a nesting territory. Copyright © 2016 Elsevier Inc. All rights reserved.

Regulated expression of the Ren-2 gene in transgenic mice derived from parental strains carrying only the Ren-1 gene.

PubMed Central

Tronik, D; Dreyfus, M; Babinet, C; Rougeon, F

1987-01-01

The Ren-2 gene encoding the mouse submaxillary gland (SMG) renin was microinjected into the pronuclei of fertilized eggs from mice carrying only the Ren-1 gene. In addition to the whole transcription unit, the injected DNA contained 2.5 and 3 kb of upstream and downstream flanking sequences, respectively. Three independent transgenic mice lines were obtained; two of them had integrated one copy of the Ren-2 gene, the last one had integrated five and eleven copies at two independent sites. Independently of the number of Ren-2 copies integrated, the pattern of Ren-2 gene expression in all the transgenic mice was identical to that observed in wild-type animals in which Ren-1 and Ren-2 are closely linked on chromosome 1. In particular, the exogenous Ren-2 gene was only transcribed in the kidney and in the SMG. In the kidney, Ren-1 and Ren-2 mRNAs were present at a comparable level, whereas in the SMG Ren-2 mRNA was at least 100-fold more abundant than Ren-1 mRNA. Moreover, Ren-2 expression in the SMG was positively regulated by androgens. Only one difference between transgenic mice and wild-type mice carrying the Ren-2 gene has been observed: the basal level of Ren-2 transcription in the SMG of transgenic females was lower than in two-gene strain females. Androgen treatment of transgenic females induced SMG renin mRNA to a level identical to that of transgenic males. This suggests that the basal level of SMG renin mRNA is dependent upon cis-acting elements which are not present in the microinjected fragment. Images Fig. 1. Fig. 2. Fig. 3. PMID:3297677

Over-expression of 3-hydroxy-3- methylglutaryl-coenzyme A reductase 1 (hmgr1) gene under super-promoter for enhanced latex biosynthesis in rubber tree (Hevea brasiliensis Muell. Arg.).

PubMed

Jayashree, R; Nazeem, P A; Rekha, K; Sreelatha, S; Thulaseedharan, A; Krishnakumar, R; Kala, R G; Vineetha, M; Leda, P; Jinu, U; Venkatachalam, P

2018-06-01

Natural rubber (cis-1, 4-polyisoprene) is being produced from bark laticifer cells of Hevea brasiliensis and the popular high latex yielding Indian rubber clones are easily prone to onset of tapping panel dryness syndrome (TPD) which is considered as a physiological syndrome affecting latex production either partially or completely. This report describes an efficient protocol for development of transgenic rubber plants by over-expression of 3-hydroxy 3-methylglutaryl Co-enzyme A reductase 1 (hmgr1) gene which is considered as rate limiting factor for latex biosynthesis via Agrobacterium-mediated transformation. The pBIB plasmid vector containing hmgr1 gene cloned under the control of a super-promoter was used for genetic transformation using embryogenic callus. Putatively transgenic cell lines were obtained on selection medium and produced plantlets with 44% regeneration efficiency. Transgene integration was confirmed by PCR amplification of 1.8 kb hmgr1 and 0.6 kb hpt genes from all putatively transformed callus lines as well as transgenic plants. Southern blot analysis showed the stable integration and presence of transgene in the transgenic plants. Over expression of hmgr1 transgene was determined by Northern blot hybridization, semi-quantitative PCR and real-time PCR (qRT-PCR) analysis. Accumulation of hmgr1 mRNA transcripts was more abundant in transgenic plants than control. Increased level of photosynthetic pigments, protein contents and HMGR enzyme activity was also noticed in transgenic plants over control. Interestingly, the latex yield was significantly enhanced in all transgenic plants compared to the control. The qRT-PCR results exhibit that the hmgr1 mRNA transcript levels was 160-fold more abundance in transgenic plants over untransformed control. These results altogether suggest that there is a positive correlation between latex yield and accumulation of mRNA transcripts level as well as HMGR enzyme activity in transgenic rubber plants. It is presumed that there is a possibility for enhanced level of latex biosynthesis in transgenic plants as the level of mRNA transcripts and HMGR enzyme activity is directly correlated with latex yield in rubber tree. Further, the present results clearly suggest that the quantification of HMGR enzyme activity in young seedlings will be highly beneficial for early selection of high latex yielding plants in rubber breeding programs. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

An algorithm for the classification of mRNA patterns in eosinophilic esophagitis: Integration of machine learning.

PubMed

Sallis, Benjamin F; Erkert, Lena; Moñino-Romero, Sherezade; Acar, Utkucan; Wu, Rina; Konnikova, Liza; Lexmond, Willem S; Hamilton, Matthew J; Dunn, W Augustine; Szepfalusi, Zsolt; Vanderhoof, Jon A; Snapper, Scott B; Turner, Jerrold R; Goldsmith, Jeffrey D; Spencer, Lisa A; Nurko, Samuel; Fiebiger, Edda

2018-04-01

Diagnostic evaluation of eosinophilic esophagitis (EoE) remains difficult, particularly the assessment of the patient's allergic status. This study sought to establish an automated medical algorithm to assist in the evaluation of EoE. Machine learning techniques were used to establish a diagnostic probability score for EoE, p(EoE), based on esophageal mRNA transcript patterns from biopsies of patients with EoE, gastroesophageal reflux disease and controls. Dimensionality reduction in the training set established weighted factors, which were confirmed by immunohistochemistry. Following weighted factor analysis, p(EoE) was determined by random forest classification. Accuracy was tested in an external test set, and predictive power was assessed with equivocal patients. Esophageal IgE production was quantified with epsilon germ line (IGHE) transcripts and correlated with serum IgE and the T h 2-type mRNA profile to establish an IGHE score for tissue allergy. In the primary analysis, a 3-class statistical model generated a p(EoE) score based on common characteristics of the inflammatory EoE profile. A p(EoE) ≥ 25 successfully identified EoE with high accuracy (sensitivity: 90.9%, specificity: 93.2%, area under the curve: 0.985) and improved diagnosis of equivocal cases by 84.6%. The p(EoE) changed in response to therapy. A secondary analysis loop in EoE patients defined an IGHE score of ≥37.5 for a patient subpopulation with increased esophageal allergic inflammation. The development of intelligent data analysis from a machine learning perspective provides exciting opportunities to improve diagnostic precision and improve patient care in EoE. The p(EoE) and the IGHE score are steps toward the development of decision trees to define EoE subpopulations and, consequently, will facilitate individualized therapy. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

The polyadenylation code: a unified model for the regulation of mRNA alternative polyadenylation*

PubMed Central

Davis, Ryan; Shi, Yongsheng

2014-01-01

The majority of eukaryotic genes produce multiple mRNA isoforms with distinct 3′ ends through a process called mRNA alternative polyadenylation (APA). Recent studies have demonstrated that APA is dynamically regulated during development and in response to environmental stimuli. A number of mechanisms have been described for APA regulation. In this review, we attempt to integrate all the known mechanisms into a unified model. This model not only explains most of previous results, but also provides testable predictions that will improve our understanding of the mechanistic details of APA regulation. Finally, we briefly discuss the known and putative functions of APA regulation. PMID:24793760

Integrated RNA-seq and sRNA-seq analysis reveals miRNA effects on secondary metabolism in Solanum tuberosum L.

PubMed

Qiao, Yan; Zhang, Jinjin; Zhang, Jinwen; Wang, Zhiwei; Ran, An; Guo, Haixia; Wang, Di; Zhang, Junlian

2017-02-01

Light is a major environmental factor that affects metabolic pathways and stimulates the production of secondary metabolites in potato. However, adaptive changes in potato metabolic pathways and physiological functions triggered by light are partly explained by gene expression changes. Regulation of secondary metabolic pathways in potato has been extensively studied at transcriptional level, but little is known about the mechanisms of post-transcriptional regulation by miRNAs. To identify light-responsive miRNAs/mRNAs and construct putative metabolism pathways regulated by the miRNA-mRNA pairs, an integrated omics (sRNAome and transcriptome) analysis was performed to potato under light stimulus. A total of 31 and 48 miRNAs were identified to be differentially expressed in the leaves and tubers, respectively. Among the DEGs, 1353 genes in the leaves and 1841 genes in the tubers were upregulated, while 1595 genes in the leaves and 897 genes in the tubers were downregulated by light. Mapman enrichment analyses showed that genes related to MVA pathway, alkaloids-like, phenylpropanoids, flavonoids, and carotenoids metabolism were significantly upregulated, while genes associated with major CHO metabolism were repressed in the leaves and tubers. Integrated miRNA and mRNA profiles revealed that light-responsive miRNAs are important regulators in alkaloids metabolism, UMP salvage, lipid biosynthesis, and cellulose catabolism. Moreover, several miRNAs may participate in glycoalkaloids metabolism via JA signaling pathway, UDP-glucose biosynthesis and hydroxylation reaction. This study provides a global view of miRNA and mRNA expression profiles in potato response to light, our results suggest that miRNAs might play important roles in secondary metabolic pathways, especially in glycoalkaloid biosynthesis. The findings will enlighten us on the genetic regulation of secondary metabolite pathways and pave the way for future application of genetically engineered potato.

GDCRNATools: an R/Bioconductor package for integrative analysis of lncRNA, miRNA, and mRNA data in GDC.

PubMed

Li, Ruidong; Qu, Han; Wang, Shibo; Wei, Julong; Zhang, Le; Ma, Renyuan; Lu, Jianming; Zhu, Jianguo; Zhong, Wei-De; Jia, Zhenyu

2018-03-02

The large-scale multidimensional omics data in the Genomic Data Commons (GDC) provides opportunities to investigate the crosstalk among different RNA species and their regulatory mechanisms in cancers. Easy-to-use bioinformatics pipelines are needed to facilitate such studies. We have developed a user-friendly R/Bioconductor package, named GDCRNATools, for downloading, organizing, and analyzing RNA data in GDC with an emphasis on deciphering the lncRNA-mRNA related competing endogenous RNAs (ceRNAs) regulatory network in cancers. Many widely used bioinformatics tools and databases are utilized in our package. Users can easily pack preferred downstream analysis pipelines or integrate their own pipelines into the workflow. Interactive shiny web apps built in GDCRNATools greatly improve visualization of results from the analysis. GDCRNATools is an R/Bioconductor package that is freely available at Bioconductor (http://bioconductor.org/packages/devel/bioc/html/GDCRNATools.html). Detailed instructions, manual and example code are also available in Github (https://github.com/Jialab-UCR/GDCRNATools). arthur.jia@ucr.edu or zhongwd2009@live.cn or doctorzhujianguo@163.com.

Gill structural integrity changes in fish deficient or excessive in dietary isoleucine: Towards the modulation of tight junction protein, inflammation, apoptosis and antioxidant defense via NF-κB, TOR and Nrf2 signaling pathways.

PubMed

Feng, Lin; Gan, Lu; Jiang, Wei-Dan; Wu, Pei; Liu, Yang; Jiang, Jun; Tang, Ling; Kuang, Sheng-Yao; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

2017-04-01

This study firstly aimed to test the impact of dietary isoleucine (Ile) on tight junction protein, inflammation, apoptosis, antioxidant defense and related signaling molecule gene expression in the gill of fish. Young grass carp (Ctenopharyngodon idella) (weighing 256.8 ± 3.5 g) were fed six diets containing graded levels of Ile, namely, 3.8, 6.6, 9.3, 12.5, 15.2 and 18.5 g/kg diet for 8 weeks. The results firstly revealed that Ile deficiency down-regulated the mRNA expressions of claudin-3, claudin-b, claudin-c, occludin and zonula occludens-1 (ZO-1) and up-regulated the mRNA expression of claudin-12, which led to the intercellular structure damage of fish gill. These effects were partially ascribed to the up-regulation of pro-inflammatory cytokines [interleukin 1β (IL-1β), interleukin 8 (IL-8) and tumor necrosis factor-α (TNF-α)] mRNA expressions that referring to up-regulated nuclear factor κB P65 (NF-κB P65) mRNA expression and down-regulated inhibitor factor κBα (IκBα) mRNA expression, and the down-regulation of anti-inflammatory cytokines [interleukin 10 (IL-10) and transforming growth factor β1 (TGF-β1)] mRNA expressions that referring to the down-regulated TOR and S6K1 mRNA expression. Interestingly, no change in claudin 15 mRNA level was observed among every treatment. At the same time, the results firstly indicated that Ile deficiency also resulted in the cellular structure damage of fish gill: (1) DNA fragmentation partially due to the up-regulation of caspase-3, caspase-8 and caspase-9 mRNA expression; (2) increase in protein carbonyl (PC), malondialdehyde (MDA) and ROS contents, which may be partially attributed to the impaired antioxidant defense [indicated by decreased glutathione (GSH) level and depressed anti-superoxide anion (ASA), anti-hydroxyl radical (a-HR), copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPx) activities] that referring to the down-regulation of corresponding antioxidant enzyme mRNA expressions and the related signaling molecules Nrf2 mRNA expression. Ile excess caused similar negative effects that observed in Ile-deficient group, whereas these negative effects were reversed with appropriate Ile supplementation. In conclusion, our results indicated that Ile deficiency or excess disrupted the structural integrity of fish gill, partially due to the trigger of apoptosis, the impairment of antioxidant defense, and the regulation of tight junction protein, inflammatory cytokines, apoptosis-related, antioxidant enzymes and related signaling molecules mRNA expressions in the fish gill. Copyright © 2017 Elsevier Ltd. All rights reserved.

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane*

PubMed Central

Tholanikunnel, Baby G.; Joseph, Kusumam; Kandasamy, Karthikeyan; Baldys, Aleksander; Raymond, John R.; Luttrell, Louis M.; McDermott, Paul J.; Fernandes, Daniel J.

2010-01-01

β2-Adrenergic receptors (β2-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β2-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β2-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β2-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β2-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β2-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals. PMID:20739277

Transcription factors Mix1 and VegT, relocalization of vegt mRNA, and conserved endoderm and dorsal specification in frogs

PubMed Central

Sudou, Norihiro; Garcés-Vásconez, Andrés; López-Latorre, María A.; Taira, Masanori

2016-01-01

Protein expression of the transcription factor genes mix1 and vegt characterized the presumptive endoderm in embryos of the frogs Engystomops randi, Epipedobates machalilla, Gastrotheca riobambae, and Eleutherodactylus coqui, as in Xenopus laevis embryos. Protein VegT was detected in the animal hemisphere of the early blastula in all frogs, and only the animal pole was VegT-negative. This finding stimulated a vegt mRNA analysis in X. laevis eggs and embryos. vegt mRNA was detected in the animal region of X. laevis eggs and early embryos, in agreement with the VegT localization observed in the analyzed frogs. Moreover, a dorso-animal relocalization of vegt mRNA occurred in the egg at fertilization. Thus, the comparative analysis indicated that vegt may participate in dorsal development besides its known roles in endoderm development, and germ-layer specification. Zygotic vegt (zvegt) mRNA was detected as a minor isoform besides the major maternal (mvegt) isoform of the X. laevis egg. In addition, α-amanitin–insensitive vegt transcripts were detected around vegetal nuclei of the blastula. Thus, accumulation of vegt mRNA around vegetal nuclei was caused by relocalization rather than new mRNA synthesis. The localization of vegt mRNA around vegetal nuclei may contribute to the identity of vegetal blastomeres. These and previously reportedly localization features of vegt mRNA and protein derive from the master role of vegt in the development of frogs. The comparative analysis indicated that the strategies for endoderm, and dorsal specification, involving vegt and mix1, have been evolutionary conserved in frogs. PMID:27140624

Relationship Between the DPD and TS mRNA Expression and the Response to S-1-Based Chemotherapy and Prognosis in Patients with Advanced Gastric Cancer.

PubMed

Shen, Xiao-Ming; Zhou, Chong; Lian, Lian; Li, Li-Qun; Li, Wei; Tao, Min

2015-04-01

The aim was to determine changes in dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) mRNAs in the blood of advanced gastric cancer (AGC) patients to see whether these enzymes affected the patients' response to S-1-based chemotherapy and prognosis. For this purpose, pretreatment DPD/TS mRNA expressions were determined in 40 AGC patients using RT-PCR. The patients were then administered with S-1-based regimen (S-1 + cisplatin) and toxicities were recorded. The relationship between the DPD/TS mRNA expressions and the chemotherapy response, drug resistance, and prognosis was analyzed. The data show that DPD mRNA expression correlated significantly with Lauren type while TS mRNA expression correlated with distant metastasis. Patients with higher DPD and/or TS mRNA expression(s) showed poor response, while those with low DPD mRNA expression showed better response to the chemotherapy. Pooled analysis showed that the patients with low DPD/TS mRNA expressions had better therapeutic response. The incidence of bone marrow suppression, diarrhea, and oral mucositis was high in patients with low DPD mRNA expression. Median overall survival (OS) in 40 patients was 13.5 months. It was 17 months for low and 10 months for high DPD (P = 0.044) and TS mRNA expression (P = 0.047). Pooled analysis showed that the patients with both low DPD/TS mRNA expressions had longer OS (P = 0.001). In conclusion, the detection of DPD and/or TS mRNA expression can be used to predict the response to S-1-based chemotherapy, drug resistance, and prognosis in AGC patients as well as to help guide the individualized treatment of gastric cancer.

Unusual RNA plant virus integration in the soybean genome leads to the production of small RNAs.

PubMed

da Fonseca, Guilherme Cordenonsi; de Oliveira, Luiz Felipe Valter; de Morais, Guilherme Loss; Abdelnor, Ricardo Vilela; Nepomuceno, Alexandre Lima; Waterhouse, Peter M; Farinelli, Laurent; Margis, Rogerio

2016-05-01

Horizontal gene transfer (HGT) is known to be a major force in genome evolution. The acquisition of genes from viruses by eukaryotic genomes is a well-studied example of HGT, including rare cases of non-retroviral RNA virus integration. The present study describes the integration of cucumber mosaic virus RNA-1 into soybean genome. After an initial metatranscriptomic analysis of small RNAs derived from soybean, the de novo assembly resulted a 3029-nt contig homologous to RNA-1. The integration of this sequence in the soybean genome was confirmed by DNA deep sequencing. The locus where the integration occurred harbors the full RNA-1 sequence followed by the partial sequence of an endogenous mRNA and another sequence of RNA-1 as an inverted repeat and allowing the formation of a hairpin structure. This region recombined into a retrotransposon located inside an exon of a soybean gene. The nucleotide similarity of the integrated sequence compared to other Cucumber mosaic virus sequences indicates that the integration event occurred recently. We described a rare event of non-retroviral RNA virus integration in soybean that leads to the production of a double-stranded RNA in a similar fashion to virus resistance RNAi plants. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Myeloperoxidase mRNA detection for lineage determination of leukemic blasts: retrospective analysis.

PubMed

Crisan, D; Anstett, M J

1995-07-01

Myeloperoxidase (MPO) mRNA is an early myeloid marker; its detection in the morphologically and immunophenotypically primitive blasts of acute undifferentiated leukemia (AUL) establishes myeloid lineage and allows reclassification as acute myelogenous leukemia with minimal differentiation (AML-MO). We have previously reported a procedure for MPO mRNA detection by RT-PCR (reverse transcription-polymerase chain reaction) and an adaptation for use of routine hematology smears. This variant procedure allows retrospective analysis of mRNA and is used in the present study to evaluate the lineage of leukemic blasts in seven cases with morphology and cytochemistry consistent with AUL. All hematology smears used in this study were air-dried, unstained or Wright-stained and stored at room temperature for periods varying between 3 days and 2 years. MPO mRNA was detected in six cases, establishing the myeloid lineage of the blasts and the diagnosis of AML-MO. In the remaining case, the blasts were MPO mRNA negative, confirming the diagnosis of AUL. The RT-PCR procedure for retrospective mRNA analysis is useful in the clinical setting, due to its high specificity and sensitivity, speed (less than 24 h), safety (no radioactivity) and convenient use of routine hematology smears; it is particularly attractive in clinical situations when fresh or frozen specimens are no longer available at the time when the need for molecular diagnostics becomes apparent.

Application of Hanging Drop Technique for Kidney Tissue Culture.

PubMed

Wang, Shaohui; Wang, Ximing; Boone, Jasmine; Wie, Jin; Yip, Kay-Pong; Zhang, Jie; Wang, Lei; Liu, Ruisheng

2017-01-01

The hanging drop technique is a well-established method used in culture of animal tissues. However, this method has not been used in adult kidney tissue culture yet. This study was to explore the feasibility of using this technique for culturing adult kidney cortex to study the time course of RNA viability in the tubules and vasculature, as well as the tissue structural integrity. In each Petri dish with the plate covered with sterile buffer, a section of mouse renal cortex was cultured within a drop of DMEM culture medium on the inner surface of the lip facing downward. The tissue were then harvested at each specific time points for Real-time PCR analysis and histological studies. The results showed that the mRNA level of most Na+ related transporters and cotransporters were stably maintained within 6 hours in culture, and that the mRNA level of most receptors found in the vasculature and glomeruli were stably maintained for up to 9 days in culture. Paraffin sections of the cultured renal cortex indicated that the tubules began to lose tubular integrity after 6 hours, but the glomeruli and vasculatures were still recognizable up to 9 days in culture. We concluded that adult kidney tissue culture by hanging drop method can be used to study gene expressions in vasculature and glomeruli. © 2017 The Author(s). Published by S. Karger AG, Basel.

Measuring mRNA copy-number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization (smFISH)

PubMed Central

Skinner, Samuel O.; Sepúlveda, Leonardo A.; Xu, Heng; Golding, Ido

2014-01-01

We present a method for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed Escherichia coli cells. A set of fluorescently-labeled oligonucleotide probes are hybridized to the target mRNA, so that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The number of target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete “spots” as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy-number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 days, with another 2-3 days typically required for image and data analysis. PMID:23680982

Messenger RNA Delivery for Tissue Engineering and Regenerative Medicine Applications.

PubMed

Patel, Siddharth; Athirasala, Avathamsa; Menezes, Paula P; Ashwanikumar, N; Zou, Ting; Sahay, Gaurav; Bertassoni, Luiz E

2018-06-07

The ability to control cellular processes and precisely direct cellular reprogramming has revolutionized regenerative medicine. Recent advances in in vitro transcribed (IVT) mRNA technology with chemical modifications have led to development of methods that control spatiotemporal gene expression. Additionally, there is a current thrust toward the development of safe, integration-free approaches to gene therapy for translational purposes. In this review, we describe strategies of synthetic IVT mRNA modifications and nonviral technologies for intracellular delivery. We provide insights into the current tissue engineering approaches that use a hydrogel scaffold with genetic material. Furthermore, we discuss the transformative potential of novel mRNA formulations that when embedded in hydrogels can trigger controlled genetic manipulation to regenerate tissues and organs in vitro and in vivo. The role of mRNA delivery in vascularization, cytoprotection, and Cas9-mediated xenotransplantation is additionally highlighted. Harmonizing mRNA delivery vehicle interactions with polymeric scaffolds can be used to present genetic cues that lead to precise command over cellular reprogramming, differentiation, and secretome activity of stem cells-an ultimate goal for tissue engineering.

Connecting rules from paired miRNA and mRNA expression data sets of HCV patients to detect both inverse and positive regulatory relationships

PubMed Central

2015-01-01

Background Intensive research based on the inverse expression relationship has been undertaken to discover the miRNA-mRNA regulatory modules involved in the infection of Hepatitis C virus (HCV), the leading cause of chronic liver diseases. However, biological studies in other fields have found that inverse expression relationship is not the only regulatory relationship between miRNAs and their targets, and some miRNAs can positively regulate a mRNA by binding at the 5' UTR of the mRNA. Results This work focuses on the detection of both inverse and positive regulatory relationships from a paired miRNA and mRNA expression data set of HCV patients through a 'change-to-change' method which can derive connected discriminatory rules. Our study uncovered many novel miRNA-mRNA regulatory modules. In particular, it was revealed that GFRA2 is positively regulated by miR-557, miR-765 and miR-17-3p that probably bind at different locations of the 5' UTR of this mRNA. The expression relationship between GFRA2 and any of these three miRNAs has not been studied before, although separate research for this gene and these miRNAs have all drawn conclusions linked to hepatocellular carcinoma. This suggests that the binding of mRNA GFRA2 with miR-557, miR-765, or miR-17-3p, or their combinations, is worthy of further investigation by experimentation. We also report another mRNA QKI which has a strong inverse expression relationship with miR-129 and miR-493-3p which may bind at the 3' UTR of QKI with a perfect sequence match. Furthermore, the interaction between hsa-miR-129-5p (previous ID: hsa-miR-129) and QKI is supported with CLIP-Seq data from starBase. Our method can be easily extended for the expression data analysis of other diseases. Conclusion Our rule discovery method is useful for integrating binding information and expression profile for identifying HCV miRNA-mRNA regulatory modules and can be applied to the study of the expression profiles of other complex human diseases. PMID:25707620

Integrated MicroRNA and mRNA Signatures Associated with Survival in Triple Negative Breast Cancer

PubMed Central

Lovat, Francesca; Carasi, Stefania; Pulvirenti, Alfredo; Ferro, Alfredo; Alder, Hansjuerg; He, Gang; Vecchione, Andrea; Croce, Carlo M.; Shapiro, Charles L.; Huebner, Kay

2013-01-01

Triple negative breast cancer (TNBC) is a heterogeneous disease at the molecular, pathologic and clinical levels. To stratify TNBCs, we determined microRNA (miRNA) expression profiles, as well as expression profiles of a cancer-focused mRNA panel, in tumor, adjacent non-tumor (normal) and lymph node metastatic lesion (mets) tissues, from 173 women with TNBCs; we linked specific miRNA signatures to patient survival and used miRNA/mRNA anti-correlations to identify clinically and genetically different TNBC subclasses. We also assessed miRNA signatures as potential regulators of TNBC subclass-specific gene expression networks defined by expression of canonical signal pathways. Tissue specific miRNAs and mRNAs were identified for normal vs tumor vs mets comparisons. miRNA signatures correlated with prognosis were identified and predicted anti-correlated targets within the mRNA profile were defined. Two miRNA signatures (miR-16, 155, 125b, 374a and miR-16, 125b, 374a, 374b, 421, 655, 497) predictive of overall survival (P = 0.05) and distant-disease free survival (P = 0.009), respectively, were identified for patients 50 yrs of age or younger. By multivariate analysis the risk signatures were independent predictors for overall survival and distant-disease free survival. mRNA expression profiling, using the cancer-focused mRNA panel, resulted in clustering of TNBCs into 4 molecular subclasses with different expression signatures anti-correlated with the prognostic miRNAs. Our findings suggest that miRNAs play a key role in triple negative breast cancer through their ability to regulate fundamental pathways such as: cellular growth and proliferation, cellular movement and migration, Extra Cellular Matrix degradation. The results define miRNA expression signatures that characterize and contribute to the phenotypic diversity of TNBC and its metastasis. PMID:23405235

Integrated microRNA and mRNA signatures associated with survival in triple negative breast cancer.

PubMed

Cascione, Luciano; Gasparini, Pierluigi; Lovat, Francesca; Carasi, Stefania; Pulvirenti, Alfredo; Ferro, Alfredo; Alder, Hansjuerg; He, Gang; Vecchione, Andrea; Croce, Carlo M; Shapiro, Charles L; Huebner, Kay

2013-01-01

Triple negative breast cancer (TNBC) is a heterogeneous disease at the molecular, pathologic and clinical levels. To stratify TNBCs, we determined microRNA (miRNA) expression profiles, as well as expression profiles of a cancer-focused mRNA panel, in tumor, adjacent non-tumor (normal) and lymph node metastatic lesion (mets) tissues, from 173 women with TNBCs; we linked specific miRNA signatures to patient survival and used miRNA/mRNA anti-correlations to identify clinically and genetically different TNBC subclasses. We also assessed miRNA signatures as potential regulators of TNBC subclass-specific gene expression networks defined by expression of canonical signal pathways.Tissue specific miRNAs and mRNAs were identified for normal vs tumor vs mets comparisons. miRNA signatures correlated with prognosis were identified and predicted anti-correlated targets within the mRNA profile were defined. Two miRNA signatures (miR-16, 155, 125b, 374a and miR-16, 125b, 374a, 374b, 421, 655, 497) predictive of overall survival (P = 0.05) and distant-disease free survival (P = 0.009), respectively, were identified for patients 50 yrs of age or younger. By multivariate analysis the risk signatures were independent predictors for overall survival and distant-disease free survival. mRNA expression profiling, using the cancer-focused mRNA panel, resulted in clustering of TNBCs into 4 molecular subclasses with different expression signatures anti-correlated with the prognostic miRNAs. Our findings suggest that miRNAs play a key role in triple negative breast cancer through their ability to regulate fundamental pathways such as: cellular growth and proliferation, cellular movement and migration, Extra Cellular Matrix degradation. The results define miRNA expression signatures that characterize and contribute to the phenotypic diversity of TNBC and its metastasis.

Long-distance transport of Gibberellic Acid Insensitive mRNA in Nicotiana benthamiana

PubMed Central

2013-01-01

Background The Gibberellic Acid (GA) signal is governed by the GAI (Gibberellic Acid Insensitive) repressor, which is characterized by a highly conserved N-terminal DELLA domain. Deletion of the DELLA domain results in constitutive suppression of GA signaling. As the GAI transcript is transportable in phloem elements, a Δ-DELLA GAI (gai) transgenic stock plant can reduce the stature of a scion through transport of gai mRNA from the stock. However, little is known about the characteristics of a scion on a gai stock. Results Arabidopsis Δ-DELLA GAI (gai) was fused with a T7 epitope tag and expressed under the control of a companion cell-specific expression promoter, Commelina yellow mottle virus promoter (CoYMVp), to enhance transport in the phloem. The CoYMVp:Atgai-T7 (CgT) transgenic Nicotiana benthamiana exhibited a dwarf phenotype and lower sensitivity to GA enhancement of shoot stature. A wild-type (WT) scion on a CgT stock contained both Atgai-T7 mRNA and the translated product. Microarray analysis to clarify the effect of the CgT stock on the gene expression pattern in the scion clearly revealed that the WT scions on CgT stocks had fewer genes whose expression was altered in response to GA treatment. An apple rootstock variety, Malus prunifolia, integrating CoYMVp:Atgai moderately reduced the tree height of the apple cultivar scion. Conclusions Our results demonstrate that Atgai mRNA can move from companion cells to sieve tubes and that the translated product remains at the sites to which it is transported, resulting in attenuation of GA responses by reducing the expression of many genes. The induction of semi-dwarfism in an apple cultivar on root stock harbouring Atgai suggests that long-distance transport of mRNA from grafts would be applicable to horticulture crops. PMID:24144190

Inflammatory Bowel Disease Therapies and Gut Function in a Colitis Mouse Model

PubMed Central

Nahidi, Lily; Leach, Steven T.; Mitchell, Hazel M.; Kaakoush, Nadeem O.; Lemberg, Daniel A.; Munday, John S.; Huinao, Karina; Day, Andrew S.

2013-01-01

Background. Exclusive enteral nutrition (EEN) is a well-established approach to the management of Crohn's disease. Aim. To determine effects of EEN upon inflammation and gut barrier function in a colitis mouse model. Methods. Interleukin-10-deficient mice (IL-10−/−) were inoculated with Helicobacter trogontum and then treated with EEN, metronidazole, hydrocortisone, or EEN and metronidazole combination. Blood and tissue were collected at 2 and 4 weeks with histology, mucosal integrity, tight junction integrity, inflammation, and H. trogontum load evaluated. Results. H. trogontum induced colitis in IL-10−/− mice with histological changes in the cecum and colon. Elevated mucosal IL-8 mRNA in infected mice was associated with intestinal barrier dysfunction indicated by decreased transepithelial electrical resistance and mRNA of tight junction proteins and increased short-circuit current, myosin light chain kinase mRNA, paracellular permeability, and tumor necrosis factor-α and myeloperoxidase plasma levels (P < 0.01 for all comparisons). EEN and metronidazole, but not hydrocortisone, treatments restored barrier function, maintained gut barrier integrity, and reversed inflammatory changes along with reduction of H. trogontum load (versus infected controls P < 0.05). Conclusion. H. trogontum infection in IL-10−/− mice induced typhlocolitis with intestinal barrier dysfunction. EEN and metronidazole, but not hydrocortisone, modulate barrier dysfunction and reversal of inflammatory changes. PMID:24027765

Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance.

PubMed

Morimoto, Shimpei; Yahara, Koji

2018-03-01

Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes ( ADC17 and KIN1 ) that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning) that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after accounting for potential time delays.

Unmasking Upstream Gene Expression Regulators with miRNA-corrected mRNA Data

PubMed Central

Bollmann, Stephanie; Bu, Dengpan; Wang, Jiaqi; Bionaz, Massimo

2015-01-01

Expressed micro-RNA (miRNA) affects messenger RNA (mRNA) abundance, hindering the accuracy of upstream regulator analysis. Our objective was to provide an algorithm to correct such bias. Large mRNA and miRNA analyses were performed on RNA extracted from bovine liver and mammary tissue. Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%). Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%) and four levels of the magnitude of miRNA effect (ME) on mRNA expression (30%, 50%, 75%, and 83% mRNA reduction), we generated 17 different datasets (including the original dataset). For each dataset, we performed upstream regulator analysis using two bioinformatics tools. We detected an increased effect on the upstream regulator analysis with larger miRNA:mRNA pair bins and higher ME. The miRNA correction allowed identification of several upstream regulators not present in the analysis of the original dataset. Thus, the proposed algorithm improved the prediction of upstream regulators. PMID:27279737

Time frequency power profile of QRS complex obtained with wavelet transform in spontaneously hypertensive rats.

PubMed

Takano, Nami K; Tsutsumi, Takeshi; Suzuki, Hiroshi; Okamoto, Yoshiwo; Nakajima, Toshiaki

2012-02-01

We evaluated whether frequency analysis could detect the development of interstitial fibrosis in rats. SHR/Izm and age-matched WKY/Izm were used. Limb lead II electrocardiograms were recorded. Continuous wavelet transform (CWT) was applied for the time-frequency analysis. The integrated time-frequency power (ITFP) between QRS complexes was measured and compared between groups. The ITFP at low-frequency bands (≤125Hz) was significantly higher in SHR/Izm. The percent change of ITFP showed the different patterns between groups. Prominent interstitial fibrosis with an increase in TIMP-1 mRNA expression was also observed in SHR/Izm. These results were partly reproduced in a computer simulation. Copyright © 2011 Elsevier Ltd. All rights reserved.

A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

PubMed Central

Rodermel, S; Haley, J; Jiang, C Z; Tsai, C H; Bogorad, L

1996-01-01

Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6 Fig. 7 Fig. 8 PMID:8632983

Expression profile of HSP genes during different seasons in goats (Capra hircus).

PubMed

Dangi, Satyaveer Singh; Gupta, Mahesh; Maurya, Divakar; Yadav, Vijay Prakash; Panda, Rudra Prasanna; Singh, Gyanendra; Mohan, Nitai Haridas; Bhure, Sanjeev Kumar; Das, Bikash Chandra; Bag, Sadhan; Mahapatra, Ramkrishna; Taru Sharma, Guttalu; Sarkar, Mihir

2012-12-01

The present study has demonstrated the expression of HSP60, HSP70, HSP90, and UBQ in peripheral blood mononuclear cells (PBMCs) during different seasons in three different age groups (Groups I, II, and III with age of 0-2, 2-5, and >5 years, respectively) of goats of tropical and temperate regions. Real-time polymerase chain reaction was applied to investigate mRNA expression of examined factors. Specificity of the desired products was documented using analysis of the melting temperature and high-resolution gel electrophoresis to verify that the transcripts are of the exact molecular size predicted. The mRNA expression of HSP60, HSP90, and UBQ was significantly higher (P < 0.05) in all age groups during peak summer season as compared with peak winter season in both tropical and temperate region goats. HSP70 mRNA expression was significantly higher (P < 0.05) during summer season as compared with winter season in tropical region goats. However, in the temperate region, in goats from all the three age groups studied, a non-significant difference of HSP70 expression between summer and winter seasons was noticed. In conclusion, results demonstrate that (1) HSP genes are expressed in caprine PBMCs and (2) higher expression of HSPs during thermal stress suggest possible involvement of them to ameliorate deleterious effect of thermal stress so as to maintain cellular integrity and homeostasis in goats.

The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus.

PubMed

Caballero, Carlos J; Menendez-Gil, Pilar; Catalan-Moreno, Arancha; Vergara-Irigaray, Marta; García, Begoña; Segura, Víctor; Irurzun, Naiara; Villanueva, Maite; Ruiz de Los Mozos, Igor; Solano, Cristina; Lasa, Iñigo; Toledo-Arana, Alejandro

2018-02-16

RNA-binding proteins (RBPs) are essential to fine-tune gene expression. RBPs containing the cold-shock domain are RNA chaperones that have been extensively studied. However, the RNA targets and specific functions for many of them remain elusive. Here, combining comparative proteomics and RBP-immunoprecipitation-microarray profiling, we have determined the regulon of the RNA chaperone CspA of Staphylococcus aureus. Functional analysis revealed that proteins involved in carbohydrate and ribonucleotide metabolism, stress response and virulence gene expression were affected by cspA deletion. Stress-associated phenotypes such as increased bacterial aggregation and diminished resistance to oxidative-stress stood out. Integration of the proteome and targetome showed that CspA post-transcriptionally modulates both positively and negatively the expression of its targets, denoting additional functions to the previously proposed translation enhancement. One of these repressed targets was its own mRNA, indicating the presence of a negative post-transcriptional feedback loop. CspA bound the 5'UTR of its own mRNA disrupting a hairpin, which was previously described as an RNase III target. Thus, deletion of the cspA 5'UTR abrogated mRNA processing and auto-regulation. We propose that CspA interacts through a U-rich motif, which is located at the RNase III cleavage site, portraying CspA as a putative RNase III-antagonist.

Coupled Evolution of Transcription and mRNA Degradation

PubMed Central

Dori-Bachash, Mally; Shema, Efrat; Tirosh, Itay

2011-01-01

mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor binding motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast. PMID:21811398

Detection of AR-V7 mRNA in whole blood may not predict the effectiveness of novel endocrine drugs for castration-resistant prostate cancer.

PubMed

Takeuchi, Takumi; Okuno, Yumiko; Hattori-Kato, Mami; Zaitsu, Masayoshi; Mikami, Koji

2016-01-01

A splice variant of androgen receptor (AR), AR-V7, lacks in androgen-binding portion and leads to aggressive cancer characteristics. Reverse transcription-polymerase chain reactions (PCRs) and subsequent nested PCRs for the amplification of AR-V7 and prostate-specific antigen (PSA) transcripts were done for whole blood of patients with prostate cancer and male controls. With primary reverse transcription PCRs, AR-V7 and PSA were detected in 4.5% and 4.7% of prostate cancer, respectively. With nested PCRs, AR-V7 messenger RNA (mRNA) was positive in 43.8% of castration-sensitive prostate cancer and 48.1% of castration-resistant prostate cancer (CRPC), while PSA mRNA was positive in 6.3% of castration-sensitive prostate cancer and 18.5% of CRPC. Whole-blood samples of controls showed AR-V7 mRNA expression by nested PCR. Based on multivariate analysis, expression of AR-V7 mRNA in whole blood was not significantly correlated with clinical parameters and PSA mRNA in blood, while univariate analysis showed a correlation between AR-V7 mRNA and metastasis at initial diagnosis. Detection of AR-V7 mRNA did not predict the reduction of serum PSA in patients with CRPC following abiraterone and enzalutamide administration. In conclusion, AR-V7 mRNA expression in normal hematopoietic cells may have annihilated the manifestation of aggressiveness of prostate cancer and the prediction of the effectiveness of abiraterone and enzalutamide by the assessment of AR-V7 mRNA in blood.

Growth hormone and Pit-1 expression in bovine fetal lymphoid cells.

PubMed

Chen, H T; Schuler, L A; Schultz, R D

1997-11-01

Bovine fetal lymphoid cells were examined for growth hormone (GH) and the transcription factor Pit-1/GHF-1 mRNA. GH and Pit-1/GHF-1 transcripts were detected in thymocytes and splenocytes from fetuses at 60, 90, 120, and 270 d of gestation using reverse transcription-polymerase chain reaction (RT-PCR). Northern analysis indicated that the lymphoid GH mRNA was approximately 350 nucleotides larger than in the pituitary. RT-PCR analysis demonstrated that the coding regions as well as 3' untranslated region of the lymphocyte GH and pituitary transcripts were the same. Analysis of the 5'-untranslated region of the lymphocyte GH mRNA showed that transcription began upstream from the start site in the pituitary gland, suggesting differences in regulation in these tissues. Fetal thymocytes and splenocytes expressed Pit-1/GHF-1 mRNA; however, they contained only the 2.5-kb transcript. The GH and Pit-1/GHF-1 mRNA in fetal lymphoid cells supports the hypothesis that lymphocyte-derived GH may function as an autocrine and/or paracrine factor during the development and maturation of the bovine fetal immune system.

Distribution of cholecystokinin mRNA and peptides in the human brain.

PubMed

Lindefors, N; Brené, S; Kopp, J; Lindén, A; Brodin, E; Sedvall, G; Persson, H

1991-01-01

Expression of preprocholecystokinin mRNA was studied in regions of post mortem human brain using RNA blot analysis (Northern blot) and in situ hybridization. Northern blot analysis using a cDNA probe showed high levels of an approximately 0.8 kb preprocholecystokinin mRNA in all regions of neocortex examined. Lower levels of preprocholecystokinin mRNA were detected in amygdaloid body and thalamus. In situ hybridization analysis using the same cDNA probe revealed numerous weakly labelled neurons in different areas of human neocortex and less numerous neurons in hippocampus and amygdaloid body. High-performance liquid-chromatography and gel-chromatography combined with radioimmunoassay of cholecystokinin-like immunoreactivity from human cerebral cortex and caudate nucleus revealed two major forms, one coeluting with sulphated cholecystokinin-8 and the other coeluting with sulphated cholecystokinin-58. Two minor components coeluting with cholecystokinin-4 and cholecystokinin-5 were also detected. The finding of cholecystokinin-like immunoreactivity corresponding to cholecystokinin-8 and cholecystokinin-58 in caudate nucleus where no preprocholecystokinin mRNA was found, indicates the presence of these peptides in afferent nerve terminals.

Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants

PubMed Central

Lattimore, Vanessa L.; Pearson, John F.; Currie, Margaret J.; Spurdle, Amanda B.; Robinson, Bridget A.; Walker, Logan C.

2018-01-01

PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates (n > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance. PMID:29774201

A novel whole tooth-in-jaw-bone culture of rat molars: morphological, immunohistochemical, and laser capture microdissection analysis.

PubMed

Chokechanachaisakul, Uraiwan; Kaneko, Tomoatsu; Yamanaka, Yusuke; Okiji, Takashi; Suda, Hideaki

2012-10-01

In conventional whole-tooth culture systems, limitation exists regarding maintenance of the vitality of the dental pulp, because this tissue is encased in rigid dentin walls that hinder nutrition supply. We here report a whole tooth-in-jaw-bone culture system of rat mandibular first molars, where transcardiac perfusion with culture medium was carried out before placement of the jaw bone into culture medium, aiming to facilitate longer time preservation of the dental pulp tissue. Following 7 days of culture, the pulp tissues were analyzed by histology and immunohistochemistry to ED2 (antiresident macrophage). ED2-positive macrophages were also analyzed for their Class II MHC, interleukin-6 (IL-6), and p53 mRNA expression levels by means of immune-laser capture microdissection (immune-LCM). Dentin sialophosphoprotein (DSPP) mRNA expression in odontobalstic layer was also examined by LCM. Teeth cultured following saline-perfusion and nonperfusion served as cultured controls. Normal teeth also served as noncultured controls. Histological examination demonstrated that the structure of the pulp tissue was well preserved in the medium-perfused explants in contrast to the cultured control groups. The Class II MHC, IL-6, and p53 mRNA expression levels of ED2-positive cells and DSPP expression levels of odontoblastic layer tissues in the pulp of medium-perfused explants were not significantly different from those in the noncultured normal teeth. In conclusion, the structural integrity and mRNA expression in the pulp were maintained at the in vivo level in the ex vivo whole tooth-in-jaw-bone culture system. The system may lay the foundation for studies aiming at defining further histological and molecular mechanism of the pulp. Copyright © 2012 Wiley Periodicals, Inc.

Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants.

PubMed

Lattimore, Vanessa L; Pearson, John F; Currie, Margaret J; Spurdle, Amanda B; Robinson, Bridget A; Walker, Logan C

2018-01-01

PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2 . The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates ( n  > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance.

Expression of a GALACTINOL SYNTHASE Gene in Tomato Seeds Is Up-Regulated before Maturation Desiccation and Again after Imbibition whenever Radicle Protrusion Is Prevented1

PubMed Central

Downie, Bruce; Gurusinghe, Sunitha; Dahal, Petambar; Thacker, Richard R.; Snyder, John C.; Nonogaki, Hiroyuki; Yim, Kyuock; Fukanaga, Keith; Alvarado, Veria; Bradford, Kent J.

2003-01-01

Raffinose family oligosaccharides (RFOs) have been implicated in mitigating the effects of environmental stresses on plants. In seeds, proposed roles for RFOs include protecting cellular integrity during desiccation and/or imbibition, extending longevity in the dehydrated state, and providing substrates for energy generation during germination. A gene encoding galactinol synthase (GOLS), the first committed enzyme in the biosynthesis of RFOs, was cloned from tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds, and its expression was characterized in tomato seeds and seedlings. GOLS (LeGOLS-1) mRNA accumulated in developing tomato seeds concomitant with maximum dry weight deposition and the acquisition of desiccation tolerance. LeGOLS-1 mRNA was present in mature, desiccated seeds but declined within 8 h of imbibition in wild-type seeds. However, LeGOLS-1 mRNA accumulated again in imbibed seeds prevented from completing germination by dormancy or water deficit. Gibberellin-deficient (gib-1) seeds maintained LeGOLS-1 mRNA amounts after imbibition unless supplied with gibberellin, whereas abscisic acid (ABA) did not prevent the loss of LeGOLS-1 mRNA from wild-type seeds. The presence of LeGOLS-1 mRNA in ABA-deficient (sitiens) tomato seeds indicated that wild-type amounts of ABA are not necessary for its accumulation during seed development. In all cases, LeGOLS-1 mRNA was most prevalent in the radicle tip. LeGOLS-1 mRNA accumulation was induced by dehydration but not by cold in germinating seeds, whereas both stresses induced LeGOLS-1 mRNA accumulation in seedling leaves. The physiological implications of LeGOLS-1 expression patterns in seeds and leaves are discussed in light of the hypothesized role of RFOs in plant stress tolerance. PMID:12644684

Comparison of the Prognostic Utility of the Diverse Molecular Data among lncRNA, DNA Methylation, microRNA, and mRNA across Five Human Cancers

PubMed Central

Xu, Li; Fengji, Liang; Changning, Liu; Liangcai, Zhang; Yinghui, Li; Yu, Li; Shanguang, Chen; Jianghui, Xiong

2015-01-01

Introduction Advances in high-throughput technologies have generated diverse informative molecular markers for cancer outcome prediction. Long non-coding RNA (lncRNA) and DNA methylation as new classes of promising markers are emerging as key molecules in human cancers; however, the prognostic utility of such diverse molecular data remains to be explored. Materials and Methods We proposed a computational pipeline (IDFO) to predict patient survival by identifying prognosis-related biomarkers using multi-type molecular data (mRNA, microRNA, DNA methylation, and lncRNA) from 3198 samples of five cancer types. We assessed the predictive performance of both single molecular data and integrated multi-type molecular data in patient survival stratification, and compared their relative importance in each type of cancer, respectively. Survival analysis using multivariate Cox regression was performed to investigate the impact of the IDFO-identified markers and traditional variables on clinical outcome. Results Using the IDFO approach, we obtained good predictive performance of the molecular datasets (bootstrap accuracy: 0.71–0.97) in five cancer types. Impressively, lncRNA was identified as the best prognostic predictor in the validated cohorts of four cancer types, followed by DNA methylation, mRNA, and then microRNA. We found the incorporating of multi-type molecular data showed similar predictive power to single-type molecular data, but with the exception of the lncRNA + DNA methylation combinations in two cancers. Survival analysis of proportional hazard models confirmed a high robustness for lncRNA and DNA methylation as prognosis factors independent of traditional clinical variables. Conclusion Our study provides insight into systematically understanding the prognostic performance of diverse molecular data in both single and aggregate patterns, which may have specific reference to subsequent related studies. PMID:26606135

Microarray Meta-Analysis of RNA-Binding Protein Functions in Alternative Polyadenylation

PubMed Central

Hu, Wenchao; Liu, Yuting; Yan, Jun

2014-01-01

Alternative polyadenylation (APA) is a post-transcriptional mechanism to generate diverse mRNA transcripts with different 3′UTRs from the same gene. In this study, we systematically searched for the APA events with differential expression in public mouse microarray data. Hundreds of genes with over-represented differential APA events and the corresponding experiments were identified. We further revealed that global APA differential expression occurred prevalently in tissues such as brain comparing to peripheral tissues, and biological processes such as development, differentiation and immune responses. Interestingly, we also observed widespread differential APA events in RNA-binding protein (RBP) genes such as Rbm3, Eif4e2 and Elavl1. Given the fact that RBPs are considered as the main regulators of differential APA expression, we constructed a co-expression network between APAs and RBPs using the microarray data. Further incorporation of CLIP-seq data of selected RBPs showed that Nova2 represses and Mbnl1 promotes the polyadenylation of closest poly(A) sites respectively. Altogether, our study is the first microarray meta-analysis in a mammal on the regulation of APA by RBPs that integrated massive mRNA expression data under a wide-range of biological conditions. Finally, we present our results as a comprehensive resource in an online website for the research community. PMID:24622240

Correlated mRNAs and miRNAs from co-expression and regulatory networks affect porcine muscle and finally meat properties.

PubMed

Ponsuksili, Siriluck; Du, Yang; Hadlich, Frieder; Siengdee, Puntita; Murani, Eduard; Schwerin, Manfred; Wimmers, Klaus

2013-08-05

Physiological processes aiding the conversion of muscle to meat involve many genes associated with muscle structure and metabolic processes. MicroRNAs regulate networks of genes to orchestrate cellular functions, in turn regulating phenotypes. We applied weighted gene co-expression network analysis to identify co-expression modules that correlated to meat quality phenotypes and were highly enriched for genes involved in glucose metabolism, response to wounding, mitochondrial ribosome, mitochondrion, and extracellular matrix. Negative correlation of miRNA with mRNA and target prediction were used to select transcripts out of the modules of trait-associated mRNAs to further identify those genes that are correlated with post mortem traits. Porcine muscle co-expression transcript networks that correlated to post mortem traits were identified. The integration of miRNA and mRNA expression analyses, as well as network analysis, enabled us to interpret the differentially-regulated genes from a systems perspective. Linking co-expression networks of transcripts and hierarchically organized pairs of miRNAs and mRNAs to meat properties yields new insight into several biological pathways underlying phenotype differences. These pathways may also be diagnostic for many myopathies, which are accompanied by deficient nutrient and oxygen supply of muscle fibers.

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

PubMed

Persson, H; Pelto-Huikko, M; Metsis, M; Söder, O; Brene, S; Skog, S; Hökfelt, T; Ritzén, E M

1990-09-01

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

PubMed Central

Persson, H; Pelto-Huikko, M; Metsis, M; Söder, O; Brene, S; Skog, S; Hökfelt, T; Ritzén, E M

1990-01-01

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility. Images PMID:1697032

Type I Glanzmann thrombasthenia caused by an apparently silent beta3 mutation that results in aberrant splicing and reduced beta3 mRNA.

PubMed

Xie, Jingli; Pabón, Dina; Jayo, Asier; Butta, Nora; González-Manchón, Consuelo

2005-05-01

We report a novel genetic defect in a patient with type I Glanzmann thrombasthenia. Flow cytometry analysis revealed undetectable levels of platelet glycoproteins alphaIIb and beta3, although residual amounts of both proteins were detectable in immunoblotting analysis. Sequence analysis of reversely transcribed platelet beta3 mRNA showed a 100-base pair deletion in the 3'-boundary of exon 11, that results in a frame shift and appearance of a premature STOP codon. Analysis of the corresponding genomic DNA fragment revealed the presence of a homozygous C1815T transition in exon 11. The mutation does not change the amino acid residue but it creates an ectopic consensus splice donor site that is used preferentially, causing splicing out of part of exon 11. The parents of the proband, heterozygous for this mutation, were asymptomatic and had reduced platelet content of alphaIIbbeta3. PCR-based relative quantification of beta3 mRNA failed to detect the mutant transcript in the parents and showed a marked reduction in the patient. The results suggest that the thrombasthenic phenotype is, mainly, the result of the reduced availability of beta3-mRNA, most probably due to activation of the nonsense-mediated mRNA decay mechanism. They also show the convenience of analyzing both genomic DNA and mRNA, in order to ascertain the functional consequences of single nucleotide substitutions.

Penalized differential pathway analysis of integrative oncogenomics studies.

PubMed

van Wieringen, Wessel N; van de Wiel, Mark A

2014-04-01

Through integration of genomic data from multiple sources, we may obtain a more accurate and complete picture of the molecular mechanisms underlying tumorigenesis. We discuss the integration of DNA copy number and mRNA gene expression data from an observational integrative genomics study involving cancer patients. The two molecular levels involved are linked through the central dogma of molecular biology. DNA copy number aberrations abound in the cancer cell. Here we investigate how these aberrations affect gene expression levels within a pathway using observational integrative genomics data of cancer patients. In particular, we aim to identify differential edges between regulatory networks of two groups involving these molecular levels. Motivated by the rate equations, the regulatory mechanism between DNA copy number aberrations and gene expression levels within a pathway is modeled by a simultaneous-equations model, for the one- and two-group case. The latter facilitates the identification of differential interactions between the two groups. Model parameters are estimated by penalized least squares using the lasso (L1) penalty to obtain a sparse pathway topology. Simulations show that the inclusion of DNA copy number data benefits the discovery of gene-gene interactions. In addition, the simulations reveal that cis-effects tend to be over-estimated in a univariate (single gene) analysis. In the application to real data from integrative oncogenomic studies we show that inclusion of prior information on the regulatory network architecture benefits the reproducibility of all edges. Furthermore, analyses of the TP53 and TGFb signaling pathways between ER+ and ER- samples from an integrative genomics breast cancer study identify reproducible differential regulatory patterns that corroborate with existing literature.

Coupling mRNA processing with transcription in time and space

PubMed Central

Bentley, David L.

2015-01-01

Maturation of mRNA precursors often occurs simultaneously with their synthesis by RNA polymerase II (Pol II). The co-transcriptional nature of mRNA processing has permitted the evolution of coupling mechanisms that coordinate transcription with mRNA capping, splicing, editing and 3′ end formation. Recent experiments using sophisticated new methods for analysis of nascent RNA have provided important insights into the relative amount of co-transcriptional and post-transcriptional processing, the relationship between mRNA elongation and processing, and the role of the Pol II carboxy-terminal domain (CTD) in regulating these processes. PMID:24514444

Application of a Master Equation for Quantitative mRNA Analysis Using qRT-PCR

USDA-ARS?s Scientific Manuscript database

The qRT-PCR has been widely accepted as the assay of choice for mRNA quantification. Gene expression as measured by mRNA dynamics varies in response to different conditions and environmental stimuli. For conventional practice, housekeeping genes have been applied as internal reference for data nor...

Increased expression of glutamic acid decarboxylase mRNA in rat substantia nigra after an ibotenic acid lesion in the caudate-putamen.

PubMed

Lindefors, N; Brené, S; Persson, H

1990-04-01

In situ hybridization histochemistry and RNA blots were used to study expression of glutamic acid decarboxylase (GAD) mRNA in rat caudate-nucleus and substantia nigra. In situ hybridization combined with computerized image analysis revealed that in the intact substantia nigra reticulata the cross-section area of GAD mRNA positive neurons were 25% larger in the dorsolateral part as compared with the ventromedial part. A unilateral ibotenic acid injection in caudate-putamen lesioned neurons, some of which project to the ipsilateral substantia nigra. An increased level of GAD mRNA was observed in substantia nigra ipsilateral to the lesion. Computerized image analysis of sections from in situ hybridization revealed an increase in the number of silver grains over GAD mRNA positive neurons in the dorsolateral substantia nigra reticulata ipsilateral to the lesion. However, no change was observed in the ventromedial part suggesting that GAD mRNA expression in this part of the nigra is less sensitive to inhibition by caudate-putamen afferents. In agreement with in situ experiments, RNA blots showed a 2-fold increased level of GAD mRNA in substantia nigra ipsilateral to the lesion. The increased GAD mRNA expression in the deafferented substantia nigra suggests a disinhibition of nigral GABA neurons, resulting in an increased utilization of GABA in these substantia nigra neurons.

miRNA and mRNA Expression Profiles Reveal Insight into Chitosan-Mediated Regulation of Plant Growth.

PubMed

Zhang, Xiaoqian; Li, Kecheng; Xing, Ronge; Liu, Song; Chen, Xiaolin; Yang, Haoyue; Li, Pengcheng

2018-04-18

Chitosan has been numerously studied as a plant growth regulator and stress tolerance inducer. To investigate the roles of chitosan as bioregulator on plant and unravel its possible metabolic responses mechanisms, we simultaneously investigated mRNAs and microRNAs (miRNAs) expression profiles of wheat seedlings in response to chitosan heptamer. We found 400 chitosan-responsive differentially expressed genes, including 268 up-regulated and 132 down-regulated mRNAs, many of which were related to photosynthesis, primary carbon and nitrogen metabolism, defense responses, and transcription factors. Moreover, miRNAs also participate in chitosan-mediated regulation on plant growth. We identified 87 known and 21 novel miRNAs, among which 56 miRNAs were induced or repressed by chitosan heptamer, such as miRNA156, miRNA159a, miRNA164, miRNA171a, miRNA319, and miRNA1127. The integrative analysis of miRNA and mRNA expression profiles in this case provides fundamental information for further investigation of regulation mechanisms of chitosan on plant growth and will facilitate its application in agriculture.

UTRdb and UTRsite: a collection of sequences and regulatory motifs of the untranslated regions of eukaryotic mRNAs

PubMed Central

Mignone, Flavio; Grillo, Giorgio; Licciulli, Flavio; Iacono, Michele; Liuni, Sabino; Kersey, Paul J.; Duarte, Jorge; Saccone, Cecilia; Pesole, Graziano

2005-01-01

The 5′ and 3′ untranslated regions of eukaryotic mRNAs play crucial roles in the post-transcriptional regulation of gene expression through the modulation of nucleo-cytoplasmic mRNA transport, translation efficiency, subcellular localization and message stability. UTRdb is a curated database of 5′ and 3′ untranslated sequences of eukaryotic mRNAs, derived from several sources of primary data. Experimentally validated functional motifs are annotated (and also collated as the UTRsite database) and cross-links to genomic and protein data are provided. The integration of UTRdb with genomic and protein data has allowed the implementation of a powerful retrieval resource for the selection and extraction of UTR subsets based on their genomic coordinates and/or features of the protein encoded by the relevant mRNA (e.g. GO term, PFAM domain, etc.). All internet resources implemented for retrieval and functional analysis of 5′ and 3′ untranslated regions of eukaryotic mRNAs are accessible at http://www.ba.itb.cnr.it/UTR/. PMID:15608165

WEREWOLF, a Regulator of Root Hair Pattern Formation, Controls Flowering Time through the Regulation of FT mRNA Stability1[C][W][OA

PubMed Central

Seo, Eunjoo; Yu, Jihyeon; Ryu, Kook Hui; Lee, Myeong Min; Lee, Ilha

2011-01-01

A key floral activator, FT, integrates stimuli from long-day, vernalization, and autonomous pathways and triggers flowering by directly regulating floral meristem identity genes in Arabidopsis (Arabidopsis thaliana). Since a small amount of FT transcript is sufficient for flowering, the FT level is strictly regulated by diverse genes. In this study, we show that WEREWOLF (WER), a MYB transcription factor regulating root hair pattern, is another regulator of FT. The mutant wer flowers late in long days but normal in short days and shows a weak sensitivity to vernalization, which indicates that WER controls flowering time through the photoperiod pathway. The expression and double mutant analyses showed that WER modulates FT transcript level independent of CONSTANS and FLOWERING LOCUS C. The histological analysis of WER shows that it is expressed in the epidermis of leaves, where FT is not expressed. Consistently, WER regulates not the transcription but the stability of FT mRNA. Our results reveal a novel regulatory mechanism of FT that is non cell autonomous. PMID:21653190

WEREWOLF, a regulator of root hair pattern formation, controls flowering time through the regulation of FT mRNA stability.

PubMed

Seo, Eunjoo; Yu, Jihyeon; Ryu, Kook Hui; Lee, Myeong Min; Lee, Ilha

2011-08-01

A key floral activator, FT, integrates stimuli from long-day, vernalization, and autonomous pathways and triggers flowering by directly regulating floral meristem identity genes in Arabidopsis (Arabidopsis thaliana). Since a small amount of FT transcript is sufficient for flowering, the FT level is strictly regulated by diverse genes. In this study, we show that WEREWOLF (WER), a MYB transcription factor regulating root hair pattern, is another regulator of FT. The mutant wer flowers late in long days but normal in short days and shows a weak sensitivity to vernalization, which indicates that WER controls flowering time through the photoperiod pathway. The expression and double mutant analyses showed that WER modulates FT transcript level independent of CONSTANS and FLOWERING LOCUS C. The histological analysis of WER shows that it is expressed in the epidermis of leaves, where FT is not expressed. Consistently, WER regulates not the transcription but the stability of FT mRNA. Our results reveal a novel regulatory mechanism of FT that is non cell autonomous.

RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA

PubMed Central

Cui, Yalei; Huang, Tianzhi; Zhang, Xiaobo

2015-01-01

MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex. PMID:26674414

Diagnostic performance of HPV E6/E7 mRNA assay for detection of cervical high-grade intraepithelial neoplasia and cancer among women with ASCUS Papanicolaou smears.

PubMed

Ren, Chenchen; Zhu, Yuanhang; Yang, Li; Zhang, Xiaoan; Liu, Ling; Ren, Chunying

2018-02-01

The aim of this study was to investigate the clinical performance of high risk (HR) HPV E6/E7 mRNA assay in detecting cervical high-grade intraepithelial neoplasia and cancer among women with atypical squamous cells of undetermined significance (ASCUS) Papanicolaou (Pap) smears. A total of 160 patients with ASCUS who underwent HR-HPV DNA assay, HR-HPV E6/E7 mRNA assay and colposcopy biopsy at Third Affiliated Hospital of Zhengzhou University, China, from December 2015 to March 2017, were enrolled. Logistic regression analysis was used to evaluate the relationship between pathological results with clinical biologic factors. Univariate analysis showed that the qualitative results of HR-HPV DNA, qualitative results of HR-HPV E6/E7 mRNA and expression levels of HR-HPV E6/E7 mRNA were risk factors of high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer (all P < 0.05). Multivariable analysis found that only the expression levels of HR-HPV E6/E7 mRNA was associated with high-grade CIN and cervical cancer (OR = 8.971, 95% CI = 2.572-31.289, P = 0.001). An optimal cut-off value of ≥ 558.26 copies/ml was determined using receiver operating characteristic curve, and specificity of cut-off value were higher than E6/E7 mRNA qualitative assay and DNA qualitative assay. HPV E6/E7 mRNA quantitative assay may be a valuable tool in triage of ASCUS pap smears. A high specificity of E6/E7 mRNA quantitative assay as a triage test in women with ASCUS can be translated into a low referral for colposcopy.

Direct and efficient transfection of mouse neural stem cells and mature neurons by in vivo mRNA electroporation.

PubMed

Bugeon, Stéphane; de Chevigny, Antoine; Boutin, Camille; Coré, Nathalie; Wild, Stefan; Bosio, Andreas; Cremer, Harold; Beclin, Christophe

2017-11-01

In vivo brain electroporation of DNA expression vectors is a widely used method for lineage and gene function studies in the developing and postnatal brain. However, transfection efficiency of DNA is limited and adult brain tissue is refractory to electroporation. Here, we present a systematic study of mRNA as a vector for acute genetic manipulation in the developing and adult brain. We demonstrate that mRNA electroporation is far more efficient than DNA electroporation, and leads to faster and more homogeneous protein expression in vivo Importantly, mRNA electroporation allows the manipulation of neural stem cells and postmitotic neurons in the adult brain using minimally invasive procedures. Finally, we show that this approach can be efficiently used for functional studies, as exemplified by transient overexpression of the neurogenic factor Myt1l and by stably inactivating Dicer nuclease in vivo in adult born olfactory bulb interneurons and in fully integrated cortical projection neurons. © 2017. Published by The Company of Biologists Ltd.

Feeder-free reprogramming of human fibroblasts with messenger RNA.

PubMed

Warren, Luigi; Wang, Jiwu

2013-11-13

This unit describes a feeder-free protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts by transfection of synthetic mRNA. The reprogramming of somatic cells requires transient expression of a set of transcription factors that collectively activate an endogenous gene regulatory network specifying the pluripotent phenotype. The necessary ectopic factor expression was first effected using retroviruses; however, as viral integration into the genome is problematic for cell therapy applications, the use of footprint-free vectors such as mRNA is increasingly preferred. Strong points of the mRNA approach include high efficiency, rapid kinetics, and obviation of a clean-up phase to purge the vector. Still, the method is relatively laborious and has, up to now, involved the use of feeder cells, which brings drawbacks including poor applicability to clinically oriented iPSC derivation. Using the methods described here, mRNA reprogramming can be performed without feeders at much-reduced labor and material costs relative to established protocols. Copyright © 2013 John Wiley & Sons, Inc.

Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation

PubMed Central

Aboulhouda, Soufiane; Di Santo, Rachael; Therizols, Gabriel; Weinberg, David

2017-01-01

The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to determine the number of ribosomes associated with an mRNA molecule, normalized to the length of the coding sequence. The primary method for this analysis of individual mRNAs is sucrose gradient fractionation, which physically separates mRNAs based on the number of bound ribosomes. Here, we describe a streamlined protocol for accurate analysis of mRNA association with ribosomes. Compared to previous protocols, our method incorporates internal controls and improved buffer conditions that together reduce artifacts caused by non-specific mRNA–ribosome interactions. Moreover, our direct-from-fraction qRT-PCR protocol eliminates the need for RNA purification from gradient fractions, which greatly reduces the amount of hands-on time required and facilitates parallel analysis of multiple conditions or gene targets. Additionally, no phenol waste is generated during the procedure. We initially developed the protocol to investigate the translationally repressed state of the HAC1 mRNA in S. cerevisiae, but we also detail adapted procedures for mammalian cell lines and tissues. PMID:29170751

A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Toki, Yasumichi; Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp; Tanaka, Hiroki

2016-08-05

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncatedmore » peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.« less

Integrated responses of Na+/HCO3- cotransporters and V-type H+-ATPases in the fish gill and kidney during respiratory acidosis.

PubMed

Perry, S F; Furimsky, M; Bayaa, M; Georgalis, T; Shahsavarani, A; Nickerson, J G; Moon, T W

2003-12-30

Using degenerate primers, followed by 3' and 5' RACE and "long" PCR, a continuous 4050-bp cDNA was obtained and sequenced from rainbow trout (Oncorhynchus mykiss) gill. The cDNA included an open reading frame encoding a deduced protein of 1088 amino acids. A BLAST search of the GenBank protein database demonstrated that the trout gene shared high sequence similarity with several vertebrate Na(+)/HCO(3)(-) cotransporters (NBCs) and in particular, NBC1. Protein alignment revealed that the trout NBC is >80% identical to vertebrate NBC1s and phylogenetic analysis provided additional evidence that the trout NBC is indeed a homolog of NBC1. Using the same degenerate primers, a partial cDNA (404 bp) for NBC was obtained from eel (Anguilla rostrata) kidney. Analysis of the tissue distribution of trout NBC, as determined by Northern blot analysis and real-time PCR, indicated high transcript levels in several absorptive/secretory epithelia including gill, kidney and intestine and significant levels in liver. NBC mRNA was undetectable in eel gill by real-time PCR. In trout, the levels of gill NBC1 mRNA were increased markedly during respiratory acidosis induced by exposure to hypercarbia; this response was accompanied by a transient increase in branchial V-type H(+)-ATPase mRNA levels. Assuming that the branchial NBC1 is localised to basolateral membranes of gill cells and operates in the influx mode (HCO(3)(-) and Na(+) entry into the cell), it would appear that in trout, the expression of branchial NBC1 is transcriptionally regulated to match the requirements of gill pHi regulation rather than to match trans-epithelial HCO(3)(-) efflux requirements for systemic acid-base balance. By analogy with mammalian systems, NBC1 in the kidney probably plays a role in the tubular reabsorption of both Na(+) and HCO(3)(-). During periods of respiratory acidosis, levels of renal NBC1 mRNA increased (after a transient reduction) in both trout and eel, presumably to increase HCO(3)(-) reabsorption. This strategy, when coupled with increased urinary acidification associated with increased vacuolar H(+)-ATPase activity, ensures that HCO(3)(-) levels accumulate in the body fluids to restore pH.

The cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons.

PubMed

García-Martínez, José; Delgado-Ramos, Lidia; Ayala, Guillermo; Pelechano, Vicent; Medina, Daniel A; Carrasco, Fany; González, Ramón; Andrés-León, Eduardo; Steinmetz, Lars; Warringer, Jonas; Chávez, Sebastián; Pérez-Ortín, José E

2016-05-05

We analyzed 80 different genomic experiments, and found a positive correlation between both RNA polymerase II transcription and mRNA degradation with growth rates in yeast. Thus, in spite of the marked variation in mRNA turnover, the total mRNA concentration remained approximately constant. Some genes, however, regulated their mRNA concentration by uncoupling mRNA stability from the transcription rate. Ribosome-related genes modulated their transcription rates to increase mRNA levels under fast growth. In contrast, mitochondria-related and stress-induced genes lowered mRNA levels by reducing mRNA stability or the transcription rate, respectively. We also detected these regulations within the heterogeneity of a wild-type cell population growing in optimal conditions. The transcriptomic analysis of sorted microcolonies confirmed that the growth rate dictates alternative expression programs by modulating transcription and mRNA decay.The regulation of overall mRNA turnover keeps a constant ratio between mRNA decay and the dilution of [mRNA] caused by cellular growth. This regulation minimizes the indiscriminate transmission of mRNAs from mother to daughter cells, and favors the response capacity of the latter to physiological signals and environmental changes. We also conclude that, by uncoupling mRNA synthesis from decay, cells control the mRNA abundance of those gene regulons that characterize fast and slow growth. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

[Effects of lipopolysaccharides extracted from Porphyromonas endodontalis on the expression of IL-1beta mRNA and IL-6 mRNA in osteoblasts].

PubMed

Yang, Di; Li, Ren; Qiu, Li-Hong; Li, Chen

2009-04-01

To quantify the IL-1 beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides (LPS)extracted from Porphyromonas endodontalis(P.e) in osteoblasts, and to relate P.e-LPS to bone absorption pathogenesis in lesions of chronical apical periodontitis. MG63 was treated with different concentrations of P.e-LPS(0-50 microg/mL) for different hours(0-24h). The expression of IL-1 beta mRNA and IL-6 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR).Statistical analysis was performed using one- way ANOVA and Dunnett t test with SPSS11.0 software package. The level of IL-1 beta mRNA and IL-6 mRNA increased significantly after treatment with P.e-LPS at more than 5 microg/mL (P<0.01)and for more than 1 hour (P<0.01), which indicated that P.e-LPS induced osteoblasts to express IL-1 beta mRNA and IL-6 mRNA in dose and time dependent manners. P.e-LPS may promote bone resorption in lesions of chronical apical periodontitis by inducing IL-1 beta mRNA and IL-6 mRNA expression in osteoblasts.

Novel RNA-binding activity of NQO1 promotes SERPINA1 mRNA translation.

PubMed

Di Francesco, Andrea; Di Germanio, Clara; Panda, Amaresh C; Huynh, Phu; Peaden, Robert; Navas-Enamorado, Ignacio; Bastian, Paul; Lehrmann, Elin; Diaz-Ruiz, Alberto; Ross, David; Siegel, David; Martindale, Jennifer L; Bernier, Michel; Gorospe, Myriam; Abdelmohsen, Kotb; de Cabo, Rafael

2016-10-01

NAD(P)H: quinone oxidoreductase (NQO1) is essential for cell defense against reactive oxidative species, cancer, and metabolic stress. Recently, NQO1 was found in ribonucleoprotein (RNP) complexes, but NQO1-interacting mRNAs and the functional impact of such interactions are not known. Here, we used ribonucleoprotein immunoprecipitation (RIP) and microarray analysis to identify comprehensively the subset of NQO1 target mRNAs in human hepatoma HepG2 cells. One of its main targets, SERPINA1 mRNA, encodes the serine protease inhibitor α-1-antitrypsin, A1AT, which is associated with disorders including obesity-related metabolic inflammation, chronic obstructive pulmonary disease (COPD), liver cirrhosis and hepatocellular carcinoma. Biotin pulldown analysis indicated that NQO1 can bind the 3' untranslated region (UTR) and the coding region (CR) of SERPINA1 mRNA. NQO1 did not affect SERPINA1 mRNA levels; instead, it enhanced the translation of SERPINA1 mRNA, as NQO1 silencing decreased the size of polysomes forming on SERPINA1 mRNA and lowered the abundance of A1AT. Luciferase reporter analysis further indicated that NQO1 regulates SERPINA1 mRNA translation through the SERPINA1 3'UTR. Accordingly, NQO1-KO mice had reduced hepatic and serum levels of A1AT and increased activity of neutrophil elastase (NE), one of the main targets of A1AT. We propose that this novel mechanism of action of NQO1 as an RNA-binding protein may help to explain its pleiotropic biological effects. Published by Elsevier Inc.

Dynamic integration of splicing within gene regulatory pathways

PubMed Central

Braunschweig, Ulrich; Gueroussov, Serge; Plocik, Alex; Graveley, Brenton R.; Blencowe, Benjamin J.

2013-01-01

Precursor mRNA splicing is one of the most highly regulated processes in metazoan species. In addition to generating vast repertoires of RNAs and proteins, splicing has a profound impact on other gene regulatory layers, including mRNA transcription, turnover, transport and translation. Conversely, factors regulating chromatin and transcription complexes impact the splicing process. This extensive cross-talk between gene regulatory layers takes advantage of dynamic spatial, physical and temporal organizational properties of the cell nucleus, and further emphasizes the importance of developing a multidimensional understanding of splicing control. PMID:23498935

Integrated Omic Analysis of a Guinea Pig Model of Heart Failure and Sudden Cardiac Death.

PubMed

Foster, D Brian; Liu, Ting; Kammers, Kai; O'Meally, Robert; Yang, Ni; Papanicolaou, Kyriakos N; Talbot, C Conover; Cole, Robert N; O'Rourke, Brian

2016-09-02

Here, we examine key regulatory pathways underlying the transition from compensated hypertrophy (HYP) to decompensated heart failure (HF) and sudden cardiac death (SCD) in a guinea pig pressure-overload model by integrated multiome analysis. Relative protein abundances from sham-operated HYP and HF hearts were assessed by iTRAQ LC-MS/MS. Metabolites were quantified by LC-MS/MS or GC-MS. Transcriptome profiles were obtained using mRNA microarrays. The guinea pig HF proteome exhibited classic biosignatures of cardiac HYP, left ventricular dysfunction, fibrosis, inflammation, and extravasation. Fatty acid metabolism, mitochondrial transcription/translation factors, antioxidant enzymes, and other mitochondrial procsses, were downregulated in HF but not HYP. Proteins upregulated in HF implicate extracellular matrix remodeling, cytoskeletal remodeling, and acute phase inflammation markers. Among metabolites, acylcarnitines were downregulated in HYP and fatty acids accumulated in HF. The correlation of transcript and protein changes in HF was weak (R(2) = 0.23), suggesting post-transcriptional gene regulation in HF. Proteome/metabolome integration indicated metabolic bottlenecks in fatty acyl-CoA processing by carnitine palmitoyl transferase (CPT1B) as well as TCA cycle inhibition. On the basis of these findings, we present a model of cardiac decompensation involving impaired nuclear integration of Ca(2+) and cyclic nucleotide signals that are coupled to mitochondrial metabolic and antioxidant defects through the CREB/PGC1α transcriptional axis.

Genome-wide analysis of Tol2 transposon reintegration in zebrafish.

PubMed

Kondrychyn, Igor; Garcia-Lecea, Marta; Emelyanov, Alexander; Parinov, Sergey; Korzh, Vladimir

2009-09-08

Tol2, a member of the hAT family of transposons, has become a useful tool for genetic manipulation of model animals, but information about its interactions with vertebrate genomes is still limited. Furthermore, published reports on Tol2 have mainly been based on random integration of the transposon system after co-injection of a plasmid DNA harboring the transposon and a transposase mRNA. It is important to understand how Tol2 would behave upon activation after integration into the genome. We performed a large-scale enhancer trap (ET) screen and generated 338 insertions of the Tol2 transposon-based ET cassette into the zebrafish genome. These insertions were generated by remobilizing the transposon from two different donor sites in two transgenic lines. We found that 39% of Tol2 insertions occurred in transcription units, mostly into introns. Analysis of the transposon target sites revealed no strict specificity at the DNA sequence level. However, Tol2 was prone to target AT-rich regions with weak palindromic consensus sequences centered at the insertion site. Our systematic analysis of sequential remobilizations of the Tol2 transposon from two independent sites within a vertebrate genome has revealed properties such as a tendency to integrate into transcription units and into AT-rich palindrome-like sequences. This information will influence the development of various applications involving DNA transposons and Tol2 in particular.

An integrated approach to characterize transcription factor and microRNA regulatory networks involved in Schwann cell response to peripheral nerve injury

PubMed Central

2013-01-01

Background The regenerative response of Schwann cells after peripheral nerve injury is a critical process directly related to the pathophysiology of a number of neurodegenerative diseases. This SC injury response is dependent on an intricate gene regulatory program coordinated by a number of transcription factors and microRNAs, but the interactions among them remain largely unknown. Uncovering the transcriptional and post-transcriptional regulatory networks governing the Schwann cell injury response is a key step towards a better understanding of Schwann cell biology and may help develop novel therapies for related diseases. Performing such comprehensive network analysis requires systematic bioinformatics methods to integrate multiple genomic datasets. Results In this study we present a computational pipeline to infer transcription factor and microRNA regulatory networks. Our approach combined mRNA and microRNA expression profiling data, ChIP-Seq data of transcription factors, and computational transcription factor and microRNA target prediction. Using mRNA and microRNA expression data collected in a Schwann cell injury model, we constructed a regulatory network and studied regulatory pathways involved in Schwann cell response to injury. Furthermore, we analyzed network motifs and obtained insights on cooperative regulation of transcription factors and microRNAs in Schwann cell injury recovery. Conclusions This work demonstrates a systematic method for gene regulatory network inference that may be used to gain new information on gene regulation by transcription factors and microRNAs. PMID:23387820

Regulated mRNA Decay in Arabidopsis: A global analysis of differential control by hormones and the circadian clock

DOE Office of Scientific and Technical Information (OSTI.GOV)

Green, Pamela J.

The long-term goal of this research was to better understand the influence of mRNA stability on gene regulation, particularly in response to hormones and the circadian clock. The primary aim of this project was to examine this using DNA microarrays, small RNA analysis and other approaches. We accomplished these objectives, although we were only able to detect small changes in mRNA stability in response to these stimuli. However, the work also contributed to a major breakthrough allowing the identification of small RNAs on a genomic scale in eukaryotes. Moreover, the project prompted us to develop a new way to analyzemore » mRNA decay genome wide. Thus, the research was hugely successful beyond our objectives.« less

Regulation of neuropeptide Y gene expression in rat brain.

PubMed

Lindefors, N; Brené, S; Herrera-Marschitz, M; Persson, H

1990-01-01

NPY mRNA expression was studied in rat brain using in situ hybridization and RNA blot analysis. Transsynaptic regulation of NPY gene expression was specifically studied in caudate-putamen and frontoparietal (somatosensory) cortex of rats with unilateral lesion of midbrain dopamine neurons and in sham-injected animals. NPY mRNA expression in these two brain regions and the regulation of midbrain dopamine neurons were compared with that of SOM, PPT, CCK and GAD mRNA expression. Neurons expressing NPY and SOM mRNA showed a similar distribution and the expression of both NPY and SOM appears to be regulated by dopamine in a similar fashion. Following a unilateral dopamine deafferentation, the numerical density of both NPY and SOM mRNA expressing neurons almost doubled in the lesioned rat caudate-putamen with no change in the average grain density over positive neurons. Hence, in the intact caudate-putamen dopamine appears to normally suppress expression of these two neuropeptide genes. An activation of both NPY and SOM mRNA expression in many non- or low-expressing neurons is seen when the level of dopamine is decreased. In the frontoparietal cortex, on the other hand, dopamine appears to stimulate NPY and SOM gene expression. RNA blot analysis shows clear-cut changes of NPY mRNA levels in both caudate-putamen and frontoparietal cortex consistent with the changes observed using in situ hybridization. No evidence was found for a change in CCK mRNA expression by the dopamine deafferentation, while PPT mRNA expression decreased in the deafferented caudate-putamen. Consequently, dopamine exerts dissimilar effects on the expression of different neuropeptide genes, that in turn do not respond in the same way in different brain regions. Indirect evidence is also presented indicating that dopamine regulates NPY mRNA expression in a subpopulation of neurons that possibly also express GAD mRNA, both in caudate-putamen and in frontoparietal cortex.

Further Confirmation of Germline Glioma Risk Variant rs78378222 in TP53 and Its Implication in Tumor Tissues via Integrative Analysis of TCGA Data

PubMed Central

Wang, Zhaoming; Rajaraman, Preetha; Melin, Beatrice S.; Chung, Charles C.; Zhang, Weijia; McKean-Cowdin, Roberta; Michaud, Dominique; Yeager, Meredith; Ahlbom, Anders; Albanes, Demetrius; Andersson, Ulrika; Beane Freeman, Laura E.; Buring, Julie E.; Butler, Mary Ann; Carreón, Tania; Feychting, Maria; Gapstur, Susan M.; Gaziano, J. Michael; Giles, Graham G.; Hallmans, Goran; Henriksson, Roger; Hoffman-Bolton, Judith; Inskip, Peter D.; Kitahara, Cari M.; Le Marchand, Loic; Linet, Martha S.; Li, Shengchao; Peters, Ulrike; Purdue, Mark P.; Rothman, Nathaniel; Ruder, Avima M.; Sesso, Howard D.; Severi, Gianluca; Stampfer, Meir; Stevens, Victoria L.; Visvanathan, Kala; Wang, Sophia S.; White, Emily; Zeleniuch-Jacquotte, Anne; Hoover, Robert; Fraumeni, Joseph F.; Chatterjee, Nilanjan; Hartge, Patricia; Chanock, Stephen J.

2016-01-01

We confirmed strong association of rs78378222:A>C (per allele odds ratio [OR] = 3.14; P = 6.48 × 10−11), a germline rare single-nucleotide polymorphism (SNP) in TP53, via imputation of a genome-wide association study of glioma (1,856 cases and 4,955 controls). We subsequently performed integrative analyses on the Cancer Genome Atlas (TCGA) data for GBM (glioblastoma multiforme) and LUAD (lung adenocarcinoma). Based on SNP data, we imputed genotypes for rs78378222 and selected individuals carrying rare risk allele (C). Using RNA sequencing data, we observed aberrant transcripts with ~3 kb longer than normal for those individuals. Using exome sequencing data, we further showed that loss of haplotype carrying common protective allele (A) occurred somatically in GBM but not in LUAD. Our bioinformatic analysis suggests rare risk allele (C) disrupts mRNA termination, and an allelic loss of a genomic region harboring common protective allele (A) occurs during tumor initiation or progression for glioma. PMID:25907361

MicroRNA regulatory networks reflective of polyhexamethylene guanidine phosphate-induced fibrosis in A549 human alveolar adenocarcinoma cells.

PubMed

Shin, Da Young; Jeong, Mi Ho; Bang, In Jae; Kim, Ha Ryong; Chung, Kyu Hyuck

2018-05-01

Polyhexamethylene guanidine phosphate (PHMG-phosphate), an active component of humidifier disinfectant, is suspected to be a major cause of pulmonary fibrosis. Fibrosis, induced by recurrent epithelial damage, is significantly affected by epigenetic regulation, including microRNAs (miRNAs). The aim of this study was to investigate the fibrogenic mechanisms of PHMG-phosphate through the profiling of miRNAs and their target genes. A549 cells were treated with 0.75 μg/mL PHMG-phosphate for 24 and 48 h and miRNA microarray expression analysis was conducted. The putative mRNA targets of the miRNAs were identified and subjected to Gene Ontology analysis. After exposure to PHMG-phosphate for 24 and 48 h, 46 and 33 miRNAs, respectively, showed a significant change in expression over 1.5-fold compared with the control. The integrated analysis of miRNA and mRNA microarray results revealed the putative targets that were prominently enriched were associated with the epithelial-mesenchymal transition (EMT), cell cycle changes, and apoptosis. The dose-dependent induction of EMT by PHMG-phosphate exposure was confirmed by western blot. We identified 13 putative EMT-related targets that may play a role in PHMG-phosphate-induced fibrosis according to the Comparative Toxicogenomic Database. Our findings contribute to the comprehension of the fibrogenic mechanism of PHMG-phosphate and will aid further study on PHMG-phosphate-induced toxicity. Copyright © 2018 Elsevier B.V. All rights reserved.

Imaging Transcriptional Regulation of Eukaryotic mRNA Genes: Advances and Outlook.

PubMed

Yao, Jie

2017-01-06

Regulation of eukaryotic transcription in vivo occurs at distinct stages. Previous research has identified many active or repressive transcription factors (TFs) and core transcription components and studied their functions in vitro and in vivo. Nonetheless, how individual TFs act in concert to regulate mRNA gene expression in a single cell remains poorly understood. Direct observation of TF assembly and disassembly and various biochemical reactions during transcription of a single-copy gene in vivo is the ideal approach to study this problem. Research in this area requires developing novel techniques for single-cell transcription imaging and integrating imaging studies into understanding the molecular biology of transcription. In the past decade, advanced cell imaging has enabled unprecedented capabilities to visualize individual TF molecules, to track single transcription sites, and to detect individual mRNA in fixed and living cells. These studies have raised several novel insights on transcriptional regulation such as the "hit-and-run" model and transcription bursting that could not be obtained by in vitro biochemistry analysis. At this point, the key question is how to achieve deeper understandings or discover novel mechanisms of eukaryotic transcriptional regulation by imaging transcription in single cells. Meanwhile, further technical advancements are likely required for visualizing distinct kinetic steps of transcription on a single-copy gene in vivo. This review article summarizes recent progress in the field and describes the challenges and opportunities ahead. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analysis of expression and prognostic significance of vimentin and the response to temozolomide in glioma patients.

PubMed

Lin, Lin; Wang, Guangzhi; Ming, Jianguang; Meng, Xiangqi; Han, Bo; Sun, Bo; Cai, Jinquan; Jiang, Chuanlu

2016-11-01

Gliomas are the most common primary intracranial malignant tumors in adults. Surgical resection followed by optional radiotherapy and chemotherapy is the current standard therapy for glioma patients. Vimentin, a protein of intermediate filament family, could maintain the cellular integrity and participate in several cell signal pathways to modulate the motility and invasion of cancer cells. The purpose of the present research was to identify the relationship between vimentin expression and clinical characteristics and detect the prognostic and predictive ability of vimentin in patients with glioma. To determine the expression of vimentin in glioma tissues, paraffin-embedded blocks from glioma patients by surgical resection were obtained and evaluated by immunohistochemistry. To further investigate the association of vimentin expression with survival, we employed mRNA expression of vimentin genes from the Chinese Glioma Genome Atlas (CGGA) and the GSE 16011 dataset. Kaplan-Meier analysis and Cox regression model were used to statistical analysis. We detected positive vimentin straining in 84 % of high-grade compared to 47 % in low-grade glioma patients. Additionally, vimentin mRNA expression was correlated with glioma grade in both CGGA and GSE16011 dataset. Patients with low vimentin expression have longer survival than high expression. In multivariate analysis, vimentin was an independent significant prognostic factor for high-grade glioma patients. We also identified that glioblastoma patients with low vimentin expression had a better response to temozolomide therapy. Vimentin expression has a significant association with tumor grade and overall survival of high-grade glioma patients. Low vimentin expression may benefit from temozolomide therapy.

The decreased growth performance and impaired immune function and structural integrity by dietary iron deficiency or excess are associated with TOR, NF-κB, p38MAPK, Nrf2 and MLCK signaling in head kidney, spleen and skin of grass carp (Ctenopharyngodon idella).

PubMed

Guo, Yan-Lin; Jiang, Wei-Dan; Wu, Pei; Liu, Yang; Zhou, Xiao-Qiu; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Feng, Lin

2017-06-01

This study was conducted to investigate the effects of dietary iron on the growth, and immune function and structural integrity in head kidney, spleen and skin as well as the underlying signaling of young grass carp (Ctenopharyngodon idella). Total 630 grass carp (242.32 ± 0.58 g) were fed diets containing graded levels of iron at 12.15 (basal diet), 35.38, 63.47, 86.43, 111.09, 136.37 mg/kg (diets 2-6 were added with ferrous fumarate) and 73.50 mg/kg (diet 7 was added with ferrous sulfate) diet for 60 days. Then, a challenge test was conducted by infection of Aeromonas hydrophila for 14 days. The results firstly showed that compared with optimal iron level, iron deficiency decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents and down-regulated the mRNA levels of antibacterial peptides, anti-inflammatory cytokines, inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1), whereas up-regulated the mRNA levels of pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) p65, IκB kinases β (IKKβ) and eIF4E-binding protein (4E-BP) in head kidney and spleen of young grass carp (P < 0.05), indicating that iron deficiency impaired immune function in head kidney and spleen of fish. Secondly, iron deficiency down-regulated the mRNA levels of B-cell lymphoma-2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), and inhibitor of apoptosis protein (IAP), and decreased activities and mRNA levels of antioxidant enzymes, down-regulated the mRNA levels of NF-E2-related factor 2 (Nrf2) and tight junction complexes, and up-regulated mRNA levels of cysteinyl aspartic acid-protease (caspase) -2, -3, -7, -8, -9, apoptotic protease activating factor-1 (Apaf-1), Bcl-2 associated X protein (Bax), Fas ligand (FasL), p38 mitogen-activated protein kinase (p38MAPK), Kelch-like ECH-associating protein (Keap) 1a, Keap1b, claudin-12 and myosin light chain kinase (MLCK), and increased malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) contents in head kidney and spleen of young grass carp (P < 0.05), indicating that iron deficiency impaired structural integrity in head kidney and spleen of fish. Thirdly, iron deficiency increased skin hemorrhage and lesion morbidity, and impaired immune function and structural integrity in skin of fish. Fourthly, iron excess decreased growth and impaired the immune function and structural integrity in head kidney, spleen and skin of fish. Besides, in young grass carp, based on PWG and ability against skin hemorrhage and lesion, the efficacy of ferrous fumarate relative to ferrous sulfate was 140.32% and 126.48%, respectively, and the iron requirements based on PWG, ability against skin hemorrhage and lesion, ACP activities and MDA contents in head kidney and spleen were estimated to be 75.65, 87.03, 79.74, 78.93, 83.17 and 82.14 mg/kg diet (based on ferrous fumarate), respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Competition between RNA-binding proteins CELF1 and HuR modulates MYC translation and intestinal epithelium renewal

PubMed Central

Liu, Lan; Ouyang, Miao; Rao, Jaladanki N.; Zou, Tongtong; Xiao, Lan; Chung, Hee Kyoung; Wu, Jing; Donahue, James M.; Gorospe, Myriam; Wang, Jian-Ying

2015-01-01

The mammalian intestinal epithelium is one of the most rapidly self-renewing tissues in the body, and its integrity is preserved through strict regulation. The RNA-binding protein (RBP) ELAV-like family member 1 (CELF1), also referred to as CUG-binding protein 1 (CUGBP1), regulates the stability and translation of target mRNAs and is implicated in many aspects of cellular physiology. We show that CELF1 competes with the RBP HuR to modulate MYC translation and regulates intestinal epithelial homeostasis. Growth inhibition of the small intestinal mucosa by fasting in mice was associated with increased CELF1/Myc mRNA association and decreased MYC expression. At the molecular level, CELF1 was found to bind the 3′-untranslated region (UTR) of Myc mRNA and repressed MYC translation without affecting total Myc mRNA levels. HuR interacted with the same Myc 3′-UTR element, and increasing the levels of HuR decreased CELF1 binding to Myc mRNA. In contrast, increasing the concentrations of CELF1 inhibited formation of the [HuR/Myc mRNA] complex. Depletion of cellular polyamines also increased CELF1 and enhanced CELF1 association with Myc mRNA, thus suppressing MYC translation. Moreover, ectopic CELF1 overexpression caused G1-phase growth arrest, whereas CELF1 silencing promoted cell proliferation. These results indicate that CELF1 represses MYC translation by decreasing Myc mRNA association with HuR and provide new insight into the molecular functions of RBPs in the regulation of intestinal mucosal growth. PMID:25808495

Zinc metallothionein (MT) induction by parenteral iron and endotoxin: A temporal analysis of hepatic MT mRNA changes

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCormick, C.C.

The present study was undertaken to compare the temporal characteristics of iron-induced hepatic MT mRNA accumulation to that effected by endotoxin. Young chicks were given (ip) either endotoxin, ferrous gluconate or an equivalent volume of saline. At various times following injections, liver was obtained from 5 chicks per treatment for total RNA extraction. Equal amounts of total hepatic RNA from each chick were pooled and 10 {mu}g separated by denaturing agarose gel electrophoresis. Hepatic MT mRNA and albumin mRNA were analyzed by Northern blot analysis using synthetic oligonucleotides. The results indicated little temporal difference in the accumulation of hepatic MTmore » mRNA as affected by either endotoxin or iron. In both treatments, MT mRNA was minimally affected at 3 hours post-injection. Maximum accumulation was achieved during a 6 h period from 6 to 12 hours post-injection. At 24 hours, MT mRNA was considerably higher in liver of endotoxin-injected chicks when compared to that of iron-injection chicks. Albumin expression appeared not to be substantially affected by either treatment. The results suggest that the induction of hepatic MT by iron injection is not substantially different than that observed following endotoxin administration. It would be speculative to suggest that the processes by which MT is induced under these conditions are also similar.« less

Association between ERCC1 and TS mRNA levels and disease free survival in colorectal cancer patients receiving oxaliplatin and fluorouracil (5-FU) adjuvant chemotherapy.

PubMed

Li, Sheng; Zhu, Liangjun; Yao, Li; Xia, Lei; Pan, Liangxi

2014-08-29

Aim was to explore the association of ERCC1 and TS mRNA levels with the disease free survival (DFS) in Chinese colorectal cancer (CRC) patients receiving oxaliplatin and 5-FU based adjuvant chemotherapy. Total 112 Chinese stage II-III CRC patients were respectively treated by four different chemotherapy regimens after curative operation. The TS and ERCC1 mRNA levels in primary tumor were measured by real-time RT-PCR. Kaplan-Meier curves and log-rank tests were used for DFS analysis. The Cox proportional hazards model was used for prognostic analysis. In univariate analysis, the hazard ratio (HR) for the mRNA expression levels of TS and ERCC1 (logTS: HR = 0.820, 95% CI = 0.600 - 1.117, P = 0.210; logERCC1: HR = 1.054, 95% CI = 0.852 - 1.304, P = 0.638) indicated no significant association of DFS with the TS and ERCC1 mRNA levels. In multivariate analyses, tumor stage (IIIc: reference, P = 0.083; IIb: HR = 0.240, 95% CI = 0.080 - 0.724, P = 0.011; IIc: HR < 0.0001, P = 0.977; IIIa: HR = 0.179, 95% CI = 0.012 - 2.593, P = 0.207) was confirmed to be the independent prognostic factor for DFS. Moreover, the Kaplan-Meier DFS curves showed that TS and ERCC1 mRNA levels were not significantly associated with the DFS (TS: P = 0.264; ERCC1: P = 0.484). The mRNA expression of ERCC1 and TS were not applicable to predict the DFS of Chinese stage II-III CRC patients receiving 5-FU and oxaliplatin based adjuvant chemotherapy.

Changes in Polysome Association of mRNA Throughout Growth and Development in Arabidopsis thaliana.

PubMed

Yamasaki, Shotaro; Matsuura, Hideyuki; Demura, Taku; Kato, Ko

2015-11-01

Translational control is a key regulatory step in the expression of genes as proteins. In plant cells, the translational efficiency of mRNAs differs for different mRNA species, and the efficiency dynamically changes in various conditions. To gain a global view of translational control throughout growth and development, we performed genome-wide analysis of polysome association of mRNA during growth and leaf development in Arabidopsis thaliana by subjecting the mRNAs in polysomes to DNA microarray. This analysis revealed that the degree of polysome association of mRNA was different depending on the mRNA species, and the polysome association changed greatly throughout growth and development for each. In the growth stage, transcripts showed varying changes in polysome association from strongly depressed to unchanged, with the majority of transcripts showing dissociation from ribosomes. On the other hand, during leaf development, the polysome association of transcripts showed a normal distribution from repressed to activated mRNAs when comparing expanding and expanded leaves. In addition, functional category analysis of the microarray data suggested that translational control has a physiological significance in the plant growth and development process, especially in the categories of signaling and protein synthesis. In addition to this, we compared changes in polysome association of mRNAs between various conditions and characterized translational controls in each. This result suggested that mRNA translation might be controlled by complicated mechanisms for response to each condition. Our results highlight the importance of dynamic changes in mRNA translation in plant development and growth. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Quantitation of TGF-beta1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: comparison with ribonuclease protection assay and in situ hybridization.

PubMed

Ceol, M; Forino, M; Gambaro, G; Sauer, U; Schleicher, E D; D'Angelo, A; Anglani, F

2001-01-01

Gene expression can be examined with different techniques including ribonuclease protection assay (RPA), in situ hybridisation (ISH), and quantitative reverse transcription-polymerase chain reaction (RT/PCR). These methods differ considerably in their sensitivity and precision in detecting and quantifying low abundance mRNA. Although there is evidence that RT/PCR can be performed in a quantitative manner, the quantitative capacity of this method is generally underestimated. To demonstrate that the comparative kinetic RT/PCR strategy-which uses a housekeeping gene as internal standard-is a quantitative method to detect significant differences in mRNA levels between different samples, the inhibitory effect of heparin on phorbol 12-myristate 13-acetate (PMA)-induced-TGF-beta1 mRNA expression was evaluated by RT/PCR and RPA, the standard method of mRNA quantification, and the results were compared. The reproducibility of RT/PCR amplification was calculated by comparing the quantity of G3PDH and TGF-beta1 PCR products, generated during the exponential phases, estimated from two different RT/PCR (G3PDH, r = 0.968, P = 0.0000; TGF-beta1, r = 0.966, P = 0.0000). The quantitative capacity of comparative kinetic RT/PCR was demonstrated by comparing the results obtained from RPA and RT/PCR using linear regression analysis. Starting from the same RNA extraction, but using only 1% of the RNA for the RT/PCR compared to RPA, significant correlation was observed (r = 0.984, P = 0.0004). Moreover the morphometric analysis of ISH signal was applied for the semi-quantitative evaluation of the expression and localisation of TGF-beta1 mRNA in the entire cell population. Our results demonstrate the close similarity of the RT/PCR and RPA methods in giving quantitative information on mRNA expression and indicate the possibility to adopt the comparative kinetic RT/PCR as reliable quantitative method of mRNA analysis. Copyright 2001 Wiley-Liss, Inc.

Cytochrome P450IA mRNA expression in feral Hudson River tomcod

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kreamer, G.L.; Squibb, K.; Gioeli, D.

1991-06-01

The authors sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, they found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached bymore » 5 days. Intraperitoneal injection of {beta}-napthoflavone in depurated Hudson tomcod resulted in a 15-fold induction of P450 gene expression within 26 hr. Exposure of depurated Hudson tomcod to natural sediment spiked with two PAHs resulted in a 7-fold induction of P450 gene expression. Exposure of depurated tomcod to sediment from a contaminated Hudson site also resulted in a 7- to 15-fold induction of P450IA mRNA expression. Northern blot analysis revealed a second polymorphic cytochrome P450IA mRNA band in some tomcod which was also detected by Southern blot analysis. Induction of cytochrome P450IA mRNA in Atlantic tomcod may provide a sensitive biomarker of environmentally relevant concentrations of some pollutants in the Hudson and other northeastern tidal rivers.« less

Messenger RNA-based therapeutics for brain diseases: An animal study for augmenting clearance of beta-amyloid by intracerebral administration of neprilysin mRNA loaded in polyplex nanomicelles.

PubMed

Lin, Chin-Yu; Perche, Federico; Ikegami, Masaru; Uchida, Satoshi; Kataoka, Kazunori; Itaka, Keiji

2016-08-10

Alzheimer's disease (AD) pathogenesis is considered to be the metabolic imbalance between anabolism and clearance of amyloid-beta (Aβ), and the strategy of breaking the equilibrium between soluble and insoluble forms of Aβ is likely to help prevent the progression of AD. Neprilysin (NEP) plays a major role in the clearance of Aβ in the brain, and its supplementation using viral vectors has shown to decrease Aβ deposition and prevent pathogenic changes in the brain. In this study, we developed a new therapeutic strategy by mRNA-based gene introduction. mRNA has the advantages of negligible risk of random integration into genome and not needing to be transcribed precludes the need for nuclear entry. This allows efficient protein expression in slowly-dividing or non-dividing cells, such as neural cells. We constructed mRNA encoding the mouse NEP protein and evaluated its ability degrade Aβ. In vitro transfection of NEP mRNA to primary neurons exhibited Amyloid Precursor Protein (APP) degradation activity superior to that of NEP encoding plasmid DNA. We then evaluated the in vivo activity of NEP mRNA by intracerebroventricular (i.c.v.) infusion using a cationic polymer-based PEGylated nanocarrier to form polyplex nanomicelles, which have been shown to have a high potential to deliver mRNA to various target tissues and organs. Nanomicelles carrying a GFP-NEP fusion mRNA produced efficient protein expression in a diffuse manner surrounding the ventricular space. An ELISA evaluation revealed that the mRNA infusion significantly augmented NEP level and effectively reduced the concentration of Aβ that had been supplemented in the mouse brain. To the best of our knowledge, this is the first study to demonstrate the therapeutic potential of introducing exogenous mRNA for the treatment of brain diseases, opening the new era of mRNA-based therapeutics. Copyright © 2016 Elsevier B.V. All rights reserved.

BioVLAB-MMIA-NGS: microRNA-mRNA integrated analysis using high-throughput sequencing data.

PubMed

Chae, Heejoon; Rhee, Sungmin; Nephew, Kenneth P; Kim, Sun

2015-01-15

It is now well established that microRNAs (miRNAs) play a critical role in regulating gene expression in a sequence-specific manner, and genome-wide efforts are underway to predict known and novel miRNA targets. However, the integrated miRNA-mRNA analysis remains a major computational challenge, requiring powerful informatics systems and bioinformatics expertise. The objective of this study was to modify our widely recognized Web server for the integrated mRNA-miRNA analysis (MMIA) and its subsequent deployment on the Amazon cloud (BioVLAB-MMIA) to be compatible with high-throughput platforms, including next-generation sequencing (NGS) data (e.g. RNA-seq). We developed a new version called the BioVLAB-MMIA-NGS, deployed on both Amazon cloud and on a high-performance publicly available server called MAHA. By using NGS data and integrating various bioinformatics tools and databases, BioVLAB-MMIA-NGS offers several advantages. First, sequencing data is more accurate than array-based methods for determining miRNA expression levels. Second, potential novel miRNAs can be detected by using various computational methods for characterizing miRNAs. Third, because miRNA-mediated gene regulation is due to hybridization of an miRNA to its target mRNA, sequencing data can be used to identify many-to-many relationship between miRNAs and target genes with high accuracy. http://epigenomics.snu.ac.kr/biovlab_mmia_ngs/. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Logic integration of mRNA signals by an RNAi-based molecular computer.

PubMed

Xie, Zhen; Liu, Siyuan John; Bleris, Leonidas; Benenson, Yaakov

2010-05-01

Synthetic in vivo molecular 'computers' could rewire biological processes by establishing programmable, non-native pathways between molecular signals and biological responses. Multiple molecular computer prototypes have been shown to work in simple buffered solutions. Many of those prototypes were made of DNA strands and performed computations using cycles of annealing-digestion or strand displacement. We have previously introduced RNA interference (RNAi)-based computing as a way of implementing complex molecular logic in vivo. Because it also relies on nucleic acids for its operation, RNAi computing could benefit from the tools developed for DNA systems. However, these tools must be harnessed to produce bioactive components and be adapted for harsh operating environments that reflect in vivo conditions. In a step toward this goal, we report the construction and implementation of biosensors that 'transduce' mRNA levels into bioactive, small interfering RNA molecules via RNA strand exchange in a cell-free Drosophila embryo lysate, a step beyond simple buffered environments. We further integrate the sensors with our RNAi 'computational' module to evaluate two-input logic functions on mRNA concentrations. Our results show how RNA strand exchange can expand the utility of RNAi computing and point toward the possibility of using strand exchange in a native biological setting.

Logic integration of mRNA signals by an RNAi-based molecular computer

PubMed Central

Xie, Zhen; Liu, Siyuan John; Bleris, Leonidas; Benenson, Yaakov

2010-01-01

Synthetic in vivo molecular ‘computers’ could rewire biological processes by establishing programmable, non-native pathways between molecular signals and biological responses. Multiple molecular computer prototypes have been shown to work in simple buffered solutions. Many of those prototypes were made of DNA strands and performed computations using cycles of annealing-digestion or strand displacement. We have previously introduced RNA interference (RNAi)-based computing as a way of implementing complex molecular logic in vivo. Because it also relies on nucleic acids for its operation, RNAi computing could benefit from the tools developed for DNA systems. However, these tools must be harnessed to produce bioactive components and be adapted for harsh operating environments that reflect in vivo conditions. In a step toward this goal, we report the construction and implementation of biosensors that ‘transduce’ mRNA levels into bioactive, small interfering RNA molecules via RNA strand exchange in a cell-free Drosophila embryo lysate, a step beyond simple buffered environments. We further integrate the sensors with our RNAi ‘computational’ module to evaluate two-input logic functions on mRNA concentrations. Our results show how RNA strand exchange can expand the utility of RNAi computing and point toward the possibility of using strand exchange in a native biological setting. PMID:20194121

Droplet Microfluidic and Magnetic Particles Platform for Cancer Typing.

PubMed

Ferraro, Davide; Champ, Jérôme; Teste, Bruno; Serra, M; Malaquin, Laurent; Descroix, Stéphanie; de Cremoux, Patricia; Viovy, Jean-Louis

2017-01-01

Analyses of nucleic acids are routinely performed in hospital laboratories to detect gene alterations for cancer diagnosis and treatment decision. Among the different possible investigations, mRNA analysis provides information on abnormal levels of genes expression. Standard laboratory methods are still not adapted to the isolation and quantitation of low mRNA amounts and new techniques needs to be developed in particular for rare subsets analysis. By reducing the volume involved, time process, and the contamination risks, droplet microfluidics provide numerous advantages to perform analysis down to the single cell level.We report on a droplet microfluidic platform based on the manipulation of magnetic particles that allows the clinical analysis of tumor tissues. In particular, it allows the extraction of mRNA from the total-RNA sample, Reverse Transcription, and cDNA amplification, all in droplets.

A Cross-Species Analysis in Pancreatic Neuroendocrine Tumors Reveals Molecular Subtypes with Distinctive Clinical, Metastatic, Developmental, and Metabolic Characteristics

PubMed Central

Sadanandam, Anguraj; Wullschleger, Stephan; Lyssiotis, Costas A.; Grötzinger, Carsten; Barbi, Stefano; Bersani, Samantha; Körner, Jan; Wafy, Ismael; Mafficini, Andrea; Lawlor, Rita T.; Simbolo, Michele; Asara, John M.; Bläker, Hendrik; Cantley, Lewis C.; Wiedenmann, Bertram; Scarpa, Aldo; Hanahan, Douglas

2016-01-01

Seeking to assess the representative and instructive value of an engineered mouse model of pancreatic neuroendocrine tumors (PanNET) for its cognate human cancer, we profiled and compared mRNA and miRNA transcriptomes of tumors from both. Mouse PanNET tumors could be classified into two distinctive subtypes, well-differentiated islet/insulinoma tumors (IT) and poorly differentiated tumors associated with liver metastases, dubbed metastasis-like primary (MLP). Human PanNETs were independently classified into these same two subtypes, along with a third, specific gene mutation–enriched subtype. The MLP subtypes in human and mouse were similar to liver metastases in terms of miRNA and mRNA transcriptome profiles and signature genes. The human/mouse MLP subtypes also similarly expressed genes known to regulate early pancreas development, whereas the IT subtypes expressed genes characteristic of mature islet cells, suggesting different tumorigenesis pathways. In addition, these subtypes exhibit distinct metabolic profiles marked by differential pyruvate metabolism, substantiating the significance of their separate identities. SIGNIFICANCE This study involves a comprehensive cross-species integrated analysis of multi-omics profiles and histology to stratify PanNETs into subtypes with distinctive characteristics. We provide support for the RIP1-TAG2 mouse model as representative of its cognate human cancer with prospects to better understand PanNET heterogeneity and consider future applications of personalized cancer therapy. PMID:26446169

A Cross-Species Analysis in Pancreatic Neuroendocrine Tumors Reveals Molecular Subtypes with Distinctive Clinical, Metastatic, Developmental, and Metabolic Characteristics.

PubMed

Sadanandam, Anguraj; Wullschleger, Stephan; Lyssiotis, Costas A; Grötzinger, Carsten; Barbi, Stefano; Bersani, Samantha; Körner, Jan; Wafy, Ismael; Mafficini, Andrea; Lawlor, Rita T; Simbolo, Michele; Asara, John M; Bläker, Hendrik; Cantley, Lewis C; Wiedenmann, Bertram; Scarpa, Aldo; Hanahan, Douglas

2015-12-01

Seeking to assess the representative and instructive value of an engineered mouse model of pancreatic neuroendocrine tumors (PanNET) for its cognate human cancer, we profiled and compared mRNA and miRNA transcriptomes of tumors from both. Mouse PanNET tumors could be classified into two distinctive subtypes, well-differentiated islet/insulinoma tumors (IT) and poorly differentiated tumors associated with liver metastases, dubbed metastasis-like primary (MLP). Human PanNETs were independently classified into these same two subtypes, along with a third, specific gene mutation-enriched subtype. The MLP subtypes in human and mouse were similar to liver metastases in terms of miRNA and mRNA transcriptome profiles and signature genes. The human/mouse MLP subtypes also similarly expressed genes known to regulate early pancreas development, whereas the IT subtypes expressed genes characteristic of mature islet cells, suggesting different tumorigenesis pathways. In addition, these subtypes exhibit distinct metabolic profiles marked by differential pyruvate metabolism, substantiating the significance of their separate identities. This study involves a comprehensive cross-species integrated analysis of multi-omics profiles and histology to stratify PanNETs into subtypes with distinctive characteristics. We provide support for the RIP1-TAG2 mouse model as representative of its cognate human cancer with prospects to better understand PanNET heterogeneity and consider future applications of personalized cancer therapy. ©2015 American Association for Cancer Research.

Differential expression of lncRNAs during the HIV replication cycle: an underestimated layer in the HIV-host interplay.

PubMed

Trypsteen, Wim; Mohammadi, Pejman; Van Hecke, Clarissa; Mestdagh, Pieter; Lefever, Steve; Saeys, Yvan; De Bleser, Pieter; Vandesompele, Jo; Ciuffi, Angela; Vandekerckhove, Linos; De Spiegelaere, Ward

2016-10-26

Studying the effects of HIV infection on the host transcriptome has typically focused on protein-coding genes. However, recent advances in the field of RNA sequencing revealed that long non-coding RNAs (lncRNAs) add an extensive additional layer to the cell's molecular network. Here, we performed transcriptome profiling throughout a primary HIV infection in vitro to investigate lncRNA expression at the different HIV replication cycle processes (reverse transcription, integration and particle production). Subsequently, guilt-by-association, transcription factor and co-expression analysis were performed to infer biological roles for the lncRNAs identified in the HIV-host interplay. Many lncRNAs were suggested to play a role in mechanisms relying on proteasomal and ubiquitination pathways, apoptosis, DNA damage responses and cell cycle regulation. Through transcription factor binding analysis, we found that lncRNAs display a distinct transcriptional regulation profile as compared to protein coding mRNAs, suggesting that mRNAs and lncRNAs are independently modulated. In addition, we identified five differentially expressed lncRNA-mRNA pairs with mRNA involvement in HIV pathogenesis with possible cis regulatory lncRNAs that control nearby mRNA expression and function. Altogether, the present study demonstrates that lncRNAs add a new dimension to the HIV-host interplay and should be further investigated as they may represent targets for controlling HIV replication.

Cytochrome P450-2C11 mRNA is not expressed in endothelial cells dissected from rat renal arterioles.

PubMed

Heil, Sandra G; De Vriese, An S; Kluijtmans, Leo A J; Dijkman, Henry; van Strien, Denise; Akkers, Robert; Blom, Henk J

2005-01-01

Cytochrome P450 (CYP) isoenzymes (CYP2C and CYP2J) are involved in the production of epoxyeicosatrienoic acids, which are postulated as endothelium-derived hyperpolarizing factors (EDHFs). We hypothesized that if CYP2C11 is involved in the EDHF-mediated responses, its mRNA should be expressed in endothelial cells. We, therefore, examined the mRNA expression of CYP2C11 in endothelial cells of renal arterioles. Laser microdissection was applied to isolate endothelial cells from the renal arterioles of 4 male and 4 female Wistar rats. As a positive control of CYP2C11 expression, hepatocytes were also dissected from these rats. RNA was isolated and real-time quantitative polymerase chain reaction (Q-PCR) analysis was applied. Q-PCR analysis showed that CYP2C11 mRNA was not expressed in laser microdissected endothelial cells of renal arterioles of male and female rats. CYP2C11 mRNA expression was highly abundant in hepatocytes dissected from male livers, but in female livers hardly any CYP2C11 mRNA was detected. We have shown that endothelial cells can be dissected from small renal arterioles by laser microdissection to study the mRNA expression of specific genes by Q-PCR. Using this novel tool, we demonstrated that the CYP2C11 mRNA was not expressed in the endothelial cells of renal arterioles. Therefore, we speculate that CYP2C11 does not contribute to the EDHF-mediated responses in renal arterioles. Copyright (c) 2005 S. Karger AG, Basel.

Age-related Differences in Dystrophin: Impact on Force Transfer Proteins, Membrane Integrity, and Neuromuscular Junction Stability.

PubMed

Hughes, David C; Marcotte, George R; Marshall, Andrea G; West, Daniel W D; Baehr, Leslie M; Wallace, Marita A; Saleh, Perrie M; Bodine, Sue C; Baar, Keith

2017-05-01

The loss of muscle strength with age has been studied from the perspective of a decline in muscle mass and neuromuscular junction (NMJ) stability. A third potential factor is force transmission. The purpose of this study was to determine the changes in the force transfer apparatus within aging muscle and the impact on membrane integrity and NMJ stability. We measured an age-related loss of dystrophin protein that was greatest in the flexor muscles. The loss of dystrophin protein occurred despite a twofold increase in dystrophin mRNA. Importantly, this disparity could be explained by the four- to fivefold upregulation of the dystromir miR-31. To compensate for the loss of dystrophin protein, aged muscle contained increased α-sarcoglycan, syntrophin, sarcospan, laminin, β1-integrin, desmuslin, and the Z-line proteins α-actinin and desmin. In spite of the adaptive increase in other force transfer proteins, over the 48 hours following lengthening contractions, the old muscles showed more signs of impaired membrane integrity (fourfold increase in immunoglobulin G-positive fibers and 70% greater dysferlin mRNA) and NMJ instability (14- to 96-fold increases in Runx1, AchRδ, and myogenin mRNA). Overall, these data suggest that age-dependent alterations in dystrophin leave the muscle membrane and NMJ more susceptible to contraction-induced damage even before changes in muscle mass are obvious. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Prognostic impact of MYC protein expression in central nervous system diffuse large B-cell lymphoma: comparison with MYC rearrangement and MYC mRNA expression.

PubMed

Son, Seung-Myoung; Ha, Sang-Yun; Yoo, Hae-Yong; Oh, Dongryul; Kim, Seok-Jin; Kim, Won-Seog; Ko, Young-Hyeh

2017-01-01

The prognostic role of MYC has been well documented in non-central nervous system diffuse large B-cell lymphoma; however, it remains controversial in central nervous system diffuse large B-cell lymphoma. To investigate the prognostic value of MYC, we analyzed the MYC protein expression by immunohistochemistry, mRNA expression by RNA in situ hybridization, and gene status by fluorescence in situ hybridization in 74 cases of central nervous system diffuse large B-cell lymphoma. Moreover, we examined the correlation between MYC translocation, mRNA expression, and protein expression. The mean percentage of MYC immunopositive cells was 49%. Using a 44% cutoff value, 49 (66%) cases showed MYC protein overexpression. The result of mRNA in situ hybridization using the RNA scope technology was obtained using the H-scoring system; the median value was 34.2. Using the cutoff value of 63.5, 16 (22%) cases showed MYC mRNA overexpression. MYC gene rearrangement was detected in five out of 68 (7%) cases. MYC translocation showed no statistically significant correlation with mRNA expression; however, all MYC translocation-positive cases showed MYC protein overexpression, with a higher mean percentage of MYC protein expression than that of translocation-negative cases (78 vs 48%, P=0.001). The level of MYC mRNA expression was moderately correlated with the level of MYC protein expression (P<0.001). The mean percentage of MYC protein expression in the high MYC mRNA group was higher than that in the low MYC mRNA group (70 vs 47%, P<0.001). A univariate analysis showed that age over 60 years, Eastern Cooperative Oncology Group (ECOG) performance status ≥2 and MYC protein overexpression were significantly associated with an increased risk of death. MYC translocation and MYC mRNA expression had no prognostic significance. On multivariate analysis, MYC protein overexpression and ECOG score retained prognostic significance.

Clinical values of AFP, GPC3 mRNA in peripheral blood for prediction of hepatocellular carcinoma recurrence following OLT: AFP, GPC3 mRNA for prediction of HCC.

PubMed

Wang, Yuliang; Shen, Zhongyang; Zhu, Zhijun; Han, Ruifa; Huai, Mingsheng

2011-03-01

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Annually, about 200,000 patients died of HCC in China. Liver transplantation (LT) holds great theoretical appeal in treating HCC. However, the high recurrence rate after transplantation is the most important limiting factor for long-term survival. To assess the value of alpha-fetoprotein (AFP) messenger RNA (mRNA), Glypican-3 (GPC3) mRNA-expressing cells in the peripheral blood (PB) for prediction of HCC recurrence following orthotopic liver transplantation (OLT). 29 patients with HCC who underwent OLT with a minimum clinical follow-up of 12 months were included in this retrospective study. We detected AFP mRNA, GPC3 mRNA-expressing cells in the PB by TaqMan real-time reverse transcriptase-polymerase chain reaction (RT-PCR), pre-, intra- and post-operatively. The early recurrence of patients was evaluated. 8 (28%), 15 (52%), and 9 (31%) patients had AFP mRNA detected pre-, intra-, and post-operatively, respectively. With 12 months of follow-up, HCC recurred in 7 (24%) patients. Univariate analysis revealed that positive pre- and post-operative AFP mRNA, TNM stage as well as vascular invasion were significant predictors for the HCC recurrence. Multivariate analysis revealed that being positive for AFP mRNA pre-operatively remained a significant risk factor for HCC recurrence after OLT. GPC3 mRNA was expressed in all PB samples. There was no significant difference in the expression levels of GPC3 mRNA between the HCC and control groups. There were no significant differences in GPC3 mRNA expression values between those patients with and without tumor recurrence. The pre-operative detection of circulating AFP mRNA-expressing cells could be a useful predictor for HCC recurrence following OLT. GPC3 mRNA-expressing cells in PB seem to have no diagnostic value.

Provirus Integration at the 3 Region of N‐myc in Cell Lines Established from Thymic Lymphomas Spontaneously Formed in AKR Mice and a [(BALB/c × B6)F1AKR] Bone Marrow Chimera

PubMed Central

Yano, Yoko; Kobayashi, Seiichi; Yasumizu, Ryoji; Tamaki, Junko; Kubo, Mitsumasa; Sasaki, Akio; Hasan, Shahid; Okuyama, Harue; Inaba, Muneo; Ikehara, Susumu; Hiai, Hiroshi; Kakinuma, Mitsuaki

1991-01-01

Among 18 thymic leukemia cell lines which have been established from spontaneous thymic lym‐phomas in AKR mice as well as in bone marrow chimeras which were constructed by transplanting allogeneic bone marrow cells into irradiated AKR mice, three proviral integration sites were identified; near c‐myc, N‐myc and pim‐l loci. No integration site specific for chimeric leukemia cell lines was found. In three thymic leukemia cell lines which contained rearranged N‐myc, genes, insertions of long terminal repeats (LTRs) of murine leukemia viruses were detected at 18 or 20 bp downstream of the translational termination codon. These results demonstrate that the 3’region of the N‐myc gene is one of the integration targets for murine leukemia viruses in spontaneous thymic lymphomas. In these three cell lines, N‐myc mRNA was stably transcribed and transcription of c‐myc mRNA was down‐regulated. The integrated murine leukemia viruses in AKR thymic leukemia were most likely AKV, though the DNA sequence of the LTR inserted in the genome of a leukemic cell line from [(BALB/c × B6)F1‐AKR], CAK20, was different from LTRs of murine leukemia viruses so far reported. PMID:1900822

Cellular localization of thrombopoietin mRNA in the liver by in situ hybridization.

PubMed

Nomura, S; Ogami, K; Kawamura, K; Tsukamoto, I; Kudo, Y; Kanakura, Y; Kitamura, Y; Miyazaki, H; Kato, T

1997-07-01

The expression of thrombopoietin (TPO) mRNA is observed in several tissues, including liver, kidney, brain, skeletal muscle, intestine, spleen, and bone marrow. Among these organs, the highest expression of TPO mRNA is detected in the liver. We identified cells producing TPO by means of in situ hybridization of adult rat liver using digoxigenin-11-UTP-labeled cRNA probes. We found that the cells expressing TPO mRNA also expressed serum albumin mRNA. TPO mRNA was detected in parenchymal cells (hepatocytes) but not in non-parenchymal cells (including endothelial cells, epithelial cells, and so forth). To determine the location of TPO expression in embryogenesis, sections of fetal mice were further analyzed by in situ hybridization. TPO mRNA was detected only in hepatocytes of fetal liver, which was also the major site of hematopoiesis. The expression of TPO mRNA in fetal liver was observed from 12.5 days postcoitus. Northern blot analysis showed that mouse liver transcribed the same size of TPO mRNA in the fetus and in the adult. These results clearly demonstrate that hepatocytes are the primary site of TPO production in the liver from fetus to adult.

Localization of mRNA in vertebrate axonal compartments by in situ hybridization.

PubMed

Sotelo-Silveira, José Roberto; Calliari, Aldo; Kun, Alejandra; Elizondo, Victoria; Canclini, Lucía; Sotelo, José Roberto

2011-01-01

The conclusive demonstration of RNA in vertebrate axons by in situ hybridization (ISH) has been elusive. We review the most important reasons for difficulties, including low concentration of axonal RNAs, localization in specific cortical domains, and the need to isolate axons. We demonstrate the importance of axon micro-dissection to obtain a whole mount perspective of mRNA distribution in the axonal territory. We describe a protocol to perform fluorescent ISH in isolated axons and guidelines for the preservation of structural and molecular integrity of cortical RNA-containing domains (e.g., Periaxoplasmic Ribosomal Plaques, or PARPs) in isolated axoplasm.

Analysis of myosin heavy chain mRNA expression by RT-PCR

NASA Technical Reports Server (NTRS)

Wright, C.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

1997-01-01

An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

Susceptibility loci of CNOT6 in the general mRNA degradation pathway and lung cancer risk-A re-analysis of eight GWASs.

PubMed

Zhou, Fei; Wang, Yanru; Liu, Hongliang; Ready, Neal; Han, Younghun; Hung, Rayjean J; Brhane, Yonathan; McLaughlin, John; Brennan, Paul; Bickeböller, Heike; Rosenberger, Albert; Houlston, Richard S; Caporaso, Neil; Landi, Maria Teresa; Brüske, Irene; Risch, Angela; Ye, Yuanqing; Wu, Xifeng; Christiani, David C; Goodman, Gary; Chen, Chu; Amos, Christopher I; Wei, Qingyi

2017-04-01

mRNA degradation is an important regulatory step for controlling gene expression and cell functions. Genetic abnormalities involved in mRNA degradation genes were found to be associated with cancer risks. Therefore, we systematically investigated the roles of genetic variants in the general mRNA degradation pathway in lung cancer risk. Meta-analyses were conducted using summary data from six lung cancer genome-wide association studies (GWASs) from the Transdisciplinary Research in Cancer of the Lung and additional two GWASs from Harvard University and deCODE in the International Lung Cancer Consortium. Expression quantitative trait loci analysis (eQTL) was used for in silico functional validation of the identified significant susceptibility loci. This pathway-based analysis included 6816 single nucleotide polymorphisms (SNP) in 68 genes in 14 463 lung cancer cases and 44 188 controls. In the single-locus analysis, we found that 20 SNPs were associated with lung cancer risk with a false discovery rate threshold of <0.05. Among the 11 newly identified SNPs in CNOT6, which were in high linkage disequilibrium, the rs2453176 with a RegulomDB score "1f" was chosen as the tagSNP for further analysis. We found that the rs2453176 T allele was significantly associated with lung cancer risk (odds ratio = 1.11, 95% confidence interval = 1.04-1.18) in the eight GWASs. In the eQTL analysis, we found that levels of CNOT6 mRNA expression were significantly correlated with the rs2453176 T allele, which provided additional biological basis for the observed positive association. The CNOT6 rs2453176 SNP may be a new functional susceptible locus for lung cancer risk. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The Shine-Dalgarno sequence of riboswitch-regulated single mRNAs shows ligand-dependent accessibility bursts

NASA Astrophysics Data System (ADS)

Rinaldi, Arlie J.; Lund, Paul E.; Blanco, Mario R.; Walter, Nils G.

2016-01-01

In response to intracellular signals in Gram-negative bacteria, translational riboswitches--commonly embedded in messenger RNAs (mRNAs)--regulate gene expression through inhibition of translation initiation. It is generally thought that this regulation originates from occlusion of the Shine-Dalgarno (SD) sequence upon ligand binding; however, little direct evidence exists. Here we develop Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) to investigate the ligand-dependent accessibility of the SD sequence of an mRNA hosting the 7-aminomethyl-7-deazaguanine (preQ1)-sensing riboswitch. Spike train analysis reveals that individual mRNA molecules alternate between two conformational states, distinguished by `bursts' of probe binding associated with increased SD sequence accessibility. Addition of preQ1 decreases the lifetime of the SD's high-accessibility (bursting) state and prolongs the time between bursts. In addition, ligand-jump experiments reveal imperfect riboswitching of single mRNA molecules. Such complex ligand sensing by individual mRNA molecules rationalizes the nuanced ligand response observed during bulk mRNA translation.

In silico analysis of miRNA-mediated gene regulation in OCA and OA genes.

PubMed

Kamaraj, Balu; Gopalakrishnan, Chandrasekhar; Purohit, Rituraj

2014-12-01

Albinism is an autosomal recessive genetic disorder due to low secretion of melanin. The oculocutaneous albinism (OCA) and ocular albinism (OA) genes are responsible for melanin production and also act as a potential targets for miRNAs. The role of miRNA is to inhibit the protein synthesis partially or completely by binding with the 3'UTR of the mRNA thus regulating gene expression. In this analysis, we predicted the genetic variation that occurred in 3'UTR of the transcript which can be a reason for low melanin production thus causing albinism. The single nucleotide polymorphisms (SNPs) in 3'UTR cause more new binding sites for miRNA which binds with mRNA which leads to inhibit the translation process either partially or completely. The SNPs in the mRNA of OCA and OA genes can create new binding sites for miRNA which may control the gene expression and lead to hypopigmentation. We have developed a computational procedure to determine the SNPs in the 3'UTR region of mRNA of OCA (TYR, OCA2, TYRP1 and SLC45A2) and OA (GPR143) genes which will be a potential cause for albinism. We identified 37 SNPs in five genes that are predicted to create 87 new binding sites on mRNA, which may lead to abrogation of the translation process. Expression analysis confirms that these genes are highly expressed in skin and eye regions. It is well supported by enrichment analysis that these genes are mainly involved in eye pigmentation and melanin biosynthesis process. The network analysis also shows how the genes are interacting and expressing in a complex network. This insight provides clue to wet-lab researches to understand the expression pattern of OCA and OA genes and binding phenomenon of mRNA and miRNA upon mutation, which is responsible for inhibition of translation process at genomic levels.

Short-term application of dexamethasone on stem cells derived from human gingiva reduces the expression of RUNX2 and β-catenin.

PubMed

Kim, Bo-Bae; Kim, Minji; Park, Yun-Hee; Ko, Youngkyung; Park, Jun-Beom

2017-06-01

Objective Next-generation sequencing was performed to evaluate the effects of short-term application of dexamethasone on human gingiva-derived mesenchymal stem cells. Methods Human gingiva-derived stem cells were treated with a final concentration of 10 -7  M dexamethasone and the same concentration of vehicle control. This was followed by mRNA sequencing and data analysis, gene ontology and pathway analysis, quantitative real-time polymerase chain reaction of mRNA, and western blot analysis of RUNX2 and β-catenin. Results In total, 26,364 mRNAs were differentially expressed. Comparison of the results of dexamethasone versus control at 2 hours revealed that 7 mRNAs were upregulated and 25 mRNAs were downregulated. The application of dexamethasone reduced the expression of RUNX2 and β-catenin in human gingiva-derived mesenchymal stem cells. Conclusion The effects of dexamethasone on stem cells were evaluated with mRNA sequencing, and validation of the expression was performed with qualitative real-time polymerase chain reaction and western blot analysis. The results of this study can provide new insights into the role of mRNA sequencing in maxillofacial areas.

Nucleotide sequence and regulatory studies of VGF, a nervous system-specific mRNA that is rapidly and relatively selectively induced by nerve growth factor.

PubMed

Salton, S R

1991-09-01

A nervous system-specific mRNA that is rapidly induced in PC12 cells to a greater extent by nerve growth factor (NGF) than by epidermal growth factor treatment has been cloned. The polypeptide deduced from the nucleic acid sequence of the NGF33.1 cDNA clone contains regions of amino acid sequence identity with that predicted by the cDNA clone VGF, and further analysis suggests that both NGF33.1 and VGF cDNA clones very likely correspond to the same mRNA (VGF). In this report both the nucleic acid sequence that corresponds to VGF mRNA and the polypeptide predicted by the NGF33.1 cDNA clone are presented. Genomic Southern analysis and database comparison did not detect additional sequences with high homology to the VGF gene. Induction of VGF mRNA by depolarization and phorbol 12-myristate 13-acetate treatment was greater than by serum stimulation or protein kinase A pathway activation. These studies suggest that VGF mRNA is induced to the greatest extent by NGF treatment and that VGF is one of the most rapidly regulated neuronal mRNAs identified in PC12 cells.

Increased expression of ADAM 9 and ADAM 15 mRNA in pancreatic cancer.

PubMed

Yamada, Daisuke; Ohuchida, Kenoki; Mizumoto, Kazuhiro; Ohhashi, Seiji; Yu, Jun; Egami, Takuya; Fujita, Hayato; Nagai, Eishi; Tanaka, Masao

2007-01-01

A disintegrin and metalloproteases (ADAMs) comprise a multifunctional family of membrane-anchored proteins. ADAM 9 and ADAM 15 are involved in cell migration and invasion. Expression of ADAM 9 and ADAM 15 was reported to be altered in several types of cancer. Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure the expression of ADAM 9 mRNA in bulk pancreatic tissues. Results showed no significant difference in the expression of ADAM 9 mRNA between pancreatic cancer and non-neoplastic pancreas. Primary cultured pancreatic fibroblasts also expressed ADAM 9 mRNA. Therefore, a laser microdissection and pressure catapulting technique was employed to isolate cancer cells from tumor tissues. The expression of ADAM 9 and ADAM 15 mRNA was measured in microdissected samples (cancer cells, n = 11; normal epithelial cells, n = 13 for ADAM 9; cancer cells, n = 9; normal epithelial cells, n = 9 for ADAM 15). Pancreatic cancer cells expressed significantly higher levels of ADAM 9 and ADAM 15 mRNA than did normal pancreatic epithelial cells (p = 0.016 for ADAM 9; p = 0.004 for ADAM 15). ADAM 9 and ADAM 15 are involved in pancreatic cancer. Microdissection-based analysis appears to be indispensable for the accurate analysis of the expression of certain ADAM family members in pancreatic cancer.

Towards Personalized Medicine Mediated by in Vitro Virus-Based Interactome Approaches

PubMed Central

Ohashi, Hiroyuki; Miyamoto-Sato, Etsuko

2014-01-01

We have developed a simple in vitro virus (IVV) selection system based on cell-free co-translation, using a highly stable and efficient mRNA display method. The IVV system is applicable to the high-throughput and comprehensive analysis of proteins and protein–ligand interactions. Huge amounts of genomic sequence data have been generated over the last decade. The accumulated genetic alterations and the interactome networks identified within cells represent a universal feature of a disease, and knowledge of these aspects can help to determine the optimal therapy for the disease. The concept of the “integrome” has been developed as a means of integrating large amounts of data. We have developed an interactome analysis method aimed at providing individually-targeted health care. We also consider future prospects for this system. PMID:24756093

Single-Cell and Single-Molecule Analysis of Gene Expression Regulation.

PubMed

Vera, Maria; Biswas, Jeetayu; Senecal, Adrien; Singer, Robert H; Park, Hye Yoon

2016-11-23

Recent advancements in single-cell and single-molecule imaging technologies have resolved biological processes in time and space that are fundamental to understanding the regulation of gene expression. Observations of single-molecule events in their cellular context have revealed highly dynamic aspects of transcriptional and post-transcriptional control in eukaryotic cells. This approach can relate transcription with mRNA abundance and lifetimes. Another key aspect of single-cell analysis is the cell-to-cell variability among populations of cells. Definition of heterogeneity has revealed stochastic processes, determined characteristics of under-represented cell types or transitional states, and integrated cellular behaviors in the context of multicellular organisms. In this review, we discuss novel aspects of gene expression of eukaryotic cells and multicellular organisms revealed by the latest advances in single-cell and single-molecule imaging technology.

Single-Cell mRNA-Seq Using the Fluidigm C1 System and Integrated Fluidics Circuits.

PubMed

Gong, Haibiao; Do, Devin; Ramakrishnan, Ramesh

2018-01-01

Single-cell mRNA-seq is a valuable tool to dissect expression profiles and to understand the regulatory network of genes. Microfluidics is well suited for single-cell analysis owing both to the small volume of the reaction chambers and easiness of automation. Here we describe the workflow of single-cell mRNA-seq using C1 IFC, which can isolate and process up to 96 cells. Both on-chip procedure (lysis, reverse transcription, and preamplification PCR) and off-chip sequencing library preparation protocols are described. The workflow generates full-length mRNA information, which is more valuable compared to 3' end counting method for many applications.

Characterization of miRNA-regulated networks, hubs of signaling, and biomarkers in obstruction-induced bladder dysfunction

PubMed Central

Kiss, Bernhard; Moltzahn, Felix; Keller, Irene; Rehrauer, Hubert; Fournier, Catharine Aquino; Burkhard, Fiona C.

2017-01-01

Bladder outlet obstruction (BOO) induces significant organ remodeling, leading to lower urinary tract symptoms accompanied by urodynamic changes in bladder function. Here, we report mRNA and miRNA transcriptome sequencing of bladder samples from human patients with different urodynamically defined states of BOO. Patients’ miRNA and mRNA expression profiles correlated with urodynamic findings. Validation of RNA sequencing results in an independent patient cohort identified combinations of 3 mRNAs (NRXN3, BMP7, UPK1A) and 3 miRNAs (miR-103a-3p, miR-10a-5p, miR-199a-3p) sufficient to discriminate between bladder functional states. All BOO patients shared cytokine and immune response pathways, TGF-β and NO signaling pathways, and hypertrophic PI3K/AKT signaling pathways. AP-1 and NFkB were dominant transcription factors, and TNF-α was the top upstream regulator. Integrated miRNA-mRNA expression analysis identified pathways and molecules targeted by differentially expressed miRNAs. Molecular changes in BOO suggest an increasing involvement of miRNAs in the control of bladder function from the overactive to underactive/acontractile states. PMID:28138557

Extensive cross-regulation of post-transcriptional regulatory networks in Drosophila

DOE PAGES

Stoiber, Marcus H.; Olson, Sara; May, Gemma E.; ...

2015-08-20

In eukaryotic cells, RNAs exist as ribonucleoprotein particles (RNPs). Despite the importance of these complexes in many biological processes, including splicing, polyadenylation, stability, transportation, localization, and translation, their compositions are largely unknown. We affinity-purified 20 distinct RNA-binding proteins (RBPs) from cultured Drosophila melanogaster cells under native conditions and identified both the RNA and protein compositions of these RNP complexes. We identified “high occupancy target” (HOT) RNAs that interact with the majority of the RBPs we surveyed. HOT RNAs encode components of the nonsense-mediated decay and splicing machinery, as well as RNA-binding and translation initiation proteins. The RNP complexes contain proteinsmore » and mRNAs involved in RNA binding and post-transcriptional regulation. Genes with the capacity to produce hundreds of mRNA isoforms, ultracomplex genes, interact extensively with heterogeneous nuclear ribonuclear proteins (hnRNPs). Our data are consistent with a model in which subsets of RNPs include mRNA and protein products from the same gene, indicating the widespread existence of auto-regulatory RNPs. Lastly, from the simultaneous acquisition and integrative analysis of protein and RNA constituents of RNPs, we identify extensive cross-regulatory and hierarchical interactions in post-transcriptional control.« less

RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA.

PubMed

Cui, Yalei; Huang, Tianzhi; Zhang, Xiaobo

2015-12-01

MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex. © 2015 The Authors.

Extensive cross-regulation of post-transcriptional regulatory networks in Drosophila

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stoiber, Marcus H.; Olson, Sara; May, Gemma E.

In eukaryotic cells, RNAs exist as ribonucleoprotein particles (RNPs). Despite the importance of these complexes in many biological processes, including splicing, polyadenylation, stability, transportation, localization, and translation, their compositions are largely unknown. We affinity-purified 20 distinct RNA-binding proteins (RBPs) from cultured Drosophila melanogaster cells under native conditions and identified both the RNA and protein compositions of these RNP complexes. We identified “high occupancy target” (HOT) RNAs that interact with the majority of the RBPs we surveyed. HOT RNAs encode components of the nonsense-mediated decay and splicing machinery, as well as RNA-binding and translation initiation proteins. The RNP complexes contain proteinsmore » and mRNAs involved in RNA binding and post-transcriptional regulation. Genes with the capacity to produce hundreds of mRNA isoforms, ultracomplex genes, interact extensively with heterogeneous nuclear ribonuclear proteins (hnRNPs). Our data are consistent with a model in which subsets of RNPs include mRNA and protein products from the same gene, indicating the widespread existence of auto-regulatory RNPs. Lastly, from the simultaneous acquisition and integrative analysis of protein and RNA constituents of RNPs, we identify extensive cross-regulatory and hierarchical interactions in post-transcriptional control.« less

Temporal and Spatial Post-Transcriptional Regulation of Zebrafish Tie1 mRNA by Long Noncoding RNA During Brain Vascular Assembly.

PubMed

Chowdhury, Tamjid A; Koceja, Chris; Eisa-Beygi, Shahram; Kleinstiver, Benjamin P; Kumar, Suresh N; Lin, Chien-Wei; Li, Keguo; Prabhudesai, Shubhangi; Joung, J Keith; Ramchandran, Ramani

2018-05-03

Tie1 (tyrosine kinase containing immunoglobulin and epidermal growth factor homology 1), an endothelial and hematopoietic cell-specific receptor tyrosine kinase, is an important regulator of angiogenesis and critical for maintaining vascular integrity. The post-transcriptional regulation of tie1 mRNA expression is not understood, but it might partly explain Tie1's differential expression pattern in endothelium. Following up on our previous work that identified natural antisense transcripts from the tie1 locus- tie1 antisense ( tie1AS ), which regulates tie1 mRNA levels in zebrafish-we attempted to identify the mechanism of this regulation. Through in vitro and in vivo ribonucleoprotein binding studies, we demonstrated that tie1AS long noncoding RNA interacts with an RNA binding protein-embryonic lethal and abnormal vision Drosophila-like 1 (Elavl1)-that regulates tie1 mRNA levels. When we disrupted the interaction between tie1AS and Elavl1 by using constitutively active antisense morpholino oligonucleotides or photoactivatable morpholino oligonucleotides, tie1 mRNA levels increased between 26 and 31 hours post-fertilization, particularly in the head. This increase correlated with dilation of primordial midbrain channels, smaller eyes, and reduced ventricular space. We also observed these phenotypes when we used CRISPR (clustered regularly interspaced short palindromic repeats)-mediated CRISPRi (CRISPR-mediated interference) to knock down tie1AS . Treatment of the morpholino oligonucleotide-injected embryos with a small molecule that decreased tie1 mRNA levels rescued all 3 abnormal phenotypes. We identified a novel mode of temporal and spatial post-transcriptional regulation of tie1 mRNA. It involves long noncoding RNA, tie1AS, and Elavl1 (an interactor of tie1AS ). © 2018 American Heart Association, Inc.

PAR-CLIP data indicate that Nrd1-Nab3-dependent transcription termination regulates expression of hundreds of protein coding genes in yeast

PubMed Central

2014-01-01

Background Nrd1 and Nab3 are essential sequence-specific yeast RNA binding proteins that function as a heterodimer in the processing and degradation of diverse classes of RNAs. These proteins also regulate several mRNA coding genes; however, it remains unclear exactly what percentage of the mRNA component of the transcriptome these proteins control. To address this question, we used the pyCRAC software package developed in our laboratory to analyze CRAC and PAR-CLIP data for Nrd1-Nab3-RNA interactions. Results We generated high-resolution maps of Nrd1-Nab3-RNA interactions, from which we have uncovered hundreds of new Nrd1-Nab3 mRNA targets, representing between 20 and 30% of protein-coding transcripts. Although Nrd1 and Nab3 showed a preference for binding near 5′ ends of relatively short transcripts, they bound transcripts throughout coding sequences and 3′ UTRs. Moreover, our data for Nrd1-Nab3 binding to 3′ UTRs was consistent with a role for these proteins in the termination of transcription. Our data also support a tight integration of Nrd1-Nab3 with the nutrient response pathway. Finally, we provide experimental evidence for some of our predictions, using northern blot and RT-PCR assays. Conclusions Collectively, our data support the notion that Nrd1 and Nab3 function is tightly integrated with the nutrient response and indicate a role for these proteins in the regulation of many mRNA coding genes. Further, we provide evidence to support the hypothesis that Nrd1-Nab3 represents a failsafe termination mechanism in instances of readthrough transcription. PMID:24393166

FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia.

PubMed

Nasilowska-Adamska, Barbara; Solarska, Iwona; Paluszewska, Monika; Malinowska, Iwona; Jedrzejczak, Wieslaw W; Warzocha, Krzysztof

2014-04-01

Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are aberrations associated with leukemia which indicate unsatisfactory prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined. We have analyzed the expression of MDR-1, multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) messenger RNA (mRNA) in relation to the mutational status of FLT3-ITD and MLL-PTD in 185 acute myeloid leukemia (AML) adult patients. The real-time quantitative polymerase chain reaction method was performed to assess the expression of the MDR-1, MRP-1, BCRP, and LRP mRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. Significantly higher expressions of MDR-1 mRNA were found in patients who did not harbor FLT3-ITD (0.20 vs. 0.05; p = 0.0001) and MRP-1 mRNA in patients with this mutation (0.96 vs. 0.70; p = 0.002) and of BCRP mRNA in patients with MLL-PTD (0.61 vs. 0.38; p = 0.03). In univariate analysis, the high expression of MDR-1 mRNA (≥0.1317) negatively influenced the outcome of induction therapy (p = 0.05), whereas the high expression of BCRP mRNA (≥1.1487) was associated with a high relapse rate (RR) (p = 0.013). We found that the high expression of MDR-1 (≥0.1317), MRP-1 (≥0.8409), and BCRP mRNA (≥1.1487) significantly influenced disease-free survival (DFS; p = 0.059, 0.032, and 0.009, respectively) and overall survival (0.048, 0.014, and 0.059, respectively). Moreover, a high expression of BCRP mRNA (≥1.1487) proved to be an independent prognostic factor for RR (p = 0.01) and DFS (p = 0.002) in multivariate analysis. The significant correlation between the expression of MDR-1, MRP-1, and BCRP mRNA and FLT3-ITD or MLL-PTD in AML patients requires further investigation.

The rapid destabilization of p53 mRNA in immortal chicken embryo fibroblast cells.

PubMed

Kim, H; You, S; Foster, L K; Farris, J; Foster, D N

2001-08-23

The steady-state levels of p53 mRNA were dramatically lower in immortal chicken embryo fibroblast (CEF) cell lines compared to primary CEF cells. In the presence of cycloheximide (CHX), the steady-state levels of p53 mRNA markedly increased in immortal CEF cell lines, similar to levels found in primary cells. The de novo synthetic rates of p53 mRNA were relatively similar in primary and immortal cells grown in the presence or absence of CHX. Destabilization of p53 mRNA was observed in the nuclei of immortal, but not primary, CEF cells. The half-life of p53 mRNA in primary cells was found to be a relatively long 23 h compared to only 3 h in immortal cells. The expression of transfected p53 cDNA was inhibited in immortal cells, but restored upon CHX treatment. The 5'-region of the p53 mRNA was shown to be involved in the rapid p53 mRNA destabilization in immortal cells by expression analysis of 5'- and 3'-deleted p53 cDNAs as well as fusion mRNA constructs of N-terminal p53 and N-terminal deleted LacZ genes. Together, it is suggestive that the downregulation of p53 mRNA in immortal CEF cells occurs through a post-transcriptional destabilizing mechanism.

Widespread promoter-mediated coordination of transcription and mRNA degradation

PubMed Central

2012-01-01

Background Previous work showed that mRNA degradation is coordinated with transcription in yeast, and in several genes the control of mRNA degradation was linked to promoter elements through two different mechanisms. Here we show at the genomic scale that the coordination of transcription and mRNA degradation is promoter-dependent in yeast and is also observed in humans. Results We first demonstrate that swapping upstream cis-regulatory sequences between two yeast species affects both transcription and mRNA degradation and suggest that while some cis-regulatory elements control either transcription or degradation, multiple other elements enhance both processes. Second, we show that adjacent yeast genes that share a promoter (through divergent orientation) have increased similarity in their patterns of mRNA degradation, providing independent evidence for the promoter-mediated coupling of transcription to mRNA degradation. Finally, analysis of the differences in mRNA degradation rates between mammalian cell types or mammalian species suggests a similar coordination between transcription and mRNA degradation in humans. Conclusions Our results extend previous studies and suggest a pervasive promoter-mediated coordination between transcription and mRNA degradation in yeast. The diverse genes and regulatory elements associated with this coordination suggest that it is generated by a global mechanism of gene regulation and modulated by gene-specific mechanisms. The observation of a similar coupling in mammals raises the possibility that coupling of transcription and mRNA degradation may reflect an evolutionarily conserved phenomenon in gene regulation. PMID:23237624

m6A-Driver: Identifying Context-Specific mRNA m6A Methylation-Driven Gene Interaction Networks

PubMed Central

Zhang, Song-Yao; Zhang, Shao-Wu; Liu, Lian; Huang, Yufei

2016-01-01

As the most prevalent mammalian mRNA epigenetic modification, N6-methyladenosine (m6A) has been shown to possess important post-transcriptional regulatory functions. However, the regulatory mechanisms and functional circuits of m6A are still largely elusive. To help unveil the regulatory circuitry mediated by mRNA m6A methylation, we develop here m6A-Driver, an algorithm for predicting m6A-driven genes and associated networks, whose functional interactions are likely to be actively modulated by m6A methylation under a specific condition. Specifically, m6A-Driver integrates the PPI network and the predicted differential m6A methylation sites from methylated RNA immunoprecipitation sequencing (MeRIP-Seq) data using a Random Walk with Restart (RWR) algorithm and then builds a consensus m6A-driven network of m6A-driven genes. To evaluate the performance, we applied m6A-Driver to build the context-specific m6A-driven networks for 4 known m6A (de)methylases, i.e., FTO, METTL3, METTL14 and WTAP. Our results suggest that m6A-Driver can robustly and efficiently identify m6A-driven genes that are functionally more enriched and associated with higher degree of differential expression than differential m6A methylated genes. Pathway analysis of the constructed context-specific m6A-driven gene networks further revealed the regulatory circuitry underlying the dynamic interplays between the methyltransferases and demethylase at the epitranscriptomic layer of gene regulation. PMID:28027310

Region specific regulation of glutamic acid decarboxylase mRNA expression by dopamine neurons in rat brain.

PubMed

Lindefors, N; Brene, S; Herrera-Marschitz, M; Persson, H

1989-01-01

In situ hybridization histochemistry and RNA blots were used to study the expression of glutamic acid decarboxylase (GAD) mRNA in rats with or without a unilateral lesion of midbrain dopamine neurons. Two populations of GAD mRNA positive neurons were found in the intact caudate-putamen, substantia nigra and fronto-parietal cortex. In caudate-putamen, only one out of ten of the GAD mRNA positive neurons expressed high levels, while in substantia nigra every second of the positive neurons expressed high levels of GAD mRNA. Relatively few, but intensively labelled neurons were found in the intact fronto-parietal cerebral cortex. In addition, one out of six of the GAD mRNA positive neurons in the fronto-parietal cortex showed a low labeling. On the ipsilateral side, the forebrain dopamine deafferentation induced an increase in the number of neurons expressing high levels of GAD mRNA in caudate-putamen, and a decrease in fronto-parietal cortex. A smaller decrease was also seen in substantia nigra. However, the total number of GAD mRNA positive neurons were not significantly changed in any of these brain regions. The changes in the levels of GAD mRNA after the dopamine lesion were confirmed by RNA blot analysis. Hence, midbrain dopamine neurons appear to control neuronal expression of GAD mRNA by a tonic down-regulation in a fraction of GAD mRNA positive neurons in caudate-putamen, and a tonic up-regulation in a fraction of GAD mRNA positive neurons in fronto-parietal cortex and substantia nigra.

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA.

PubMed

Wang, Hongyang; Owens, James D; Shih, Joanna H; Li, Ming-Chung; Bonner, Robert F; Mushinski, J Frederic

2006-04-27

Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA

PubMed Central

Wang, Hongyang; Owens, James D; Shih, Joanna H; Li, Ming-Chung; Bonner, Robert F; Mushinski, J Frederic

2006-01-01

Background Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. Results The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. Conclusion RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts. PMID:16643667

Whole-genome analysis of mRNA decay in Plasmodium falciparum reveals a global lengthening of mRNA half-life during the intra-erythrocytic development cycle.

PubMed

Shock, Jennifer L; Fischer, Kael F; DeRisi, Joseph L

2007-01-01

The rate of mRNA decay is an essential element of post-transcriptional regulation in all organisms. Previously, studies in several organisms found that the specific half-life of each mRNA is precisely related to its physiologic role, and plays an important role in determining levels of gene expression. We used a genome-wide approach to characterize mRNA decay in Plasmodium falciparum. We found that, globally, rates of mRNA decay increase dramatically during the asexual intra-erythrocytic developmental cycle. During the ring stage of the cycle, the average mRNA half-life was 9.5 min, but this was extended to an average of 65 min during the late schizont stage of development. Thus, a major determinant of mRNA decay rate appears to be linked to the stage of intra-erythrocytic development. Furthermore, we found specific variations in decay patterns superimposed upon the dominant trend of progressive half-life lengthening. These variations in decay pattern were frequently enriched for genes with specific cellular functions or processes. Elucidation of Plasmodium mRNA decay rates provides a key element for deciphering mechanisms of genetic control in this parasite, by complementing and extending previous mRNA abundance studies. Our results indicate that progressive stage-dependent decreases in mRNA decay rate function are a major determinant of mRNA accumulation during the schizont stage of intra-erythrocytic development. This type of genome-wide change in mRNA decay rate has not been observed in any other organism to date, and indicates that post-transcriptional regulation may be the dominant mechanism of gene regulation in P. falciparum.

Susceptibility loci of CNOT6 in the general mRNA degradation pathway and lung cancer risk - a re-analysis of eight GWASs

PubMed Central

Zhou, Fei; Wang, Yanru; Liu, Hongliang; Ready, Neal; Han, Younghun; Hung, Rayjean J.; Brhane, Yonathan; McLaughlin, John; Brennan, Paul; Bickeböller, Heike; Rosenberger, Albert; Houlston, Richard S.; Caporaso, Neil; Landi, Maria Teresa; Brüske, Irene; Risch, Angela; Ye, Yuanqing; Wu, Xifeng; Christiani, David C.; Goodman, Gary; Chen, Chu; Amos, Christopher I.; Qingyi, Wei

2017-01-01

Purpose mRNA degradation is an important regulatory step for controlling gene expression and cell functions. Genetic abnormalities of the genes involved in mRNA degradation were found to be associated with cancer risks. Therefore, we systematically investigated the roles of genetic variants of genes in the general mRNA degradation pathway in lung cancer risk. Experimental design Meta-analyses were conducted in six lung cancer genome-wide association studies (GWASs) from the Transdisciplinary Research in Cancer of the Lung and additional two GWASs from Harvard University and deCODE in the International Lung Cancer Consortium. Expression quantitative trait loci analysis (eQTL) was used for in silico functional validation of the identified significant susceptibility loci. Results This pathway-based analysis included 4,603 single nucleotide polymorphisms (SNP) in 68 genes in 14,463 lung cancer cases and 44,188 controls, of which 20 SNPs were found to be associated with lung cancer risk with a false discovery rate threshold of <0.05. Among the 11 newly identified SNPs in CNOT6, which were in high linkage disequilibrium, the rs2453176 with a RegulomDB score “1f” was chosen as the tag SNP for further analysis. We found that the rs2453176 T allele was significantly associated with lung cancer risk (odds ratio=1.11, 95% confidence interval=1.04–1.18, P=0.001) in the eight GWASs. In the eQTL analysis, we found that levels of CNOT6 mRNA expression were significantly correlated with the rs2453176 T allele, which provided additional biological basis for the observed positive association. Conclusion The CNOT6 rs2453176 SNP may be a new functional susceptible locus for lung cancer risk. PMID:27805284

Integrated microRNA and mRNA network analysis of the human myometrial transcriptome in the transition from quiescence to labor.

PubMed

Ackerman, William E; Buhimschi, Irina A; Brubaker, Douglas; Maxwell, Sean; Rood, Kara M; Chance, Mark R; Jing, Hongwu; Mesiano, Sam; Buhimschi, Catalin S

2018-02-13

We conducted integrated transcriptomics network analyses of miRNA and mRNA interactions in human myometrium to identify novel molecular candidates potentially involved in human parturition. Myometrial biopsies were collected from women undergoing primary Cesarean deliveries in well-characterized clinical scenarios: 1) spontaneous term labor (TL, n = 5); 2) term non-labor (TNL, n = 5); 3) spontaneous preterm birth (PTB) with histologic chorioamnionitis (PTB-HCA, n = 5); and 4) indicated PTB non-labor (PTB-NL, n = 5). MicroRNAs and long RNAs were profiled using RNA sequencing, and miRNA-target interaction networks were mined for key discriminatory subnetworks. Forty miRNAs differed between TL and TNL myometrium while seven miRNAs differed between PTB-HCA vs. PTB-NL specimens; six of these miRNAs were cross-validated using quantitative PCR. Based on the combined sequencing data, unsupervised clustering revealed two non-overlapping cohorts that differed primarily by absence or presence of uterine quiescence, rather than gestational age or original clinical cohort. The intersection of differentially expressed miRNAs and their mRNA targets predicted 22 subnetworks with enriched representation of miR-146b-5p, miR-223-3p, and miR-150-5p among miRNAs, and of myocyte enhancer factor-2C (MEF2C) among mRNAs. Of four known MEF2 transcription factors, decreased MEF2A and MEF2C expression in women with uterine non-quiescence was observed in the transcriptome profiling data, and validated in a second cohort by quantitative PCR. Immunohistochemistry localized MEF2A and MEF2C to myometrial smooth muscle cells and confirmed decreased abundance with labor. Collectively, these results suggest that repression of MEF2 expression may represent a previously unrecognized process through which miRNAs contribute to the phenotypic switch from quiescence to labor in human myometrium. © The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Glucocorticoid receptor interacts with PNRC2 in a ligand-dependent manner to recruit UPF1 for rapid mRNA degradation.

PubMed

Cho, Hana; Park, Ok Hyun; Park, Joori; Ryu, Incheol; Kim, Jeonghan; Ko, Jesang; Kim, Yoon Ki

2015-03-31

Glucocorticoid receptor (GR), which was originally known to function as a nuclear receptor, plays a role in rapid mRNA degradation by acting as an RNA-binding protein. The mechanism by which this process occurs remains unknown. Here, we demonstrate that GR, preloaded onto the 5'UTR of a target mRNA, recruits UPF1 through proline-rich nuclear receptor coregulatory protein 2 (PNRC2) in a ligand-dependent manner, so as to elicit rapid mRNA degradation. We call this process GR-mediated mRNA decay (GMD). Although GMD, nonsense-mediated mRNA decay (NMD), and staufen-mediated mRNA decay (SMD) share upstream frameshift 1 (UPF1) and PNRC2, we find that GMD is mechanistically distinct from NMD and SMD. We also identify de novo cellular GMD substrates using microarray analysis. Intriguingly, GMD functions in the chemotaxis of human monocytes by targeting chemokine (C-C motif) ligand 2 (CCL2) mRNA. Thus, our data provide molecular evidence of a posttranscriptional role of the well-studied nuclear hormone receptor, GR, which is traditionally considered a transcription factor.

Development of Lead Hammerhead Ribozyme Candidates against Human Rod Opsin mRNA for Retinal Degeneration Therapy

PubMed Central

Abdelmaksoud, Heba E.; Yau, Edwin H.; Zuker, Michael; Sullivan, Jack M.

2011-01-01

To identify lead candidate allele-independent hammerhead ribozymes (hhRz) for the treatment of autosomal dominant mutations in the human rod opsin (RHO) gene, we tested a series of hhRzs for potential to significantly knockdown human RHO gene expression in a human cell expression system. Multiple computational criteria were used to select target mRNA regions likely to be single stranded and accessible to hhRz annealing and cleavage. Target regions are tested for accessibility in a human cell culture expression system where the hhRz RNA and target mRNA and protein are coexpressed. The hhRz RNA is embedded in an adenoviral VAI RNA chimeric RNA of established structure and properties which are critical to the experimental paradigm. The chimeric hhRz-VAI RNA is abundantly transcribed so that the hhRzs are expected to be in great excess over substrate mRNA. HhRz-VAI traffics predominantly to the cytoplasm to colocalize with the RHO mRNA target. Colocalization is essential for second-order annealing reactions. The VAI chimera protects the hhRz RNA from degradation and provides for a long half life. With cell lines chosen for high transfection efficiency and a molar excess of hhRz plasmid over target plasmid, the conditions of this experimental paradigm are specifically designed to evaluate for regions of accessibility of the target mRNA in cellulo. Western analysis was used to measure the impact of hhRz expression on RHO protein expression. Three lead candidate hhRz designs were identified that significantly knockdown target protein expression relative to control (p < 0.05). Successful lead candidates (hhRz CUC↓ 266, hhRz CUC↓ 1411, hhRz AUA↓ 1414) targeted regions of human RHO mRNA that were predicted to be accessible by a bioinformatics approach, whereas regions predicted to be inaccessible supported no knockdown. The maximum opsin protein level knockdown is approximately 30% over a 48 hr paradigm of testing. These results validate a rigorous computational bioinformatics approach to detect accessible regions of target mRNAs in cellulo. The opsin knockdown effect could prove to be clinically significant when integrated over longer periods in photoreceptors. Further optimization and animal testing is the next step in this stratified RNA drug discovery program. A recently developed novel and efficient screening assay based upon expression of a dicistronic mRNA (RHO-IRES-SEAP) containing both RHO and reporter (SEAP) cDNAs was used to compare the hhRz 266 lead candidate to another agent (Rz525/hhRz485) already known to partially rescue retinal degeneration in a rodent model. Lead hhRz 266 CUC↓ proved more efficacious than Rz525/hhRz485 which infers viability for rescue of retinal degeneration in appropriate preclinical models of disease. PMID:19094986

Highly accessible AU-rich regions in 3' untranslated regions are hotspots for binding of regulatory factors.

PubMed

Plass, Mireya; Rasmussen, Simon H; Krogh, Anders

2017-04-01

Post-transcriptional regulation is regarded as one of the major processes involved in the regulation of gene expression. It is mainly performed by RNA binding proteins and microRNAs, which target RNAs and typically affect their stability. Recent efforts from the scientific community have aimed at understanding post-transcriptional regulation at a global scale by using high-throughput sequencing techniques such as cross-linking and immunoprecipitation (CLIP), which facilitates identification of binding sites of these regulatory factors. However, the diversity in the experimental procedures and bioinformatics analyses has hindered the integration of multiple datasets and thus limited the development of an integrated view of post-transcriptional regulation. In this work, we have performed a comprehensive analysis of 107 CLIP datasets from 49 different RBPs in HEK293 cells to shed light on the complex interactions that govern post-transcriptional regulation. By developing a more stringent CLIP analysis pipeline we have discovered the existence of conserved regulatory AU-rich regions in the 3'UTRs where miRNAs and RBPs that regulate several processes such as polyadenylation or mRNA stability bind. Analogous to promoters, many factors have binding sites overlapping or in close proximity in these hotspots and hence the regulation of the mRNA may depend on their relative concentrations. This hypothesis is supported by RBP knockdown experiments that alter the relative concentration of RBPs in the cell. Upon AGO2 knockdown (KD), transcripts containing "free" target sites show increased expression levels compared to those containing target sites in hotspots, which suggests that target sites within hotspots are less available for miRNAs to bind. Interestingly, these hotspots appear enriched in genes with regulatory functions such as DNA binding and RNA binding. Taken together, our results suggest that hotspots are functional regulatory elements that define an extra layer of regulation of post-transcriptional regulatory networks.

Highly accessible AU-rich regions in 3’ untranslated regions are hotspots for binding of regulatory factors

PubMed Central

2017-01-01

Post-transcriptional regulation is regarded as one of the major processes involved in the regulation of gene expression. It is mainly performed by RNA binding proteins and microRNAs, which target RNAs and typically affect their stability. Recent efforts from the scientific community have aimed at understanding post-transcriptional regulation at a global scale by using high-throughput sequencing techniques such as cross-linking and immunoprecipitation (CLIP), which facilitates identification of binding sites of these regulatory factors. However, the diversity in the experimental procedures and bioinformatics analyses has hindered the integration of multiple datasets and thus limited the development of an integrated view of post-transcriptional regulation. In this work, we have performed a comprehensive analysis of 107 CLIP datasets from 49 different RBPs in HEK293 cells to shed light on the complex interactions that govern post-transcriptional regulation. By developing a more stringent CLIP analysis pipeline we have discovered the existence of conserved regulatory AU-rich regions in the 3’UTRs where miRNAs and RBPs that regulate several processes such as polyadenylation or mRNA stability bind. Analogous to promoters, many factors have binding sites overlapping or in close proximity in these hotspots and hence the regulation of the mRNA may depend on their relative concentrations. This hypothesis is supported by RBP knockdown experiments that alter the relative concentration of RBPs in the cell. Upon AGO2 knockdown (KD), transcripts containing “free” target sites show increased expression levels compared to those containing target sites in hotspots, which suggests that target sites within hotspots are less available for miRNAs to bind. Interestingly, these hotspots appear enriched in genes with regulatory functions such as DNA binding and RNA binding. Taken together, our results suggest that hotspots are functional regulatory elements that define an extra layer of regulation of post-transcriptional regulatory networks. PMID:28410363

Calcium Signaling Pathway Genes RUNX2 and CACNA1C Are Associated With Calcific Aortic Valve Disease

PubMed Central

Guauque-Olarte, Sandra; Messika-Zeitoun, David; Droit, Arnaud; Lamontagne, Maxime; Tremblay-Marchand, Joël; Lavoie-Charland, Emilie; Gaudreault, Nathalie; Arsenault, Benoit J.; Dubé, Marie-Pierre; Tardif, Jean-Claude; Body, Simon C.; Seidman, Jonathan G.; Boileau, Catherine; Mathieu, Patrick; Pibarot, Philippe; Bossé, Yohan

2016-01-01

Background Calcific aortic valve stenosis (AS) is a life-threatening disease with no medical therapy. The genetic architecture of AS remains elusive. This study combines genome-wide association studies, gene expression, and expression quantitative trait loci mapping in human valve tissues to identify susceptibility genes of AS. Methods and Results A meta-analysis was performed combining the results of 2 genome-wide association studies in 474 and 486 cases from Quebec City (Canada) and Paris (France), respectively. Corresponding controls consisted of 2988 and 1864 individuals with European ancestry from the database of genotypes and phenotypes. mRNA expression levels were evaluated in 9 calcified and 8 normal aortic valves by RNA sequencing. The results were integrated with valve expression quantitative trait loci data obtained from 22 AS patients. Twenty-five single-nucleotide polymorphisms had P<5×10−6 in the genome-wide association studies meta-analysis. The calcium signaling pathway was the top gene set enriched for genes mapped to moderately AS-associated single-nucleotide polymorphisms. Genes in this pathway were found differentially expressed in valves with and without AS. Two single-nucleotide polymorphisms located in RUNX2 (runt-related transcription factor 2), encoding an osteogenic transcription factor, demonstrated some association with AS (genome-wide association studies P=5.33×10−5). The mRNA expression levels of RUNX2 were upregulated in calcified valves and associated with eQTL-SNPs. CACNA1C encoding a subunit of a voltage-dependent calcium channel was upregulated in calcified valves. The eQTL-SNP with the most significant association with AS located in CACNA1C was associated with higher expression of the gene. Conclusions This integrative genomic study confirmed the role of RUNX2 as a potential driver of AS and identified a new AS susceptibility gene, CACNA1C, belonging to the calcium signaling pathway. PMID:26553695

Diversity in mRNA expression of the serine-type carboxypeptidase ocpG in Aspergillus oryzae through intron retention.

PubMed

Ishida, Ken; Kuboshima, Megumi; Morita, Hiroto; Maeda, Hiroshi; Okamoto, Ayako; Takeuchi, Michio; Yamagata, Youhei

2014-01-01

Alternative splicing is thought to be a means for diversification of products by mRNA modification. Although some intron retentions are predicted by transcriptome analysis in Aspergillus oryzae, its physiological significance remains unknown. We found that intron retention occurred occasionally in the serine-type carboxypeptidase gene, ocpG. Analysis under various culture conditions revealed that extracellular nitrogen conditions influence splicing patterns; this suggested that there might be a correlation between splicing efficiency and the necessity of OcpG activity for obtaining a nitrogen source. Since further analysis showed that splicing occurred independently in each intron, we constructed ocpG intron-exchanging strain by interchanging the positions of intron-1 and intron-2. The splicing pattern indicated the probability that ocpG intron retention was affected by the secondary structures of intronic mRNA.

DIANA-microT web server v5.0: service integration into miRNA functional analysis workflows.

PubMed

Paraskevopoulou, Maria D; Georgakilas, Georgios; Kostoulas, Nikos; Vlachos, Ioannis S; Vergoulis, Thanasis; Reczko, Martin; Filippidis, Christos; Dalamagas, Theodore; Hatzigeorgiou, A G

2013-07-01

MicroRNAs (miRNAs) are small endogenous RNA molecules that regulate gene expression through mRNA degradation and/or translation repression, affecting many biological processes. DIANA-microT web server (http://www.microrna.gr/webServer) is dedicated to miRNA target prediction/functional analysis, and it is being widely used from the scientific community, since its initial launch in 2009. DIANA-microT v5.0, the new version of the microT server, has been significantly enhanced with an improved target prediction algorithm, DIANA-microT-CDS. It has been updated to incorporate miRBase version 18 and Ensembl version 69. The in silico-predicted miRNA-gene interactions in Homo sapiens, Mus musculus, Drosophila melanogaster and Caenorhabditis elegans exceed 11 million in total. The web server was completely redesigned, to host a series of sophisticated workflows, which can be used directly from the on-line web interface, enabling users without the necessary bioinformatics infrastructure to perform advanced multi-step functional miRNA analyses. For instance, one available pipeline performs miRNA target prediction using different thresholds and meta-analysis statistics, followed by pathway enrichment analysis. DIANA-microT web server v5.0 also supports a complete integration with the Taverna Workflow Management System (WMS), using the in-house developed DIANA-Taverna Plug-in. This plug-in provides ready-to-use modules for miRNA target prediction and functional analysis, which can be used to form advanced high-throughput analysis pipelines.

DIANA-microT web server v5.0: service integration into miRNA functional analysis workflows

PubMed Central

Paraskevopoulou, Maria D.; Georgakilas, Georgios; Kostoulas, Nikos; Vlachos, Ioannis S.; Vergoulis, Thanasis; Reczko, Martin; Filippidis, Christos; Dalamagas, Theodore; Hatzigeorgiou, A.G.

2013-01-01

MicroRNAs (miRNAs) are small endogenous RNA molecules that regulate gene expression through mRNA degradation and/or translation repression, affecting many biological processes. DIANA-microT web server (http://www.microrna.gr/webServer) is dedicated to miRNA target prediction/functional analysis, and it is being widely used from the scientific community, since its initial launch in 2009. DIANA-microT v5.0, the new version of the microT server, has been significantly enhanced with an improved target prediction algorithm, DIANA-microT-CDS. It has been updated to incorporate miRBase version 18 and Ensembl version 69. The in silico-predicted miRNA–gene interactions in Homo sapiens, Mus musculus, Drosophila melanogaster and Caenorhabditis elegans exceed 11 million in total. The web server was completely redesigned, to host a series of sophisticated workflows, which can be used directly from the on-line web interface, enabling users without the necessary bioinformatics infrastructure to perform advanced multi-step functional miRNA analyses. For instance, one available pipeline performs miRNA target prediction using different thresholds and meta-analysis statistics, followed by pathway enrichment analysis. DIANA-microT web server v5.0 also supports a complete integration with the Taverna Workflow Management System (WMS), using the in-house developed DIANA-Taverna Plug-in. This plug-in provides ready-to-use modules for miRNA target prediction and functional analysis, which can be used to form advanced high-throughput analysis pipelines. PMID:23680784

Kinetic Induction of Oat Shoot Pulvinus Invertase mRNA by Gravistimulation and Partial cDNA Cloning by the Polymerase Chain Reaction

NASA Technical Reports Server (NTRS)

Wu, Liu-Lai; Song, Il; Karuppiah, Nadarajah; Kaufman, Peter B.

1993-01-01

An asymmetric (top vs. bottom halves of pulvini) induction of invertase mRNA by gravistimulation was analyzed in oat shoot pulvini. Total RNA and poly(A)(+) RNA, isolated from oat pulvini, and two oli-gonucleotide primers, corresponding to two conserved amino acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the polymerase chain reaction (PCR). A partial length cDNA (550 bp) was obtained and characterized. A 62% nucleotide sequence homology and 58% deduced amino acid sequence homology, as compared to beta-fructosidase of carrot cell wall, was found. Northern blot analysis showed that there was an obviously transient induction of invertase mRNA by gravistimulation in the oat pulvinus system. The mRNA was rapidly induced to a maximum level at 1 hour after gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher than that in the top half of the pulvinus tissue. The kinetic induction of invertase mRNA was consistent with the transient accumulation of invertase activity during the graviresponse of the pulvinus. This indicates that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional level. Southern blot analysis showed that there were two to three genomic DNA fragments which hybridized with the partial-length invertase cDNA.

Creatine kinase and alpha-actin mRNA levels decrease in diabetic rat hearts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popovich, B.; Barrieux, A.; Dillmann, W.H.

1987-05-01

Diabetic cardiomyopathy is associated with cardiac atrophy and isoenzyme redistribution. To determine if tissue specific changes occur in mRNAs coding for ..cap alpha..-actin and creatine kinase (CK), they performed RNA blot analysis. Total ventricular RNA from control (C) and 4 wk old diabetic (D) rats were hybridized with /sup 32/P cDNA probes for ..cap alpha..-actin and CK. A tissue independent cDNA probe, CHOA was also used. Signal intensity was quantified by photodensitometry. D CK mRNA was 47 +/- 16% lower in D vs C. Insulin increases CK mRNA by 20% at 1.5 hs, and completely reverses the deficit after 4more » wks. D ..cap alpha..-actin mRNA is 66 +/- 18% lower in D vs C. Insulin normalized ..cap alpha..-actin mRNA by 5 hs. CHOA mRNA is unchanged in D vs C, but D + insulin CHOA mRNA is 30 +/- 2% lower than C. In rats with diabetic cardiomyopathy, muscle specific CK and ..cap alpha..-actin mRNAs are decreased. Insulin treatment reverses these changes.« less

Detection of active human papilloma virus-16 in head and neck cancers of Asian North Indian patients.

PubMed

Sannigrahi, M K; Singh, V; Sharma, R; Panda, N K; Radotra, B D; Khullar, M

2016-01-01

Head and neck cancers (HNC) are one of the most common cancers in India. Human papillomavirus (HPV) has been identified as an emerging risk factor for HNC. The present study was carried out to determine the active form of HPV-16 using a combination of PCR, viral load determination, HPV-16 E7 mRNA expression, p16, p53, and pRB immuno-histochemistry (IHC). A total of 226 HNC patients were enrolled in the present study. Sixty-seven (29.7%) of HNC cases were found to be HPV DNA positive. Thirty-two (14%) cases were HPV-16 DNA positive and 20 (9%) cases expressed HPV-16 E7 mRNA. HPV-16 mRNA/p16 positive cases had significantly increased viral load and integrated HPV-16 DNA. In summary, of total HNC patients, 6% cases were positive for both HPV-16 DNA and p16, and 5% were positive for both E7 mRNA and p16 IHC. We observed similar HPV-16 DNA/E7mRNA prevalence in oropharynx and oral cavity sites, however, oropharynx SCC had significantly higher viral load. Our results show low prevalence of active HPV-16 in North Indian HNC patients. HPV-16 E7 mRNA expression correlated with p16 nuclear positivity and increased viral load. Therefore, E7 mRNA expression may be used as a good surrogate indicator for active form of HPV-16 infection. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Modulation of tumor necrosis factor (TNF) receptor expression during monocytic differentiation by glucocorticoids.

PubMed

Goppelt-Struebe, M; Reiser, C O; Schneider, N; Grell, M

1996-10-01

Regulation of tumor necrosis factor receptors by glucocorticoids was investigated during phorbol ester-induced monocytic differentiation. As model system the human monocytic cell lines U937 and THP-1, which express both types of TNF receptors (TNF-R60 and TNF-R80), were differentiated with tetradecanoyl phorbol-13-acetate (TPA, 5 x 10(-9) M) in the presence or absence of dexamethasone (10(-9) - 10(-6) M). Expression of TNF receptors was determined at the mRNA level by Northern blot analysis and at the protein level by FACS analysis. During differentiation, TNF-R60 mRNA was down-regulated, whereas TNF-R80 mRNA levels were increased. Dexamethasone had no effect on TNF-R60 mRNA expression but attenuated TNF-R80 mRNA expression in both cell lines. Cell surface expression of TNF-R60 protein remained essentially unchanged during differentiation of THP-1 cells, whereas a rapid down-regulation of TNF-R80 was observed that was followed by a slow recovery. Surface expression of TNF-R80 was not affected by dexamethasone, whereas TNF-R60 expression was reduced by about 25%. These results indicate differential regulation of the two types of TNF receptors at the mRNA and protein level during monocytic differentiation. Glucocorticoids interfered with mRNA expression of TNF-R80 and protein expression of TNF-R60, but the rather limited effect leaves the question of its functional relevance open. In contrast to other cytokine systems, TNF receptors do not appear to be major targets of glucocorticoid action.

Changes in beta-actin mRNA expression in remodeling canine myocardium.

PubMed

Carlyle, W C; Toher, C A; Vandervelde, J R; McDonald, K M; Homans, D C; Cohn, J N

1996-01-01

Beta-actin, a cytoskeletal protein important in the maintenance of cytoarchitecture, has long been thought to be expressed constitutively in myocardial tissue. As such, beta-actin mRNA has been used as a control gene in a wide range of experiments. However, we have uncovered consistent changes in beta-actin mRNA expression in canine myocardium remodeling as a result of insult to the left ventricle. The experimental canine models used were either DC shock damage to the left ventricle or volume overload resulting from severe mitral regurgitation. The remodeling process in both canine models is characterized by an increase in left ventricular mass. PCR amplification using primers designed to selectively amplify the 3' end and a portion of the 3' untranslated region of beta-actin mRNA resulted in the generation of a 297 base pair product predominant only in normal canine myocardium and a 472 base pair product that became increasingly prominent from 1 to 30 days after DC shock damage to the left ventricle and from 10 to 90 days after creation of mitral regurgitation. Northern analysis showed a three-fold increase in beta-actin mRNA after either DC shock or creation of mitral regurgitation. Western analysis revealed an early increase in beta-actin protein followed by an apparent decrease to below baseline levels. These observations suggest that changes in beta-actin mRNA expression accompany the structural alterations that occur in response to myocardial damage. Whether or not the changes in beta-actin mRNA expression play a role in mediating these structural alterations remains to be determined.

Human microRNA target analysis and gene ontology clustering by GOmir, a novel stand-alone application

PubMed Central

Roubelakis, Maria G; Zotos, Pantelis; Papachristoudis, Georgios; Michalopoulos, Ioannis; Pappa, Kalliopi I; Anagnou, Nicholas P; Kossida, Sophia

2009-01-01

Background microRNAs (miRNAs) are single-stranded RNA molecules of about 20–23 nucleotides length found in a wide variety of organisms. miRNAs regulate gene expression, by interacting with target mRNAs at specific sites in order to induce cleavage of the message or inhibit translation. Predicting or verifying mRNA targets of specific miRNAs is a difficult process of great importance. Results GOmir is a novel stand-alone application consisting of two separate tools: JTarget and TAGGO. JTarget integrates miRNA target prediction and functional analysis by combining the predicted target genes from TargetScan, miRanda, RNAhybrid and PicTar computational tools as well as the experimentally supported targets from TarBase and also providing a full gene description and functional analysis for each target gene. On the other hand, TAGGO application is designed to automatically group gene ontology annotations, taking advantage of the Gene Ontology (GO), in order to extract the main attributes of sets of proteins. GOmir represents a new tool incorporating two separate Java applications integrated into one stand-alone Java application. Conclusion GOmir (by using up to five different databases) introduces miRNA predicted targets accompanied by (a) full gene description, (b) functional analysis and (c) detailed gene ontology clustering. Additionally, a reverse search initiated by a potential target can also be conducted. GOmir can freely be downloaded BRFAA. PMID:19534746

Human microRNA target analysis and gene ontology clustering by GOmir, a novel stand-alone application.

PubMed

Roubelakis, Maria G; Zotos, Pantelis; Papachristoudis, Georgios; Michalopoulos, Ioannis; Pappa, Kalliopi I; Anagnou, Nicholas P; Kossida, Sophia

2009-06-16

microRNAs (miRNAs) are single-stranded RNA molecules of about 20-23 nucleotides length found in a wide variety of organisms. miRNAs regulate gene expression, by interacting with target mRNAs at specific sites in order to induce cleavage of the message or inhibit translation. Predicting or verifying mRNA targets of specific miRNAs is a difficult process of great importance. GOmir is a novel stand-alone application consisting of two separate tools: JTarget and TAGGO. JTarget integrates miRNA target prediction and functional analysis by combining the predicted target genes from TargetScan, miRanda, RNAhybrid and PicTar computational tools as well as the experimentally supported targets from TarBase and also providing a full gene description and functional analysis for each target gene. On the other hand, TAGGO application is designed to automatically group gene ontology annotations, taking advantage of the Gene Ontology (GO), in order to extract the main attributes of sets of proteins. GOmir represents a new tool incorporating two separate Java applications integrated into one stand-alone Java application. GOmir (by using up to five different databases) introduces miRNA predicted targets accompanied by (a) full gene description, (b) functional analysis and (c) detailed gene ontology clustering. Additionally, a reverse search initiated by a potential target can also be conducted. GOmir can freely be downloaded BRFAA.

Multi-omics facilitated variable selection in Cox-regression model for cancer prognosis prediction.

PubMed

Liu, Cong; Wang, Xujun; Genchev, Georgi Z; Lu, Hui

2017-07-15

New developments in high-throughput genomic technologies have enabled the measurement of diverse types of omics biomarkers in a cost-efficient and clinically-feasible manner. Developing computational methods and tools for analysis and translation of such genomic data into clinically-relevant information is an ongoing and active area of investigation. For example, several studies have utilized an unsupervised learning framework to cluster patients by integrating omics data. Despite such recent advances, predicting cancer prognosis using integrated omics biomarkers remains a challenge. There is also a shortage of computational tools for predicting cancer prognosis by using supervised learning methods. The current standard approach is to fit a Cox regression model by concatenating the different types of omics data in a linear manner, while penalty could be added for feature selection. A more powerful approach, however, would be to incorporate data by considering relationships among omics datatypes. Here we developed two methods: a SKI-Cox method and a wLASSO-Cox method to incorporate the association among different types of omics data. Both methods fit the Cox proportional hazards model and predict a risk score based on mRNA expression profiles. SKI-Cox borrows the information generated by these additional types of omics data to guide variable selection, while wLASSO-Cox incorporates this information as a penalty factor during model fitting. We show that SKI-Cox and wLASSO-Cox models select more true variables than a LASSO-Cox model in simulation studies. We assess the performance of SKI-Cox and wLASSO-Cox using TCGA glioblastoma multiforme and lung adenocarcinoma data. In each case, mRNA expression, methylation, and copy number variation data are integrated to predict the overall survival time of cancer patients. Our methods achieve better performance in predicting patients' survival in glioblastoma and lung adenocarcinoma. Copyright © 2017. Published by Elsevier Inc.

Cooperative gene regulation by microRNA pairs and their identification using a computational workflow

PubMed Central

Schmitz, Ulf; Lai, Xin; Winter, Felix; Wolkenhauer, Olaf; Vera, Julio; Gupta, Shailendra K.

2014-01-01

MicroRNAs (miRNAs) are an integral part of gene regulation at the post-transcriptional level. Recently, it has been shown that pairs of miRNAs can repress the translation of a target mRNA in a cooperative manner, which leads to an enhanced effectiveness and specificity in target repression. However, it remains unclear which miRNA pairs can synergize and which genes are target of cooperative miRNA regulation. In this paper, we present a computational workflow for the prediction and analysis of cooperating miRNAs and their mutual target genes, which we refer to as RNA triplexes. The workflow integrates methods of miRNA target prediction; triplex structure analysis; molecular dynamics simulations and mathematical modeling for a reliable prediction of functional RNA triplexes and target repression efficiency. In a case study we analyzed the human genome and identified several thousand targets of cooperative gene regulation. Our results suggest that miRNA cooperativity is a frequent mechanism for an enhanced target repression by pairs of miRNAs facilitating distinctive and fine-tuned target gene expression patterns. Human RNA triplexes predicted and characterized in this study are organized in a web resource at www.sbi.uni-rostock.de/triplexrna/. PMID:24875477

Visualization of RNA structure models within the Integrative Genomics Viewer.

PubMed

Busan, Steven; Weeks, Kevin M

2017-07-01

Analyses of the interrelationships between RNA structure and function are increasingly important components of genomic studies. The SHAPE-MaP strategy enables accurate RNA structure probing and realistic structure modeling of kilobase-length noncoding RNAs and mRNAs. Existing tools for visualizing RNA structure models are not suitable for efficient analysis of long, structurally heterogeneous RNAs. In addition, structure models are often advantageously interpreted in the context of other experimental data and gene annotation information, for which few tools currently exist. We have developed a module within the widely used and well supported open-source Integrative Genomics Viewer (IGV) that allows visualization of SHAPE and other chemical probing data, including raw reactivities, data-driven structural entropies, and data-constrained base-pair secondary structure models, in context with linear genomic data tracks. We illustrate the usefulness of visualizing RNA structure in the IGV by exploring structure models for a large viral RNA genome, comparing bacterial mRNA structure in cells with its structure under cell- and protein-free conditions, and comparing a noncoding RNA structure modeled using SHAPE data with a base-pairing model inferred through sequence covariation analysis. © 2017 Busan and Weeks; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Finding similar nucleotide sequences using network BLAST searches.

PubMed

Ladunga, Istvan

2009-06-01

The Basic Local Alignment Search Tool (BLAST) is a keystone of bioinformatics due to its performance and user-friendliness. Beginner and intermediate users will learn how to design and submit blastn and Megablast searches on the Web pages at the National Center for Biotechnology Information. We map nucleic acid sequences to genomes, find identical or similar mRNA, expressed sequence tag, and noncoding RNA sequences, and run Megablast searches, which are much faster than blastn. Understanding results is assisted by taxonomy reports, genomic views, and multiple alignments. We interpret expected frequency thresholds, biological significance, and statistical significance. Weak hits provide no evidence, but hints for further analyses. We find genes that may code for homologous proteins by translated BLAST. We reduce false positives by filtering out low-complexity regions. Parsed BLAST results can be integrated into analysis pipelines. Links in the output connect to Entrez, PUBMED, structural, sequence, interaction, and expression databases. This facilitates integration with a wide spectrum of biological knowledge.

Transterm—extended search facilities and improved integration with other databases

PubMed Central

Jacobs, Grant H.; Stockwell, Peter A.; Tate, Warren P.; Brown, Chris M.

2006-01-01

Transterm has now been publicly available for >10 years. Major changes have been made since its last description in this database issue in 2002. The current database provides data for key regions of mRNA sequences, a curated database of mRNA motifs and tools to allow users to investigate their own motifs or mRNA sequences. The key mRNA regions database is derived computationally from Genbank. It contains 3′ and 5′ flanking regions, the initiation and termination signal context and coding sequence for annotated CDS features from Genbank and RefSeq. The database is non-redundant, enabling summary files and statistics to be prepared for each species. Advances include providing extended search facilities, the database may now be searched by BLAST in addition to regular expressions (patterns) allowing users to search for motifs such as known miRNA sequences, and the inclusion of RefSeq data. The database contains >40 motifs or structural patterns important for translational control. In this release, patterns from UTRsite and Rfam are also incorporated with cross-referencing. Users may search their sequence data with Transterm or user-defined patterns. The system is accessible at . PMID:16381889

Altered sperm chromatin structure in mice exposed to sodium fluoride through drinking water.

PubMed

Sun, Zilong; Niu, Ruiyan; Wang, Bin; Wang, Jundong

2014-06-01

This study investigated the effects of sodium fluoride (NaF) on sperm abnormality, sperm chromatin structure, protamine 1 and protamine 2 (P1 and P2) mRNA expression, and histones expression in sperm in male mice. NaF was orally administrated to male mice at 30, 70, and 150 mg/l for 49 days (more than one spermatogenic cycle). Sperm head and tail abnormalities were significantly enhanced at middle and high doses. Similarly, sperm chromatin structure was also adversely affected by NaF exposure, indicating DNA integrity damage. Furthermore, middle and high NaF significantly reduced the mRNA expressions of P1 and P2, and P1/P2 ratio, whereas the sperm histones level was increased, suggesting the abnormal histone-protamine replacement. Therefore, we concluded that the mechanism by which F induced mice sperm abnormality and DNA integrity damage may involved in the alterations in P1, P2, and histones expression in sperm of mice. Copyright © 2012 Wiley Periodicals, Inc.

Relationship between variant forms of estrogen receptor RNA and an apoptosis-related RNA, TRPM-2, with survival in patients with breast cancer.

PubMed

Rennie, P S; Mawji, N R; Coldman, A J; Godolphin, W; Jones, E C; Vielkind, J R; Bruchovsky, N

1993-12-15

Although smaller variant forms of estrogen receptor (ER) messenger RNA (mRNA) have been detected in breast tumors, neither their prevalence nor their prognostic significance have been evaluated. Similarly, TRPM-2 mRNA, the product of a gene induced principally during the onset of apoptosis, is present in mouse and human breast cancer cell lines, but whether it also occurs in primary breast tumors and is related to disease outcome is unknown. The relative expression and transcript size of ER mRNA and TRPM-2 mRNA in 126 primary breast tumors were measured by Northern analysis and compared with tumor grade, hormone receptor status, extent of tumor necrosis, and survival. In ER-positive tumors, 64% of the tumors had only the normal 6.5 kb ER mRNA, an additional 9% had the normal plus smaller ER mRNA, and 2% had variant forms. Only 8% of ER-negative tumors had ER mRNA transcripts. There were significant relationships between the occurrence of ER mRNA and low tumor grade, ER-positive receptor status, and better survival. In contrast, TRPM-2 mRNA was found in only 17% of breast tumors, none of which could be grouped with respect to grade, hormone receptor status, or survival. The presence of smaller variant forms of ER mRNA either alone or in association with the normal ER transcript is not indicative of an unfavorable prognosis, whereas TRPM-2 mRNA occurs in many primary breast tumors, but has no apparent relationship to survival.

Transcriptional bursting explains the noise–versus–mean relationship in mRNA and protein levels

DOE PAGES

Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai; ...

2016-07-28

Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

Thymidylate synthase (TS) protein expression as a prognostic factor in advanced colorectal cancer: a comparison with TS mRNA expression.

PubMed

Nakagawa, Tateo; Shimada, Mitsuo; Kurita, Nobuhiro; Iwata, Takashi; Nishioka, Masanori; Yoshikawa, Kozo; Higashijima, Jun; Utsunomiya, Tohru

2012-06-01

The role of intratumoral thymidylate synthase (TS) mRNA or protein expression is still controversial and little has been reported regarding relation of them in colorectal cancer. Forty-six patients with advanced colorectal cancer who underwent surgical resection were included. TS mRNA expression was determined by the Danenberg tumor profile method based on laser-captured micro-dissection of the tumor cells. TS protein expression was evaluated using immunohistochemical staining. TS mRNA expression tended to relate TS protein expression. Statistical significance was not found in overall survival between the TS mRNA high group and low group regardless of performing adjuvant chemotherapy. The overall survival in the TS protein negative group was significantly higher than that in positive group in all and the patients without adjuvant chemotherapy. Multivariate analysis showed TS protein expression was as an independent prognostic factor. TS protein expression tends to be related TS mRNA expression and is an independent prognostic factor in advanced colorectal cancer.

The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans

PubMed Central

Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hau; Florens, Laurence; Mak, Ho Yi

2012-01-01

The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans. PMID:22508728

The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans.

PubMed

Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hua; Li, Hau; Florens, Laurence; Mak, Ho Yi

2012-04-15

The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.

Transcriptome In Vivo Analysis (TIVA) of spatially defined single cells in intact live mouse and human brain tissue

PubMed Central

Lovatt, Ditte; Ruble, Brittani K.; Lee, Jaehee; Dueck, Hannah; Kim, Tae Kyung; Fisher, Stephen; Francis, Chantal; Spaethling, Jennifer M.; Wolf, John A.; Grady, M. Sean; Ulyanova, Alexandra V.; Yeldell, Sean B.; Griepenburg, Julianne C.; Buckley, Peter T.; Kim, Junhyong; Sul, Jai-Yoon; Dmochowski, Ivan J.; Eberwine, James

2014-01-01

Transcriptome profiling is an indispensable tool in advancing the understanding of single cell biology, but depends upon methods capable of isolating mRNA at the spatial resolution of a single cell. Current capture methods lack sufficient spatial resolution to isolate mRNA from individual in vivo resident cells without damaging adjacent tissue. Because of this limitation, it has been difficult to assess the influence of the microenvironment on the transcriptome of individual neurons. Here, we engineered a Transcriptome In Vivo Analysis (TIVA)-tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA-tag in combination with RNA-seq to analyze transcriptome variance among single dispersed cells and in vivo resident mouse and human neurons, we show that the tissue microenvironment shapes the transcriptomic landscape of individual cells. The TIVA methodology provides the first noninvasive approach for capturing mRNA from single cells in their natural microenvironment. PMID:24412976

Improving the Transduction of Bone Marrow–Derived Cells with an Integrase-Defective Lentiviral Vector

PubMed Central

Pay, S. Louise; Qi, Xiaoping; Willard, Jeffrey F.; Godoy, Juliana; Sankhavaram, Kavya; Horton, Ranier; Mitter, Sayak K.; Quigley, Judith L.; Chang, Lung-Ji; Grant, Maria B.; Boulton, Michael E.

2018-01-01

In lentiviral vector (LV) applications where transient transgene expression is sufficient, integrase-defective lentiviral vectors (IDLVs) are beneficial for reducing the potential for off-target effects associated with insertional mutagenesis. It was previously demonstrated that human RPE65 mRNA expression from an integrating lentiviral vector (ILV) induces endogenous Rpe65 and Cralbp mRNA expression in murine bone marrow–derived cells (BMDCs), initiating programming of the cells to retinal pigment epithelium (RPE)-like cells. These cells regenerate RPE in retinal degeneration models when injected systemically. As transient expression of RPE65 is sufficient to activate endogenous RPE-associated genes for programming BMDCs, use of an ILV is an unnecessary risk. In this study, an IDLV expressing RPE65 (IDLV3-RPE65) was generated. Transduction with IDLV3-RPE65 is less efficient than the integrating vector (ILV3-RPE65). Therefore, IDLV3-RPE65 transduction was enhanced with a combination of preloading 20 × -concentrated viral supernatant on RetroNectin at a multiplicity of infection of 50 and transduction of BMDCs by low-speed centrifugation. RPE65 mRNA levels increased from ∼12-fold to ∼25-fold (p < 0.05) after modification of the IDLV3-RPE65 transduction protocol, achieving expression similar to the ∼27-fold (p < 0.05) increase observed with ILV3-RPE65. Additionally, the study shows that the same preparation of RetroNectin can be used to coat up to three wells with no reduction in transduction. Critically, IDLV3-RPE65 transduction initiates endogenous Rpe65 mRNA expression in murine BMDCs and Cralbp/CRALBP mRNA in both murine and human BMDCs, similar to expression observed in ILV3-RPE65-transduced cells. Systemic administration of ILV3-RPE65 or IDLV3-RPE65 programmed BMDCs in a mouse model of retinal degeneration is sufficient to retain visual function and reduce retinal degeneration compared to mice receiving no treatment or naïve BMDC. It is concluded that IDLV3-RPE65 is appropriate for programming BMDCs to RPE-like cells. PMID:29160102

Primary induction of vitellogenin mRNA in the rooster by 17beta-estradiol.

PubMed Central

Burns, A T; Deeley, R G; Gordon, J I; Udell, D S; Mullinix, K P; Goldberger, R F

1978-01-01

We have studied the kinetics of vitellogenin mRNA accumulation in rooster liver after a primary injection of 17beta-estradiol. The levels of vitellogenin mRNA have been determined both by hybridization of total cellular RNA to vitellogenin cDNA and by translation of vitellogenin mRNA in a wheat germ cell-free system. The results obtained by both methods of analysis are in good agreement and indicate that vitellogenin mRNA is present in the liver of normal roosters at a level of 0-5 molecules per liver cell and increases in amount during the 3 days following injection of estrogen, reaching a level of almost 6000 molecules per cell at the peak of the response. The level of vitellogenin mRNA declined exponentially during the next 14 days with a half-life of 29 hr, reaching a level of less than 10 molecules per cell at 17 days after injection of the hormone. The levels of vitellogenin mRNA after stimulation with estrogen have been correlated with the in vivo rate of synthesis of the vitellogenin polypeptide. The results indicate that the rate of vitellogenin synthesis is closely correlated with the level of vitellogenin mRNA. On the basis of these findings, we conclude that vitellogenin mRNA does not exist in the liver in an untranslated form after withdrawal from estrogen. PMID:273910

Integrated Phloem Sap mRNA and Protein Expression Analysis Reveals Phytoplasma-infection Responses in Mulberry.

PubMed

Gai, Yingping; Yuan, Shuo-Shuo; Liu, Zhao-Yang; Zhao, Huai-Ning; Liu, Qi; Qin, Yong-Li; Fang, Li-Jing; Ji, Xian-Ling

2018-05-30

To gain insight into the response of mulberry to phytoplasma-infection, the expression profiles of mRNAs and proteins in mulberry phloem sap were examined. A total of 955 unigenes and 136 proteins were found to be differentially expressed between the healthy and infected phloem sap. These differentially expressed mRNAs and proteins are involved in signalling, hormone metabolism, stress responses, etc. Interestingly, we found that both the mRNA and protein levels of the major latex protein-like 329 ( MuMLPL329 ) gene were increased in the infected phloem saps. Expression of the MuMLPL329 gene was induced by pathogen inoculation and was responsive to jasmonic acid. Ectopic expression of MuMLPL329 in Arabidopsis enhances transgenic plant resistance to Botrytis cinerea, Pseudomonas syringae pv tomato DC3000 ( Pst. DC3000 ) and phytoplasma. Further analysis revealed that MuMLPL329 can enhance the expression of some defense genes and might be involved in altering flavonoid content resulting in increased resistance of plants to pathogen infection. Finally, the roles of the differentially expressed mRNAs and proteins and the potential molecular mechanisms of their changes were discussed. It was likely that the phytoplasma-responsive mRNAs and proteins in the phloem saps were involved in multiple pathways of mulberry responses to phytoplasma-infection, and their changes may be partially responsible for some symptoms in the phytoplasma infected plants. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

MC EMiNEM maps the interaction landscape of the Mediator.

PubMed

Niederberger, Theresa; Etzold, Stefanie; Lidschreiber, Michael; Maier, Kerstin C; Martin, Dietmar E; Fröhlich, Holger; Cramer, Patrick; Tresch, Achim

2012-01-01

The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between proteins that have pleiotropic effects on mRNA transcription. MC EMiNEM is based on Nested Effects Models (NEMs), a class of probabilistic graphical models that extends the idea of hierarchical clustering. It combines mode-hopping Monte Carlo (MC) sampling with an Expectation-Maximization (EM) algorithm for NEMs to increase sensitivity compared to existing methods. A meta-analysis of four Mediator perturbation studies in Saccharomyces cerevisiae, three of which are unpublished, provides new insight into the Mediator signaling network. In addition to the known modular organization of the Mediator subunits, MC EMiNEM reveals a hierarchical ordering of its internal information flow, which is putatively transmitted through structural changes within the complex. We identify the N-terminus of Med7 as a peripheral entity, entailing only local structural changes upon perturbation, while the C-terminus of Med7 and Med19 appear to play a central role. MC EMiNEM associates Mediator subunits to most directly affected genes, which, in conjunction with gene set enrichment analysis, allows us to construct an interaction map of Mediator subunits and transcription factors.

Polyadenylation state microarray (PASTA) analysis.

PubMed

Beilharz, Traude H; Preiss, Thomas

2011-01-01

Nearly all eukaryotic mRNAs terminate in a poly(A) tail that serves important roles in mRNA utilization. In the cytoplasm, the poly(A) tail promotes both mRNA stability and translation, and these functions are frequently regulated through changes in tail length. To identify the scope of poly(A) tail length control in a transcriptome, we developed the polyadenylation state microarray (PASTA) method. It involves the purification of mRNA based on poly(A) tail length using thermal elution from poly(U) sepharose, followed by microarray analysis of the resulting fractions. In this chapter we detail our PASTA approach and describe some methods for bulk and mRNA-specific poly(A) tail length measurements of use to monitor the procedure and independently verify the microarray data.

UAP56 is a conserved crucial component of a divergent mRNA export pathway in Toxoplasma gondii.

PubMed

Serpeloni, Mariana; Jiménez-Ruiz, Elena; Vidal, Newton Medeiros; Kroeber, Constanze; Andenmatten, Nicole; Lemgruber, Leandro; Mörking, Patricia; Pall, Gurman S; Meissner, Markus; Ávila, Andréa R

2016-11-01

Nucleo-cytoplasmic RNA export is an essential post-transcriptional step to control gene expression in eukaryotic cells and is poorly understood in apicomplexan parasites. With the exception of UAP56, a component of TREX (Transcription Export) complex, other components of mRNA export machinery are not well conserved in divergent supergroups. Here, we use Toxoplasma gondii as a model system to functionally characterize TgUAP56 and its potential interaction factors. We demonstrate that TgUAP56 is crucial for mRNA export and that functional interference leads to significant accumulation of mRNA in the nucleus. It was necessary to employ bioinformatics and phylogenetic analysis to identify orthologs related to mRNA export, which show a remarkable low level of conservation in T. gondii. We adapted a conditional Cas9/CRISPR system to carry out a genetic screen to verify if these factors were involved in mRNA export in T. gondii. Only the disruption of TgRRM_1330 caused accumulation of mRNA in the nucleus as found with TgUAP56. This protein is potentially a divergent partner of TgUAP56, and provides insight into a divergent mRNA export pathway in apicomplexans. © 2016 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

UAP56 is a conserved crucial component of a divergent mRNA export pathway in Toxoplasma gondii

PubMed Central

Serpeloni, Mariana; Jiménez‐Ruiz, Elena; Vidal, Newton Medeiros; Kroeber, Constanze; Andenmatten, Nicole; Lemgruber, Leandro; Mörking, Patricia; Pall, Gurman S.

2016-01-01

Summary Nucleo‐cytoplasmic RNA export is an essential post‐transcriptional step to control gene expression in eukaryotic cells and is poorly understood in apicomplexan parasites. With the exception of UAP56, a component of TREX (Transcription Export) complex, other components of mRNA export machinery are not well conserved in divergent supergroups. Here, we use Toxoplasma gondii as a model system to functionally characterize TgUAP56 and its potential interaction factors. We demonstrate that TgUAP56 is crucial for mRNA export and that functional interference leads to significant accumulation of mRNA in the nucleus. It was necessary to employ bioinformatics and phylogenetic analysis to identify orthologs related to mRNA export, which show a remarkable low level of conservation in T. gondii. We adapted a conditional Cas9/CRISPR system to carry out a genetic screen to verify if these factors were involved in mRNA export in T. gondii. Only the disruption of TgRRM_1330 caused accumulation of mRNA in the nucleus as found with TgUAP56. This protein is potentially a divergent partner of TgUAP56, and provides insight into a divergent mRNA export pathway in apicomplexans. PMID:27542978

Heat Shock Response in Yeast Involves Changes in Both Transcription Rates and mRNA Stabilities

PubMed Central

Castells-Roca, Laia; García-Martínez, José; Moreno, Joaquín; Herrero, Enrique; Bellí, Gemma; Pérez-Ortín, José E.

2011-01-01

We have analyzed the heat stress response in the yeast Saccharomyces cerevisiae by determining mRNA levels and transcription rates for the whole transcriptome after a shift from 25°C to 37°C. Using an established mathematical algorithm, theoretical mRNA decay rates have also been calculated from the experimental data. We have verified the mathematical predictions for selected genes by determining their mRNA decay rates at different times during heat stress response using the regulatable tetO promoter. This study indicates that the yeast response to heat shock is not only due to changes in transcription rates, but also to changes in the mRNA stabilities. mRNA stability is affected in 62% of the yeast genes and it is particularly important in shaping the mRNA profile of the genes belonging to the environmental stress response. In most cases, changes in transcription rates and mRNA stabilities are homodirectional for both parameters, although some interesting cases of antagonist behavior are found. The statistical analysis of gene targets and sequence motifs within the clusters of genes with similar behaviors shows that both transcriptional and post-transcriptional regulons apparently contribute to the general heat stress response by means of transcriptional factors and RNA binding proteins. PMID:21364882

Systems Genetics as a Tool to Identify Master Genetic Regulators in Complex Disease.

PubMed

Moreno-Moral, Aida; Pesce, Francesco; Behmoaras, Jacques; Petretto, Enrico

2017-01-01

Systems genetics stems from systems biology and similarly employs integrative modeling approaches to describe the perturbations and phenotypic effects observed in a complex system. However, in the case of systems genetics the main source of perturbation is naturally occurring genetic variation, which can be analyzed at the systems-level to explain the observed variation in phenotypic traits. In contrast with conventional single-variant association approaches, the success of systems genetics has been in the identification of gene networks and molecular pathways that underlie complex disease. In addition, systems genetics has proven useful in the discovery of master trans-acting genetic regulators of functional networks and pathways, which in many cases revealed unexpected gene targets for disease. Here we detail the central components of a fully integrated systems genetics approach to complex disease, starting from assessment of genetic and gene expression variation, linking DNA sequence variation to mRNA (expression QTL mapping), gene regulatory network analysis and mapping the genetic control of regulatory networks. By summarizing a few illustrative (and successful) examples, we highlight how different data-modeling strategies can be effectively integrated in a systems genetics study.

Advances in the integration of transcriptional regulatory information into genome-scale metabolic models.

PubMed

Vivek-Ananth, R P; Samal, Areejit

2016-09-01

A major goal of systems biology is to build predictive computational models of cellular metabolism. Availability of complete genome sequences and wealth of legacy biochemical information has led to the reconstruction of genome-scale metabolic networks in the last 15 years for several organisms across the three domains of life. Due to paucity of information on kinetic parameters associated with metabolic reactions, the constraint-based modelling approach, flux balance analysis (FBA), has proved to be a vital alternative to investigate the capabilities of reconstructed metabolic networks. In parallel, advent of high-throughput technologies has led to the generation of massive amounts of omics data on transcriptional regulation comprising mRNA transcript levels and genome-wide binding profile of transcriptional regulators. A frontier area in metabolic systems biology has been the development of methods to integrate the available transcriptional regulatory information into constraint-based models of reconstructed metabolic networks in order to increase the predictive capabilities of computational models and understand the regulation of cellular metabolism. Here, we review the existing methods to integrate transcriptional regulatory information into constraint-based models of metabolic networks. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Effects of hypothermia and cerebral ischemia on cold-inducible RNA-binding protein mRNA expression in rat brain.

PubMed

Liu, Aijun; Zhang, Zhiwen; Li, Anmin; Xue, Jinghui

2010-08-06

CIRP (cold-inducible RNA-binding protein) mRNA is highly expressed in hypothermic conditions in mammalian cells, and the relationship between CIRP and neuroprotection for cerebral ischemia under hypothermia has been focused upon. At present, however, the expression characteristics of CIRP under hypothermia and cerebral ischemia in vivo are not clearly elucidated. In this study, CIRP mRNA expression in various regions of rat brain was examined by reverse transcriptase polymerase chain reaction (RT-PCR). CIRP expression levels were found to be similar in the hippocampus and cortex. Real-time quantitative PCR analysis revealed increasing CIRP mRNA expression in the cortex during the 24-h observation period following treatment with hypothermia or cerebral ischemia, with a greater increase in the hypothermia group. When cerebral ischemia was induced following hypothermia, CIRP mRNA expression in the cortex again showed a significant increasing tendency, but ischemia delayed the appearance of this increase. To reveal the relationship between CIRP and energy metabolism in the rat brain, lactate and pyruvate concentrations in the cortex of the rats treated with hypothermia, ischemia and ischemia after hypothermia were determined by spectrophotometric assay, and levels of phosphofructokinas-1 (PFK-1), the major regulatory enzyme of the glycolytic pathway, in the rat cortex in the three groups was also analyzed by Western blot. Using linear correlation, lactate and pyruvate concentrations, and PFK-1 levels, were each analyzed in the three groups in association with CIRP mRNA expression levels. The analysis did not reveal any correlation between the three metabolic parameters and CIRP mRNA expression induced by hypothermia, suggesting that while playing a role in neuroprotection under hypothermia, CIRP does not affect cerebral energy metabolism. Copyright 2010. Published by Elsevier B.V.

Detection of circulating Bmi-1 mRNA in plasma and its potential diagnostic and prognostic value for uterine cervical cancer.

PubMed

Zhang, Xin; Wang, Chuanxin; Wang, Lili; Du, Lutao; Wang, Shun; Zheng, Guixi; Li, Wei; Zhuang, Xuewei; Zhang, Xuhua; Dong, Zhaogang

2012-07-01

Bmi-1 is overexpressed in uterine cervical cancer (UCC) and is found to be associated with adverse clinical characteristics and poor prognosis. However, little information is available on the status of circulating Bmi-1 mRNA in UCC. Because circulating cell-free nucleic acids have emerged as a novel class of markers for cancer detection, our research aims to address this question by detecting the circulating Bmi-1 mRNA and to assess its diagnostic and prognostic potential in UCC. Reverse transcription quantitative real-time PCR method was established to detect the circulating Bmi-1 mRNA in plasma of 109 patients with UCC, 138 patients with cervical intraepithelial neoplasia (CIN) and 80 healthy volunteers, and found that it was significantly increased in UCC compared with CINs and healthy controls (all at p < 0.001). Moreover, its high level was significantly correlated with advanced clinical stage (p < 0.001) and positive lymph nodes metastasis (p = 0.002). The area under the receiver operating characteristic curve (AUC) was 0.881, and the optimal cut-off value was 0.057, providing a sensitivity of 69.7% and a specificity of 95.9%. The AUC for circulating Bmi-1 mRNA showed higher diagnosis capability than that for SCC-Ag (p = 0.035) or CA125 (p < 0.001) currently utilized. Kaplan-Meier analysis demonstrated a correlation between increased circulating Bmi-1 mRNA level and reduced disease-free survival (DFS) (p = 0.001) and overall survival (OS) (p = 0.015). And, Cox analysis indicated that it was an independent prognostic factor for DFS and OS. We conclude that circulating Bmi-1 mRNA may be a potential noninvasive molecular marker for diagnosis and prognosis of UCC. Copyright © 2011 UICC.

Cloning and tissue distribution of rat hear fatty acid binding protein mRNA: identical forms in heart and skeletal muscle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Claffey, K.P.; Herrera, V.L.; Brecher, P.

1987-12-01

A fatty acid binding protein (FABP) as been identified and characterized in rat heart, but the function and regulation of this protein are unclear. In this study the cDNA for rat heart FABP was cloned from a lambda gt11 library. Sequencing of the cDNA showed an open reading frame coding for a protein with 133 amino acids and a calculated size of 14,776 daltons. Several differences were found between the sequence determined from the cDNA and that reported previously by protein sequencing techniques. Northern blot analysis using rat heart FABP cDNA as a probe established the presence of an abundantmore » mRNA in rat heart about 0.85 kilobases in length. This mRNA was detected, but was not abundant, in fetal heart tissue. Tissue distribution studies showed a similar mRNA species in red, but not white, skeletal muscle. In general, the mRNA tissue distribution was similar to that of the protein detected by Western immunoblot analysis, suggesting that heart FABP expression may be regulated at the transcriptional level. S1 nuclease mapping studies confirmed that the mRNA hybridized to rat heart FABP cDNA was identical in heart and red skeletal muscle throughout the entire open reading frame. The structural differences between heart FABP and other members of this multigene family may be related to the functional requirements of oxidative muscle for fatty acids as a fuel source.« less

Inhibition of hepatic gluconeogenesis and enhanced glucose uptake contribute to the development of hypoglycemia in mice bearing interleukin-1beta- secreting tumor.

PubMed

Metzger, Shulamit; Nusair, Samir; Planer, David; Barash, Varda; Pappo, Orit; Shilyansky, Joel; Chajek-Shaul, Tova

2004-11-01

Mice bearing IL-1beta-secreting tumor were used to study the chronic effect of IL-1beta on glucose metabolism. Mice were injected with syngeneic tumor cells transduced with the human IL-1beta gene. Serum IL-1beta levels increased exponentially with time. Secretion of IL-1beta from the developed tumors was associated with decreased food consumption, reduced body weight, and reduced blood glucose levels. Body composition analysis revealed that IL-1beta caused a significant loss in fat tissue without affecting lean body mass and water content. Hepatic phosphoenolpyruvate carboxykinase and glucose-6-phosphatase activities and mRNA levels of these enzymes were reduced, and 2-deoxy-glucose uptake by peripheral tissues was enhanced. mRNA levels of glucose transporters (Gluts) in the liver were determined by real-time PCR analysis. Glut-3 mRNA levels were up-regulated by IL-1beta. Glut-1 and Glut-4 mRNA levels in IL-1beta mice were similar to mRNA levels in pair-fed mice bearing nonsecreting tumor. mRNA level of Glut-2, the major Glut of the liver, was down-regulated by IL-1beta. We concluded that both decreased glucose production by the liver and enhanced glucose disposal lead to the development of hypoglycemia in mice bearing IL-1beta-secreting tumor. The observed changes in expression of hepatic Gluts that are not dependent on insulin may contribute to the increased glucose uptake.

Quantitative analysis of fluorouracil-related genes in chronic viral hepatitis using microdissection.

PubMed

Kakinuma, Daisuke; Yoshida, Hiroshi; Mamada, Yasuhiro; Taniai, Nobuhiko; Mizuguchi, Yoshiaki; Takahashi, Tsubasa; Shimizu, Tetsuya; Ishikawa, Yoshinori; Akimaru, Koho; Naito, Zenya; Tajiri, Takashi

2008-01-01

Dihydropyrimidine dehydrogenase is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil. The aim of this study was to determine the levels of messenger RNA for 5-fluorouracil-related metabolic enzymes in cirrhotic liver and to assess the correlation between these mRNA levels and clinicopathological features. The study material consisted of 33 liver samples. The levels of mRNA for the 5- fluorouracil-related metabolic enzymes were quantified by real-time reverse transcription polymerase chain reaction combined with laser-captured microdissection. The Dihydropyrimidine dehydrogenase mRNA level in patients with grade B liver damage was significantly lower than that in patients with grade A liver damage (p=0.009). The Dihydropyrimidine dehydrogenase and orotate phosphoribosyl transferase mRNA level in al samples was higher than that in a2 and a3 samples (p= 0.01 and 0.013, respectively). Statistically significant correlations were found between the hyaluronic acid and the thymidylate phosphorylase mRNA level (p= 0.0001), and the T-BIL and the dihydropyrimidine dehydrogenase mRNA level (p=0.01). The level of Dihydropyrimidine dehydrogenase mRNA may be affected by the clinicopathological status of patients with cirrhosis.

Spiny Nanorod and Upconversion Nanoparticle Satellite Assemblies for Ultrasensitive Detection of Messenger RNA in Living Cells.

PubMed

Gao, Rui; Hao, Changlong; Xu, Liguang; Xu, Chuanlai; Kuang, Hua

2018-04-17

Quantitation and in situ monitoring of target mRNA (mRNA) in living cells remains a significant challenge for the chemical and biomedical communities. To quantitatively detect mRNA expression levels in living cells, we have developed DNA-driven gold nanorod coated platinum-upconversion nanoparticle satellite assemblies (termed Au NR@Pt-UCNP satellites) for intracellular thymidine kinase 1 (TK1) mRNA analysis. The nanostructures were capable of recognizing target mRNA in a sequence-specific manner as luminescence of UCNPs was effectively quenched by Au NR@Pt within the assemblies. Following recognition, UCNPs detached from Au NR@Pt, resulting in luminescence restoration to achieve effective in situ imaging and quantifiable detection of target mRNA. The upconversional luminescence intensity of confocal images showed a good linear relationship with intracellular TK1 mRNA ranging from 1.17 to 65.21 fmol/10 μg RNA and a limit of detection (LOD) of 0.67 fmol/10 μg RNA. We believe that our present assay can be broadly applied for detection of endogenous biomolecules at the cellular and tissue levels and restoration of tissue homeostasis in vivo.

Nucleotide sequence determination of guinea-pig casein B mRNA reveals homology with bovine and rat alpha s1 caseins and conservation of the non-coding regions of the mRNA.

PubMed Central

Hall, L; Laird, J E; Craig, R K

1984-01-01

Nucleotide sequence analysis of cloned guinea-pig casein B cDNA sequences has identified two casein B variants related to the bovine and rat alpha s1 caseins. Amino acid homology was largely confined to the known bovine or predicted rat phosphorylation sites and within the 'signal' precursor sequence. Comparison of the deduced nucleotide sequence of the guinea-pig and rat alpha s1 casein mRNA species showed greater sequence conservation in the non-coding than in the coding regions, suggesting a functional and possibly regulatory role for the non-coding regions of casein mRNA. The results provide insight into the evolution of the casein genes, and raise questions as to the role of conserved nucleotide sequences within the non-coding regions of mRNA species. Images Fig. 1. PMID:6548375

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai

Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

The Ribosome Shape Directs mRNA Translocation through Entrance and Exit Dynamics

USDA-ARS?s Scientific Manuscript database

The protein-synthesizing ribosome undergoes large motions to effect the translocation of tRNAs (transfer ribonucleic acids) and mRNA (messenger ribonucleic acid); here the domain motions of this system are explored with a coarse-grained elastic network model using normal mode analysis. Crystal struc...

The importance of mRNA structure in determining the pathogenicity of synonymous and non-synonymous mutations in haemophilia

PubMed Central

Hamasaki-Katagiri, Nobuko; Lin, Brian C.; Simon, Jonathan; Hunt, Ryan C.; Schiller, Tal; Russek-Cohen, Estelle; Komar, Anton A.; Bar, Haim; Kimchi-Sarfaty, Chava

2016-01-01

Introduction Mutational analysis is commonly used to support the diagnosis and management of haemophilia. This has allowed for the generation of large mutation databases which provide unparalleled insight into genotype-phenotype relationships. Haemophilia is associated with inversions, deletions, insertions, nonsense and missense mutations. Both synonymous and non-synonymous mutations influence the base pairing of messenger RNA (mRNA), which can alter mRNA structure, cellular half-life and ribosome processivity/elongation. However, the role of mRNA structure in determining the pathogenicity of point mutations in haemophilia has not been evaluated. Aim To evaluate mRNA thermodynamic stability and associated RNA prediction software as a means to distinguish between neutral and disease-associated mutations in haemophilia. Methods Five mRNA structure prediction software programs were used to assess the thermodynamic stability of mRNA fragments carrying neutral vs. disease-associated and synonymous vs. non-synonymous point mutations in F8, F9 and a third X-linked gene, DMD (dystrophin). Results In F8 and DMD, disease-associated mutations tend to occur in more structurally stable mRNA regions, represented by lower MFE (minimum free energy) levels. In comparing multiple software packages for mRNA structure prediction, a 101–151 nucleotide fragment length appears to be a feasible range for structuring future studies. Conclusion mRNA thermodynamic stability is one predictive characteristic, which when combined with other RNA and protein features, may offer significant insight when screening sequencing data for novel disease-associated mutations. Our results also suggest potential utility in evaluating the mRNA thermodynamic stability profile of a gene when determining the viability of interchanging codons for biological and therapeutic applications. PMID:27933712

Nonsense mutations in the alcohol dehydrogenase gene of Drosophila melanogaster correlate with an abnormal 3' end processing of the corresponding pre-mRNA.

PubMed Central

Brogna, S

1999-01-01

From bacteria to mammals, mutations that generate premature termination codons have been shown to result in the reduction in the abundance of the corresponding mRNA. In mammalian cells, more often than not, the reduction happens while the RNA is still associated with the nucleus. Here, it is reported that mutations in the alcohol dehydrogenase gene (Adh) of Drosophila melanogaster that generate premature termination codons lead to reduced levels of cytoplasmic and nuclear mRNA. Unexpectedly, it has been found that the poly(A) tails of Adh mRNAs and pre-mRNAs that carry a premature termination codon are longer than in the wild-type transcript. The more 5' terminal the mutation is, the longer is the poly(A) tail of the transcript. These findings suggest that the integrity of the coding region may be required for accurate mRNA 3' end processing. PMID:10199572

Transcription of the pst Operon of Clostridium acetobutylicum Is Dependent on Phosphate Concentration and pH

PubMed Central

Fischer, Ralf-Jörg; Oehmcke, Sonja; Meyer, Uta; Mix, Maren; Schwarz, Katrin; Fiedler, Tomas; Bahl, Hubert

2006-01-01

The pst operon of Clostridium acetobutylicum ATCC 824 comprises five genes, pstS, pstC, pstA, pstB, and phoU, and shows a gene architecture identical to that of Escherichia coli. Deduced proteins are predicted to represent a high-affinity phosphate-specific ABC (ATP-binding cassette) transport system (Pst) and a protein homologous to PhoU, a negative phosphate regulon regulator. We analyzed the expression patterns of the pst operon in Pi-limited chemostat cultures during acid production at pH 5.8 or solvent production at pH 4.5 and in response to Pi pulses. Specific mRNA transcripts were found only when external Pi concentrations had dropped below 0.2 mM. Two specific transcripts were detected, a 4.7-kb polycistronic mRNA spanning the whole operon and a quantitatively dominating 1.2-kb mRNA representing the first gene, pstS. The mRNA levels clearly differed depending on the external pH. The amounts of the full-length mRNA detected were about two times higher at pH 5.8 than at pH 4.5. The level of pstS mRNA increased by a factor of at least 8 at pH 5.8 compared to pH 4.5 results. Primer extension experiments revealed only one putative transcription start point 80 nucleotides upstream of pstS. Thus, additional regulatory sites are proposed in the promoter region, integrating two different extracellular signals, namely, depletion of inorganic phosphate and the pH of the environment. After phosphate pulses were applied to a phosphate-limited chemostat we observed faster phosphate consumption at pH 5.8 than at pH 4.5, although higher optical densities were recorded at pH 4.5. PMID:16855236

A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811 + 1.6kbA {yields} G, produces a new exon: High frequency in spanish cystic fibrosis chromosomes and association with severe phenotype

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chillon, M.; Casals, T.; Gimenez, J.

1995-03-01

mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6bA{yields}G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA{r_arrow}G-mRNA was 5-10-fold less abundant than {triangle}F508 mRNA. Mutations 1811+1.6kbA{yields}G was found in 21 Spanish and 1 German CF chromosome(s), making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype {triangle}F508/1811+1.6kbA{yields}G have only 1%-3% of normal CFTRmore » mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients. 30 refs., 3 figs., 2 tabs.« less

Programmable oligonucleotide probes design and applications for in situ and in vivo RNA imaging in cells

NASA Astrophysics Data System (ADS)

Cheglakov, Zoya

Unequal spreading of mRNA is a frequent experience observed in varied cell lines. The study of cellular processes dynamics and precise localization of mRNAs offers a vital toolbox to target specific proteins in precise cytoplasmic areas and provides a convenient instrument to uncover their mechanisms and functions. Latest methodological innovations have allowed imaging of a single mRNA molecule in situ and in vivo. Today, Fluorescent In Situ Hybridization (FISH) methods allow the studying of mRNA expression and offer a vital toolbox for accurate biological models. Studies enable analysis of the dynamics of an individual mRNA, have uncovered the multiplex RNA transport systems. With all current approaches, a single mRNA tracking in the mammalian cells is still challenging. This thesis describes mRNA detection methods based on programmable fluorophore-labeled DNA structures complimentary to native targets providing an accurate mRNA imaging in mammalian cells. First method represents beta-actin (ACTB) transcripts in situ detection in human cells, the technique strategy is based on programmable DNA probes, amplified by rolling circle amplification (RCA). The method reports precise localization of molecule of interest with an accuracy of a single-cell. Visualization and localization of specific endogenous mRNA molecules in real-time in vivo has the promising to innovate cellular biology studies, medical analysis and to provide a vital toolbox in drugs invention area. Second method described in this thesis represents miR-21 miRNA detection within a single live-cell resolution. The method using fluorophore-labeled short synthetic DNAs probes forming a stem-loop shape and generating Fluorescent Resonance Energy Transfer (FRET) as a result of target-probes hybridization. Catalytic nucleic acid (DNAzymes) probes are cooperative tool for precise detection of different mRNA targets. With assistance of a complementary fluorophore-quencher labeled substrate, the DNAzymes provide a beneficial strategy for simultaneous tracking readily accomplished by multicolor imaging with diverse fluorescent tags. The third method in this thesis will demonstrate the advantage of DNAzymes probes amplification, and offers potential strategy to monitor miRNAs in mammalian live cells.

RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes

PubMed Central

Cao, Yan; Du, Juan; Chen, Dandan; Wang, Qian; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Weng, Jing; Liang, Yuanjing; Ma, Wei

2016-01-01

ABSTRACT Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes. PMID:27433972

RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes.

PubMed

Cao, Yan; Du, Juan; Chen, Dandan; Wang, Qian; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Weng, Jing; Liang, Yuanjing; Ma, Wei

2016-10-01

Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.

Combined RT-qPCR of mRNA and microRNA Targets within One Fluidigm Integrated Fluidic Circuit.

PubMed

Baldwin, Don A; Horan, Annamarie D; Hesketh, Patrick J; Mehta, Samir

2016-07-01

The ability to profile expression levels of a large number of mRNAs and microRNAs (miRNAs) within the same sample, using a single assay method, would facilitate investigations of miRNA effects on mRNA abundance and streamline biomarker screening across multiple RNA classes. A protocol is described for reverse transcription of long RNA and miRNA targets, followed by preassay amplification of the pooled cDNAs and quantitative PCR (qPCR) detection for a mixed panel of candidate RNA biomarkers. The method provides flexibility for designing custom target panels, is robust over a range of input RNA amounts, and demonstrated a high assay success rate.

Analysis of thyroid hormone receptor {beta}A mRNA expression in Xenopus laevis tadpoles as a means to detect agonism and antagonism of thyroid hormone action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Opitz, Robert; Lutz, Ilka; Nguyen, Ngoc-Ha

2006-04-01

Amphibian metamorphosis represents a unique biological model to study thyroid hormone (TH) action in vivo. In this study, we examined the utility of thyroid hormone receptors {alpha} (TR{alpha}) and {beta}A (TR{beta}A) mRNA expression patterns in Xenopus laevis tadpoles as molecular markers indicating modulation of TH action. During spontaneous metamorphosis, only moderate changes were evident for TR{alpha} gene expression whereas a marked up-regulation of TR{beta}A mRNA occurred in hind limbs (prometamorphosis), head (late prometamorphosis), and tail tissue (metamorphic climax). Treatment of premetamorphic tadpoles with 1 nM 3,5,3'-triiodothyronine (T3) caused a rapid induction of TR{beta}A mRNA in head and tail tissue withinmore » 6 to 12 h which was maintained for at least 72 h after initiation of T3 treatment. Developmental stage had a strong influence on the responsiveness of tadpole tissues to induce TR{beta}A mRNA during 24 h treatment with thyroxine (0, 1, 5, 10 nM T4) or T3 (0, 1, 5, 10 nM). Premetamorphic tadpoles were highly sensitive in their response to T4 and T3 treatments, whereas sensitivity to TH was decreased in early prometamorphic tadpoles and strongly diminished in late prometamorphic tadpoles. To examine the utility of TR{beta}A gene expression analysis for detection of agonistic and antagonistic effects on T3 action, mRNA expression was assessed in premetamorphic tadpoles after 48 h of treatment with the synthetic agonist GC-1 (0, 10, 50, 250 nM), the synthetic antagonist NH-3 (0, 40, 200, 1000 nM), and binary combinations of NH-3 (0, 40, 200, 1000 nM) and T3 (1 nM). All tested concentrations of GC-1 as well as the highest concentration of NH-3 caused an up-regulation of TR{beta}A expression. Co-treatment with NH-3 and T3 revealed strong antagonistic effects by NH-3 on T3-induced TR{beta}A mRNA up-regulation. Results of this study suggest that TR{beta}A mRNA expression analysis could serve as a sensitive molecular testing approach to study effects of environmental compounds on the thyroid system in X. laevis tadpoles.« less

Topical Administration of Oxygenated Hemoglobin Improved Wound Healing in an Ischemic Rabbit Ear Model.

PubMed

Xie, Ping; Jia, Shengxian; Tye, Ross; Xu, Wei; Zhong, Aimei; Hong, Seok J; Galiano, Robert D; Mustoe, Thomas A

2016-02-01

Localized oxygen deficiency plays a central role in the pathogenesis of chronic wounds; thus, rectifying localized ischemia with oxygen therapy has been postulated to be an integral aspect of the management of chronic wounds. The efficacy of a novel approach for oxygen therapy on chronic wound healing was evaluated. Oxygen was delivered to ischemic wounds by means of the topical application of oxygenated, chemically modified bovine hemoglobin (IKOR 2084) in a validated rabbit ear ischemic wound model. The wound healing was evaluated histologically by measuring epithelial gap and neo-granulation tissue area. In situ expression of endothelial cells (CD31) and proliferative cells (Ki-67) was examined by immunohistochemistry analysis. The mRNA of vascular endothelial growth factor, endothelial nitric oxide synthase, and matrix metalloproteinase-9 was quantified by real-time reverse-transcriptase polymerase chain reaction. The collagen was detected by Sirius red staining. In comparison with topical application of saline, the administration of oxygenated IKOR 2084 increases wound reepithelialization and formation of neo-granulation tissue in a dose-dependent manner, and cellular proliferation (Ki-67). Conversely, the administration of deoxygenated IKOR 2084 aggravated the ischemic wound healing process. Moreover, the topical administration of oxygenated IKOR 2084 induces angiogenesis as evidenced by concomitant increases in CD31 protein and vascular endothelial growth factor and endothelial nitric oxide synthase mRNA expression in treated wounds. Oxygenated IKOR 2084 administration also increased collagen deposition in wounds, with decreases in the expression of matrix metalloproteinase-9 mRNA. This study suggests that the topical application of oxygenated IKOR 2084 ameliorates the reparative progress of ischemic wounds through enhanced angiogenesis, cellular proliferation, and collagen deposition.

Shiga Toxin 2 Subtypes of Enterohemorrhagic E. coli O157:H- E32511 Analyzed by RT-qPCR and Top-Down Proteomics Using MALDI-TOF-TOF-MS

NASA Astrophysics Data System (ADS)

Fagerquist, Clifton K.; Zaragoza, William J.

2015-05-01

We have measured the relative abundance of the B-subunits and mRNA transcripts of two Stx2 subtypes present in Shiga toxin-producing Escherichia coli (STEC) O157:H- strain E32511 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) with post source decay (PSD) and real time-quantitative polymerase chain reaction (RT-qPCR). Stx2a and Stx2c in STEC strain E32511 were quantified from the integrated peak area of their singly charged disulfide-intact B-subunit ions at m/z ~7819 and m/z ~7774, respectively. We found that the Stx2a subtype was 21-fold more abundant than the Stx2c subtype. The two amino acid substitutions (16D ↔ 16 N and 24D ↔ 24A) that distinguish Stx2a from Stx2c not only result in a mass difference of 45 Da between their respective B-subunits but also result in distinctly different fragmentation channels by MS/MS-PSD because both substitutions involve an aspartic acid (D) residue. Importantly, these two substitutions have also been linked to differences in subtype toxicity. We measured the relative abundances of mRNA transcripts using RT-qPCR and determined that the stx2a transcript is 13-fold more abundant than stx2c transcript. In silico secondary structure analysis of the full mRNA operons of stx2a and stx2c suggest that transcript structural differences may also contribute to a relative increase of Stx2a over Stx2c. In consequence, toxin expression may be under both transcriptional and post-transcriptional control.

Heterogeneity in Pseudomonas aeruginosa Biofilms Includes Expression of Ribosome Hibernation Factors in the Antibiotic-Tolerant Subpopulation and Hypoxia-Induced Stress Response in the Metabolically Active Population

PubMed Central

Williamson, Kerry S.; Richards, Lee A.; Perez-Osorio, Ailyn C.; Pitts, Betsey; McInnerney, Kathleen; Stewart, Philip S.

2012-01-01

Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptome responses of Pseudomonas aeruginosa growing in biofilms by using a microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNA levels for these housekeeping genes were low in cells at the bottom of the biofilms. Selective green fluorescent protein (GFP) labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNA levels for genes regulated by the hypoxia-induced regulator Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-term anoxia. Transcripts for ribosomal proteins were associated primarily with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (the rmf and PA4463 genes) at the bottom of the biofilms. The dormant biofilm cells of a P. aeruginosa Δrmf strain had decreased membrane integrity, as shown by propidium iodide staining. Using selective GFP labeling and cell sorting, we show that the dividing cells are more susceptible to killing by tobramycin and ciprofloxacin. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state. PMID:22343293

Fasting for 21days leads to changes in adipose tissue and liver physiology in juvenile checkered garter snakes (Thamnophis marcianus).

PubMed

Davis, Mary; Jessee, Renee; Close, Matthew; Fu, Xiangping; Settlage, Robert; Wang, Guoqing; Cline, Mark A; Gilbert, Elizabeth R

2015-12-01

Snakes often undergo periods of prolonged fasting and, under certain conditions, can survive years without food. Despite this unique phenomenon, there are relatively few reports of the physiological adaptations to fasting in snakes. At post-prandial day 1 (fed) or 21 (fasting), brain, liver, and adipose tissues were collected from juvenile checkered garter snakes (Thamnophis marcianus). There was greater glycerol-3-phosphate dehydrogenase (G3PDH)-specific activity in the liver of fasted than fed snakes (P=0.01). The mRNA abundance of various fat metabolism-associated factors was measured in brain, liver, and adipose tissue. Lipoprotein lipase (LPL) mRNA was greater in fasted than fed snakes in the brain (P=0.04). Adipose triglyceride lipase (ATGL; P=0.006) mRNA was greater in the liver of fasted than fed snakes. In adipose tissue, expression of peroxisome proliferator-activated receptor (PPAR)γ (P=0.01), and fatty acid binding protein 4 (P=0.03) was greater in fed than fasted snakes. Analysis of adipocyte morphology revealed that cross-sectional area (P=0.095) and diameter (P=0.27) were not significantly different between fed and fasted snakes. Results suggest that mean adipocyte area can be preserved during fasting by dampening lipid biosynthesis while not changing rates of lipid hydrolysis. In the liver, however, extensive lipid remodeling may provide energy and lipoproteins to maintain lipid structural integrity during energy restriction. Because the duration of fasting was not sufficient to change adipocyte size, results suggest that the liver is important as a short-term provider of energy in the snake. Copyright © 2015 Elsevier Inc. All rights reserved.

Amino Acid Flux from Metabolic Network Benefits Protein Translation: the Role of Resource Availability.

PubMed

Hu, Xiao-Pan; Yang, Yi; Ma, Bin-Guang

2015-06-09

Protein translation is a central step in gene expression and affected by many factors such as codon usage bias, mRNA folding energy and tRNA abundance. Despite intensive previous studies, how metabolic amino acid supply correlates with protein translation efficiency remains unknown. In this work, we estimated the amino acid flux from metabolic network for each protein in Escherichia coli and Saccharomyces cerevisiae by using Flux Balance Analysis. Integrated with the mRNA expression level, protein abundance and ribosome profiling data, we provided a detailed description of the role of amino acid supply in protein translation. Our results showed that amino acid supply positively correlates with translation efficiency and ribosome density. Moreover, with the rank-based regression model, we found that metabolic amino acid supply facilitates ribosome utilization. Based on the fact that the ribosome density change of well-amino-acid-supplied genes is smaller than poorly-amino-acid-supply genes under amino acid starvation, we reached the conclusion that amino acid supply may buffer ribosome density change against amino acid starvation and benefit maintaining a relatively stable translation environment. Our work provided new insights into the connection between metabolic amino acid supply and protein translation process by revealing a new regulation strategy that is dependent on resource availability.

Integrative Analysis of Sex-Specific microRNA Networks Following Stress in Mouse Nucleus Accumbens.

PubMed

Pfau, Madeline L; Purushothaman, Immanuel; Feng, Jian; Golden, Sam A; Aleyasin, Hossein; Lorsch, Zachary S; Cates, Hannah M; Flanigan, Meghan E; Menard, Caroline; Heshmati, Mitra; Wang, Zichen; Ma'ayan, Avi; Shen, Li; Hodes, Georgia E; Russo, Scott J

2016-01-01

Adult women are twice as likely as men to suffer from affective and anxiety disorders, although the mechanisms underlying heightened female stress susceptibility are incompletely understood. Recent findings in mouse Nucleus Accumbens (NAc) suggest a role for DNA methylation-driven sex differences in genome-wide transcriptional profiles. However, the role of another epigenetic process-microRNA (miR) regulation-has yet to be explored. We exposed male and female mice to Subchronic Variable Stress (SCVS), a stress paradigm that produces depression-like behavior in female, but not male, mice, and performed next generation mRNA and miR sequencing on NAc tissue. We applied a combination of differential expression, miR-mRNA network and functional enrichment analyses to characterize the transcriptional and post-transcriptional landscape of sex differences in NAc stress response. We find that male and female mice exhibit largely non-overlapping miR and mRNA profiles following SCVS. The two sexes also show enrichment of different molecular pathways and functions. Collectively, our results suggest that males and females mount fundamentally different transcriptional and post-transcriptional responses to SCVS and engage sex-specific molecular processes following stress. These findings have implications for the pathophysiology and treatment of stress-related disorders in women.

An integrated mRNA and microRNA expression signature for glioblastoma multiforme prognosis.

PubMed

Xiong, Jie; Bing, Zhitong; Su, Yanlin; Deng, Defeng; Peng, Xiaoning

2014-01-01

Although patients with Glioblastoma multiforme (GBM) have grave prognosis, significant variability in patient outcome is observed. The objective of this study is to identify a molecular signature for GBM prognosis. We subjected 355 mRNA and microRNA expression profiles to elastic net-regulated Cox regression for identification of an integrated RNA signature for GBM prognosis. A prognostic index (PI) was generated for patient stratification. Survival comparison was conducted by Kaplan-Meier method and a general multivariate Cox regression procedure was applied to evaluate the independence of the PI. The abilities and efficiencies of signatures to predict GBM patient outcome was assessed and compared by the area under the curve (AUC) of the receiver-operator characteristic (ROC). An integrated RNA prognostic signature consisted by 4 protective mRNAs, 12 risky mRNAs, and 1 risky microRNA was identified. Decreased survival was associated with being in the high-risk group (hazard ratio = 2.864, P<0.0001). The prognostic value of the integrated signature was validated in five independent GBM expression datasets (n = 201, hazard ratio = 2.453, P<0.0001). The PI outperformed the known clinical factors, mRNA-only, and miRNA-only prognostic signatures for GBM prognosis (area under the ROC curve for the integrated RNA, mRNA-only, and miRNA-only signatures were 0.828, 0.742, and 0.757 at 3 years of overall survival, respectively, P<0.0001 by permutation test). We describe the first, to our knowledge, robust transcriptome-based integrated RNA signature that improves the current GBM prognosis based on clinical variables, mRNA-only, and miRNA-only signatures.

An Integrated mRNA and microRNA Expression Signature for Glioblastoma Multiforme Prognosis

PubMed Central

Xiong, Jie; Bing, Zhitong; Su, Yanlin; Deng, Defeng; Peng, Xiaoning

2014-01-01

Although patients with Glioblastoma multiforme (GBM) have grave prognosis, significant variability in patient outcome is observed. The objective of this study is to identify a molecular signature for GBM prognosis. We subjected 355 mRNA and microRNA expression profiles to elastic net-regulated Cox regression for identification of an integrated RNA signature for GBM prognosis. A prognostic index (PI) was generated for patient stratification. Survival comparison was conducted by Kaplan-Meier method and a general multivariate Cox regression procedure was applied to evaluate the independence of the PI. The abilities and efficiencies of signatures to predict GBM patient outcome was assessed and compared by the area under the curve (AUC) of the receiver-operator characteristic (ROC). An integrated RNA prognostic signature consisted by 4 protective mRNAs, 12 risky mRNAs, and 1 risky microRNA was identified. Decreased survival was associated with being in the high-risk group (hazard ratio = 2.864, P<0.0001). The prognostic value of the integrated signature was validated in five independent GBM expression datasets (n = 201, hazard ratio = 2.453, P<0.0001). The PI outperformed the known clinical factors, mRNA-only, and miRNA-only prognostic signatures for GBM prognosis (area under the ROC curve for the integrated RNA, mRNA-only, and miRNA-only signatures were 0.828, 0.742, and 0.757 at 3 years of overall survival, respectively, P<0.0001 by permutation test). We describe the first, to our knowledge, robust transcriptome-based integrated RNA signature that improves the current GBM prognosis based on clinical variables, mRNA-only, and miRNA-only signatures. PMID:24871302

2',4'-Dihydroxychalcone-induced apoptosis of human gastric cancer MGC-803 cells via down-regulation of survivin mRNA.

PubMed

Lou, Chenghua; Yang, Guangming; Cai, Hao; Zou, Mingchang; Xu, Zisheng; Li, Yu; Zhao, Fengming; Li, Weidong; Tong, Li; Wang, Mingyan; Cai, Baochang

2010-08-01

2',4'-Dihydroxychalcone (TFC), a main component in Herba Oxytropis, is grouped under flavonoids, which are well known to have antitumor activities in vitro. In this study, the possible antitumor mechanism of TFC in human gastric cancer MGC-803 cells is examined. Hoechst 33258 staining analysis indicates that TFC causes MGC-803 cell shrinkage and apoptotic body formation, typical characteristics of apoptosis. Flow cytometric analysis demonstrates that TFC causes cell cycle arrest in the G2/M phase. Furthermore, TFC significantly increases caspase-3 activity but decreases survivin mRNA expression. Therefore, TFC can induce the apoptosis of MGC-803 cells via down-regulation of survivin mRNA expression. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

An Integrated Prognostic Classifier for Stage I Lung Adenocarcinoma based on mRNA, microRNA and DNA Methylation Biomarkers

PubMed Central

Robles, Ana I.; Arai, Eri; Mathé, Ewy A.; Okayama, Hirokazu; Schetter, Aaron J.; Brown, Derek; Petersen, David; Bowman, Elise D.; Noro, Rintaro; Welsh, Judith A.; Edelman, Daniel C.; Stevenson, Holly S.; Wang, Yonghong; Tsuchiya, Naoto; Kohno, Takashi; Skaug, Vidar; Mollerup, Steen; Haugen, Aage; Meltzer, Paul S.; Yokota, Jun; Kanai, Yae

2015-01-01

Introduction Up to 30% Stage I lung cancer patients suffer recurrence within 5 years of curative surgery. We sought to improve existing protein-coding gene and microRNA expression prognostic classifiers by incorporating epigenetic biomarkers. Methods Genome-wide screening of DNA methylation and pyrosequencing analysis of HOXA9 promoter methylation were performed in two independently collected cohorts of Stage I lung adenocarcinoma. The prognostic value of HOXA9 promoter methylation alone and in combination with mRNA and miRNA biomarkers was assessed by Cox regression and Kaplan-Meier survival analysis in both cohorts. Results Promoters of genes marked by Polycomb in Embryonic Stem Cells were methylated de novo in tumors and identified patients with poor prognosis. The HOXA9 locus was methylated de novo in Stage I tumors (P < 0.0005). High HOXA9 promoter methylation was associated with worse cancer-specific survival (Hazard Ratio [HR], 2.6; P = 0.02) and recurrence-free survival (HR, 3.0; P = 0.01), and identified high-risk patients in stratified analysis of Stage IA and IB. Four protein-coding gene (XPO1, BRCA1, HIF1α, DLC1), miR-21 expression and HOXA9 promoter methylation were each independently associated with outcome (HR, 2.8; P = 0.002; HR, 2.3; P = 0.01; and HR, 2.4; P = 0.005, respectively), and, when combined, identified high-risk, therapy naïve, Stage I patients (HR, 10.2; P = 3x10−5). All associations were confirmed in two independently collected cohorts. Conclusion A prognostic classifier comprising three types of genomic and epigenomic data may help guide the postoperative management of Stage I lung cancer patients at high risk of recurrence. PMID:26134223

Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

DTIC Science & Technology

2016-09-07

sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

Amplified in Breast Cancer Regulates Transcription and Translation in Breast Cancer Cells.

PubMed

Ochnik, Aleksandra M; Peterson, Mark S; Avdulov, Svetlana V; Oh, Annabell S; Bitterman, Peter B; Yee, Douglas

2016-02-01

Control of mRNA translation is fundamentally altered in cancer. Insulin-like growth factor-I (IGF-I) signaling regulates key translation mediators to modulate protein synthesis (e.g. eIF4E, 4E-BP1, mTOR, and S6K1). Importantly the Amplified in Breast Cancer (AIB1) oncogene regulates transcription and is also a downstream mediator of IGF-I signaling. To determine if AIB1 also affects mRNA translation, we conducted gain and loss of AIB1 function experiments in estrogen receptor alpha (ERα)(+) (MCF-7L) and ERα(-) (MDA-MB-231, MDA-MB-435 and LCC6) breast cancer cells. AIB1 positively regulated IGF-I-induced mRNA translation in both ERα(+) and ERα(-) cells. Formation of the eIF4E-4E-BP1 translational complex was altered in the AIB1 ERα(+) and ERα(-) knockdown cells, leading to a reduction in the eIF4E/4E-BP1 and eIF4G/4E-BP1 ratios. In basal and IGF-I stimulated MCF-7 and LCC6 cells, knockdown of AIB1 decreased the integrity of the cap-binding complex, reduced global IGF-I stimulated polyribosomal mRNA recruitment with a concomitant decrease in ten of the thirteen genes tested in polysome-bound mRNAs mapping to proliferation, cell cycle, survival, transcription, translation and ribosome biogenesis ontologies. Specifically, knockdown of AIB1 decreased ribosome-bound mRNA and steady-state protein levels of the transcription factors ERα and E2F1 in addition to reduced ribosome-bound mRNA of the ribosome biogenesis factor BYSL in a cell-line specific manner to regulate mRNA translation. The oncogenic transcription factor AIB1 has a novel role in the regulation of polyribosome recruitment and formation of the translational complex. Combinatorial therapies targeting IGF signaling and mRNA translation in AIB1 expressing breast cancers may have clinical benefit and warrants further investigation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Label-Free Isolation and mRNA Detection of Circulating Tumor Cells from Patients with Metastatic Lung Cancer for Disease Diagnosis and Monitoring Therapeutic Efficacy.

PubMed

Wang, Jidong; Lu, Wenjing; Tang, Chuanhao; Liu, Yi; Sun, Jiashu; Mu, Xuan; Zhang, Lin; Dai, Bo; Li, Xiaoyan; Zhuo, Hailong; Jiang, Xingyu

2015-12-01

We develop an inertial-based microfluidic cell sorter combined with an integrated membrane filter, allowing for size-based, label-free, and high-efficiency separation and enrichment of circulating tumor cells (CTCs) in whole blood. The cell sorter is composed of a double spiral microchannel that hydrodynamically focuses and separates large CTCs from small blood cells. The focused CTCs with the equilibrium position around the midline of microchannel are further captured and enriched by a membrane filter (pore size of 8 μm) attached at the middle outlet. This integrated microfluidic device can process 1 mL of whole blood containing spiked tumor cells (A549, human lung adenocarcinoma epithelial cell line) within 15 min, with the capture efficiency of 74.4% at the concentration as low as tens of A549 cells per mL of whole blood. This microfluidic cell sorter is further adopted for isolation of CTCs from peripheral blood samples of patients with metastatic lung cancer. The immunostaining and CK-19 mRNA detection are applied for identification of captured CTCs, showing that our method can detect 90% of metastatic lung cancer patients before therapy, whereas the commercially used system can only detect 40% of the same patients. We also use the expression of CK-19 mRNA from captured CTCs as an indicator for monitoring the therapeutic efficiency, which correlates well with X-ray computed tomography (CT) assessment of the disease.

Integrating discrete stochastic models and single-cell experiments to infer predictive models of MAPK-induced transcription dynamics

NASA Astrophysics Data System (ADS)

Munsky, Brian

2015-03-01

MAPK signal-activated transcription plays central roles in myriad biological processes including stress adaptation responses and cell fate decisions. Recent single-cell and single-molecule experiments have advanced our ability to quantify the spatial, temporal, and stochastic fluctuations for such signals and their downstream effects on transcription regulation. This talk explores how integrating such experiments with discrete stochastic computational analyses can yield quantitative and predictive understanding of transcription regulation in both space and time. We use single-molecule mRNA fluorescence in situ hybridization (smFISH) experiments to reveal locations and numbers of multiple endogenous mRNA species in 100,000's of individual cells, at different times and under different genetic and environmental perturbations. We use finite state projection methods to precisely and efficiently compute the full joint probability distributions of these mRNA, which capture measured spatial, temporal and correlative fluctuations. By combining these experimental and computational tools with uncertainty quantification, we systematically compare models of varying complexity and select those which give optimally precise and accurate predictions in new situations. We use these tools to explore two MAPK-activated gene regulation pathways. In yeast adaptation to osmotic shock, we analyze Hog1 kinase activation of transcription for three different genes STL1 (osmotic stress), CTT1 (oxidative stress) and HSP12 (heat shock). In human osteosarcoma cells under serum induction, we analyze ERK activation of c-Fos transcription.

Inventory of high-abundance mRNAs in skeletal muscle of normal men.

PubMed

Welle, S; Bhatt, K; Thornton, C A

1999-05-01

G42875rial analysis of gene expression (SAGE) method was used to generate a catalog of 53,875 short (14 base) expressed sequence tags from polyadenylated RNA obtained from vastus lateralis muscle of healthy young men. Over 12,000 unique tags were detected. The frequency of occurrence of each tag reflects the relative abundance of the corresponding mRNA. The mRNA species that were detected 10 or more times, each comprising >/=0.02% of the mRNA population, accounted for 64% of the mRNA mass but <10% of the total number of mRNA species detected. Almost all of the abundant tags matched mRNA or EST sequences cataloged in GenBank. Mitochondrial transcripts accounted for approximately 20% of the polyadenylated RNA. Transcripts encoding proteins of the myofibrils were the most abundant nuclear-encoded mRNAs. Transcripts encoding ribosomal proteins, and those encoding proteins involved in energy metabolism, also were very abundant. The database can be used as a reference for investigations of alterations in gene expression associated with conditions that influence muscle function, such as muscular dystrophies, aging, and exercise.

High-level mRNA quantification of proliferation marker pKi-67 is correlated with favorable prognosis in colorectal carcinoma.

PubMed

Ihmann, Thomas; Liu, Jian; Schwabe, Wolfgang; Häusler, Peter; Behnke, Detlev; Bruch, Hans-Peter; Broll, Rainer; Windhövel, Ute; Duchrow, Michael

2004-12-01

The present study retrospectively examines the expression of pKi-67 mRNA and protein in colorectal carcinoma and their correlation to the outcome of patients. Immunohistochemistry and quantitative RT-PCR were used to analyze the expression of pKi-67 in 43 archival specimens of patients with curatively resected primary colorectal carcinoma, who were not treated with neo-adjuvant therapy. We determined a median pKi-67 (MIB-1) labeling index of 31.3% (range 10.3-66.4%), and a mean mRNA level of 0.1769 (DeltaC(T): range 0.01-0.69); indices and levels did not correlate. High pKi-67 mRNA DeltaC(T) values were associated with a significantly favorable prognosis, while pKi-67 labeling indices were not correlated to prognostic outcome. A multivariate analysis of clinical and biological factors indicated that tumor stage (UICC) and pKi-67 mRNA expression level were independent prognostic factors. Quantitatively determined pKi-67 mRNA can be a good and new prognostic indicator for primary resected colorectal carcinoma.

Integrating microRNA and mRNA expression profiles of acute promyelocytic leukemia cells to explore the occurrence mechanisms of differentiation syndrome

PubMed Central

Ge, Fei; Cao, Fenglin; Li, Haitao; Wang, Ping; Xu, Mengyuan; Song, Peng; Li, Xiaoxia; Wang, Shuye; Li, Jinmei; Han, Xueying; Zhao, Yanhong; Su, Yanhua; Li, Yinghua; Fan, Shengjin; Li, Limin; Zhou, Jin

2016-01-01

The pathogenesis of therapy-induced differentiation syndrome (DS) in patients with acute promyelocytic leukemia (APL) remains unclear. In this study, mRNA and microRNA (miRNA) expression profiling of peripheral blood APL cells from patients complicated with vs. without DS were integratively analyzed to explore the mechanisms underlying arsenic trioxide treatment-associated DS. By integrating the differentially expressed data with the data of differentially expressed microRNAs and their computationally predicted target genes, as well as the data of transcription factors and differentially expressed target microRNAs obtained from a literature search, a DS-related genetic regulatory network was constructed. Then using an EAGLE algorithm in clusterViz, the network was subdivided into 10 modules. Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database the modules were annotated functionally, and three functionally active modules were recognized. The further in-depth analyses on the annotated functions of the three modules and the expression and roles of the related genes revealed that proliferation, differentiation, apoptosis and infiltration capability of APL cells might play important roles in the DS pathogenesis. The results could improve our understanding of DS pathogenesis from a more overall perspective, and could provide new clues for future research. PMID:27634874

Evidences for involvement of growth hormone and insulin-like growth factor in ovarian development of starry flounder (Platichthys stellatus).

PubMed

Xu, Yongjiang; Wang, Bin; Liu, Xuezhou; Shi, Bao; Zang, Kun

2017-04-01

Although gonadotrophins are major regulators of ovarian function in teleosts and other vertebrates, accumulating evidence indicates that the growth hormone (GH)-insulin-like growth factor (IGF) axis also plays an important role in fish reproduction. As a first step to understand the physiological role of the GH-IGF system in the ovarian development of starry flounder (Platichthys stellatus), the expression profiles of GH and IGF messenger RNAs (mRNAs) and plasma GH, IGF-I, estradiol-17β (E2), and testosterone (T) levels during the ovarian development were investigated. The developmental stages of ovaries were divided into five stages (II, III, IV, V, and VI) by histological analysis. The hepatosomatic index (HSI) and gonadosomatic index (GSI) values increased and peaked at stage IV and stage V, respectively, and then declined at stage VI. Pituitary GH mRNA levels decreased sharply at stage III and raised to top level at stage VI. The hepatic IGF-I mRNA levels ascended to maximum value at stage V and then declined significantly at stage VI. However, the hepatic IGF-II mRNA levels remained stable and increased significantly at stage VI. In contrast, the ovarian IGF-I mRNA levels increased gradually and peaked at stage VI. The ovarian IGF-II mRNA levels were initially stable and increased significantly at stage V until the top level at stage VI. Consistent with the pituitary GH mRNA levels, plasma GH levels reduced sharply at stage III and remained depressed until stage V and then raised remarkably at stage VI. Plasma IGF-I level peaked at stage V and then declined to initial level. Plasma E2 level peaked at stage IV and then dramatically descended to the basal level. Plasma T level peaked at stage V and then declined significantly back to the basal level. Based on statistical analysis, significant positive correlations between hepatic IGF-I mRNA and GSI, ovarian IGF-II mRNA and hepatic IGF-II mRNA, ovarian IGF-I mRNA and ovarian IGF-II mRNA, and plasma IGF-I and plasma T were observed, respectively. These results suggest that the GH-IGF system may be involved in the ovarian development of starry flounder; GH and IGFs appear to play distinct roles in the regulation of the ovarian development in paracrine/autocrine manners. These findings extend our knowledge of the roles of the GH-IGF axis on reproduction regulation in fish.

Genome wide assessment of mRNA in astrocyte protrusions by direct RNA sequencing reveals mRNA localization for the intermediate filament protein nestin.

PubMed

Thomsen, Rune; Pallesen, Jonatan; Daugaard, Tina F; Børglum, Anders D; Nielsen, Anders L

2013-11-01

Subcellular RNA localization plays an important role in development, cell differentiation, and cell migration. For a comprehensive description of the population of protrusion localized mRNAs in astrocytes we separated protrusions from cell bodies in a Boyden chamber and performed high-throughput direct RNA sequencing. The mRNAs with localization in astrocyte protrusions encode proteins belonging to a variety of functional groups indicating involvement of RNA localization for a palette of cellular functions. The mRNA encoding the intermediate filament protein Nestin was among the identified mRNAs. By RT-qPCR and RNA FISH analysis we confirmed Nestin mRNA localization in cell protrusions and also protrusion localization of Nestin protein. Nestin mRNA localization was dependent of Fragile X mental retardation syndrome proteins Fmrp and Fxr1, and the Nestin 3'-UTR was sufficient to mediate protrusion mRNA localization. The mRNAs for two other intermediate filament proteins in astrocytes, Gfap and Vimentin, have moderate and no protrusion localization, respectively, showing that individual intermediate filament components have different localization mechanisms. The correlated localization of Nestin mRNA with Nestin protein in cell protrusions indicates the presence of a regulatory mechanism at the mRNA localization level for the Nestin intermediate filament protein with potential importance for astrocyte functions during brain development and maintenance. Copyright © 2013 Wiley Periodicals, Inc.

Unproductively spliced ribosomal protein mRNAs are natural targets of mRNA surveillance in C. elegans

PubMed Central

Mitrovich, Quinn M.; Anderson, Philip

2000-01-01

Messenger RNA surveillance, the selective and rapid degradation of mRNAs containing premature stop codons, occurs in all eukaryotes tested. The biological role of this decay pathway, however, is not well understood. To identify natural substrates of mRNA surveillance, we used a cDNA-based representational difference analysis to identify mRNAs whose abundance increases in Caenorhabditis elegans smg(−) mutants, which are deficient for mRNA surveillance. Alternatively spliced mRNAs of genes encoding ribosomal proteins L3, L7a, L10a, and L12 are abundant natural targets of mRNA surveillance. Each of these genes expresses two distinct mRNAs. A productively spliced mRNA, whose abundance does not change in smg(−) mutants, encodes a normal, full-length, ribosomal protein. An unproductively spliced mRNA, whose abundance increases dramatically in smg(−) mutants, contains premature stop codons because of incomplete removal of an alternatively spliced intron. In transgenic animals expressing elevated quantities of RPL-12, a greater proportion of endogenous rpl-12 transcript is spliced unproductively. Thus, RPL-12 appears to autoregulate its own splicing, with unproductively spliced mRNAs being degraded by mRNA surveillance. We demonstrate further that alternative splicing of rpl introns is conserved among widely diverged nematodes. Our results suggest that one important role of mRNA surveillance is to eliminate unproductive by-products of gene regulation. PMID:10970881

Multiple Transcript Properties Related to Translation Affect mRNA Degradation Rates in Saccharomyces cerevisiae

PubMed Central

Neymotin, Benjamin; Ettorre, Victoria; Gresham, David

2016-01-01

Degradation of mRNA contributes to variation in transcript abundance. Studies of individual mRNAs have shown that both cis and trans factors affect mRNA degradation rates. However, the factors underlying transcriptome-wide variation in mRNA degradation rates are poorly understood. We investigated the contribution of different transcript properties to transcriptome-wide degradation rate variation in the budding yeast, Saccharomyces cerevisiae, using multiple regression analysis. We find that multiple transcript properties are significantly associated with variation in mRNA degradation rates, and that a model incorporating these properties explains ∼50% of the genome-wide variance. Predictors of mRNA degradation rates include transcript length, ribosome density, biased codon usage, and GC content of the third position in codons. To experimentally validate these factors, we studied individual transcripts expressed from identical promoters. We find that decreasing ribosome density by mutating the first translational start site of a transcript increases its degradation rate. Using coding sequence variants of green fluorescent protein (GFP) that differ only at synonymous sites, we show that increased GC content of the third position of codons results in decreased rates of mRNA degradation. Thus, in steady-state conditions, a large fraction of genome-wide variation in mRNA degradation rates is determined by inherent properties of transcripts, many of which are related to translation, rather than specific regulatory mechanisms. PMID:27633789

PTB and TIAR binding to insulin mRNA 3'- and 5'UTRs; implications for insulin biosynthesis and messenger stability.

PubMed

Fred, Rikard G; Mehrabi, Syrina; Adams, Christopher M; Welsh, Nils

2016-09-01

Insulin expression is highly controlled on the posttranscriptional level. The RNA binding proteins (RBPs) responsible for this result are still largely unknown. To identify RBPs that bind to insulin mRNA we performed mass spectrometry analysis on proteins that bound synthetic oligonucloetides mimicing the 5'- and the 3'-untranslated regions (UTRs) of rat and human insulin mRNA in vitro . We observed that the RBPs heterogeneous nuclear ribonucleoprotein (hnRNP) U, polypyrimidine tract binding protein (PTB), hnRNP L and T-cell restricted intracellular antigen 1-related protein (TIA-1-related protein; TIAR) bind to insulin mRNA sequences, and that the in vitro binding affinity of these RBPs changed when INS-1 cells were exposed to glucose, 3-isobutyl-1-methylxanthine (IBMX) or nitric oxide. High glucose exposure resulted in a modest increase in PTB and TIAR binding to an insulin mRNA sequence. The inducer of nitrosative stress DETAnonoate increased markedly hnRNP U and TIAR mRNA binding. An increased PTB to TIAR binding ratio in vitro correlated with higher insulin mRNA levels and insulin biosynthesis rates in INS-1 cells. To further investigate the importance of RNA-binding proteins for insulin mRNA stability, we decreased INS-1 and EndoC-βH1 cell levels of PTB and TIAR by RNAi. In both cell lines, decreased levels of PTB resulted in lowered insulin mRNA levels while decreased levels of TIAR resulted in increased insulin mRNA levels. Thapsigargin-induced stress granule formation was associated with a redistribution of TIAR from the cytosol to stress granules. These experiments indicate that alterations in insulin mRNA stability and translation correlate with differential RBP binding. We propose that the balance between PTB on one hand and TIAR on the other participates in the control of insulin mRNA stability and utilization for insulin biosynthesis.

Sequences within the 5' untranslated region regulate the levels of a kinetoplast DNA topoisomerase mRNA during the cell cycle.

PubMed Central

Pasion, S G; Hines, J C; Ou, X; Mahmood, R; Ray, D S

1996-01-01

Gene expression in trypanosomatids appears to be regulated largely at the posttranscriptional level and involves maturation of mRNA precursors by trans splicing of a 39-nucleotide miniexon sequence to the 5' end of the mRNA and cleavage and polyadenylation at the 3' end of the mRNA. To initiate the identification of sequences involved in the periodic expression of DNA replication genes in trypanosomatids, we have mapped splice acceptor sites in the 5' flanking region of the TOP2 gene, which encodes the kinetoplast DNA topoisomerase, and have carried out deletion analysis of this region on a plasmid-encoded TOP2 gene. Block deletions within the 5' untranslated region (UTR) identified two regions (-608 to -388 and -387 to -186) responsible for periodic accumulation of the mRNA. Deletion of one or the other of these sequences had no effect on periodic expression of the mRNA, while deletion of both regions resulted in constitutive expression of the mRNA throughout the cell cycle. Subcloning of these sequences into the 5' UTR of a construct lacking both regions of the TOP2 5' UTR has shown that an octamer consensus sequence present in the 5' UTR of the TOP2, RPA1, and DHFR-TS mRNAs is required for normal cycling of the TOP2 mRNA. Mutation of the consensus octamer sequence in the TOP2 5' UTR in a plasmid construct containing only a single consensus octamer and that shows normal cycling of the plasmid-encoded TOP2 mRNA resulted in substantial reduction of the cycling of the mRNA level. These results imply a negative regulation of TOP2 mRNA during the cell cycle by a mechanism involving redundant elements containing one or more copies of a conserved octamer sequence within the 5' UTR of TOP2 mRNA. PMID:8943327

Sequences within the 5' untranslated region regulate the levels of a kinetoplast DNA topoisomerase mRNA during the cell cycle.

PubMed

Pasion, S G; Hines, J C; Ou, X; Mahmood, R; Ray, D S

1996-12-01

Gene expression in trypanosomatids appears to be regulated largely at the posttranscriptional level and involves maturation of mRNA precursors by trans splicing of a 39-nucleotide miniexon sequence to the 5' end of the mRNA and cleavage and polyadenylation at the 3' end of the mRNA. To initiate the identification of sequences involved in the periodic expression of DNA replication genes in trypanosomatids, we have mapped splice acceptor sites in the 5' flanking region of the TOP2 gene, which encodes the kinetoplast DNA topoisomerase, and have carried out deletion analysis of this region on a plasmid-encoded TOP2 gene. Block deletions within the 5' untranslated region (UTR) identified two regions (-608 to -388 and -387 to -186) responsible for periodic accumulation of the mRNA. Deletion of one or the other of these sequences had no effect on periodic expression of the mRNA, while deletion of both regions resulted in constitutive expression of the mRNA throughout the cell cycle. Subcloning of these sequences into the 5' UTR of a construct lacking both regions of the TOP2 5' UTR has shown that an octamer consensus sequence present in the 5' UTR of the TOP2, RPA1, and DHFR-TS mRNAs is required for normal cycling of the TOP2 mRNA. Mutation of the consensus octamer sequence in the TOP2 5' UTR in a plasmid construct containing only a single consensus octamer and that shows normal cycling of the plasmid-encoded TOP2 mRNA resulted in substantial reduction of the cycling of the mRNA level. These results imply a negative regulation of TOP2 mRNA during the cell cycle by a mechanism involving redundant elements containing one or more copies of a conserved octamer sequence within the 5' UTR of TOP2 mRNA.

Modulation of transferrin receptor mRNA by transferrin-gallium in human myeloid HL60 and lymphoid CCRF-CEM leukaemic cells.

PubMed Central

Ul-Haq, R; Chitambar, C R

1993-01-01

Gallium binds to the iron transport protein transferrin (Tf), is incorporated into cells through transferrin receptors (TfR) and inhibits iron-dependent DNA synthesis. Since cellular TfR expression is tightly regulated by the availability of iron, we investigated the effects of transferrin-gallium (Tf-Ga) on TfR mRNA levels in myeloid HL60 and lymphoid CCRF-CEM cells. In HL60 cells, Tf-Ga increased TfR mRNA levels in a dose-dependent fashion. This increase in TfR mRNA was blocked by Tf-Fe and by cycloheximide. Analysis of the rate of mRNA decay in the presence of actinomycin D revealed that the half-life of TfR mRNA was increased in HL60 cells incubated with Tf-Ga. The rate of transcription of TfR mRNA was not increased by Tf-Ga. In contrast with HL60 cells, CCRF-CEM cells displayed a decrease in the level of TfR mRNA after incubation with Tf-Ga. Tf-Ga inhibited iron uptake in both HL60 and CCRF-CEM cells but increased the level of TfR mRNA only in HL60 cells, suggesting that the Tf-Ga induction of TfR mRNA was not solely due to inhibition of cellular iron uptake. At growth-inhibitory concentrations, Tf-Ga increased the TfR mRNA level in HL60 cells but decreased it in CCRF-CEM cells. Our studies suggest that in HL60 cells, gallium regulates TfR expression at the post-transcriptional level by mechanisms which require de novo protein synthesis and involve interaction with iron. The divergent effects of Tf-Ga on TfR mRNA in myeloid HL60 and lymphoid CCRF-CEM cells suggest that differences exist in the regulation of TfR expression between these two cell types. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8379943

Regional distribution of neuropeptide Y mRNA in postmortem human brain.

PubMed

Brené, S; Lindefors, N; Kopp, J; Sedvall, G; Persson, H

1989-12-01

The distribution of messenger RNA encoding neuropeptide Y (NPY) was studied in 11 different postmortem human brain regions using in situ hybridization histochemistry, and RNA blot analysis. In situ hybridization data revealed that the highest numerical density of labeled cells corresponded to neurons in accumbens area, caudate nucleus, putamen, and substantia innominata. Significantly fewer NPY mRNA-containing neurons were found in frontal and parietal cortex, amygdaloid body and dentate gyrus. No NPY mRNA-containing cells were found in substantia nigra. NPY mRNA-positive neurons from all regions studied showed relatively similar labeling, as revealed by computerized image analysis. Blot analysis showed an approximately 0.8 kb NPY mRNA in all brain regions studied, except in substantia nigra and cerebellum. Densitometric scanning of the autoradiograms revealed levels of NPY mRNA in the following order: putamen greater than caudate nucleus greater than frontal cortex (Brodmann areas 4 and 6) greater than temporal cortex (Brodmann area 38) greater than parietal cortex (Brodmann areas 5 and 7) greater than frontal cortex (Brodmann area 11). Hence, although NPY mRNA is widely distributed in neurons of the human brain large regional variation exists, with the highest expression in accumbens area and parts of the basal ganglia.

Transforming growth factor-beta stimulates wound healing and modulates extracellular matrix gene expression in pig skin. I. Excisional wound model.

PubMed

Quaglino, D; Nanney, L B; Kennedy, R; Davidson, J M

1990-09-01

The effect of transforming growth factor-beta 1 (TGF-beta 1) on matrix gene expression has been investigated during the process of wound repair, where the formation of new connective tissue represents a critical step in restoring tissue integrity. Split-thickness excisional wounds in the pig were studied by in situ hybridization in order to obtain subjective findings on the activity and location of cells involved in matrix gene expression after the administration of recombinant TGF-beta 1. Data focus on the stimulatory role of this growth factor in granulation tissue formation, on the enhanced mRNA content of collagen types I and III, fibronectin, TGF-beta 1 itself, and on the reduction in stromelysin mRNA, suggesting that increased matrix formation measured after treatment with TGF-beta 1 is due to fibroplasia regulated by the abundance of mRNAs for several different structural, matrix proteins as well as inhibition of proteolytic phenomena elicited by metalloproteinases. These studies reveal elastin mRNA early in the repair process, and elastin mRNA expression is enhanced by administration of TGF-beta 1. Moreover, we show that TGF-beta 1 was auto-stimulating in wounds, accounting, at least in part, for the persistent effects of single doses of this multipotential cytokine.

Quality control of mRNP biogenesis: networking at the transcription site.

PubMed

Eberle, Andrea B; Visa, Neus

2014-08-01

Eukaryotic cells carry out quality control (QC) over the processes of RNA biogenesis to inactivate or eliminate defective transcripts, and to avoid their production. In the case of protein-coding transcripts, the quality controls can sense defects in the assembly of mRNA-protein complexes, in the processing of the precursor mRNAs, and in the sequence of open reading frames. Different types of defect are monitored by different specialized mechanisms. Some of them involve dedicated factors whose function is to identify faulty molecules and target them for degradation. Others are the result of a more subtle balance in the kinetics of opposing activities in the mRNA biogenesis pathway. One way or another, all such mechanisms hinder the expression of the defective mRNAs through processes as diverse as rapid degradation, nuclear retention and transcriptional silencing. Three major degradation systems are responsible for the destruction of the defective transcripts: the exosome, the 5'-3' exoribonucleases, and the nonsense-mediated mRNA decay (NMD) machinery. This review summarizes recent findings on the cotranscriptional quality control of mRNA biogenesis, and speculates that a protein-protein interaction network integrates multiple mRNA degradation systems with the transcription machinery. Copyright © 2014 Elsevier Ltd. All rights reserved.

In Silico Estimation of Translation Efficiency in Human Cell Lines: Potential Evidence for Widespread Translational Control

PubMed Central

Stevens, Stewart G.; Brown, Chris M

2013-01-01

Recently large scale transcriptome and proteome datasets for human cells have become available. A striking finding from these studies is that the level of an mRNA typically predicts no more than 40% of the abundance of protein. This correlation represents the overall figure for all genes. We present here a bioinformatic analysis of translation efficiency – the rate at which mRNA is translated into protein. We have analysed those human datasets that include genome wide mRNA and protein levels determined in the same study. The analysis comprises five distinct human cell lines that together provide comparable data for 8,170 genes. For each gene we have used levels of mRNA and protein combined with protein stability data from the HeLa cell line to estimate translation efficiency. This was possible for 3,990 genes in one or more cell lines and 1,807 genes in all five cell lines. Interestingly, our analysis and modelling shows that for many genes this estimated translation efficiency has considerable consistency between cell lines. Some deviations from this consistency likely result from the regulation of protein degradation. Others are likely due to known translational control mechanisms. These findings suggest it will be possible to build improved models for the interpretation of mRNA expression data. The results we present here provide a view of translation efficiency for many genes. We provide an online resource allowing the exploration of translation efficiency in genes of interest within different cell lines (http://bioanalysis.otago.ac.nz/TranslationEfficiency). PMID:23460887

Genome-wide expression profiling of in vivo-derived bloodstream parasite stages and dynamic analysis of mRNA alterations during synchronous differentiation in Trypanosoma brucei

PubMed Central

Kabani, Sarah; Fenn, Katelyn; Ross, Alan; Ivens, Al; Smith, Terry K; Ghazal, Peter; Matthews, Keith

2009-01-01

Background Trypanosomes undergo extensive developmental changes during their complex life cycle. Crucial among these is the transition between slender and stumpy bloodstream forms and, thereafter, the differentiation from stumpy to tsetse-midgut procyclic forms. These developmental events are highly regulated, temporally reproducible and accompanied by expression changes mediated almost exclusively at the post-transcriptional level. Results In this study we have examined, by whole-genome microarray analysis, the mRNA abundance of genes in slender and stumpy forms of T.brucei AnTat1.1 cells, and also during their synchronous differentiation to procyclic forms. In total, five biological replicates representing the differentiation of matched parasite populations derived from five individual mouse infections were assayed, with RNAs being derived at key biological time points during the time course of their synchronous differentiation to procyclic forms. Importantly, the biological context of these mRNA profiles was established by assaying the coincident cellular events in each population (surface antigen exchange, morphological restructuring, cell cycle re-entry), thereby linking the observed gene expression changes to the well-established framework of trypanosome differentiation. Conclusion Using stringent statistical analysis and validation of the derived profiles against experimentally-predicted gene expression and phenotypic changes, we have established the profile of regulated gene expression during these important life-cycle transitions. The highly synchronous nature of differentiation between stumpy and procyclic forms also means that these studies of mRNA profiles are directly relevant to the changes in mRNA abundance within individual cells during this well-characterised developmental transition. PMID:19747379

Differential regulation of preprotachykinin-A mRNA expression in striatum by excitation of hippocampal neurons.

PubMed

Brené, S; Lindefors, N; Herrera-Marschitz, M; Persson, H

1993-07-01

In this report we have studied the influence of hippocampal neurons on neuropeptide mRNA expression in both dorsal and ventral striatum in the rat. Intrahippocampal unilateral kainic acid injections were performed in control animals and in animals with a unilateral 6-hydroxydopamine-induced dopamine deafferentation of the striatum. In situ hybridization combined with quantitative image analysis was used to study the expression of preprotachykinin A mRNA encoding the neuropeptides substance P and neurokinin A. The 6-hydroxydopamine-induced lesion caused a decrease of preprotachykinin A mRNA levels in the ipsilateral dorsal striatum and in both sides of the ventral striatum. In normal rats, the intrahippocampal kainic acid injection caused a twofold increase in preprotachykinin A mRNA in the limbic parts of the striatum, which are innervated by the hippocampus. No effect of the kainic acid injection was seen in the lateral parts of the dorsal striatum, a region which does not appear to be innervated by the hippocampus. Animals with a 6-hydroxydopamine lesion showed a similar kainic acid-mediated increase in preprotachykinin A mRNA in parts of the ventral striatum. In the dopamine-lesioned dorsal striatum and ventral striatum the decreased preprotachykinin A mRNA levels were normalized by the intrahippocampal kainic acid injection. These results show that kainic acid-mediated excitation of hippocampal neurons causes a dopamine-independent induction of preprotachykinin A mRNA expression in parts of the ventral striatum, and reverses the dopamine deafferentation-induced decrease of preprotachykinin A mRNA in both dorsal and ventral striatum. Combined, our results suggest that hippocampal neurons can regulate preprotachykinin A mRNA expression in both the ventral and the dorsal striatum.

Reversible Ca2+-induced fast-to-slow transition in primary skeletal muscle culture cells at the mRNA level

PubMed Central

Meißner, Joachim D; Kubis, Hans-Peter; Scheibe, Renate J; Gros, Gerolf

2000-01-01

The adult fast character and a Ca2+-inducible reversible transition from a fast to a slow type of rabbit myotube in a primary culture were demonstrated at the mRNA level by Northern blot analysis with probes specific for different myosin heavy chain (MyHC) isoforms and enzymes of energy metabolism. No non-adult MyHC isoform mRNA was detected after 22 days of culture. After 4 weeks of culture the fast MyHCIId mRNA was strongly expressed while MyHCI mRNA was virtually absent, indicating the fast adult character of the myotubes in the primary skeletal muscle culture. The data show that a fast-to-slow transition occurred in the myotubes at the level of MyHC isoform gene expression after treatment with the Ca2+ ionophore A23187. The effects of ionophore treatment were decreased levels of fast MyHCII mRNA and an augmented expression of the slow MyHCI gene. Changes in gene expression started very rapidly 1 day after the onset of ionophore treatment. Levels of citrate synthase mRNA increased and levels of glyceraldehyde 3-phosphate dehydrogenase mRNA decreased during ionophore treatment. This points to a shift from anaerobic to oxidative energy metabolism in the primary skeletal muscle culture cells at the level of gene expression. Withdrawal of the Ca2+ ionophore led to a return to increased levels of MyHCII mRNA and decreased levels of MyHCI mRNA, indicating a slow-to-fast transition in the myotubes and the reversibility of the effect of ionophore on MyHC isoform gene expression. PMID:10673542

Depletion of mRNA export regulator DBP5/DDX19, GLE1 or IPPK that is a key enzyme for the production of IP6, resulting in differentially altered cytoplasmic mRNA expression and specific cell defect

PubMed Central

Okamura, Masumi; Yamanaka, Yasutaka; Shigemoto, Maki; Kitadani, Yuya; Kobayashi, Yuhko; Kambe, Taiho; Nagao, Masaya; Kobayashi, Issei; Okumura, Katsuzumi

2018-01-01

DBP5, also known as DDX19, GLE1 and inositol hexakisphosphate (IP6) function in messenger RNA (mRNA) export at the cytoplasmic surface of the nuclear pore complex in eukaryotic cells. DBP5 is a DEAD-box RNA helicase, and its activity is stimulated by interactions with GLE1 and IP6. In addition, these three factors also have unique role(s). To investigate how these factors influenced the cytoplasmic mRNA expression and cell phenotype change, we performed RNA microarray analysis to detect the effect and function of DBP5, GLE1 and IP6 on the cytoplasmic mRNA expression. The expression of some cytoplasmic mRNA subsets (e.g. cell cycle, DNA replication) was commonly suppressed by the knock-down of DBP5, GLE1 and IPPK (IP6 synthetic enzyme). The GLE1 knock-down selectively reduced the cytoplasmic mRNA expression required for mitotic progression, results in an abnormal spindle phenotype and caused the delay of mitotic process. Meanwhile, G1/S cell cycle arrest was observed in DBP5 and IPPK knock-down cells. Several factors that function in immune response were also down-regulated in DBP5 or IPPK knock-down cells. Thereby, IFNβ-1 mRNA transcription evoked by poly(I:C) treatment was suppressed. These results imply that DBP5, GLE1 and IP6 have a conserved and individual function in the cytoplasmic mRNA expression. Variations in phenotype are due to the difference in each function of DBP5, GLE1 and IPPK in intracellular mRNA metabolism. PMID:29746542

Isoenzyme-specific up-regulation of glutathione transferase and aldo-keto reductase mRNA expression by dietary quercetin in rat liver.

PubMed

Odbayar, Tseye-Oidov; Kimura, Toshinori; Tsushida, Tojiro; Ide, Takashi

2009-05-01

The impact of quercetin on the mRNA expression of hepatic enzymes involved in drug metabolism was evaluated with a DNA microarray and real-time PCR. Male Sprague-Dawley rats were fed an experimental diet containing either 0, 2.5, 5, 10, or 20 g/kg of quercetin for 15 days. The DNA microarray analysis of the gene expression profile in pooled RNA samples from rats fed diets containing 0, 5, and 20 g/kg of quercetin revealed genes of some isoenzymes of glutathione transferase (Gst) and aldo-keto reductase (Akr) to be activated by this flavonoid. Real-time PCR conducted with RNA samples from individual rats fed varying amounts of quercetin together with the microarray analysis showed that quercetin caused marked dose-dependent increases in the mRNA expression of Gsta3, Gstp1, and Gstt3. Some moderate increases were also noted in the mRNA expression of isoenzymes belonging to the Gstm class. Quercetin also dose-dependently increased the mRNA expression of Akr1b8 and Akr7a3. However, it did not affect the parameters of the other Gst and Akr isoenzymes. It is apparent that quercetin increases the mRNA expression of Gst and Akr involved in drug metabolism in an isoenzyme-specific manner. Inasmuch as Gst and Akr isoenzymes up-regulated in their gene expression are involved in the prevention and attenuation of cancer development, this consequence may account for the chemopreventive propensity of quercetin.

Differences in expression of retinal pigment epithelium mRNA between normal canines

PubMed Central

2004-01-01

Abstract A reference database of differences in mRNA expression in normal healthy canine retinal pigment epithelium (RPE) has been established. This database identifies non-informative differences in mRNA expression that can be used in screening canine RPE for mutations associated with clinical effects on vision. Complementary DNA (cDNA) pools were prepared from mRNA harvested from RPE, amplified by PCR, and used in a subtractive hybridization protocol (representational differential analysis) to identify differences in RPE mRNA expression between canines. The effect of relatedness of the test canines on the frequency of occurrence of differences was evaluated by using 2 unrelated canines for comparison with 2 female sibling canines of blue heeler/bull terrier lineage. Differentially expressed cDNA species were cloned, sequenced, and identified by comparison to public database entries. The most frequently observed differentially expressed sequence from the unrelated canine comparison was cDNA with 21 base pairs (bp) identical to the human epithelial membrane protein 1 gene (present in 8 of 20 clones). Different clones from the same-sex sibling RPE contained repetitions of several short sequence motifs including the human epithelial membrane protein 1 (4 of 25 clones). Other prevalent differences between sibling RPE included sequences similar to a chicken genetic marker sequence motif (5 of 25), and 6 clones with homology to porcine major histocompatibility loci. In addition to identifying several repetitively occurring, noninformative, differentially expressed RPE mRNA species, the findings confirm that fewer differences occurred between siblings, highlighting the importance of using closely related subjects in representational difference analysis studies. PMID:15352545

Mutational Inactivation of Herpes Simplex Virus 1 MicroRNAs Identifies Viral mRNA Targets and Reveals Phenotypic Effects in Culture

PubMed Central

Flores, Omar; Nakayama, Sanae; Whisnant, Adam W.; Javanbakht, Hassan; Cullen, Bryan R.

2013-01-01

Herpes simplex virus 1 (HSV-1), a ubiquitous human pathogen, expresses several viral microRNAs (miRNAs). These, along with the latency-associated transcript, represent the only viral RNAs detectable in latently infected neuronal cells. Here, for the first time, we analyze which HSV-1 miRNAs are loaded into the RNA-induced silencing complex (RISC), the key effector of miRNA function. Only 9 of the 17 reported HSV-1 miRNAs, i.e., miR-H1 to miR-H8 plus miR-H11, were found to actually load into the RISC. Surprisingly, this analysis also revealed that HSV-1 miRNAs loaded into the RISC with efficiencies that differed widely; <1% of the miR-H1-3p miRNA detectable in HSV-1-infected cells was loaded into the RISC. Analysis of HSV-1 mutants individually lacking the viral miR-H2, miR-H3, or miR-H4 miRNA revealed that loss of these miRNAs affected the rate of replication of HSV-1 in neuronal cells but not in fibroblasts. Analysis of mRNA and protein expression, as well as assays mapping viral miRNA binding sites in infected cells, showed that endogenous HSV-1 miR-H2 binds to viral ICP0 mRNA and inhibits its expression, while endogenous miR-H4 inhibits the expression of the viral ICP34.5 gene. In contrast, no viral mRNA target for miR-H3 could be detected, even though miR-H3, like miR-H4, is perfectly complementary to ICP34.5 mRNA. Together, these data demonstrate that endogenous HSV-1 miRNA expression can significantly alter viral replication in culture, and they also identify two viral mRNA targets for miR-H2 and miR-H4 that can partially explain this phenotype. PMID:23536669

metaseq: a Python package for integrative genome-wide analysis reveals relationships between chromatin insulators and associated nuclear mRNA.

PubMed

Dale, Ryan K; Matzat, Leah H; Lei, Elissa P

2014-08-01

Here we introduce metaseq, a software library written in Python, which enables loading multiple genomic data formats into standard Python data structures and allows flexible, customized manipulation and visualization of data from high-throughput sequencing studies. We demonstrate its practical use by analyzing multiple datasets related to chromatin insulators, which are DNA-protein complexes proposed to organize the genome into distinct transcriptional domains. Recent studies in Drosophila and mammals have implicated RNA in the regulation of chromatin insulator activities. Moreover, the Drosophila RNA-binding protein Shep has been shown to antagonize gypsy insulator activity in a tissue-specific manner, but the precise role of RNA in this process remains unclear. Better understanding of chromatin insulator regulation requires integration of multiple datasets, including those from chromatin-binding, RNA-binding, and gene expression experiments. We use metaseq to integrate RIP- and ChIP-seq data for Shep and the core gypsy insulator protein Su(Hw) in two different cell types, along with publicly available ChIP-chip and RNA-seq data. Based on the metaseq-enabled analysis presented here, we propose a model where Shep associates with chromatin cotranscriptionally, then is recruited to insulator complexes in trans where it plays a negative role in insulator activity. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Improved Brain Expression of Anti-Amyloid β scFv by Complexation of mRNA Including a Secretion Sequence with PEG-based Block Catiomer.

PubMed

Perche, Federico; Uchida, Satoshi; Akiba, Hiroki; Lin, Chin-Yu; Ikegami, Masaru; Dirisala, Anjaneyulu; Nakashima, Toshihiro; Itaka, Keiji; Tsumoto, Kohei; Kataoka, Kazunori

2017-01-01

The ever-increasing number of people living with Alzheimer's disease urges to develop more effective therapies. Despite considerable success, anti-Alzheimer immunotherapy still faces the challenge of intracerebral and intracellular delivery. This work introduces in situ production of anti-amyloid beta (Aβ) antibody after intracerebral injection of PEG-PAsp(DET)/mRNA polyplexes as a novel immunotherapy approach and a safer alternative compared to high systemic antibodies doses or administration of adenovirus encoding anti- Aβ antibodies. We used mRNA encoding three different Aβ-specific scFV with a secretion signal for passive immunotherapy. scFv contained a 6xHis-tag for immuno-detection. The secretion signal from IL2 (IL2ss) was added to allow extracellular engagement of senile plaques. Aβ affinity of scFv was measured by surface plasmon resonance. To allow intracellular delivery, scFv were administered as polyplexes formed with our smart copolymer polyethylene glycol-poly[N'-[N-(2-aminoethyl)-2-aminoethyl] aspartamide] [PEG-PAsp (DET)]. We evaluated scFv expression in cellulo by Western blot and ELISA, their ability to disaggregate amyloid aggregates by thioflavine T assay. Moreover, in vivo expression and therapeutic activity were evaluated in a murine amyloidosis model, by anti-6xHis-tag ELISA and anti- Aβ ELISA, respectively. The selected anti-amyloid beta scFv showed affinity towards Aβ and disaggregated Aβ fibers in vitro. Whereas both DNA and mRNA transfection led to scFV expression in cancer cells, only mRNA led to detectable scFv expression in primary neurons. In addition, the use of IL2ss increased by 3.4-fold scFv secretion by primary neurons over mRNA polyplexes devoid of secretion signal. In vivo, a 3 to 11- fold of intracranial scFv levels was measured for mRNA compared to DNA polyplexes and higher in vivo scFv levels were obtained with mRNA containing IL2ss over non-secreted mRNA. Intracranial injection of anti-Aβ mRNA polyplexes with IL2ss resulted in 40 % Aβ decrease in an acute amyloidosis model; with no decrease detected with control scFv mRNA nor DNA polyplexes. However, no Aβ decrease was detected in a more challenging transgenic model of Alzheimer's disease. Our results introduce a concerted approach not only for Alzheimer's disease treatment but also for immunotherapy against neurological diseases. The effectivity of our platform required the intracranial delivery of anti-Aβ scFv as mRNA not DNA, as mRNA with an IL2ss secretion sequence to favor engagement of Aβ in the amyloidosis model, complexation with a smart copolymer for efficient transfection of primary neurons and to achieve detectable mRNA expression in the brain during 48h. Amyloid burden decrease in an acute amyloidosis model was only achieved when these three factors (mRNA coding scFv, smart copolymer, IL2ss) were integrated into a single formulation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Kyoung Mi; Cho, Hana; Kim, Yoon Ki, E-mail: yk-kim@korea.ac.kr

Highlights: Black-Right-Pointing-Pointer CDKN1A mRNA is a bona fide NMD substrate. Black-Right-Pointing-Pointer The uORF of CDKN1A mRNA is efficiently translated. Black-Right-Pointing-Pointer Translation of downstream main ORF is negatively regulated by translation of uORF in CDKN1A mRNA. -- Abstract: The first round of translation occurs on mRNAs bound by nuclear cap-binding complex (CBC), which is composed of nuclear cap-binding protein 80 and 20 (CBP80/20). During this round of translation, aberrant mRNAs are recognized and downregulated in abundance by nonsense-mediated mRNA decay (NMD), which is one of the mRNA quality control mechanisms. Here, our microarray analysis reveals that the level of cyclin-dependent kinasemore » inhibitor 1A (CDKN1A; also known as Waf1/p21) mRNAs increases in cells depleted of cellular NMD factors. Intriguingly, CDKN1A mRNA contains an upstream open reading frame (uORF), which is a NMD-inducing feature. Using chimeric reporter constructs, we find that the uORF of CDKN1A mRNA negatively modulates translation of the main downstream ORF. These findings provide biological insights into the possible role of NMD in diverse biological pathways mediated by CDKN1A.« less

Codon-Resolution Analysis Reveals a Direct and Context-Dependent Impact of Individual Synonymous Mutations on mRNA Level

PubMed Central

Chen, Siyu; Li, Ke; Cao, Wenqing; Wang, Jia; Zhao, Tong; Huan, Qing; Yang, Yu-Fei; Wu, Shaohuan; Qian, Wenfeng

2017-01-01

Abstract Codon usage bias (CUB) refers to the observation that synonymous codons are not used equally frequently in a genome. CUB is stronger in more highly expressed genes, a phenomenon commonly explained by stronger natural selection on translational accuracy and/or efficiency among these genes. Nevertheless, this phenomenon could also occur if CUB regulates gene expression at the mRNA level, a hypothesis that has not been tested until recently. Here, we attempt to quantify the impact of synonymous mutations on mRNA level in yeast using 3,556 synonymous variants of a heterologous gene encoding green fluorescent protein (GFP) and 523 synonymous variants of an endogenous gene TDH3. We found that mRNA level was positively correlated with CUB among these synonymous variants, demonstrating a direct role of CUB in regulating transcript concentration, likely via regulating mRNA degradation rate, as our additional experiments suggested. More importantly, we quantified the effects of individual synonymous mutations on mRNA level and found them dependent on 1) CUB and 2) mRNA secondary structure, both in proximal sequence contexts. Our study reveals the pleiotropic effects of synonymous codon usage and provides an additional explanation for the well-known correlation between CUB and gene expression level. PMID:28961875

CXCL1 and CXCR2 as potential markers for vital reactions in skin contusions.

PubMed

He, Jie-Tao; Huang, Hong-Yan; Qu, Dong; Xue, Ye; Zhang, Kai-Kai; Xie, Xiao-Li; Wang, Qi

2018-06-01

Detection of the vitality of wounds is one of the most important issues in forensic practice. This study investigated mRNA and protein levels of CXCL1 and CXCR2 in skin wounds in mice and humans. Western blot analysis of CXCL1 and CXCR2 protein levels showed no difference between wounded and intact skin. However, mRNA levels demonstrated higher expression of CXCL1 and CXCR2 in contused mouse and human skin, compared with intact skin. At postmortem there were no remarkable changes in CXCL1 and CXCR2 mRNA levels in contused mouse skin. Increased mRNA expression was observed in contused mouse skin up to 96 h and 72 h after death for CXCL1 and CXCR2 respectively. In human samples of wounded skin, increased CXCL1 mRNA levels were detected up to 48 h after autopsy in all 5 cases, while increased CXCR2 mRNA levels were observed 48 h after autopsy in 4 of 5 cases. These findings suggest that the levels of CXCL1 and CXCR2 mRNA present in contused skin can be used as potential markers for a vital reaction in forensic practice.

Sequence features and phylogenetic analysis of the stress protein Hsp90α in chinook salmon Oncorhynchus tshawytscha, a poikilothermic vertebrate

USGS Publications Warehouse

Palmisano, Aldo N.; Winton, James R.; Dickhoff, Walton W.

1999-01-01

We cloned and sequenced a chinook salmon Hsp90 cDNA; sequence analysis shows it to be Hsp90??. Phylogenetic analysis supports the hypothesis that ?? and ?? paralogs of Hsp90 arose as a result of a gene duplication event and that they diverged early in the evolution of vertebrates, before tetrapods separated from the teleost lineage. Among several differences distinguishing poikilothermic Hsp90?? sequences from their bird and mammal orthologs, the teleost versions specifically lack a characteristic QTQDQP phosphorylation site near the N-terminus. We used the cDNA to develop an RNA (Northern) blot to quantify cellular Hsp90 mRNA levels. Chinook salmon embryonic (CHSE-214) cells responded to heat shock with a rapid rise in Hsp90 mRNA through 4 h, followed by a gradual decline over the next 20 h. Hsp90 mRNA level may be useful as a stress indicator, especially in a laboratory setting or in response to acute heat stress.

Integrating De Novo Transcriptome Assembly and Cloning to Obtain Chicken Ovocleidin-17 Full-Length cDNA

PubMed Central

Ning, ZhongHua; Hincke, Maxwell T.; Yang, Ning; Hou, ZhuoCheng

2014-01-01

Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not ‘finished’. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length cDNA sequences. PMID:24676480

Integrating de novo transcriptome assembly and cloning to obtain chicken Ovocleidin-17 full-length cDNA.

PubMed

Zhang, Quan; Liu, Long; Zhu, Feng; Ning, ZhongHua; Hincke, Maxwell T; Yang, Ning; Hou, ZhuoCheng

2014-01-01

Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not 'finished'. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length cDNA sequences.

mRNA transcripts as molecular biomarkers in medicine and nutrition

PubMed Central

Sunde, Roger A.

2010-01-01

In medicine, mRNA transcripts are being developed as molecular biomarkers for the diagnosis and treatment of a number of diseases. These biomarkers offer early and more accurate prediction and diagnosis of disease and disease progression, and ability to identify individuals at risk. Use of microarrays also offers opportunity to identify orthogonal (uncorrelated) biomarkers not known to be linked with conventional biomarkers. Investigators are increasingly using blood as a surrogate tissue for biopsy and analysis; total RNA isolated from whole blood is predominantly from erythroid cells, and whole blood mRNA share more than 80% of the transcriptome with major tissues. Thus blood mRNA biomarkers for individualized disease prediction and diagnosis are an exciting area in medicine; mRNA biomarkers in nutrition have potential application that parallel these opportunities. Assessment of selenium (Se) status and requirements is one area where tissue mRNA levels have been used successfully. Selenoprotein-H and selenoprotein-W as well as glutathione peroxidase-1 (Gpx1) mRNAs are highly down-regulated in Se deficiency in rat liver, and the minimum dietary Se requirement is 0.06–0.07 μg Se/g based on these biomarkers, similar to requirements determined using conventional biomarkers. Blood Gpx1 mRNA can also be used to determine Se requirements in rats, showing that blood mRNA has potential for assessment of nutrient status. Future research is needed to develop mRNA biomarker panels for all nutrients that will discriminate between deficient, marginal, adequate, and supernutritional individuals and populations, and differentiate between individuals that will benefit versus be adversely affected by nutrient supplementation. PMID:20303730

The poly(A) tail length of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of milk.

PubMed Central

Kuraishi, T; Sun, Y; Aoki, F; Imakawa, K; Sakai, S

2000-01-01

The length of casein mRNA from the lactating mouse mammary gland, as assessed on Northern blots, is shorter after weaning, but is elongated following the removal of milk. In order to investigate this phenomenon, the molecular structures of beta- and gamma-casein mRNAs were analysed. The coding and non-coding regions of the two forms were the same length, but the long form of casein mRNA had a longer poly(A) tail than the short form (P<0.05). In order to examine the stability of casein mRNA under identical conditions, casein mRNAs with the long and short poly(A) tails were incubated in the rabbit reticulocyte lysate (RRL) cell-free translation system. Casein mRNA with the long poly(A) tail had a longer half-life than that with the short tail (P<0.05). The beta- and gamma-casein mRNAs were first degraded into 0.92 and 0.81 kb fragments respectively. With undegraded mRNA, the poly(A) tail shortening by exoribonuclease was not observed until the end of the incubation. Northern blot analysis showed that casein mRNA with the long poly(A) tail was protected efficiently from endoribonucleases. We conclude that the length of the poly(A) tail of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of the gland's milk, and we show that the longer poly(A) tail potentially protects the mRNA from degradation by endoribonucleases. PMID:10749689

Changes in mRNA expression for gluconeogenic enzymes in liver of dairy cattle during the transition to lactation.

PubMed

Greenfield, R B; Cecava, M J; Donkin, S S

2000-06-01

The objective of this study was to profile phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC) mRNA expression in the liver of dairy cattle during the peripartum transition and determine changes in abundance of these mRNA in response to protein fed during the prepartum period. Thirty-eight multiparous Holstein cows were fed diets containing either 12% crude protein (CP) and 26% rumen undegradable protein (RUP), 16% CP and 26% RUP, 16% CP and 33% RUP, or 16% CP and 40% RUP on a dry-matter basis beginning 28 d before expected calving. After calving, all cows were fed a common diet through 56 d in milk (DIM). Northern analysis of RNA from liver biopsy samples obtained on days -28, -14, +1, +28, and +56 relative to calving indicated that PC and PEPCK mRNA expression were responsive to onset of lactation but not to prepartum protein or RUP concentration. Abundance of PEPCK mRNA was similar at -28, -14, and +1 DIM but was elevated by +28 and +56 DIM relative to precalving levels. Liver PC mRNA abundance was elevated on +1 DIM, remained elevated through 28 DIM, and declined to precalving levels by 56 DIM. The activity of PC enzyme was correlated (r2 = 0.89) with PC mRNA abundance. The data demonstrate increased abundance of PC mRNA during the early transition period followed by increased abundance of PEPCK mRNA during the postpartum period and suggest increased potential metabolism of lactate, pyruvate, and amino acids that contribute to the liver pyruvate pool.

Oxytocin receptor mRNA expression in rat brain: implications for behavioral integration and reproductive success.

PubMed

Ostrowski, N L

1998-11-01

The nonapeptide, oxytocin (OT), has been implicated in a wide range of physiological, behavioral and pharmacological effects related to learning and memory, parturition and lactation, maternal and sexual behavior, and the formation of social attachments. Specific G-protein linked membrane bound OT receptors mediate OTs effects. The unavailability of highly selective pharmacological ligands that discriminate the OT receptor from the highly homologous vasopressin receptors (V1a, V1b and V2 subtypes) has made it difficult to confirm specific effects of oxytocin, particularly in brain regions where OT and multiple AVP receptor subtypes may be coexpressed. Here, data on the oxytocin receptor (OTR) messenger ribonucleic acid (mRNA) localization in brain are presented in the context of a model that proposes a reproductive state-dependent role for steroid-hormone restructuring of neural circuits, and a role for oxytocin in the integration of neural transmission in pathways subserving: (1) steroid-sensitive reproductive behaviors; (2) learning; and (3) reinforcement. It is hypothesized that social attachments emerge as a consequence of a conditioned association between OT-related activity in these pathways and the eliciting stimulus.

[Intergration and epression of porcine endogenous retrovinus in the immortal cell line of Banna Minipig Inberd Line-Mesenhymal Stem Cells].

PubMed

Yu, Ping; Liu, Jin; Zhang, Li; Li, Shrng-Fu; Bu, Hong; Li, You-Ping; Cheng, Jing-Qui; Lu, Yan-Rong; Long, Dan

2005-11-01

To detect the integration and expression of porcine endogenous retrovirus (PERV) in the immortal cell line of Banna Minipig Inbred Line-Mesenchymal Stem Cells (BMI-MSCs). DNA and total RNA of the immortal cell line of BMI-MSCs were extracted and PCR, RT-PCR were performed to detect PERV-gag, pol and env gene, and the type of PERV was also detected. PERV-gag, pol and env gene were all detected in the primary culture and immortal cell line (passage 150 and passage 180) of BMI-MSCs, and the type of PERV was PERV-A, B. Functional expression of PERV-gag and pol mRNA was also detected. In this laboratory, PERV was not lost during the proceeding of pig inbred and since has been in long-term culture of pig cells in vitro. PERV has integrated into the genome of its natural host, and virus mRNA can effectively express. So it is very essential to evaluate the possibility of xenozoonoses in pig-to-human xenotransplantation.

Postage for the messenger: Designating routes for Nuclear mRNA Export

PubMed Central

Natalizio, Barbara J.; Wente, Susan R.

2013-01-01

Transcription of messenger(m) RNA occurs in the nucleus, making the translocation of mRNA across the nuclear envelope (NE) boundary a critical determinant of proper gene expression and cell survival. A major mRNA export route occurs via the NXF1-dependent pathway through the nuclear pore complexes (NPCs) embedded in the NE. However, recent findings have discovered new evidence supporting the existence of multiple mechanisms for crossing the NE, including both NPC-mediated and NE budding-mediated pathways. An analysis of the trans-acting factors and cis components that define these pathways reveals shared elements as well as mechanistic differences. We review here the current understanding of the mechanisms that characterize each pathway and highlight the determinants that influence mRNA transport fate. PMID:23583578

Elevation of D4 dopamine receptor mRNA in postmortem schizophrenic brain.

PubMed

Stefanis, N C; Bresnick, J N; Kerwin, R W; Schofield, W N; McAllister, G

1998-01-01

The D4 dopamine (DA) receptor has been proposed to be a target for the development of a novel antipsychotic drug based on its pharmacological and distribution profile. There is much interest in whether D4 DA receptor levels are altered in schizophrenia, but the lack of an available receptor subtype-specific radioligand made this difficult to quantitate. In this study, we examined whether D4 mRNA levels are altered in different brain regions of schizophrenics compared to controls. Ribonuclease protection assays were carried out on total RNA samples isolated postmortem from frontal cortex and caudate brain regions of schizophrenics and matched controls. 32P-labelled RNA probes to the D4 DA receptor and to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), were hybridised with the RNA samples, digested with ribonucleases to remove unhybridised probe, and separated on 6% sequencing gels. Densitometer analysis on the subsequent autoradiogams was used to calculate the relative optical density of D4 mRNA compared to G3PDH mRNA. Statistical analysis of the data revealed a 3-fold higher level (P<0.011) of D4 mRNA in the frontal cortex of schizophrenics compared to controls. No increase was seen in caudate. D4 receptors could play a role in mediating dopaminergic activity in frontal cortex, an activity which may be malfunctioning in schizophrenia.

LC3-mediated fibronectin mRNA translation induces fibrosarcoma growth by increasing connective tissue growth factor

PubMed Central

Ying, Lihua; Lau, Agatha; Alvira, Cristina M.; West, Robert; Cann, Gordon M.; Zhou, Bin; Kinnear, Caroline; Jan, Eric; Sarnow, Peter; Van de Rijn, Matt; Rabinovitch, Marlene

2009-01-01

Summary Previously, we related fibronectin (Fn1) mRNA translation to an interaction between an AU-rich element in the Fn1 3′ UTR and light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce little fibronectin and LC3, we used these cells to investigate how LC3-mediated Fn1 mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced fibronectin mRNA translation. Using polysome analysis and RNA-binding assays, we show that elevated levels of translation depend on an interaction between a triple arginine motif in LC3 and the AU-rich element in Fn1 mRNA. Wild-type but not mutant LC3 accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of WT LC3 with vector-transfected HT1080 cells revealed increased fibronectin-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in WT and vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show that enhanced expression of CTGF is fibronectin dependent and that LC3-mediated adhesion, invasion and proliferation are CTGF dependent. Expression profiling of soft tissue tumors revealed increased expression of both LC3 and CTGF in some locally invasive tumor types. PMID:19366727

Low-level lasers alter mRNA levels from traditional reference genes used in breast cancer cells

NASA Astrophysics Data System (ADS)

Teixeira, A. F.; Canuto, K. S.; Rodrigues, J. A.; Fonseca, A. S.; Mencalha, A. L.

2017-07-01

Cancer is among the leading causes of mortality worldwide, increasing the importance of treatment development. Low-level lasers are used in several diseases, but some concerns remains on cancers. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a technique used to understand cellular behavior through quantification of mRNA levels. Output data from target genes are commonly relative to a reference that cannot vary according to treatment. This study evaluated reference genes levels from MDA-MB-231 cells exposed to red or infrared lasers at different fluences. Cultures were exposed to red and infrared lasers, incubated (4 h, 37 °C), total RNA was extracted and cDNA synthesis was performed to evaluate mRNA levels from ACTB, GUSB and TRFC genes by RT-qPCR. Specific amplification was verified by melting curves and agarose gel electrophoresis. RefFinder enabled data analysis by geNorm, NormFinder and BestKeeper. Specific amplifications were obtained and, although mRNA levels from ACTB, GUSB or TRFC genes presented no significant variation through traditional statistical analysis, Excel-based tools revealed that the use of these reference genes are dependent of laser characteristics. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from ACTB, GUSB and TRFC in MDA-MB-231 cells.

LncSubpathway: a novel approach for identifying dysfunctional subpathways associated with risk lncRNAs by integrating lncRNA and mRNA expression profiles and pathway topologies.

PubMed

Xu, Yanjun; Li, Feng; Wu, Tan; Xu, Yingqi; Yang, Haixiu; Dong, Qun; Zheng, Meiyu; Shang, Desi; Zhang, Chunlong; Zhang, Yunpeng; Li, Xia

2017-02-28

Long non-coding RNAs (lncRNAs) play important roles in various biological processes, including the development of many diseases. Pathway analysis is a valuable aid for understanding the cellular functions of these transcripts. We have developed and characterized LncSubpathway, a novel method that integrates lncRNA and protein coding gene (PCG) expression with interactome data to identify disease risk subpathways that functionally associated with risk lncRNAs. LncSubpathway identifies the most relevance regions which are related with risk lncRNA set and implicated with study conditions through simultaneously considering the dysregulation extent of lncRNAs, PCGs and their correlations. Simulation studies demonstrated that the sensitivity and false positive rates of LncSubpathway were within acceptable ranges, and that LncSubpathway could accurately identify dysregulated regions that related with disease risk lncRNAs within pathways. When LncSubpathway was applied to colorectal carcinoma and breast cancer subtype datasets, it identified cancer type- and breast cancer subtype-related meaningful subpathways. Further, analysis of its robustness and reproducibility indicated that LncSubpathway was a reliable means of identifying subpathways that functionally associated with lncRNAs. LncSubpathway is freely available at http://www.bio-bigdata.com/lncSubpathway/.

LncSubpathway: a novel approach for identifying dysfunctional subpathways associated with risk lncRNAs by integrating lncRNA and mRNA expression profiles and pathway topologies

PubMed Central

Wu, Tan; Xu, Yingqi; Yang, Haixiu; Dong, Qun; Zheng, Meiyu; Shang, Desi; Zhang, Chunlong; Zhang, Yunpeng; Li, Xia

2017-01-01

Long non-coding RNAs (lncRNAs) play important roles in various biological processes, including the development of many diseases. Pathway analysis is a valuable aid for understanding the cellular functions of these transcripts. We have developed and characterized LncSubpathway, a novel method that integrates lncRNA and protein coding gene (PCG) expression with interactome data to identify disease risk subpathways that functionally associated with risk lncRNAs. LncSubpathway identifies the most relevance regions which are related with risk lncRNA set and implicated with study conditions through simultaneously considering the dysregulation extent of lncRNAs, PCGs and their correlations. Simulation studies demonstrated that the sensitivity and false positive rates of LncSubpathway were within acceptable ranges, and that LncSubpathway could accurately identify dysregulated regions that related with disease risk lncRNAs within pathways. When LncSubpathway was applied to colorectal carcinoma and breast cancer subtype datasets, it identified cancer type- and breast cancer subtype-related meaningful subpathways. Further, analysis of its robustness and reproducibility indicated that LncSubpathway was a reliable means of identifying subpathways that functionally associated with lncRNAs. LncSubpathway is freely available at http://www.bio-bigdata.com/lncSubpathway/. PMID:28152521

Proteogenomics connects somatic mutations to signalling in breast cancer.

PubMed

Mertins, Philipp; Mani, D R; Ruggles, Kelly V; Gillette, Michael A; Clauser, Karl R; Wang, Pei; Wang, Xianlong; Qiao, Jana W; Cao, Song; Petralia, Francesca; Kawaler, Emily; Mundt, Filip; Krug, Karsten; Tu, Zhidong; Lei, Jonathan T; Gatza, Michael L; Wilkerson, Matthew; Perou, Charles M; Yellapantula, Venkata; Huang, Kuan-lin; Lin, Chenwei; McLellan, Michael D; Yan, Ping; Davies, Sherri R; Townsend, R Reid; Skates, Steven J; Wang, Jing; Zhang, Bing; Kinsinger, Christopher R; Mesri, Mehdi; Rodriguez, Henry; Ding, Li; Paulovich, Amanda G; Fenyö, David; Ellis, Matthew J; Carr, Steven A

2016-06-02

Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. Here we describe quantitative mass-spectrometry-based proteomic and phosphoproteomic analyses of 105 genomically annotated breast cancers, of which 77 provided high-quality data. Integrated analyses provided insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. Interrogation of the 5q trans-effects against the Library of Integrated Network-based Cellular Signatures, connected loss of CETN3 and SKP1 to elevated expression of epidermal growth factor receptor (EGFR), and SKP1 loss also to increased SRC tyrosine kinase. Global proteomic data confirmed a stromal-enriched group of proteins in addition to basal and luminal clusters, and pathway analysis of the phosphoproteome identified a G-protein-coupled receptor cluster that was not readily identified at the mRNA level. In addition to ERBB2, other amplicon-associated highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. We demonstrate that proteogenomic analysis of breast cancer elucidates the functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets.

iTRAQ-Based Proteomics Analysis and Network Integration for Kernel Tissue Development in Maize

PubMed Central

Dong, Yongbin; Wang, Qilei; Du, Chunguang; Xiong, Wenwei; Li, Xinyu; Zhu, Sailan; Li, Yuling

2017-01-01

Grain weight is one of the most important yield components and a developmentally complex structure comprised of two major compartments (endosperm and pericarp) in maize (Zea mays L.), however, very little is known concerning the coordinated accumulation of the numerous proteins involved. Herein, we used isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative proteomic method to analyze the characteristics of dynamic proteomics for endosperm and pericarp during grain development. Totally, 9539 proteins were identified for both components at four development stages, among which 1401 proteins were non-redundant, 232 proteins were specific in pericarp and 153 proteins were specific in endosperm. A functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the tissue development. Three and 76 proteins involved in 49 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were integrated for the specific endosperm and pericarp proteins, respectively, reflecting their complex metabolic interactions. In addition, four proteins with important functions and different expression levels were chosen for gene cloning and expression analysis. Different concordance between mRNA level and the protein abundance was observed across different proteins, stages, and tissues as in previous research. These results could provide useful message for understanding the developmental mechanisms in grain development in maize. PMID:28837076

MC EMiNEM Maps the Interaction Landscape of the Mediator

PubMed Central

Niederberger, Theresa; Etzold, Stefanie; Lidschreiber, Michael; Maier, Kerstin C.; Martin, Dietmar E.; Fröhlich, Holger; Cramer, Patrick; Tresch, Achim

2012-01-01

The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between proteins that have pleiotropic effects on mRNA transcription. MC EMiNEM is based on Nested Effects Models (NEMs), a class of probabilistic graphical models that extends the idea of hierarchical clustering. It combines mode-hopping Monte Carlo (MC) sampling with an Expectation-Maximization (EM) algorithm for NEMs to increase sensitivity compared to existing methods. A meta-analysis of four Mediator perturbation studies in Saccharomyces cerevisiae, three of which are unpublished, provides new insight into the Mediator signaling network. In addition to the known modular organization of the Mediator subunits, MC EMiNEM reveals a hierarchical ordering of its internal information flow, which is putatively transmitted through structural changes within the complex. We identify the N-terminus of Med7 as a peripheral entity, entailing only local structural changes upon perturbation, while the C-terminus of Med7 and Med19 appear to play a central role. MC EMiNEM associates Mediator subunits to most directly affected genes, which, in conjunction with gene set enrichment analysis, allows us to construct an interaction map of Mediator subunits and transcription factors. PMID:22737066

MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype.

PubMed

Blenkiron, Cherie; Goldstein, Leonard D; Thorne, Natalie P; Spiteri, Inmaculada; Chin, Suet-Feung; Dunning, Mark J; Barbosa-Morais, Nuno L; Teschendorff, Andrew E; Green, Andrew R; Ellis, Ian O; Tavaré, Simon; Caldas, Carlos; Miska, Eric A

2007-01-01

MicroRNAs (miRNAs), a class of short non-coding RNAs found in many plants and animals, often act post-transcriptionally to inhibit gene expression. Here we report the analysis of miRNA expression in 93 primary human breast tumors, using a bead-based flow cytometric miRNA expression profiling method. Of 309 human miRNAs assayed, we identify 133 miRNAs expressed in human breast and breast tumors. We used mRNA expression profiling to classify the breast tumors as luminal A, luminal B, basal-like, HER2+ and normal-like. A number of miRNAs are differentially expressed between these molecular tumor subtypes and individual miRNAs are associated with clinicopathological factors. Furthermore, we find that miRNAs could classify basal versus luminal tumor subtypes in an independent data set. In some cases, changes in miRNA expression correlate with genomic loss or gain; in others, changes in miRNA expression are likely due to changes in primary transcription and or miRNA biogenesis. Finally, the expression of DICER1 and AGO2 is correlated with tumor subtype and may explain some of the changes in miRNA expression observed. This study represents the first integrated analysis of miRNA expression, mRNA expression and genomic changes in human breast cancer and may serve as a basis for functional studies of the role of miRNAs in the etiology of breast cancer. Furthermore, we demonstrate that bead-based flow cytometric miRNA expression profiling might be a suitable platform to classify breast cancer into prognostic molecular subtypes.

cDNA cloning and characterization of UDP-glucose: anthocyanidin 3-O-glucosyltransferase in Freesia hybrida.

PubMed

Sui, Xin; Gao, Xiang; Ao, Man; Wang, Qinmei; Yang, Dan; Wang, Meng; Fu, Yang; Wang, Li

2011-07-01

The enzyme that catalyzes the formation of the first stable anthocyanin in the biosynthesis of natural compounds is UDP-glucose: anthocyanidin 3-O-glucosyltransferase (UF3GT). A cDNA clone (Fh3GT1) encoding UF3GT was isolated from Freesia hybrida. Phylogenetic tree analysis indicated that Fh3GT1 was a novel member of glycosyltransferase, which was classified into monocot subgroups. Semi-quantitative RT-PCR analysis detected transcripts of Fh3GT1 in different organs of F. hybrida and in petals of Freesia cultivars of different colors, and the expression level reached the maximum at the fully opened stage of petals. Characterization of the enzymatic assays indicated that Fh3GT1 had a role in anthocyanin glycoside biosyntheses in vitro. To elucidate the function of Fh3GT1, RNA interference vector (pART-Fh3GT1i) was constructed, and introduced into Petunia grandiflora by Agrobacterium-mediated transformation. Integration of the Fh3GT1 in petunia genome was confirmed by PCR and Southern blotting. SqRT-PCR revealed that the endogenous Ph3GT1 mRNA expression levels decreased in transgenic lines compared with the wild-type. The content of total anthocyanin pigments also decreased with the reduction of mRNA transcript levels, and the transgenic petunia plants had significant changes on their flower colors. In summary, this work identified a UF3GT gene from Freesia hybrida and demonstrated a method to modify plant flower color by redirecting the anthocyanin biosynthesis.

Detection of high mobility group A2 specific mRNA in the plasma of patients affected by epithelial ovarian cancer

PubMed Central

Pasquinelli, Rosa; Pignata, Sandro; Greggi, Stefano; Vuttariello, Emilia; Bello, Anna Maria; Calise, Celeste; Scaffa, Cono; Pisano, Carmela; Losito, Nunzia Simona; Fusco, Alfredo; Califano, Daniela; Chiappetta, Gennaro

2015-01-01

Ovarian cancer is the most lethal gynecological malignancy and the high mortality rate is associated with advanced-stage disease at the time of the diagnosis. In order to find new tools to make diagnosis of Epithelial Ovarian Cancer (EOC) at early stages we have analyzed the presence of specific HMGA2 mRNA in the plasma of patients affected by this neoplasm. HMGA2 overexpression represents a feature of several malignances including ovarian carcinomas. Notably, we detected HMGA2 specific mRNA in the plasma of 40 out 47 patients with EOC, but not in the plasma of healthy donors. All cases found positive for HMGA2 mRNA in the plasma showed HMGA2 protein expression in EOC tissues. Therefore, on the basis of these results, the analysis of circulating HMGA2 specific mRNA might be considered a very promising tool for the early diagnosis of EOC. PMID:25749380

Light-regulated protein and mRNA synthesis in root caps of maize

NASA Technical Reports Server (NTRS)

Feldman, L. J.; Piechulla, B.; Sun, P. S.

1988-01-01

Illumination of maize roots initiates changes in mRNA levels and in the activities of proteins within the root cap. Using Northern analysis we showed a 5-6 fold increase in the levels of three specific mRNAs and a 14-fold increase in plastid mRNA. This increase is rapid, occurring within 30 minutes of illumination. With prolonged periods of darkness following illumination, messages return to levels observed in dark, control caps. For two species of mRNA illumination results in a reduction in message levels. Light-stimulated increases in the levels of specific mRNAs are proportionally greater than are increases in the activities of corresponding proteins. We suggest that the light-stimulated increase in protein activity in root caps may be preceded by and occur as a consequence of enhanced levels of mRNA. Our work suggests that photomorphogenesis in roots could involve changes in the levels of a wide variety of mRNAs within the root cap.

Detection of low-concentration host mRNA transcripts in Malawian children at risk for environmental enteropathy

USDA-ARS?s Scientific Manuscript database

Transcriptomic analysis of fecal samples is an emerging method for the diagnosis of gastrointestinal pathology because it is noninvasive and requires minute volumes of analyte; however, detection of mRNA in low copy numbers in human stool is challenging. Our objective was to develop a method for det...

Mismatch DNA repair mRNA expression profiles in oral melanin pigmentation lesion and hamartomatous polyp of a child with Peutz-Jeghers syndrome.

PubMed

Vageli, Dimitra P; Doukas, Sotirios G; Markou, Andreas

2013-10-01

Mismatch DNA repair (MMR) mRNA expression analysis was performed on a biopsy of oral mucosa melanin pigmentation lesion, a hamartomatous polyp and peripheral blood derived from a 12-year-old child with Peutz-Jeghers Syndrome (PJS). We present a deficient MMR system, in a PJS patient, which demonstrated low mRNA levels of hMSH6 and hPMS2 and an increasing MMR deficiency from the non-dysplastic lesion to hamartomatous polyp of PJS with a high risk of cancer. Copyright © 2013 Wiley Periodicals, Inc.

Latest development on RNA-based drugs and vaccines.

PubMed

Lundstrom, Kenneth

2018-06-01

Drugs and vaccines based on mRNA and RNA viruses show great potential and direct translation in the cytoplasm eliminates chromosomal integration. Limitations are associated with delivery and stability issues related to RNA degradation. Clinical trials on RNA-based drugs have been conducted in various disease areas. Likewise, RNA-based vaccines for viral infections and various cancers have been subjected to preclinical and clinical studies. RNA delivery and stability improvements include RNA structure modifications, targeting dendritic cells and employing self-amplifying RNA. Single-stranded RNA viruses possess self-amplifying RNA, which can provide extreme RNA replication in the cytoplasm to support RNA-based drug and vaccine development. Although oligonucleotide-based approaches have demonstrated potential, the focus here is on mRNA- and RNA virus-based methods.

Systems analysis identifies miR-29b regulation of invasiveness in melanoma.

PubMed

Andrews, Miles C; Cursons, Joseph; Hurley, Daniel G; Anaka, Matthew; Cebon, Jonathan S; Behren, Andreas; Crampin, Edmund J

2016-11-16

In many cancers, microRNAs (miRs) contribute to metastatic progression by modulating phenotypic reprogramming processes such as epithelial-mesenchymal plasticity. This can be driven by miRs targeting multiple mRNA transcripts, inducing regulated changes across large sets of genes. The miR-target databases TargetScan and DIANA-microT predict putative relationships by examining sequence complementarity between miRs and mRNAs. However, it remains a challenge to identify which miR-mRNA interactions are active at endogenous expression levels, and of biological consequence. We developed a workflow to integrate TargetScan and DIANA-microT predictions into the analysis of data-driven associations calculated from transcript abundance (RNASeq) data, specifically the mutual information and Pearson's correlation metrics. We use this workflow to identify putative relationships of miR-mediated mRNA repression with strong support from both lines of evidence. Applying this approach systematically to a large, published collection of unique melanoma cell lines - the Ludwig Melbourne melanoma (LM-MEL) cell line panel - we identified putative miR-mRNA interactions that may contribute to invasiveness. This guided the selection of interactions of interest for further in vitro validation studies. Several miR-mRNA regulatory relationships supported by TargetScan and DIANA-microT demonstrated differential activity across cell lines of varying matrigel invasiveness. Strong negative statistical associations for these putative regulatory relationships were consistent with target mRNA inhibition by the miR, and suggest that differential activity of such miR-mRNA relationships contribute to differences in melanoma invasiveness. Many of these relationships were reflected across the skin cutaneous melanoma TCGA dataset, indicating that these observations also show graded activity across clinical samples. Several of these miRs are implicated in cancer progression (miR-211, -340, -125b, -221, and -29b). The specific role for miR-29b-3p in melanoma has not been well studied. We experimentally validated the predicted miR-29b-3p regulation of LAMC1 and PPIC and LASP1, and show that dysregulation of miR-29b-3p or these mRNA targets can influence cellular invasiveness in vitro. This analytic strategy provides a comprehensive, systems-level approach to identify miR-mRNA regulation in high-throughput cancer data, identifies novel putative interactions with functional phenotypic relevance, and can be used to direct experimental resources for subsequent experimental validation. Computational scripts are available: http://github.com/uomsystemsbiology/LMMEL-miR-miner.

Connective tissue growth factor is overexpressed in human hepatocellular carcinoma and promotes cell invasion and growth.

PubMed

Xiu, Ming; Liu, Ya-Hui; Brigstock, David R; He, Fang-Hui; Zhang, Rui-Juan; Gao, Run-Ping

2012-12-21

To determine the expression characteristics of connective tissue growth factor (CTGF/CCN2) in human hepatocellular carcinoma (HCC) in histology and to elucidate the roles of CCN2 on hepatoma cell cycle progression and metastasis in vitro. Liver samples from 36 patients (who underwent hepatic resection for the first HCC between 2006 and 2011) and 6 normal individuals were examined for transforming growth factor β1 (TGF-β1) or CCN2 mRNA by in situ hybridization. Computer image analysis was performed to measure integrated optimal density of CCN2 mRNA-positive cells in carcinoma foci and the surrounding stroma. Fibroblast-specific protein-1 (FSP-1) and E-cadherin were examined to evaluate the process of epithelial to mesenchymal transition, α-smooth muscle actin and FSP-1 were detected to identify hepatic stellate cells, and CD34 was measured to evaluate the extent of vascularization in liver tissues by immunohistochemical staining. CCN2 was assessed for its stimulation of HepG2 cell migration and invasion using commercial kits while flow cytometry was used to determine CCN2 effects on HepG2 cell-cycle. In situ hybridization analysis showed that TGF-β1 mRNA was mainly detected in connective tissues and vasculature around carcinoma foci. In comparison to normal controls, CCN2 mRNA was enhanced 1.9-fold in carcinoma foci (12.36 ± 6.08 vs 6.42 ± 2.35) or 9.4-fold in the surrounding stroma (60.27 ± 28.71 vs 6.42 ± 2.35), with concomitant expression of CCN2 and TGF-β1 mRNA in those areas. Epithelial-mesenchymal transition phenotype related with CCN2 was detected in 12/36 (33.3%) of HCC liver samples at the edges between carcinoma foci and vasculature. Incubation of HepG2 cells with CCN2 (100 ng/mL) resulted in more of the cells transitioning into S phase (23.85 ± 2.35 vs 10.94 ± 0.23), and induced a significant migratory (4.0-fold) and invasive (5.7-fold) effect. TGF-β1-induced cell invasion was abrogated by a neutralizing CCN2 antibody showing that CCN2 is a downstream mediator of TGF-β1-induced hepatoma cell invasion. These data support a role for CCN2 in the growth and metastasis of HCC and highlight CCN2 as a potential novel therapeutic target.

The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation.

PubMed Central

Zubiaga, A M; Belasco, J G; Greenberg, M E

1995-01-01

Labile mRNAs that encode cytokine and immediate-early gene products often contain AU-rich sequences within their 3' untranslated region (UTR). These AU-rich sequences appear to be key determinants of the short half-lives of these mRNAs, although the sequence features of these elements and the mechanism by which they target mRNAs for rapid decay have not been fully defined. We have examined the features of AU-rich elements (AREs) that are crucial for their function as determinants of mRNA instability in mammalian cells by testing the ability of various mutant c-fos AREs and synthetic AREs to direct rapid mRNA deadenylation and decay when inserted within the 3' UTR of the normally stable beta-globin mRNA. Evidence is presented that the pentamer AUUUA, which previously was suggested to be the minimal determinant of instability present in mammalian AREs, cannot direct rapid mRNA deadenylation and decay. Instead, the nonomer UUAUUUAUU is the elemental AU-rich sequence motif that destabilizes mRNA. Removal of one uridine residue from either end of the nonamer (UUAUUUAU or UAUUUAUU) results in a decrease of potency of the element, while removal of a uridine residue from both ends of the nonamer (UAUUUAU) eliminates detectable destabilizing activity. The inclusion of an additional uridine residue at both ends of the nonamer (UUUAUUUAUUU) does not further increase the efficacy of the element. Taken together, these findings suggest that the nonamer UUAUUUAUU is the minimal AU-rich motif that effectively destabilizes mRNA. Additional ARE potency is achieved by combining multiple copies of this nonamer in a single mRNA 3' UTR. Furthermore, analysis of poly(A) shortening rates for ARE-containing mRNAs reveals that the UUAUUUAUU sequence also accelerates mRNA deadenylation and suggests that the UUAUUUAUU motif targets mRNA for rapid deadenylation as an early step in the mRNA decay process. PMID:7891716

Interleukin-1β induces tumor necrosis factor-α secretion from rat hepatocytes.

PubMed

Yoshigai, Emi; Hara, Takafumi; Inaba, Hiroyuki; Hashimoto, Iwao; Tanaka, Yoshito; Kaibori, Masaki; Kimura, Tominori; Okumura, Tadayoshi; Kwon, A-Hon; Nishizawa, Mikio

2014-05-01

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine involved in various inflammatory diseases. The only production of TNF-α in the liver is thought to be from hepatic macrophages known as Kupffer cells, predominantly in response to bacterial lipopolysaccharide (LPS). Primary cultured rat hepatocytes were used to analyze TNF-α expression in response to the pro-inflammatory cytokine, interleukin-1β (IL-1β). Livers of rats subjected to LPS-induced endotoxemia were analyzed. Immunocytochemistry and enzyme-linked immunosorbent assays demonstrated that IL-1β-treated rat hepatocytes secreted TNF-α, and RNA analyses indicated that TNF-α mRNA was induced specifically by IL-1β. Northern blot analysis showed that not only mRNA, but also a natural antisense transcript (asRNA), was transcribed from the rat Tnf gene in IL-1β-treated hepatocytes. TNF-α was detected in the hepatocytes of LPS-treated rats. Both TNF-α mRNA and asRNA were expressed in the hepatocytes of LPS-treated rats, human hepatocellular carcinoma and human monocyte/macrophage cells. To disrupt the interaction between TNF-α asRNA and TNF-α mRNA, sense oligonucleotides corresponding to TNF-α mRNA were introduced into rat hepatocytes resulting in significantly increased levels of TNF-α mRNA. One of these sense oligonucleotides increased a half-life of TNF-α mRNA, suggesting that the TNF-α asRNA may reduce the stability of TNF-α mRNA. IL-1β-stimulated rat hepatocytes are a newly identified source of TNF-α in the liver. TNF-α mRNA and asRNA are expressed in rats and humans, and the TNF-α asRNA reduces the stability of the TNF-α mRNA. Hepatocytes and TNF-α asRNA may be therapeutic targets to regulate levels of TNF-α mRNA. © 2013 The Japan Society of Hepatology.

Epithelial–mesenchymal transition-associated secretory phenotype predicts survival in lung cancer patients

PubMed Central

Reka, Ajaya Kumar; Chen, Guoan; Keshamouni, Venkateshwar G.

2014-01-01

In cancer cells, the process of epithelial–mesenchymal transition (EMT) confers migratory and invasive capacity, resistance to apoptosis, drug resistance, evasion of host immune surveillance and tumor stem cell traits. Cells undergoing EMT may represent tumor cells with metastatic potential. Characterizing the EMT secretome may identify biomarkers to monitor EMT in tumor progression and provide a prognostic signature to predict patient survival. Utilizing a transforming growth factor-β-induced cell culture model of EMT, we quantitatively profiled differentially secreted proteins, by GeLC-tandem mass spectrometry. Integrating with the corresponding transcriptome, we derived an EMT-associated secretory phenotype (EASP) comprising of proteins that were differentially upregulated both at protein and mRNA levels. Four independent primary tumor-derived gene expression data sets of lung cancers were used for survival analysis by the random survival forests (RSF) method. Analysis of 97-gene EASP expression in human lung adenocarcinoma tumors revealed strong positive correlations with lymph node metastasis, advanced tumor stage and histological grade. RSF analysis built on a training set (n = 442), including age, sex and stage as variables, stratified three independent lung cancer data sets into low-, medium- and high-risk groups with significant differences in overall survival. We further refined EASP to a 20 gene signature (rEASP) based on variable importance scores from RSF analysis. Similar to EASP, rEASP predicted survival of both adenocarcinoma and squamous carcinoma patients. More importantly, it predicted survival in the early-stage cancers. These results demonstrate that integrative analysis of the critical biological process of EMT provides mechanism-based and clinically relevant biomarkers with significant prognostic value. PMID:24510113

Phenome-genome association studies of pancreatic cancer: new targets for therapy and diagnosis.

PubMed

Narayanan, Ramaswamy

2015-01-01

Pancreatic cancer, has a very high mortality rate and requires novel molecular targets for diagnosis and therapy. Genetic association studies over databases offer an attractive starting point for gene discovery. The National Center for Biotechnology Information (NCBI) Phenome Genome Integrator (PheGenI) tool was enriched for pancreatic cancer-associated traits. The genes associated with the trait were characterized using diverse bioinformatics tools for Genome-Wide Association (GWA), transcriptome and proteome profile and protein classes for motif and domain. Two hundred twenty-six genes were identified that had a genetic association with pancreatic cancer in the human genome. This included 25 uncharacterized open reading frames (ORFs). Bioinformatics analysis of these ORFs identified putative druggable proteins and biomarkers including enzymes, transporters and G-protein-coupled receptor signaling proteins. Secreted proteins including a neuroendocrine factor and a chemokine were identified. Five out of these ORFs encompassed non coding RNAs. The ORF protein expression was detected in numerous body fluids, such as ascites, bile, pancreatic juice, milk, plasma, serum and saliva. Transcriptome and proteome analyses showed a correlation of mRNA and protein expression for nine ORFs. Analysis of the Catalogue of Somatic Mutations in Cancer (COSMIC) database revealed a strong correlation across copy number variations and mRNA over-expression for four ORFs. Mining of the International Cancer Gene Consortium (ICGC) database identified somatic mutations in a significant number of pancreatic patients' tumors for most of these ORFs. The pancreatic cancer-associated ORFs were also found to be genetically associated with other neoplasms, including leukemia, malignant melanoma, neuroblastoma and prostate carcinomas, as well as other unrelated diseases and disorders, such as Alzheimer's disease, Crohn's disease, coronary diseases, attention deficit disorder and addiction. Based on Genome-Wide Association Studies (GWAS), copy number variations, somatic mutational status and correlation of gene expression in pancreatic tumors at the mRNA and protein level, expression specificity in normal tissues and detection in body fluids, six ORFs emerged as putative leads for pancreatic cancer. These six targets provide a basis for accelerated drug discovery and diagnostic marker development for pancreatic cancer. Copyright© 2015, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

CB-02MiRNA EXPRESSION PROFILES OF GLIOMA STEM CELLS AND THEIR ASSOCIATION WITH THE MESENCHYMAL TRANSFORMATION OF THESE CELLS

PubMed Central

Bier, Ariel; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Lee, Hae Kyung; Rand, Daniel; Yalon, Michal; Toren, Amos; Poisson, Laila; Brodie, Chaya

2014-01-01

Glioblastoma, are characterized by increased infiltration into the surrounding brain tissue, resistance to therapies, and poor prognosis. A major pathway that contributes to these characteristics is the mesenchymal phenotype of these tumors. A small subpopulation of cancer stem cells (GSCs) have been implicated in the enhanced infiltration, radio-resistance and tumor recurrence. GSCs share some similarities with neural stem cells (NSCs) but exhibit deregulated differentiation ability and enhanced oncogenic potential. Recent studies documented miRNAs as important regulators of GSC functions and of the malignant and stemness features of these cells. In this study we performed miRNA and mRNA integrated analysis of GSCs compared to human NSCs and mesenchymal stromal cells (MSCs) to identify significant miRNA-mRNA signatures associated with the mesenchymal signature of GSCs, using miRNA and mRNA microarray analysis. The comparison of GSCs and NSCs identified 79 miRNAs that were upregulated in GSCs and 21 miRNAs that were increased in MSCs. Twenty six miRNAs were downregulated in GSCs compared to NSCs and 21 miRNAs from this group were further downregulated in MSCs. The comparison of mRNA expression of GSCs and NSCs identified gene clusters associated with glioma cell invasiveness, axonal guidance signaling and TGF-b signaling. miR-504 is one of the miRNAs that was significantly downregulated in GSCs compared to NSCs. The expression of miR-504 was also decreased in mesenchymal GBM and highly increased in the G-CIMP subset of GBM. miR-504 promoted the neural differentiation of GSC, inhibited their self-renewal, migration and the mesenchymal signature of these cells, by downregulating CD44, BCAN, ZRB1 and ZEB2. In conclusion, these results reveal novel miRNAs and potential target networks that play a role in the oncogenic potential and stemness of GSCs and in their mesenchymal transformation and may lead to the identification of therapeutic targets for the eradication of GSCs and the treatment of GBM.

A Multi-layered Quantitative In Vivo Expression Atlas of the Podocyte Unravels Kidney Disease Candidate Genes.

PubMed

Rinschen, Markus M; Gödel, Markus; Grahammer, Florian; Zschiedrich, Stefan; Helmstädter, Martin; Kretz, Oliver; Zarei, Mostafa; Braun, Daniela A; Dittrich, Sebastian; Pahmeyer, Caroline; Schroder, Patricia; Teetzen, Carolin; Gee, HeonYung; Daouk, Ghaleb; Pohl, Martin; Kuhn, Elisa; Schermer, Bernhard; Küttner, Victoria; Boerries, Melanie; Busch, Hauke; Schiffer, Mario; Bergmann, Carsten; Krüger, Marcus; Hildebrandt, Friedhelm; Dengjel, Joern; Benzing, Thomas; Huber, Tobias B

2018-05-22

Damage to and loss of glomerular podocytes has been identified as the culprit lesion in progressive kidney diseases. Here, we combine mass spectrometry-based proteomics with mRNA sequencing, bioinformatics, and hypothesis-driven studies to provide a comprehensive and quantitative map of mammalian podocytes that identifies unanticipated signaling pathways. Comparison of the in vivo datasets with proteomics data from podocyte cell cultures showed a limited value of available cell culture models. Moreover, in vivo stable isotope labeling by amino acids uncovered surprisingly rapid synthesis of mitochondrial proteins under steady-state conditions that was perturbed under autophagy-deficient, disease-susceptible conditions. Integration of acquired omics dimensions suggested FARP1 as a candidate essential for podocyte function, which could be substantiated by genetic analysis in humans and knockdown experiments in zebrafish. This work exemplifies how the integration of multi-omics datasets can identify a framework of cell-type-specific features relevant for organ health and disease. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Kinetics of lipid-nanoparticle-mediated intracellular mRNA delivery and function

NASA Astrophysics Data System (ADS)

Zhdanov, Vladimir P.

2017-10-01

mRNA delivery into cells forms the basis for one of the new and promising ways to treat various diseases. Among suitable carriers, lipid nanoparticles (LNPs) with a size of about 100 nm are now often employed. Despite high current interest in this area, the understanding of the basic details of LNP-mediated mRNA delivery and function is limited. To clarify the kinetics of mRNA release from LNPs, the author uses three generic models implying (i) exponential, (ii) diffusion-controlled, and (iii) detachment-controlled kinetic regimes, respectively. Despite the distinct differences in these kinetics, the associated transient kinetics of mRNA translation to the corresponding protein and its degradation are shown to be not too sensitive to the details of the mRNA delivery by LNPs (or other nanocarriers). In addition, the author illustrates how this protein may temporarily influence the expression of one gene or a few equivalent genes. The analysis includes positive or negative regulation of the gene transcription via the attachment of the protein without or with positive or negative feedback in the gene expression. Stable, bistable, and oscillatory schemes have been scrutinized in this context.

Identification of CRASH, a gene deregulated in gynecological tumors.

PubMed

Evtimova, Vesna; Zeillinger, Robert; Kaul, Sepp; Weidle, Ulrich H

2004-01-01

We have identified CRASH, a human asparaginase-like protein which is composed of 308 amino acids and exhibits 32% homology to human aspartylglucosaminadase at the amino acid level. Database analysis revealed that the gene corresponding to CRASH is composed of 7 exons and 6 introns. Steady-state level of CRASH mRNA was found to be increased in 5 cell lines derived from metastatic lesions compared with 2 cell lines derived from primary mammary carcinoma and HMEC (human mammary epithelial cells). We found that the mRNA level of CRASH correlates with the metastatic propensity of several isogenic human colon cancer and pancreatic carcinoma cell lines. CRASH corresponds to a recently identified sperm autoantigen and furthermore we have demonstrated inducibility of CRASH mRNA by androgen and progesterone. Investigation of several types of human cancers and their corresponding normal tissues revealed high levels of CRASH mRNA in uterine, mammary and ovarian tumors compared with the corresponding normal tissues. CRASH mRNA expression was analysed in breast cancer samples with disclosed clinico-pathological features and corresponding normal tissues. The levels of CRASH mRNA were significantly up-regulated in tumors compared with normal breast tissues and correlate with lack of estrogen receptor expression of the tumors.

Upregulation of estrogen receptor subtypes and vitellogenin mRNA in cinnamon clownfish Amphiprion melanopus during the sex change process: profiles on effects of 17beta-estradiol.

PubMed

Kim, Na Na; Jin, Deuk-Hee; Lee, Jehee; Kil, Gyung-Suk; Choi, Cheol Young

2010-10-01

In the present study, we investigated the expression pattern of estrogen receptors (esr) and vitellogenin (vtg) mRNA in the gonads and liver during sex change in cinnamon clownfish by using quantitative polymerase chain reaction. We divided gonadal development during the sex change from male to female into 3 stages (mature male, male at 90days after removing female, and mature female) and investigated esr and vtg mRNA expressions during the sex change. With female, the esr and vtg mRNA expressions increased. In western blot analysis, Esr1 protein was detected only in the ovaries of female cinnamon clownfish. Also, to understand the effect of 17beta-estradiol (E(2)), we investigated the esr and vtg mRNA expression patterns in the gonads and liver, and the changes in plasma E(2) level after E(2) injection. E(2) treatment increased both mRNA expression levels of esr and vtg and plasma E(2) levels. The present study describes the molecular characterization of esr subtypes and the interactions between esr and vtg after E(2) treatment in cinnamon clownfish. 2010 Elsevier Inc. All rights reserved.

Cold-Induced Accumulation of hsp90 Transcripts in Brassica napus.

PubMed Central

Krishna, P.; Sacco, M.; Cherutti, J. F.; Hill, S.

1995-01-01

Characterization of the expression of hsp90 genes of Brassica napus by northern blot analysis and immunoblotting showed that the hsp90 mRNA and protein are present in all B. napus tissues examined, albeit at different levels. High levels of hsp90 mRNA and protein were found in young and rapidly dividing tissues such as shoot apices and flower buds, suggesting that hsp90 may have an important role in plant growth and development. A significant increase in hsp90 mRNA levels was detected in seedlings exposed to 5[deg]C. The transcript levels reached a maximum within 1 d of cold treatment and remained elevated for the entire duration of cold treatment. The levels of hsp90 mRNA rapidly decreased to the level found in control plants upon return to 20[deg]C. The cold-induced accumulation of hsp90 mRNA closely resembles the expression of two previously identified cold-regulated genes of B. napus. We have also confirmed cold regulation of hsp90 mRNA in spinach (Spinacea oleracea). Our results suggest a role for hsp90 in adaptation to cold temperature stress. PMID:12228411

Chemical and structural characterization of a model Post-Termination Complex (PoTC) for the ribosome recycling reaction: Evidence for the release of the mRNA by RRF and EF-G

PubMed Central

Iwakura, Nobuhiro; Yokoyama, Takeshi; Quaglia, Fabio; Mitsuoka, Kaoru; Mio, Kazuhiro; Shigematsu, Hideki; Shirouzu, Mikako; Kaji, Akira; Kaji, Hideko

2017-01-01

A model Post-Termination Complex (PoTC) used for the discovery of Ribosome Recycling Factor (RRF) was purified and characterized by cryo-electron microscopic analysis and biochemical methods. We established that the model PoTC has mostly one tRNA, at the P/E or P/P position, together with one mRNA. The structural studies were supported by the biochemical measurement of bound tRNA and mRNA. Using this substrate, we establish that the release of tRNA, release of mRNA and splitting of ribosomal subunits occur during the recycling reaction. Order of these events is tRNA release first followed by mRNA release and splitting almost simultaneously. Moreover, we demonstrate that IF3 is not involved in any of the recycling reactions but simply prevents the re-association of split ribosomal subunits. Our finding demonstrates that the important function of RRF includes the release of mRNA, which is often missed by the use of a short ORF with the Shine-Dalgarno sequence near the termination site. PMID:28542628

MiR-506 suppresses liver cancer angiogenesis through targeting sphingosine kinase 1 (SPHK1) mRNA.

PubMed

Lu, Zhanping; Zhang, Weiying; Gao, Shan; Jiang, Qiulei; Xiao, Zelin; Ye, Lihong; Zhang, Xiaodong

MicroRNAs acting as oncogenes or tumor suppressor genes play crucial roles in human cancers. Sphingosine kinase 1 (SPHK1) and its metabolite sphingosine 1-phosphate (S1P) contribute to tumor angiogenesis. We have reported that the down-regulation of miR-506 targeting YAP mRNA results in the hepatocarcinogenesis. In the present study, we report a novel function of miR-506, which suppresses tumor angiogenesis through targeting SPHK1 mRNA in liver cancer. Bioinformatics analysis showed that miR-506 might target 3'-untranslated region (3'UTR) of SPHK1 mRNA. Then, we validated that by luciferase reporter gene assays. MiR-506 was able to reduce the expression of SPHK1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis in hepatoma HepG2 cells. Functionally, human umbilical vein endothelial cell (HUVEC) tube formation assays demonstrated that the forced miR-506 expression remarkably inhibited the production of S1P in the supernatant of hepatoma cells. The supernatant resulted in the inhibition of tumor angiogenesis. Interestingly, the supernatant with overexpression of SPHK1 could rescue the inhibition of angiogenesis of liver cancer mediated by miR-506. Anti-miR-506 increased the production of S1P in the supernatant of hepatoma cells, but the supernatant with silencing of SPHK1 abolished anti-miR-506-induced acceleration of tumor angiogenesis. Clinically, we observed that the levels of miR-506 were negatively related to those of SPHK1 mRNA in liver cancer tissues. Thus, we conclude that miR-506 depresses the angiogenesis of liver cancer through targeting 3'UTR of SPHK1 mRNA. Our finding provides new insights into the mechanism of tumor angiogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

[Effect of TUBB3, TS and ERCC1 mRNA expression on chemoresponse and clinical outcome of advanced gastric cancer by multiplex branched-DNA liquid chip technology].

PubMed

Huang, Jin; Hu, Huabin; Xie, Yangchun; Tang, Youhong; Liu, Wei; Zhong, Meizuo

2013-06-01

To analyze the impact of β-tubulin-III (TUBB3), thymidylate synthase (TS) and excision repair cross complementation group 1 (ERCC1) mRNA expression on chemoresponse and clinical outcome of patients with advanced gastric cancer treated with TXT/CDDP/FU (DCF) regimen chemotherapy. The study population consisted of 48 patients with advanced gastric cancer. All patients were treated with DCF regimen palliative chemotherapy. The mRNA expressions of TUBB3, TS and ERCC1 of primary tumors were examined by multiplex branched-DNA liquid chip technology. The patients with low TUBB3 mRNA expression had higher response rate to chemotherapy than patients with high TUBB3 expression (P=0.011). There were no significant differences between response rate and TS or ERCC1 expression pattern. Median overall survival (OS) and median time to progression (TTP) were significantly longer in patients with low TUBB3 mRNA expression (P=0.002, P<0.001). TS or ERCC1 expression was not correlated with TTP and OS. In the combined analysis including TUBB3, TS and ERCC1, the patients with 0 or 1 high expression gene had better response rate, TTP and OS than the remaining patients (all P<0.001). Multivariate analysis revealed that ECOG (Eastern Cooperative Oncology Group)≥2 (HR=2.42, P=0.009) and TUBB3 (HR=2.34, P=0.036) mRNA expression significantly impacted on OS. High TUBB3 mRNA expression is correlated with resistance to DCF regimen chemotherapy. TUBB3 might be a predictive and prognostic factor in patients with advanced gastric cancer treated with TXT-based chemotherapy. The combined evaluation of TUBB3, TS and ERCC1 expression can promote the individual treatment in advanced gastric cancer.

Endothelial glucocorticoid receptor promoter methylation according to dexamethasone sensitivity

PubMed Central

Mata-Greenwood, Eugenia; Jackson, P Naomi; Pearce, William J; Zhang, Lubo

2016-01-01

We have previously shown that in vitro sensitivity to dexamethasone (DEX) stimulation in human endothelial cells is positively regulated by the glucocorticoid receptor (NR3C1, GR). The present study determined the role of differential GR transcriptional regulation in glucocorticoid sensitivity. We studied 25 human umbilical vein endothelial cells (HUVECs) that had been previously characterized as DEX-sensitive (n = 15), or resistant (n = 10). Real-time PCR analysis of GR 5′UTR mRNA isoforms showed that all HUVECs expressed isoforms 1B, 1C, 1D, 1F, and 1H, and isoforms 1B and 1C were predominantly expressed. DEX-resistant cells expressed higher basal levels of the 5′UTR mRNA isoforms 1C and 1D, but lower levels of the 5′UTR mRNA isoform 1F than DEX-sensitive cells. DEX treatment significantly decreased GRα and GR-1C mRNA isoform expression in DEX-resistant cells only. Reporter luciferase assays indicated that differential GR mRNA isoform expression was not due to differential promoter usage between DEX-sensitive and DEX-resistant cells. Analysis of promoter methylation, however, showed that DEX-sensitive cells have higher methylation levels of promoter 1D and lower methylation levels of promoter 1F than DEX-resistant cells. Treatment with 5-aza-2-deoxycytidine abolished the differential 5′UTR mRNA isoform expression between DEX-sensitive and DEX-resistant cells. Finally, both GRα overexpression and 5-aza-2-deoxycytidine treatment eliminated the differences between sensitivity groups to DEX-mediated downregulation of endothelial nitric oxide synthase (NOS3), and upregulation of plasminogen activator inhibitor 1 (SERPINE1). In sum, human endothelial GR 5′UTR mRNA expression is regulated by promoter methylation with DEX-sensitive and DEX-resistant cells having different GR promoter methylation patterns. PMID:26242202

Impact of target mRNA structure on siRNA silencing efficiency: A large-scale study.

PubMed

Gredell, Joseph A; Berger, Angela K; Walton, S Patrick

2008-07-01

The selection of active siRNAs is generally based on identifying siRNAs with certain sequence and structural properties. However, the efficiency of RNA interference has also been shown to depend on the structure of the target mRNA, primarily through studies using exogenous transcripts with well-defined secondary structures in the vicinity of the target sequence. While these studies provide a means for examining the impact of target sequence and structure independently, the predicted secondary structures for these transcripts are often not reflective of structures that form in full-length, native mRNAs where interactions can occur between relatively remote segments of the mRNAs. Here, using a combination of experimental results and analysis of a large dataset, we demonstrate that the accessibility of certain local target structures on the mRNA is an important determinant in the gene silencing ability of siRNAs. siRNAs targeting the enhanced green fluorescent protein were chosen using a minimal siRNA selection algorithm followed by classification based on the predicted minimum free energy structures of the target transcripts. Transfection into HeLa and HepG2 cells revealed that siRNAs targeting regions of the mRNA predicted to have unpaired 5'- and 3'-ends resulted in greater gene silencing than regions predicted to have other types of secondary structure. These results were confirmed by analysis of gene silencing data from previously published siRNAs, which showed that mRNA target regions unpaired at either the 5'-end or 3'-end were silenced, on average, approximately 10% more strongly than target regions unpaired in the center or primarily paired throughout. We found this effect to be independent of the structure of the siRNA guide strand. Taken together, these results suggest minimal requirements for nucleation of hybridization between the siRNA guide strand and mRNA and that both mRNA and guide strand structure should be considered when choosing candidate siRNAs. (c) 2008 Wiley Periodicals, Inc.

Differential expression of type X collagen in a mechanically active 3-D chondrocyte culture system: a quantitative study

PubMed Central

Yang, Xu; Vezeridis, Peter S; Nicholas, Brian; Crisco, Joseph J; Moore, Douglas C; Chen, Qian

2006-01-01

Objective Mechanical loading of cartilage influences chondrocyte metabolism and gene expression. The gene encoding type X collagen is expressed specifically by hypertrophic chondrocytes and up regulated during osteoarthritis. In this study we tested the hypothesis that the mechanical microenvironment resulting from higher levels of local strain in a three dimensional cell culture construct would lead to an increase in the expression of type X collagen mRNA by chondrocytes in those areas. Methods Hypertrophic chondrocytes were isolated from embryonic chick sterna and seeded onto rectangular Gelfoam sponges. Seeded sponges were subjected to various levels of cyclic uniaxial tensile strains at 1 Hz with the computer-controlled Bio-Stretch system. Strain distribution across the sponge was quantified by digital image analysis. After mechanical loading, sponges were cut and the end and center regions were separated according to construct strain distribution. Total RNA was extracted from the cells harvested from these regions, and real-time quantitative RT-PCR was performed to quantify mRNA levels for type X collagen and a housing-keeping gene 18S RNA. Results Chondrocytes distributed in high (9%) local strain areas produced more than two times type X collagen mRNA compared to the those under no load conditions, while chondrocytes located in low (2.5%) local strain areas had no appreciable difference in type X collagen mRNA production in comparison to non-loaded samples. Increasing local strains above 2.5%, either in the center or end regions of the sponge, resulted in increased expression of Col X mRNA by chondrocytes in that region. Conclusion These findings suggest that the threshold of chondrocyte sensitivity to inducing type X collagen mRNA production is more than 2.5% local strain, and that increased local strains above the threshold results in an increase of Col X mRNA expression. Such quantitative analysis has important implications for our understanding of mechanosensitivity of cartilage and mechanical regulation of chondrocyte gene expression. PMID:17150098

Effect of dietary α-lipoic acid on the mRNA expression of genes involved in drug metabolism and antioxidation system in rat liver.

PubMed

Ide, Takashi

2014-08-14

In the present study, the mRNA levels of hepatic proteins involved in the drug metabolism of rats fed α-lipoic acid were evaluated by DNA microarray and real-time PCR analyses. Experimental diets containing 0, 0·1, 0·25 and 0·5 % (w/w) α-lipoic acid were fed to four groups of rats consisting of seven animals each for 21 d. DNA microarray analysis revealed that the diet containing 0·5 % α-lipoic acid significantly (P< 0·05) increased the mRNA levels of various phase I drug-metabolising enzymes up to 15-fold and phase II enzymes up to 52-fold in an isoenzyme-specific manner. α-Lipoic acid also up-regulated the mRNA levels of some members of the ATP-binding cassette transporter superfamily, presumed to be involved in the exportation of xenobiotics, up to 6·6-fold. In addition, we observed that α-lipoic acid increased the mRNA levels of many proteins involved in antioxidation, such as members of the thiol redox system (up to 5·5-fold), metallothioneins (up to 12-fold) and haeme oxygenase 1 (1·5-fold). These results were confirmed using real-time PCR analysis, and α-lipoic acid dose dependently increased the mRNA levels of various proteins involved in drug metabolism and antioxidation. Consistent with these observations, α-lipoic acid dose dependently increased the hepatic concentration of glutathione and the activities of glutathione reductase and glutathione transferase measured using 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates, but decreased the hepatic and serum concentrations of malondialdehyde. In conclusion, the present study unequivocally demonstrated that α-lipoic acid increases the mRNA expression of proteins involved in drug metabolism and antioxidation in the liver.

Impact of target mRNA structure on siRNA silencing efficiency: a large-scale study

PubMed Central

Gredell, Joseph A.; Berger, Angela K.; Walton, S. Patrick

2009-01-01

The selection of active siRNAs is generally based on identifying siRNAs with certain sequence and structural properties. However, the efficiency of RNA interference has also been shown to depend on the structure of the target mRNA, primarily through studies using exogenous transcripts with well-defined secondary structures in the vicinity of the target sequence. While these studies provide a means for examining the impact of target sequence and structure independently, the predicted secondary structures for these transcripts are often not reflective of structures that form in full-length, native mRNAs where interactions can occur between relatively remote segments of the mRNAs. Here, using a combination of experimental results and analysis of a large dataset, we demonstrate that the accessibility of certain local target structures on the mRNA is an important determinant in the gene silencing ability of siRNAs. siRNAs targeting the enhanced green fluorescent protein were chosen using a minimal siRNA selection algorithm followed by classification based on the predicted minimum free energy structures of the target transcripts. Transfection into HeLa and HepG2 cells revealed that siRNAs targeting regions of the mRNA predicted to have unpaired 5’- and 3’-ends resulted in greater gene silencing than regions predicted to have other types of secondary structure. These results were confirmed by analysis of gene silencing data from previously published siRNAs, which showed that mRNA target regions unpaired at either the 5’-end or 3’-end were silenced, on average, ~10% more strongly than target regions unpaired in the center or primarily paired throughout. We found this effect to be independent of the structure of the siRNA guide strand. Taken together, these results suggest minimal requirements for nucleation of hybridization between the siRNA guide strand and mRNA and that both mRNA and guide strand structure should be considered when choosing candidate siRNAs. PMID:18306428

Cost-Effectiveness of High-Risk Human Papillomavirus Testing With Messenger RNA Versus DNA Under United States Guidelines for Cervical Cancer Screening.

PubMed

Ting, Jie; Smith, Jennifer S; Myers, Evan R

2015-10-01

To compare the cost-effectiveness of high-risk human papillomavirus (hrHPV) testing using a hrHPV DNA and a hrHPV messenger RNA (mRNA) assay under current US cervical cancer screening guidelines. We constructed a Markov model for stochastic cost-effectiveness analysis using published data. We compared screening efficiency using DNA and mRNA testing for the following: (1) cotesting with cytology in women 30 to 65 years, and (2) triage of women with mild cervical cytological abnormalities (atypical squamous cells of undetermined significance [ASC-US]) in the United States. Screening end point is histologically confirmed high-grade lesions (cervical intraepithelial neoplasia grade 2, 3, or invasive cancer). Sensitivity and specificity estimates of DNA and mRNA testing to detect cervical intraepithelial neoplasia grade 2, 3, or invasive cancer were obtained from 2 published trials: the US Clinical Evaluation of APTIMA mRNA (CLEAR) study for ASC-US triage and the French APTIMA Screening Evaluation (FASE) study for cotesting. Costs of DNA and mRNA testing were assumed identical. Costs of screening, diagnosis, and treatment of cervical neoplasia and cancer were from previously published estimates, adjusted to 2012 US dollars. Inputs were modeled as distributions for Monte Carlo probabilistic sensitivity analysis. Model outcomes were costs per life-year saved for each strategy, discounted at 3% annually. For both cotesting and ASC-US triage, mRNA testing cost less than DNA testing, whereas life expectancies were widely overlapping. There was a 100% probability that DNA testing was not cost-effective at $100,000/life-year saved threshold for ASC-US triage and a 55% probability that DNA testing was not cost-effective at the same threshold for cotesting. Based on the available evidence, mRNA testing for cotesting or ASC-US triage is likely to be more efficient than DNA testing under current US cervical cancer screening guidelines.

Identifier mapping performance for integrating transcriptomics and proteomics experimental results

PubMed Central

2011-01-01

Background Studies integrating transcriptomic data with proteomic data can illuminate the proteome more clearly than either separately. Integromic studies can deepen understanding of the dynamic complex regulatory relationship between the transcriptome and the proteome. Integrating these data dictates a reliable mapping between the identifier nomenclature resultant from the two high-throughput platforms. However, this kind of analysis is well known to be hampered by lack of standardization of identifier nomenclature among proteins, genes, and microarray probe sets. Therefore data integration may also play a role in critiquing the fallible gene identifications that both platforms emit. Results We compared three freely available internet-based identifier mapping resources for mapping UniProt accessions (ACCs) to Affymetrix probesets identifications (IDs): DAVID, EnVision, and NetAffx. Liquid chromatography-tandem mass spectrometry analyses of 91 endometrial cancer and 7 noncancer samples generated 11,879 distinct ACCs. For each ACC, we compared the retrieval sets of probeset IDs from each mapping resource. We confirmed a high level of discrepancy among the mapping resources. On the same samples, mRNA expression was available. Therefore, to evaluate the quality of each ACC-to-probeset match, we calculated proteome-transcriptome correlations, and compared the resources presuming that better mapping of identifiers should generate a higher proportion of mapped pairs with strong inter-platform correlations. A mixture model for the correlations fitted well and supported regression analysis, providing a window into the performance of the mapping resources. The resources have added and dropped matches over two years, but their overall performance has not changed. Conclusions The methods presented here serve to achieve concrete context-specific insight, to support well-informed decisions in choosing an ID mapping strategy for "omic" data merging. PMID:21619611

Study of formation of green eggshell color in ducks through global gene expression.

PubMed

Xu, Fa Qiong; Li, Ang; Lan, Jing Jing; Wang, Yue Ming; Yan, Mei Jiao; Lian, Sen Yang; Wu, Xu

2018-01-01

The green eggshell color produced by ducks is a threshold trait that can be influenced by various factors, such as hereditary, environment and nutrition. The aim of this study was to investigate the genetic regulation of the formation of eggs with green shells in Youxian ducks. We performed integrative analysis of mRNAs and miRNAs expression profiling in the shell gland samples from ducks by RNA-Seq. We found 124 differentially expressed genes that were associated with various pathways, such as the ATP-binding cassette (ABC) transporter and solute carrier supper family pathways. A total of 31 differentially expressed miRNAs were found between ducks laying green eggs and white eggs. KEGG pathway analysis of the predicted miRNA target genes also indicated the functional characteristics of these miRNAs; they were involved in the ABC transporter pathway and the solute carrier (SLC) supper family. Analysis with qRT-PCR was applied to validate the results of global gene expression, which showed a correlation between results obtained by RNA-seq and RT-qPCR. Moreover, a miRNA-mRNA interaction network was established using correlation analysis of differentially expressed mRNA and miRNA. Compared to ducks that lay white eggs, ducks that lay green eggs include six up-regulated miRNAs that had regulatory effects on 35 down-regulated genes, and seven down-regulated miRNAs which influenced 46 up-regulated genes. For example, the ABC transporter pathway could be regulated by expressing gga-miR-144-3p (up-regulated) with ABCG2 (up-regulated) and other miRNAs and genes. This study provides valuable information about mRNA and miRNA regulation in duck shell gland tissues, and provides foundational information for further study on the eggshell color formation and marker-assisted selection for Youxian duck breeding.

Systematic transcriptome-wide analysis of mRNA-miRNA interactions reveals the involvement of miR-142-5p and its target (FOXO3) in skeletal muscle growth in chickens.

PubMed

Li, Zhenhui; Abdalla, Bahareldin Ali; Zheng, Ming; He, Xiaomei; Cai, Bolin; Han, Peigong; Ouyang, Hongjia; Chen, Biao; Nie, Qinghua; Zhang, Xiquan

2018-02-01

The goal of this study was to perform a systematic transcriptome-wide analysis of mRNA-miRNA interactions and to identify candidates involved in the interplay between miRNAs and mRNAs that regulate chicken muscle growth. We used our previously published mRNA (GSE72424) and miRNA (GSE62971) deep sequencing data from two-tailed samples [i.e., the highest (h) and lowest (l) body weights] of Recessive White Rock (WRR) and Xinghua (XH) chickens to conduct integrative analyses of the miRNA-mRNA interactions involved in chicken skeletal muscle growth. A total of 162, 15, 173, and 27 miRNA-mRNA pairs with negatively correlated expression patterns were identified in miRNA-mRNA networks constructed on the basis of the WRR h vs. XH h , WRR h vs. WRR l , WRR l vs. XH l , and XH h vs. XH l comparisons, respectively. Ingenuity Pathway Analysis revealed that gene networks identified for the WRR h vs. XH h contrast were associated with developmental disorders. Importantly, the WRR h vs. XH h contrast miRNA-mRNA network was enriched in IGF-1 signaling pathway genes, including FOXO3. A dual-luciferase reporter assay showed that FOXO3 was a target of miR-142-5p. Furthermore, miR-142-5p overexpression significantly decreased FOXO3 mRNA levels and promoted the expression of growth-related genes. These data demonstrated that miR-142-5p targets FOXO3 and promotes growth-related gene expression and regulates skeletal muscle growth in chicken. Comprehensive analysis facilitated the identification of miRNAs and target genes that might contribute to the regulation of skeletal muscle development. Our results provide new clues for understanding the molecular basis of chicken growth.

Analysis of miRNA and mRNA Expression Profiles Highlights Alterations in Ionizing Radiation Response of Human Lymphocytes under Modeled Microgravity

PubMed Central

Casara, Silvia; Sales, Gabriele; Lanfranchi, Gerolamo; Celotti, Lucia; Mognato, Maddalena

2012-01-01

Background Ionizing radiation (IR) can be extremely harmful for human cells since an improper DNA-damage response (DDR) to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation, being microRNAs (miRNAs) small noncoding RNA that act as post-transcriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity, a condition of weightlessness experienced by astronauts during space missions, which could have a synergistic action on cells, increasing the risk of radiation exposure. Methodology/Principal Findings We analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL) incubated for 4 and 24 h in normal gravity (1 g) and in modeled microgravity (MMG) during the repair time after irradiation with 0.2 and 2Gy of γ-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover, let-7i*, miR-7, miR-7-1*, miR-27a, miR-144, miR-200a, miR-598, miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles, carried out on PBL of the same donors, identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of “Response to DNA damage” is enriched when PBL are incubated in 1 g but not in MMG. Moreover, some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses. Conclusions/Significance On the whole, by integrating the transcriptome and microRNome, we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL. PMID:22347458

Editing of the grapevine mitochondrial cytochrome b mRNA and molecular modeling of the protein.

PubMed

Islas-Osuna, María A; Silva-Moreno, Begonia; Caceres-Carrizosa, Nidia; García-Robles, Jesús M; Sotelo-Mundo, Rogerio R; Yepiz-Plascencia, Gloria M

2006-05-01

Cytochrome b (COB), the central catalytic subunit of ubiquinol cytochrome c reductase, is a component of the transmembrane electron transfer chain that generates proton motive force. Some plant COB mRNAs are processed by RNA editing, which changes the gene coding sequence. This report presents the sequences of the grapevine (Vitis vinifera L.) mitochondrial gene for apocytochrome b (cob), the edited mRNA and the deduced protein. Grapevine COB is 393 amino acids long and is 98% identical to homologs in rapeseed, Arabidopsis thaliana and Oenothera sp. Twenty-one C-U editing sites were identified in the grapevine cob mRNA, resulting in 20 amino acid changes. These changes increase the overall hydrophobicity of the protein and result in a more conserved protein. Molecular modeling of grapevine COB shows that residues changed by RNA editing fit the secondary structure characteristic of an integral membrane protein. This is the first complete mitochondrial gene reported for grapevine. Novel RNA editing sites were identified in grapevine cob, which have not been previously reported for other plants.

RNA Surveillance: Molecular Approaches in Transcript Quality Control and their Implications in Clinical Diseases

PubMed Central

Moraes, Karen CM

2010-01-01

Production of mature mRNAs that encode functional proteins involves highly complex pathways of synthesis, processing and surveillance. At numerous steps during the maturation process, the mRNA transcript undergoes scrutiny by cellular quality control machinery. This extensive RNA surveillance ensures that only correctly processed mature mRNAs are translated and precludes production of aberrant transcripts that could encode mutant or possibly deleterious proteins. Recent advances in elucidating the molecular mechanisms of mRNA processing have demonstrated the existence of an integrated network of events, and have revealed that a variety of human diseases are caused by disturbances in the well-coordinated molecular equilibrium of these events. From a medical perspective, both loss and gain of function are relevant, and a considerable number of different diseases exemplify the importance of the mechanistic function of RNA surveillance in a cell. Here, mechanistic hallmarks of mRNA processing steps are reviewed, highlighting the medical relevance of their deregulation and how the understanding of such mechanisms can contribute to the development of therapeutic strategies. PMID:19829759

Multi-Scale Modeling to Improve Single-Molecule, Single-Cell Experiments

NASA Astrophysics Data System (ADS)

Munsky, Brian; Shepherd, Douglas

2014-03-01

Single-cell, single-molecule experiments are producing an unprecedented amount of data to capture the dynamics of biological systems. When integrated with computational models, observations of spatial, temporal and stochastic fluctuations can yield powerful quantitative insight. We concentrate on experiments that localize and count individual molecules of mRNA. These high precision experiments have large imaging and computational processing costs, and we explore how improved computational analyses can dramatically reduce overall data requirements. In particular, we show how analyses of spatial, temporal and stochastic fluctuations can significantly enhance parameter estimation results for small, noisy data sets. We also show how full probability distribution analyses can constrain parameters with far less data than bulk analyses or statistical moment closures. Finally, we discuss how a systematic modeling progression from simple to more complex analyses can reduce total computational costs by orders of magnitude. We illustrate our approach using single-molecule, spatial mRNA measurements of Interleukin 1-alpha mRNA induction in human THP1 cells following stimulation. Our approach could improve the effectiveness of single-molecule gene regulation analyses for many other process.

p62/SQSTM1 enhances breast cancer stem-like properties by stabilizing MYC mRNA

PubMed Central

Xu, L-Z; Li, S-S; Zhou, W; Kang, Z-J; Zhang, Q-X; Kamran, M; Xu, J; Liang, D-P; Wang, C-L; Hou, Z-J; Wan, X-B; Wang, H-J; Lam, E W-F; Zhao, Z-W; Liu, Q

2017-01-01

Aberrant p62 overexpression has been implicated in breast cancer development. Here, we found that p62 expression was elevated in breast cancer stem cells (BCSCs), including CD44+CD24− fractions, mammospheres, ALDH1+ populations and side population cells. Indeed, short-hairpin RNA (shRNA)-mediated knockdown of p62 impaired breast cancer cells from self-renewing under anchorage-independent conditions, whereas ectopic overexpression of p62 enhanced the self-renewal ability of breast cancer cells in vitro. Genetic depletion of p62 robustly inhibited tumor-initiating frequencies, as well as growth rates of BCSC-derived tumor xenografts in immunodeficient mice. Consistently, immunohistochemical analysis of clinical breast tumor tissues showed that high p62 expression levels were linked to poorer clinical outcome. Further gene expression profiling analysis revealed that p62 was positively correlated with MYC expression level, which mediated the function of p62 in promoting breast cancer stem-like properties. MYC mRNA level was reduced upon p62 deletion by siRNA and increased with p62 overexpression in breast cancer cells, suggesting that p62 positively regulated MYC mRNA. Interestingly, p62 did not transactivate MYC promoter. Instead, p62 delayed the degradation of MYC mRNA by repressing the expression of let-7a and let-7b, thus promoting MYC mRNA stabilization at the post-transcriptional level. Consistently, let-7a and let-7b mimics attenuated p62-mediated MYC mRNA stabilization. Together, these findings unveiled a previously unappreciated role of p62 in the regulation of BCSCs, assigning p62 as a promising therapeutic target for breast cancer treatments. PMID:27345399

Molecular Diagnosis for Nodal Metastasis in Endoscopically Managed Cervical Cancer: The Accuracy of the APTIMA Test to Detect High-risk Human Papillomavirus Messenger RNA in Sentinel Lymph Nodes.

PubMed

Köhler, Christhardt; Le, Xin; Dogan, Nasuh Utku; Pfiffer, Tatiana; Schneider, Achim; Marnitz, Simone; Bertolini, Julia; Favero, Giovanni

2016-01-01

To evaluate the feasibility and accuracy of a commercially available test to detect E6/E7 mRNA of 14 subtypes of high-risk HPVs (APTIMA; Hologic, Bedford, MA) in the sentinel lymph nodes of CC patients laparoscopically operated. Prospective pilot study. The study was conducted in the Department of Advanced Operative and Oncologic Gynecology, Asklepios Hospital, Hamburg, Germany. 54 women with HPV-positive CC submitted to laparoscopic sentinel node biopsy alone or sentinel node biopsy followed by systematic pelvic and/or para-aortic endoscopic lymphadenectomy. All removed sentinel lymph nodes (SLNs) underwent sample collection by cytobrush for the APTIMA assay before frozen section. Results obtained with the HPV mRNA test were compared with the definitive histopathological analysis of the SLNs and additional lymph nodes removed. A total of 125 SLNs (119 pelvic and 6 paraaortic) were excised with a mean number of 2.3 SLNs per patient. Final histopathologic analysis confirmed nodal metastases in 10 SLNs from 10 different patients (18%). All the histologically confirmed metastatic lymph nodes were also HPV E6/E7 mRNA positive, resulting in a sensitivity of 100%. Four histologically free sentinel nodes were positive for HPV E6/E7 mRNA, resulting in a specificity of 96.4%. The HPV E6/E7 mRNA assay in the SLNs of patients with CC is feasible and highly accurate. The detection of HPV mRNA in 4 women with negative SLNs might denote a shift from microscopic identification of metastasis to the molecular level. The prognostic value of this findings awaits further verification. Copyright © 2016. Published by Elsevier Inc.

Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood.

PubMed

Zheng, Zhi; Luo, Yuling; McMaster, Gary K

2006-07-01

Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format. We performed single- and multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection. The single- and multiplex assays could quantitatively measure as few as 6000 and 24,000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 microL of whole blood. Both formats had CVs < 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar to those obtained by quantitative reverse transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAXgene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood. Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood.

Combined microRNA and mRNA expression analysis in pediatric multiple sclerosis: an integrated approach to uncover novel pathogenic mechanisms of the disease.

PubMed

Liguori, Maria; Nuzziello, Nicoletta; Licciulli, Flavio; Consiglio, Arianna; Simone, Marta; Viterbo, Rosa Gemma; Creanza, Teresa Maria; Ancona, Nicola; Tortorella, Carla; Margari, Lucia; Grillo, Giorgio; Giordano, Paola; Liuni, Sabino; Trojano, Maria

2018-01-01

Multiple sclerosis (MS) is a complex disease of the CNS that usually affects young adults, although 3-5% of cases are diagnosed in childhood and adolescence (hence called pediatric MS, PedMS). Genetic predisposition, among other factors, seems to contribute to the risk of the onset, in pediatric as in adult ages, but few studies have investigated the genetic 'environmentally naïve' load of PedMS. The main goal of this study was to identify circulating markers (miRNAs), target genes (mRNAs) and functional pathways associated with PedMS; we also verified the impact of miRNAs on clinical features, i.e. disability and cognitive performances. The investigation was performed in 19 PedMS and 20 pediatric controls (PCs) using a High-Throughput Next-generation Sequencing (HT-NGS) approach followed by an integrated bioinformatics/biostatistics analysis. Twelve miRNAs were significantly upregulated (let-7a-5p, let-7b-5p, miR-25-3p, miR-125a-5p, miR-942-5p, miR-221-3p, miR-652-3p, miR-182-5p, miR-185-5p, miR-181a-5p, miR-320a, miR-99b-5p) and 1 miRNA was downregulated (miR-148b-3p) in PedMS compared with PCs. The interactions between the significant miRNAs and their targets uncovered predicted genes (i.e. TNFSF13B, TLR2, BACH2, KLF4) related to immunological functions, as well as genes involved in autophagy-related processes (i.e. ATG16L1, SORT1, LAMP2) and ATPase activity (i.e. ABCA1, GPX3). No significant molecular profiles were associated with any PedMS demographic/clinical features. Both miRNAs and mRNA expressions predicted the phenotypes (PedMS-PC) with an accuracy of 92% and 91%, respectively. In our view, this original strategy of contemporary miRNA/mRNA analysis may help to shed light in the genetic background of the disease, suggesting further molecular investigations in novel pathogenic mechanisms. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Evidence for Alteration of Gene Regulatory Networks through MicroRNAs of the HIV-infected brain: novel analysis of retrospective cases.

PubMed

Tatro, Erick T; Scott, Erick R; Nguyen, Timothy B; Salaria, Shahid; Banerjee, Sugato; Moore, David J; Masliah, Eliezer; Achim, Cristian L; Everall, Ian P

2010-04-26

HIV infection disturbs the central nervous system (CNS) through inflammation and glial activation. Evidence suggests roles for microRNA (miRNA) in host defense and neuronal homeostasis, though little is known about miRNAs' role in HIV CNS infection. MiRNAs are non-coding RNAs that regulate gene translation through post-transcriptional mechanisms. Messenger-RNA profiling alone is insufficient to elucidate the dynamic dance of molecular expression of the genome. We sought to clarify RNA alterations in the frontal cortex (FC) of HIV-infected individuals and those concurrently infected and diagnosed with major depressive disorder (MDD). This report is the first published study of large-scale miRNA profiling from human HIV-infected FC. The goals of this study were to: 1. Identify changes in miRNA expression that occurred in the frontal cortex (FC) of HIV individuals, 2. Determine whether miRNA expression profiles of the FC could differentiate HIV from HIV/MDD, and 3. Adapt a method to meaningfully integrate gene expression data and miRNA expression data in clinical samples. We isolated RNA from the FC (n = 3) of three separate groups (uninfected controls, HIV, and HIV/MDD) and then pooled the RNA within each group for use in large-scale miRNA profiling. RNA from HIV and HIV/MDD patients (n = 4 per group) were also used for non-pooled mRNA analysis on Affymetrix U133 Plus 2.0 arrays. We then utilized a method for integrating the two datasets in a Target Bias Analysis. We found miRNAs of three types: A) Those with many dysregulated mRNA targets of less stringent statistical significance, B) Fewer dysregulated target-genes of highly stringent statistical significance, and C) unclear bias. In HIV/MDD, more miRNAs were downregulated than in HIV alone. Specific miRNA families at targeted chromosomal loci were dysregulated. The dysregulated miRNAs clustered on Chromosomes 14, 17, 19, and X. A small subset of dysregulated genes had many 3' untranslated region (3'UTR) target-sites for dysregulated miRNAs. We provide evidence that certain miRNAs serve as key elements in gene regulatory networks in HIV-infected FC and may be implicated in neurobehavioral disorder. Finally, our data indicates that some genes may serve as hubs of miRNA activity.

A prior-based integrative framework for functional transcriptional regulatory network inference

PubMed Central

Siahpirani, Alireza F.

2017-01-01

Abstract Transcriptional regulatory networks specify regulatory proteins controlling the context-specific expression levels of genes. Inference of genome-wide regulatory networks is central to understanding gene regulation, but remains an open challenge. Expression-based network inference is among the most popular methods to infer regulatory networks, however, networks inferred from such methods have low overlap with experimentally derived (e.g. ChIP-chip and transcription factor (TF) knockouts) networks. Currently we have a limited understanding of this discrepancy. To address this gap, we first develop a regulatory network inference algorithm, based on probabilistic graphical models, to integrate expression with auxiliary datasets supporting a regulatory edge. Second, we comprehensively analyze our and other state-of-the-art methods on different expression perturbation datasets. Networks inferred by integrating sequence-specific motifs with expression have substantially greater agreement with experimentally derived networks, while remaining more predictive of expression than motif-based networks. Our analysis suggests natural genetic variation as the most informative perturbation for network inference, and, identifies core TFs whose targets are predictable from expression. Multiple reasons make the identification of targets of other TFs difficult, including network architecture and insufficient variation of TF mRNA level. Finally, we demonstrate the utility of our inference algorithm to infer stress-specific regulatory networks and for regulator prioritization. PMID:27794550

Induction of the Immunoproteasome Subunit Lmp7 Links Proteostasis and Immunity in α-Synuclein Aggregation Disorders.

PubMed

Ugras, Scott; Daniels, Malcolm J; Fazelinia, Hossein; Gould, Neal S; Yocum, Anastasia K; Luk, Kelvin C; Luna, Esteban; Ding, Hua; McKennan, Chris; Seeholzer, Steven; Martinez, Dan; Evans, Perry; Brown, Daniel; Duda, John E; Ischiropoulos, Harry

2018-05-01

Accumulation of aggregated α-synuclein into Lewy bodies is thought to contribute to the onset and progression of dopaminergic neuron degeneration in Parkinson's disease (PD) and related disorders. Although protein aggregation is associated with perturbation of proteostasis, how α-synuclein aggregation affects the brain proteome and signaling remains uncertain. In a mouse model of α-synuclein aggregation, 6% of 6215 proteins and 1.6% of 8183 phosphopeptides changed in abundance, indicating conservation of proteostasis and phosphorylation signaling. The proteomic analysis confirmed changes in abundance of proteins that regulate dopamine synthesis and transport, synaptic activity and integrity, and unearthed changes in mRNA binding, processing and protein translation. Phosphorylation signaling changes centered on axonal and synaptic cytoskeletal organization and structural integrity. Proteostatic responses included a significant increase in the levels of Lmp7, a component of the immunoproteasome. Increased Lmp7 levels and activity were also quantified in postmortem human brains with PD and dementia with Lewy bodies. Functionally, the immunoproteasome degrades α-synuclein aggregates and generates potentially antigenic peptides. Expression and activity of the immunoproteasome may represent testable targets to induce adaptive responses that maintain proteome integrity and modulate immune responses in protein aggregation disorders. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Low-level laser irradiation modulates brain-derived neurotrophic factor mRNA transcription through calcium-dependent activation of the ERK/CREB pathway.

PubMed

Yan, Xiaodong; Liu, Juanfang; Zhang, Zhengping; Li, Wenhao; Sun, Siguo; Zhao, Jian; Dong, Xin; Qian, Jixian; Sun, Honghui

2017-01-01

Low-level laser (LLL) irradiation has been reported to promote neuronal differentiation, but the mechanism remains unclear. Brain-derived neurotrophic factor (BDNF) has been confirmed to be one of the most important neurotrophic factors because it is critical for the differentiation and survival of neurons during development. Thus, this study aimed to investigate the effects of LLL irradiation on Bdnf messenger RNA (mRNA) transcription and the molecular pathway involved in LLL-induced Bdnf mRNA transcription in cultured dorsal root ganglion neurons (DRGNs) using Ca 2+ imaging, pharmacological detections, RNA interference, immunocytochemistry assay, Western blot, and qPCR analysis. We show here that LLL induced increases in the [Ca 2+ ] i level, Bdnf mRNA transcription, cAMP-response element-binding protein (CREB) phosphorylation, and extracellular signal-regulated kinase (ERK) phosphorylation, mediated by Ca 2+ release via inositol triphosphate receptor (IP3R)-sensitive calcium (Ca 2+ ) stores. Blockade of Ca 2+ increase suppressed Bdnf mRNA transcription, CREB phosphorylation, and ERK phosphorylation. Downregulation of phosphorylated (p)-CREB reduced Bdnf mRNA transcription triggered by LLL. Furthermore, blockade of ERK using PD98059 inhibitor reduced p-CREB and Bdnf mRNA transcription induced by LLL. Taken together, these findings establish the Ca 2+ -ERK-CREB cascade as a potential signaling pathway involved in LLL-induced Bdnf mRNA transcription. To our knowledge, this is the first report of the mechanisms of Ca 2+ -dependent Bdnf mRNA transcription triggered by LLL. These findings may help further explore the complex molecular signaling networks in LLL-triggered nerve regeneration in vivo and may also provide experimental evidence for the development of LLL for clinical applications.

Quantivirus® HPV E6/E7 RNA 3.0 assay (bDNA) is as sensitive, but less specific than Hybrid Capture 2 test.

PubMed

Shen, Yong; Gong, Jiaomei; He, Yanxia; Cheng, Guomei; Okunieff, Paul; Li, Xiaofu

2013-02-01

Human papillomavirus (HPV) infection is the primary cause of cervical cancer. The Quantivirus(®) HPV E6/E7 RNA 3.0 assay (DiaCarta, CA, USA) detects E6/E7 mRNA of 13 high risk subtypes and 6 low risk subtypes. Cervical specimens collected in PreservCyt were processed for HPV detection. Cervical biopsies were taken only from those women with abnormal colposcopy. 200 out of 272 (73.5%) cases were mRNA positive. The percentage of HPV E6/E7 mRNA positive samples increases with the severity of the cytological diagnosis, but not in histological diagnosis. In 146 patients with both tests, the E6/E7 mRNA assay had significant higher positivity rate than the Hybrid Capture 2 assay (75.3% versus 62.3%). The HPV mRNA assay and the HC2 assay had the same sensitivity of high grade cervical intraepithelial neoplasia (CIN 2+), 82.4% (14/17) (95% confidence interval [CI], 64.3, 100). However, the specificity of CIN 2+ for the HPV mRNA assay was significantly lower than HC2 assay. Receiver operating characteristic curve analysis was used to compare the diagnostic performance of the E6/E7 mRNA and HC2. E6/E7 mRNA achieved 58.8% sensitivity with 74.1% specificity, HC2, achieved 47.1% sensitivity with 70.7% specificity. The overall performance of HPV E6/E7 mRNA assay for detecting CIN 2+ was lower than HC2. This study does not support the use of this assay in screening for cervical cancer prevention alone. Copyright © 2012 Elsevier B.V. All rights reserved.

Airways in smooth muscle α-actin null mice experience a compensatory mechanism that modulates their contractile response.

PubMed

Shardonofsky, Felix R; Moore, Joan; Schwartz, Robert J; Boriek, Aladin M

2012-03-01

We hypothesized that ablation of smooth muscle α-actin (SM α-A), a contractile-cytoskeletal protein expressed in airway smooth muscle (ASM) cells, abolishes ASM shortening capacity and decreases lung stiffness. In both SM α-A knockout and wild-type (WT) mice, airway resistance (Raw) determined by the forced oscillation technique rose in response to intravenous methacholine (Mch). However, the slope of Raw (cmH(2)O·ml(-1)·s) vs. log(2) Mch dose (μg·kg(-1)·min(-1)) was lower (P = 0.007) in mutant (0.54 ± 0.14) than in WT mice (1.23 ± 0.19). RT-PCR analysis performed on lung tissues confirmed that mutant mice lacked SM α-A mRNA and showed that these mice had robust expressions of both SM γ-A mRNA and skeletal muscle (SKM) α-A mRNA, which were not expressed in WT mice, and an enhanced SM22 mRNA expression relative to that in WT mice. Compared with corresponding spontaneously breathing mice, mechanical ventilation-induced lung mechanical strain increased the expression of SM α-A mRNA in WT lungs; in mutant mice, it augmented the expressions of SM γ-A mRNA and SM22 mRNA and did not alter that of SKM α-A mRNA. In mutant mice, the expression of SM γ-A mRNA in the lung during spontaneous breathing and its enhanced expression following mechanical ventilation are consistent with the likely possibility that in the absence of SM α-A, SM γ-A underwent polymerization and interacted with smooth muscle myosin to produce ASM shortening during cholinergic stimulation. Thus our data are consistent with ASM in mutant mice experiencing compensatory mechanisms that modulated its contractile muscle capacity.

Inactivation of parkin by promoter methylation correlated with lymph node metastasis and genomic instability in nasopharyngeal carcinoma.

PubMed

Ni, Haifeng; Zhou, Zhen; Jiang, Bo; Yuan, Xiaoyang; Cao, Xiaolin; Huang, Guangwu; Li, Yong

2017-03-01

This study aimed to investigate the inactivation of the parkin gene by promoter methylation and its relationship with genome instability in nasopharyngeal carcinoma. Parkin was considered as a tumor suppressor gene in various types of cancers. However, its role in nasopharyngeal carcinoma is unexplored. Genomic instabilities were detected in nasopharyngeal carcinoma tissues by the random amplified polymorphic DNA. The methylation-specific polymerase chain reaction, semi-quantitative reverse transcription polymerase chain reaction, and immunohistochemical analysis were used to detect methylation and mRNA and protein expression of parkin in 54 cases of nasopharyngeal carcinoma tissues and 16 cases of normal nasopharyngeal epithelia tissues, and in 5 nasopharyngeal carcinoma cell lines (CNE1, CNE2, TWO3, C666, and HONE1) and 1 normal nasopharyngeal epithelia cell line (NP69). mRNA expression of parkin in CNE1 and CNE2 was analyzed before and after methyltransferase inhibitor 5-aza-2-deoxycytidine treatment. The relationship between promoter methylation and mRNA expression, demethylation and mRNA expression, and mRNA and protein expression of the gene and clinical factors and genomic instabilities were analyzed. The mRNA and protein expression levels were significantly reduced in 54 cases of human nasopharyngeal carcinoma compared with 16 cases of normal nasopharyngeal epithelia. Parkin-methylated cases showed significantly lower mRNA and protein expression levels compared with unmethylated cases. After 5-aza-2-deoxycytidine treatment, parkin mRNA expression was restored in CNE1 and CNE2; 92.59% (50/54) of nasopharyngeal carcinoma demonstrated genomic instability. Parkin is frequently inactivated by promoter methylation, and its mRNA and protein expression correlate with lymph node metastasis and genomic instability. Parkin deficiency probably promotes tumorigenesis in nasopharyngeal carcinoma.

The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons.

PubMed

Gervasi, Noreen M; Scott, Shane S; Aschrafi, Armaz; Gale, Jenna; Vohra, Sanah N; MacGibeny, Margaret A; Kar, Amar N; Gioio, Anthony E; Kaplan, Barry B

2016-06-01

Synthesis and regulation of catecholamine neurotransmitters in the central nervous system are implicated in the pathogenesis of a number of neuropsychiatric disorders. To identify factors that regulate the presynaptic synthesis of catecholamines, we tested the hypothesis that the rate-limiting enzyme of the catecholamine biosynthetic pathway, tyrosine hydroxylase (TH), is locally synthesized in axons and presynaptic nerve terminals of noradrenergic neurons. To isolate pure axonal mRNA and protein, rat superior cervical ganglion sympathetic neurons were cultured in compartmentalized Campenot chambers. qRT-PCR and RNA in situ hybridization analyses showed that TH mRNA is present in distal axons. Colocalization experiments with nerve terminal marker proteins suggested that both TH mRNA and protein localize in regions of the axon that resemble nerve terminals (i.e., synaptic boutons). Analysis of polysome-bound RNA showed that TH mRNA is present in polysomes isolated from distal axons. Metabolic labeling of axonally synthesized proteins labeled with the methionine analog, L-azidohomoalanine, showed that TH is locally synthesized in axons. Moreover, the local transfection and translation of exogenous TH mRNA into distal axons facilitated axonal dopamine synthesis. Finally, using chimeric td-Tomato-tagged constructs, we identified a sequence element within the TH 3'UTR that is required for the axonal localization of the reporter mRNA. Taken together, our results provide the first direct evidence that TH mRNA is trafficked to the axon and that the mRNA is locally translated. These findings raise the interesting possibility that the biosynthesis of the catecholamine neurotransmitters is locally regulated in the axon and/or presynaptic nerve terminal. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

The Extent of mRNA Editing Is Limited in Chicken Liver and Adipose, but Impacted by Tissular Context, Genotype, Age, and Feeding as Exemplified with a Conserved Edited Site in COG3.

PubMed

Roux, Pierre-François; Frésard, Laure; Boutin, Morgane; Leroux, Sophie; Klopp, Christophe; Djari, Anis; Esquerré, Diane; Martin, Pascal G P; Zerjal, Tatiana; Gourichon, David; Pitel, Frédérique; Lagarrigue, Sandrine

2015-12-04

RNA editing is a posttranscriptional process leading to differences between genomic DNA and transcript sequences, potentially enhancing transcriptome diversity. With recent advances in high-throughput sequencing, many efforts have been made to describe mRNA editing at the transcriptome scale, especially in mammals, yielding contradictory conclusions regarding the extent of this phenomenon. We show, by detailed description of the 25 studies focusing so far on mRNA editing at the whole-transcriptome scale, that systematic sequencing artifacts are considered in most studies whereas biological replication is often neglected and multi-alignment not properly evaluated, which ultimately impairs the legitimacy of results. We recently developed a rigorous strategy to identify mRNA editing using mRNA and genomic DNA sequencing, taking into account sequencing and mapping artifacts, and biological replicates. We applied this method to screen for mRNA editing in liver and white adipose tissue from eight chickens and confirm the small extent of mRNA recoding in this species. Among the 25 unique edited sites identified, three events were previously described in mammals, attesting that this phenomenon is conserved throughout evolution. Deeper investigations on five sites revealed the impact of tissular context, genotype, age, feeding conditions, and sex on mRNA editing levels. More specifically, this analysis highlighted that the editing level at the site located on COG3 was strongly regulated by four of these factors. By comprehensively characterizing the mRNA editing landscape in chickens, our results highlight how this phenomenon is limited and suggest regulation of editing levels by various genetic and environmental factors. Copyright © 2016 Roux et al.

The Extent of mRNA Editing Is Limited in Chicken Liver and Adipose, but Impacted by Tissular Context, Genotype, Age, and Feeding as Exemplified with a Conserved Edited Site in COG3

PubMed Central

Roux, Pierre-François; Frésard, Laure; Boutin, Morgane; Leroux, Sophie; Klopp, Christophe; Djari, Anis; Esquerré, Diane; Martin, Pascal GP; Zerjal, Tatiana; Gourichon, David; Pitel, Frédérique; Lagarrigue, Sandrine

2015-01-01

RNA editing is a posttranscriptional process leading to differences between genomic DNA and transcript sequences, potentially enhancing transcriptome diversity. With recent advances in high-throughput sequencing, many efforts have been made to describe mRNA editing at the transcriptome scale, especially in mammals, yielding contradictory conclusions regarding the extent of this phenomenon. We show, by detailed description of the 25 studies focusing so far on mRNA editing at the whole-transcriptome scale, that systematic sequencing artifacts are considered in most studies whereas biological replication is often neglected and multi-alignment not properly evaluated, which ultimately impairs the legitimacy of results. We recently developed a rigorous strategy to identify mRNA editing using mRNA and genomic DNA sequencing, taking into account sequencing and mapping artifacts, and biological replicates. We applied this method to screen for mRNA editing in liver and white adipose tissue from eight chickens and confirm the small extent of mRNA recoding in this species. Among the 25 unique edited sites identified, three events were previously described in mammals, attesting that this phenomenon is conserved throughout evolution. Deeper investigations on five sites revealed the impact of tissular context, genotype, age, feeding conditions, and sex on mRNA editing levels. More specifically, this analysis highlighted that the editing level at the site located on COG3 was strongly regulated by four of these factors. By comprehensively characterizing the mRNA editing landscape in chickens, our results highlight how this phenomenon is limited and suggest regulation of editing levels by various genetic and environmental factors. PMID:26637431

In vivo modulation of androgen receptor by androgens.

PubMed

Kumar, V L; Majumder, P K; Kumar, V

2002-09-01

To study the effect of androgen and antiandrogen on the level of androgen receptor (AR) mRNA. The total RNA was extracted from the prostate and analyzed by slot blot analysis. The blots were hybridized with AR cDNA probe and 1A probe (internal control) and autoradiography was performed. The intensity of signal was measured with a densitometer and the ratio of AR RNA and 1A RNA was calculated. Androgenic deprivation produced by castration decreased the weight of the prostate and increased the levels of AR mRNA. Treatment of the castrated rats with testostrone increased the weight of prostate and decreased the levels of AR mRNA. Treatment of normal rats with flutamide decreased the weight of the gland and increased the levels of AR mRNA. Androgens produce proliferative effect on the prostate and negatively regulate the AR transcription.

A mRNA and cognate microRNAs localize in the nucleolus.

PubMed

Reyes-Gutierrez, Pablo; Ritland Politz, Joan C; Pederson, Thoru

2014-01-01

We previously discovered that a set of 5 microRNAs are concentrated in the nucleolus of rat myoblasts. We now report that several mRNAs are also localized in the nucleoli of these cells as determined by microarray analysis of RNA from purified nucleoli. Among the most abundant of these nucleolus-localized mRNAs is that encoding insulin-like growth factor 2 (IGF2), a regulator of myoblast proliferation and differentiation. The presence of IGF2 mRNA in nucleoli was confirmed by fluorescence in situ hybridization, and RT-PCR experiments demonstrated that these nucleolar transcripts are spliced, thus arriving from the nucleoplasm. Bioinformatics analysis predicted canonically structured, highly thermodynamically stable interactions between IGF2 mRNA and all 5 of the nucleolus-localized microRNAs. These results raise the possibility that the nucleolus is a staging site for setting up particular mRNA-microRNA interactions prior to export to the cytoplasm.

Differential accumulation of nif structural gene mRNA in Azotobacter vinelandii.

PubMed

Hamilton, Trinity L; Jacobson, Marty; Ludwig, Marcus; Boyd, Eric S; Bryant, Donald A; Dean, Dennis R; Peters, John W

2011-09-01

Northern analysis was employed to investigate mRNA produced by mutant strains of Azotobacter vinelandii with defined deletions in the nif structural genes and in the intergenic noncoding regions. The results indicate that intergenic RNA secondary structures effect the differential accumulation of transcripts, supporting the high Fe protein-to-MoFe protein ratio required for optimal diazotrophic growth.

Dmrt1 Expression Is Regulated by Follicle-Stimulating Hormone and Phorbol Esters in Postnatal Sertoli Cells*

PubMed Central

CHEN, JIANG KAI; HECKERT, LESLIE L.

2006-01-01

Dmrt1 is a recently described gene that is expressed exclusively in the testis and is required for postnatal testis differentiation. Here we describe the expression of Dmrt1 in postnatal rat testis and Sertoli cells. RNase protection analysis was used to examine Dmrt1 messenger RNA (mRNA) levels in intact testis during postnatal development and in primary cultures of Sertoli cells under various culture conditions. We show that Dmrt1 mRNA levels rise significantly beginning approximately 10 days after birth and remain elevated until after the third postnatal week. Thereafter, mRNA levels drop coincident with the proliferation of germ cells in the testis. In freshly isolated Sertoli cells, Dmrt1 mRNA levels were robust but decreased significantly when the cells were placed in culture for 24 h. Treatment of Sertoli cells with either FSH or 8-bromo-cAMP resulted in a significant rise in Dmrt1 mRNA levels. This cAMP response was sensitive to treatment with the transcriptional inhibitor actinomycin D but not to the translational inhibitor cycloheximide. The cAMP-dependent rise in Dmrt1 mRNA also required activation of protein kinase A, as mRNA induction was sensitive to the inhibitor H89. Studies also show that Dmrt1 expression was inhibited by phorbol esters (PMA) but only modestly effected by serum. PMID:11181532

Single-cell mRNA profiling reveals transcriptional heterogeneity among pancreatic circulating tumour cells.

PubMed

Lapin, Morten; Tjensvoll, Kjersti; Oltedal, Satu; Javle, Milind; Smaaland, Rune; Gilje, Bjørnar; Nordgård, Oddmund

2017-05-31

Single-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process. In addition, such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer. Single circulating tumour cells were isolated from patients with locally advanced or metastatic pancreatic cancer with immuno-magnetic depletion and immuno-fluorescence microscopy. mRNA expression was analysed with single-cell multiplex RT-qPCR. Hierarchical clustering and principal component analysis were performed to identify expression patterns. Circulating tumour cells were detected in 33 of 56 (59%) examined blood samples. Single-cell mRNA profiling of intact isolated circulating tumour cells revealed both epithelial-like and mesenchymal-like subpopulations, which were distinct from leucocytes. The profiled circulating tumour cells also expressed elevated levels of stem cell markers, and the extracellular matrix protein, SPARC. The expression of SPARC might correspond to an epithelial-mesenchymal transition in pancreatic circulating tumour cells. The analysis of single pancreatic circulating tumour cells identified distinct subpopulations and revealed elevated expression of transcripts relevant to the dissemination of circulating tumour cells to distant organ sites.

Integrated analysis of long noncoding RNA and mRNA expression profile in children with obesity by microarray analysis.

PubMed

Liu, Yuesheng; Ji, Yuqiang; Li, Min; Wang, Min; Yi, Xiaoqing; Yin, Chunyan; Wang, Sisi; Zhang, Meizhen; Zhao, Zhao; Xiao, Yanfeng

2018-06-08

Long noncoding RNAs (lncRNAs) have an important role in adipose tissue function and energy metabolism homeostasis, and abnormalities may lead to obesity. To investigate whether lncRNAs are involved in childhood obesity, we investigated the differential expression profile of lncRNAs in obese children compared with non-obese children. A total number of 1268 differentially expressed lncRNAs and 1085 differentially expressed mRNAs were identified. Gene Ontology (GO) and pathway analysis revealed that these lncRNAs were involved in varied biological processes, including the inflammatory response, lipid metabolic process, osteoclast differentiation and fatty acid metabolism. In addition, the lncRNA-mRNA co-expression network and the protein-protein interaction (PPI) network were constructed to identify hub regulatory lncRNAs and genes based on the microarray expression profiles. This study for the first time identifies an expression profile of differentially expressed lncRNAs in obese children and indicated hub lncRNA RP11-20G13.3 attenuated adipogenesis of preadipocytes, which is conducive to the search for new diagnostic and therapeutic strategies of childhood obesity.

Regression Analysis of Combined Gene Expression Regulation in Acute Myeloid Leukemia

PubMed Central

Li, Yue; Liang, Minggao; Zhang, Zhaolei

2014-01-01

Gene expression is a combinatorial function of genetic/epigenetic factors such as copy number variation (CNV), DNA methylation (DM), transcription factors (TF) occupancy, and microRNA (miRNA) post-transcriptional regulation. At the maturity of microarray/sequencing technologies, large amounts of data measuring the genome-wide signals of those factors became available from Encyclopedia of DNA Elements (ENCODE) and The Cancer Genome Atlas (TCGA). However, there is a lack of an integrative model to take full advantage of these rich yet heterogeneous data. To this end, we developed RACER (Regression Analysis of Combined Expression Regulation), which fits the mRNA expression as response using as explanatory variables, the TF data from ENCODE, and CNV, DM, miRNA expression signals from TCGA. Briefly, RACER first infers the sample-specific regulatory activities by TFs and miRNAs, which are then used as inputs to infer specific TF/miRNA-gene interactions. Such a two-stage regression framework circumvents a common difficulty in integrating ENCODE data measured in generic cell-line with the sample-specific TCGA measurements. As a case study, we integrated Acute Myeloid Leukemia (AML) data from TCGA and the related TF binding data measured in K562 from ENCODE. As a proof-of-concept, we first verified our model formalism by 10-fold cross-validation on predicting gene expression. We next evaluated RACER on recovering known regulatory interactions, and demonstrated its superior statistical power over existing methods in detecting known miRNA/TF targets. Additionally, we developed a feature selection procedure, which identified 18 regulators, whose activities clustered consistently with cytogenetic risk groups. One of the selected regulators is miR-548p, whose inferred targets were significantly enriched for leukemia-related pathway, implicating its novel role in AML pathogenesis. Moreover, survival analysis using the inferred activities identified C-Fos as a potential AML prognostic marker. Together, we provided a novel framework that successfully integrated the TCGA and ENCODE data in revealing AML-specific regulatory program at global level. PMID:25340776

Supplementation of direct-fed microbial as an alternative to antibiotic on growth performance, intestinal immune status and epithelial barrier integrity in broiler chicken

USDA-ARS?s Scientific Manuscript database

The objective of this study was to investigate the effects of supplementation of broiler diets with Bacillus subtilis-based probiotics on growth performance, feed efficiency, intestinal cytokine and tight junction (TJ) protein mRNA expression. Day-old broiler chicks (n = 140) were randomly assigned...

Linking nuclear mRNP assembly and cytoplasmic destiny.

PubMed

Kuersten, Scott; Goodwin, Elizabeth B

2005-06-01

From the very beginning, mRNAs have a complex existence. They are transcribed, capped, spliced, modified at the 3'end, exported from the nucleus, translated, and eventually degraded. These many events not only affect the overall survival and properties of an mRNA, but are also carefully co-ordinated and integrated with quality control mechanisms that function to ensure that only 'proper' mRNAs are translated at the correct developmental time and place. This does not mean that all mRNAs follow a single or uniform path from synthesis to death. Instead, there are diverse means by which the activities of specific mRNAs are regulated, and these controls often depend upon multiple events in the mRNA's life. mRNAs are not found naked in the cell, instead they are part of complex RNPs (ribonucleoproteins) that consist of many factors. These RNPs are highly dynamic structures that change during the lifetime of a given RNA; linking events such as synthesis and processing to the final fate of the mRNA. Here, we will discuss what is known of the assembly of RNPs in general, with specific reference to the myriad of connections between different nuclear events and the cytoplasmic activity of an mRNA. Due to space limitations this review is not comprehensive, instead we focus on specific examples to illustrate these emerging themes in gene expression.

Blood cell mRNAs and microRNAs: optimized protocols for extraction and preservation.

PubMed

Eikmans, Michael; Rekers, Niels V; Anholts, Jacqueline D H; Heidt, Sebastiaan; Claas, Frans H J

2013-03-14

Assessing messenger RNA (mRNA) and microRNA levels in peripheral blood cells may complement conventional parameters in clinical practice. Working with small, precious samples requires optimal RNA yields and minimal RNA degradation. Several procedures for RNA extraction and complementary DNA (cDNA) synthesis were compared for their efficiency. The effect on RNA quality of freeze-thawing peripheral blood cells and storage in preserving reagents was investigated. In terms of RNA yield and convenience, quality quantitative polymerase chain reaction signals per nanogram of total RNA and using NucleoSpin and mirVana columns is preferable. The SuperScript III protocol results in the highest cDNA yields. During conventional procedures of storing peripheral blood cells at -180°C and thawing them thereafter, RNA integrity is maintained. TRIzol preserves RNA in cells stored at -20°C. Detection of mRNA levels significantly decreases in degraded RNA samples, whereas microRNA molecules remain relatively stable. When standardized to reference targets, mRNA transcripts and microRNAs can be reliably quantified in moderately degraded (quality index 4-7) and severely degraded (quality index <4) RNA samples, respectively. We describe a strategy for obtaining high-quality and quantity RNA from fresh and stored cells from blood. The results serve as a guideline for sensitive mRNA and microRNA expression assessment in clinical material.

LINE-1 Retroelements Complexed and Inhibited by Activation Induced Cytidine Deaminase

PubMed Central

Metzner, Mirjam; Jäck, Hans-Martin; Wabl, Matthias

2012-01-01

LINE-1 (abbreviated L1) is a major class of retroelements in humans and mice. If unrestricted, retroelements accumulate in the cytoplasm and insert their DNA into the host genome, with the potential to cause autoimmune disease and cancer. Retroviruses and other retroelements are inhibited by proteins of the APOBEC family, of which activation-induced cytidine deaminase (AID) is a member. Although AID is mainly known for being a DNA mutator shaping the antibody repertoire in B lymphocytes, we found that AID also restricts de novo L1 integrations in B- and non-B-cell lines. It does so by decreasing the protein level of open reading frame 1 (ORF1) of both exogenous and endogenous L1. In activated B lymphocytes, AID deficiency increased L1 mRNA 1.6-fold and murine leukemia virus (MLV) mRNA 2.7-fold. In cell lines and activated B lymphocytes, AID forms cytoplasmic high-molecular-mass complexes with L1 mRNA, which may contribute to L1 restriction. Because AID-deficient activated B lymphocytes do not express ORF1 protein, we suggest that ORF1 protein expression is inhibited by additional restriction factors in these cells. The greater increase in MLV compared to L1 mRNA in AID-deficient activated B lymphocytes may indicate less strict surveillance of retrovirus. PMID:23133680

Alpha-lipoic acid impairs body weight gain of young broiler chicks via modulating peripheral AMPK.

PubMed

Wang, Yufeng; Everaert, Nadia; Song, Zhigang; Decuypere, Eddy; Vermeulen, Daniel; Buyse, Johan

2017-09-01

In mammals, the AMP-activated protein kinase (AMPK) pathways in the central and peripheral tissues coordinately integrate inputs from multiple sources to regulate energy balance. The present study was aimed to explore the potential role of hepatic AMPK in the energy homeostasis of broiler chickens. Diets with 0, 0.05% or 0.1% alpha-lipoic acid (α-LA), a known AMPK inhibitor were provided to broiler chicks for 7days. As a result, α-LA supplementation decreased the relative growth rate of broiler chicks. Hepatic AMPKα2 mRNA levels were significantly upregulated by dietary α-LA, in concert with the increased phosphorylated AMPKα protein levels. In addition, hepatic FAS mRNA levels together with the malonyl-CoA to total CoA ester ratio were reduced by α-LA supplementation. Moreover, the hepatic phosphorylated glycogen synthase levels were increased resulting in a markedly decreased hepatic glycogen content. In conclusion, dietary α-LA supplementation decreased the in vivo hepatic glycogenesis and lipogenesis via stimulating hepatic AMPKα mRNA levels and the phosphorylated gene product. The stimulatory effect of α-LA on hepatic AMPK mRNA and pAMPKα protein levels together with our previous observations regarding its inhibitory effect on hypothalamic AMPK may have altered the energy balance and hence impaired body weight gain of broiler chicks. Copyright © 2017 Elsevier Inc. All rights reserved.

Dopaminergic Modulation of Risky Decision-Making

PubMed Central

Simon, Nicholas W.; Montgomery, Karienn S.; Beas, Blanca S.; Mitchell, Marci R.; LaSarge, Candi L.; Mendez, Ian A.; Bañuelos, Cristina; Vokes, Colin M.; Taylor, Aaron B.; Haberman, Rebecca P.; Bizon, Jennifer L.; Setlow, Barry

2012-01-01

Many psychiatric disorders are characterized by abnormal risky decision-making and dysregulated dopamine receptor expression. The current study was designed to determine how different dopamine receptor subtypes modulate risk-taking in young adult rats, using a “Risky Decision-making Task” that involves choices between small “safe” rewards and large “risky” rewards accompanied by adverse consequences. Rats showed considerable, stable individual differences in risk preference in the task, which were not related to multiple measures of reward motivation, anxiety, or pain sensitivity. Systemic activation of D2-like receptors robustly attenuated risk-taking, whereas drugs acting on D1-like receptors had no effect. Systemic amphetamine also reduced risk-taking, an effect which was attenuated by D2-like (but not D1-like) receptor blockade. Dopamine receptor mRNA expression was evaluated in a separate cohort of drug-naive rats characterized in the task. D1 mRNA expression in both nucleus accumbens shell and insular cortex was positively associated with risk-taking, while D2 mRNA expression in orbitofrontal and medial prefrontal cortex predicted risk preference in opposing nonlinear patterns. Additionally, lower levels of D2 mRNA in dorsal striatum were associated with greater risk-taking. These data strongly implicate dopamine signaling in prefrontal corticalstriatal circuitry in modulating decision-making processes involving integration of reward information with risks of adverse consequences. PMID:22131407

Effects of environmental stress on mRNA expression levels of seven genes related to oxidative stress and growth in Atlantic salmon Salmo salar L. of farmed, hybrid and wild origin.

PubMed

Solberg, Monica F; Kvamme, Bjørn Olav; Nilsen, Frank; Glover, Kevin A

2012-12-05

Ten generations of domestication selection has caused farmed Atlantic salmon Salmo salar L. to deviate from wild salmon in a range of traits. Each year hundreds of thousands of farmed salmon escape into the wild. Thus, interbreeding between farmed escapees and wild conspecifics represents a significant threat to the genetic integrity of wild salmon populations. In a previous study we demonstrated how domestication has inadvertently selected for reduced responsiveness to stress in farmed salmon. To complement that study, we have evaluated the expression of seven stress-related genes in head kidney of salmon of farmed, hybrid and wild origin exposed to environmentally induced stress. In general, the crowding stressor used to induce environmental stress did not have a strong impact on mRNA expression levels of the seven genes, except for insulin-like growth factor-1 (IGF-1) that was downregulated in the stress treatment relative to the control treatment. mRNA expression levels of glutathione reductase (GR), Cu/Zn superoxide dismutase (Cu/Zn SOD), Mn superoxide dismutase (Mn SOD), glutathione peroxidase (GP) and IGF-1 were affected by genetic origin, thus expressed significantly different between the salmon of farmed, hybrid or wild origin. A positive relationship was detected between body size of wild salmon and mRNA expression level of the IGF-1 gene, in both environments. No such relationship was observed for the hybrid or farmed salmon. Farmed salmon in this study displayed significantly elevated mRNA levels of the IGF-1 gene relative to the wild salmon, in both treatments, while hybrids displayed a non additive pattern of inheritance. As IGF-1 mRNA levels are positively correlated to growth rate, the observed positive relationship between body size and IGF-1 mRNA levels detected in the wild but neither in the farmed nor the hybrid salmon, could indicate that growth selection has increased IGF-1 levels in farmed salmon to the extent that they may not be limiting growth rate.

Effects of environmental stress on mRNA expression levels of seven genes related to oxidative stress and growth in Atlantic salmon Salmo salar L. of farmed, hybrid and wild origin

PubMed Central

2012-01-01

Background Ten generations of domestication selection has caused farmed Atlantic salmon Salmo salar L. to deviate from wild salmon in a range of traits. Each year hundreds of thousands of farmed salmon escape into the wild. Thus, interbreeding between farmed escapees and wild conspecifics represents a significant threat to the genetic integrity of wild salmon populations. In a previous study we demonstrated how domestication has inadvertently selected for reduced responsiveness to stress in farmed salmon. To complement that study, we have evaluated the expression of seven stress-related genes in head kidney of salmon of farmed, hybrid and wild origin exposed to environmentally induced stress. Results In general, the crowding stressor used to induce environmental stress did not have a strong impact on mRNA expression levels of the seven genes, except for insulin-like growth factor-1 (IGF-1) that was downregulated in the stress treatment relative to the control treatment. mRNA expression levels of glutathione reductase (GR), Cu/Zn superoxide dismutase (Cu/Zn SOD), Mn superoxide dismutase (Mn SOD), glutathione peroxidase (GP) and IGF-1 were affected by genetic origin, thus expressed significantly different between the salmon of farmed, hybrid or wild origin. A positive relationship was detected between body size of wild salmon and mRNA expression level of the IGF-1 gene, in both environments. No such relationship was observed for the hybrid or farmed salmon. Conclusion Farmed salmon in this study displayed significantly elevated mRNA levels of the IGF-1 gene relative to the wild salmon, in both treatments, while hybrids displayed a non additive pattern of inheritance. As IGF-1 mRNA levels are positively correlated to growth rate, the observed positive relationship between body size and IGF-1 mRNA levels detected in the wild but neither in the farmed nor the hybrid salmon, could indicate that growth selection has increased IGF-1 levels in farmed salmon to the extent that they may not be limiting growth rate. PMID:23217180

Relative Abundance of Integral Plasma Membrane Proteins in Arabidopsis Leaf and Root Tissue Determined by Metabolic Labeling and Mass Spectrometry

PubMed Central

Bernfur, Katja; Larsson, Olaf; Larsson, Christer; Gustavsson, Niklas

2013-01-01

Metabolic labeling of proteins with a stable isotope (15N) in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins. Forty-one of the integral proteins, including nine of the 13 isoforms of the PIP (plasma membrane intrinsic protein) aquaporin subfamily, could be identified by peptides unique to these proteins, which made it possible to determine their relative abundance in leaf and root tissue. In addition, peptides shared between isoforms gave information on the proportions of these isoforms. A comparison between our data for protein levels and corresponding data for mRNA levels in the widely used database Genevestigator showed an agreement for only about two thirds of the proteins. By contrast, localization data available in the literature for 21 of the 41 proteins show a much better agreement with our data, in particular data based on immunostaining of proteins and GUS-staining of promoter activity. Thus, although mRNA levels may provide a useful approximation for protein levels, detection and quantification of isoform-specific peptides by proteomics should generate the most reliable data for the proteome. PMID:23990937

Dietary vitamin C deficiency depresses the growth, head kidney and spleen immunity and structural integrity by regulating NF-κB, TOR, Nrf2, apoptosis and MLCK signaling in young grass carp (Ctenopharyngodon idella).

PubMed

Xu, Hui-Jun; Jiang, Wei-Dan; Feng, Lin; Liu, Yang; Wu, Pei; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

2016-05-01

This study investigated the effects of dietary vitamin C on the growth, and head kidney, spleen and skin immunity, structural integrity and related signaling molecules mRNA expression levels of young grass carp (Ctenopharyngodon idella). A total of 540 grass carp (264.37 ± 0.66 g) were fed six diets with graded levels of vitamin C (2.9, 44.2, 89.1, 133.8, 179.4 and 224.5 mg/kg diet) for 10 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila and the survival rate recorded for 14 days. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) decreased lysozyme (LA) and acid phosphatase (ACP) activities, and complement 3 and complement 4 (C4) contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides [liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, hepcidin, β-defensin] and anti-inflammatory cytokines-related factors, interleukin (IL) 4/13A, IL-4/13B (only in head kidney), IL-10, IL-11, transforming growth factor (TGF) β1, TGF-β2, inhibitor of κBα and eIF4E-binding protein 1 (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors, tumor necrosis factor α, interferon γ2, IL-1β, IL-6, IL-8, IL-12 P35 (only in spleen), IL-12 P40, IL-15, IL-17D, nuclear factor κB p65, IκB kinases (IKKα, IKKβ, IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head kidney and spleen immunity and cause inflammation. Meanwhile, compared with optimal vitamin C supplementation, vitamin C deficiency decreased the activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase, glutathione S-transferases and glutathione reductase (P < 0.05), and down-regulated zonula occludens (ZO) 1, ZO-2, Claudin-b, -c, -3c, -7a, -7b, B-cell lymphoma-2, inhibitor of apoptosis protein, NF-E2-related factor 2 mRNA levels (P < 0.05), increased reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl contents (P < 0.05), and up-regulated Claudin-12, 15a, -15b, Fas ligand, mitogen-activated protein kinase kinase 6, p38 mitogen-activated protein kinase, B-cell lymphoma protein 2 associated X protein, apoptotic protease activating factor-1, caspase-3, -7, -8, -9, Kelch-like ECH-associating protein (Keap) 1a and Keap 1b mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head kidney and spleen structural integrity through depression of antioxidative ability, induction of apoptosis and disruption of tight junctional complexes. In addition, except the activities of ACP and MnSOD, and mRNA expression levels of TGF-β1, Occludin and MnSOD, the effect of vitamin C on fish head kidney, spleen and skin immunity and structural integrity other indicators model are similar under infection of A. hydrophila. Finally, the vitamin C requirement for the growth performance (PWG) of young grass carp was estimated to be 92.8 mg/kg diet. Meanwhile, the vitamin C requirement for against skin lesion morbidity of young grass carp was estimated to be 122.9 mg/kg diet. In addition, based on the biochemical indices [immune indices (LA activity in the head kidney and C4 content in the spleen) and antioxidant indices (MDA content in the head kidney and ROS content in the spleen)] the vitamin C requirements for young grass carp were estimated to be 131.2, 137.5, 135.8 and 129.8 mg/kg diet, respectively. Copyright © 2016. Published by Elsevier Ltd.

Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells

PubMed Central

Carlile, Thomas M.; Rojas-Duran, Maria F.; Zinshteyn, Boris; Shin, Hakyung; Bartoli, Kristen M.; Gilbert, Wendy V.

2014-01-01

Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs1, enhances the function of transfer RNA and ribosomal RNA by stabilizing RNA structure2–8. mRNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function – it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding center9,10. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological was unclear. Here we present a comprehensive analysis of pseudouridylation in yeast and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as 100 novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1–4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease11–13. PMID:25192136

Tissue culture specificity of the tobacco ASA2 promoter driving hpt as a selectable marker for soybean transformation selection.

PubMed

Zernova, Olga; Zhong, Wei; Zhang, Xing-Hai; Widholm, Jack

2008-11-01

This study was carried out to determine if the tobacco anthranilate synthase ASA2 2.3 kb promoter drives tissue culture specific expression and if it is strong enough to drive hpt (hygromycin phosphotransferase) gene expression at a level sufficient to allow selection of transformed soybean embryogenic culture lines. A number of transformed cell lines were selected showing that the promoter was strong enough. Northern blot analysis of plant tissues did not detect hpt mRNA in the untransformed control or in the ASA2-hpt plants except in developing seeds while hpt mRNA was detected in all tissues of the CaMV35S-hpt positive control line plants. However, when the more sensitive RT-PCR assay was used all tissues of the ASA2-hpt plants except roots and mature seeds were found to contain detectable hpt mRNA. Embryogenic tissue cultures initiated from the ASA2-hpt plants contained hpt mRNA detectable by both northern and RT-PCR analysis and the cultures were hygromycin resistant. Friable callus initiated from leaves of ASA2-hpt plants did in some cases contain hpt mRNA that was only barely detectable by northern hybridization even though the callus was very hygromycin resistant. Thus the ASA2 promoter is strong enough to drive sufficient hpt expression in soybean embryogenic cultures for hygromycin selection and only very low levels of expression were found in most plant tissues with none in mature seeds.

Limitations of commonly used internal controls for real-time RT-PCR analysis of renal epithelial-mesenchymal cell transition.

PubMed

Elberg, Gerard; Elberg, Dorit; Logan, Charlotte J; Chen, Lijuan; Turman, Martin A

2006-01-01

Progressive renal fibrotic disease is accompanied by the massive accumulation of myofibroblasts as defined by alpha smooth muscle actin (alphaSMA) expression. We quantitated gene expression using real-time RT-PCR analysis during conversion of primary cultured human renal tubular cells (RTC) to myofibroblasts after treatment with transforming growth factor-beta1 (TGF-beta1). We report herein the limitations of commonly used reference genes for mRNA quantitation. We determined the expression of alphaSMA and megakaryoblastic leukemia-1 (MKL1), a transcriptional regulator of alphaSMA, by quantitative real-time PCR using three common internal controls, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A and 18S rRNA. Expression of GAPDH mRNA and cyclophilin A mRNA, and to a lesser extent, 18S rRNA levels varied over time in culture and with exposure to TGF-beta1. Thus, depending on which reference gene was used, TGF-beta1 appeared to have different effects on expression of MKL1 and alphaSMA. RTC converting to myofibroblasts in primary culture is a valuable system to study renal fibrosis in humans. However, variability in expression of reference genes with TGF-beta1 treatment illustrates the need to validate mRNA quantitation with multiple reference genes to provide accurate interpretation of fibrosis studies in the absence of a universal internal standard for mRNA expression. 2006 S. Karger AG, Basel.

A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18

PubMed Central

Chi, Binkai; Wang, Ke; Du, Yanhua; Gui, Bin; Chang, Xingya; Wang, Lantian; Fan, Jing; Chen, She; Wu, Xudong; Li, Guohui; Cheng, Hong

2014-01-01

Viral RNA elements that facilitate mRNA export are useful tools for identifying cellular RNA export factors. Here we show that hepatitis B virus post-transcriptional element (PRE) is one such element, and using PRE several new cellular mRNA export factors were identified. We found that PRE drastically enhances the cytoplasmic accumulation of cDNA transcripts independent of any viral protein. Systematic deletion analysis revealed the existence of a 116 nt functional Sub-Element of PRE (SEP1). The RNP that forms on the SEP1 RNA was affinity purified, in which TREX components as well as several other proteins were identified. TREX components and the SEP1-associating protein ZC3H18 are required for SEP1-mediated mRNA export. Significantly, ZC3H18 directly binds to the SEP1 RNA, interacts with TREX and is required for stable association of TREX with the SEP1-containing mRNA. Requirements for SEP1-mediated mRNA export are similar to those for splicing-dependent mRNA export. Consistent with these similarities, several SEP1-interacting proteins, including ZC3H18, ARS2, Acinus and Brr2, are required for efficient nuclear export of polyA RNAs. Together, our data indicate that SEP1 enhances mRNA export by recruiting TREX via ZC3H18. The new mRNA export factors that we identified might be involved in cap- and splicing-dependent TREX recruitment to cellular mRNAs. PMID:24782531

A novel mechanism of protamine expression deregulation highlighted by abnormal protamine transcript retention in infertile human males with sperm protamine deficiency.

PubMed

Aoki, V W; Liu, L; Carrell, D T

2006-01-01

Sperm protamine deficiency has been associated with human male infertility. However, the aetiology of deregulated protamine expression remains elusive. The objective of this study was to evaluate the underlying aetiology of protamine deficiency in male infertility patients with deregulated protamine expression. Protamine-1 (P1) and protamine-2 (P2) protein concentrations were compared against P1 and P2 mRNA levels in the sperm of 166 male infertility patients and 27 men of known fertility. Protamine protein concentrations were quantified by nuclear protein extraction, gel electrophoresis and densitometry analysis. Semi-quantitative real-time RT-PCR was used to quantify P1 and P2 mRNA levels. P1 mRNA concentrations were significantly increased in patients underexpressing P1 protein versus those with normal and increased P1 levels. In patients with an abnormally low ratio of P1 to P2 (P1/P2 <0.8), there was a significant increase in P1 mRNA retention. Patients underexpressing P2 also had significantly increased mean P2 mRNA levels, although the majority of these P2-deficient patients showed an increased frequency of significantly reduced P2 mRNA levels. This is the first study to concomitantly evaluate P1 and P2 protein and mRNA levels in mature human sperm. Abnormally elevated protamine mRNA retention appears to be associated with aberrant protamine expression in infertile human males. These data suggest that defects in protamine translation regulation may contribute to protamine deficiency in infertile males.

Inflammation-Mediated Regulation of MicroRNA Expression in Transplanted Pancreatic Islets

PubMed Central

Bravo-Egana, Valia; Rosero, Samuel; Klein, Dagmar; Jiang, Zhijie; Vargas, Nancy; Tsinoremas, Nicholas; Doni, Marco; Podetta, Michele; Ricordi, Camillo; Molano, R. Damaris; Pileggi, Antonello; Pastori, Ricardo L.

2012-01-01

Nonspecific inflammation in the transplant microenvironment results in β-cell dysfunction and death influencing negatively graft outcome. MicroRNA (miRNA) expression and gene target regulation in transplanted islets are not yet well characterized. We evaluated the impact of inflammation on miRNA expression in transplanted rat islets. Islets exposed in vitro to proinflammatory cytokines and explanted syngeneic islet grafts were evaluated by miRNA arrays. A subset of 26 islet miRNAs was affected by inflammation both in vivo and in vitro. Induction of miRNAs was dependent on NF-κB, a pathway linked with cytokine-mediated islet cell death. RT-PCR confirmed expression of 8 miRNAs. The association between these miRNAs and mRNA target-predicting algorithms in genome-wide RNA studies of β-cell inflammation identified 238 potential miRNA gene targets. Several genes were ontologically associated with regulation of insulin signaling and secretion, diabetes, and islet physiology. One of the most activated miRNAs was miR-21. Overexpression of miR-21 in insulin-secreting MIN6 cells downregulated endogenous expression of the tumor suppressor Pdcd4 and of Pclo, a Ca2+ sensor protein involved in insulin secretion. Bioinformatics identified both as potential targets. The integrated analysis of miRNA and mRNA expression profiles revealed potential targets that may identify molecular targets for therapeutic interventions. PMID:22655170

Genome-wide DNA Methylation Profiles and Their Relationships with mRNA and the microRNA Transcriptome in Bovine Muscle Tissue (Bos taurine)

PubMed Central

Huang, Yong-Zhen; Sun, Jia-Jie; Zhang, Liang-Zhi; Li, Cong-Jun; Womack, James E.; Li, Zhuan-Jian; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Zhao, Xin; Chen, Hong

2014-01-01

DNA methylation is a key epigenetic modification in mammals and plays important roles in muscle development. We sampled longissimus dorsi muscle (LDM) from a well-known elite native breed of Chinese Qinchuan cattle living within the same environment but displaying distinct skeletal muscle at the fetal and adult stages. We generated and provided a genome-wide landscape of DNA methylomes and their relationship with mRNA and miRNA for fetal and adult muscle studies. Integration analysis revealed a total of 77 and 1,054 negatively correlated genes with methylation in the promoter and gene body regions, respectively, in both the fetal and adult bovine libraries. Furthermore, we identified expression patterns of high-read genes that exhibit a negative correlation between methylation and expression from nine different tissues at multiple developmental stages of bovine muscle-related tissue or organs. In addition, we validated the MeDIP-Seq results by bisulfite sequencing PCR (BSP) in some of the differentially methylated promoters. Together, these results provide valuable data for future biomedical research and genomic and epigenomic studies of bovine skeletal muscle that may help uncover the molecular basis underlying economically valuable traits in cattle. This comprehensive map also provides a solid basis for exploring the epigenetic mechanisms of muscle growth and development. PMID:25306978

Expression of group III metabotropic glutamate receptors in the reproductive system of male mice.

PubMed

Marciniak, Marcin; Chruścicka, Barbara; Lech, Tomasz; Burnat, Grzegorz; Pilc, Andrzej

2016-03-01

Although the presence of metabotropic glutamate (mGlu) receptors in the central nervous system is well documented, they have recently been found in peripheral and non-neuronal tissues. In the present study we investigated the expression of group III mGlu receptors in the reproductive system of male mice. Reverse transcription-polymerase chain reaction analysis revealed the presence of mGlu6, mGlu7 and mGlu8 (but not mGlu4) receptor transcripts in testes and epididymides from adult mice. In addition, expression of mGlu6 (Grm6) and mGlu8 receptor (Grm8) mRNA was detected in spermatozoa isolated from the vas deferens. The vas deferens was found to contain only mGlu7 receptor (Grm7) mRNA, which was particularly intense in 21-day-old male mice. In penile homogenates, only the mGlu7 receptor signal was detected. Genetic ablation of the mGlu7 receptor in males led to fertility disorders manifested by decreased insemination capability as well as deterioration of sperm parameters, particularly sperm motility, vitality, sperm membrane integrity and morphology, with a simultaneous increase in sperm concentration. These results indicate that constitutively expressed mGlu receptors in the male reproductive system may play an important role in ejaculation and/or erection processes, as well as in the formation and maturation of spermatozoa.

A Systematic Analysis on mRNA and MicroRNA Expression in Runting and Stunting Chickens

PubMed Central

Xu, Haiping; Xu, Zhenqiang; Ma, Jinge; Li, Bixiao; Lin, Shudai; Nie, Qinghua; Luo, Qingbin; Zhang, Xiquan

2015-01-01

Runting and stunting syndrome (RSS), which is characterized by lower body weight, widely occurs in broilers. Some RSS chickens simply exhibit slow growth without pathological changes. An increasing number of studies indicate that broiler strains differ in susceptibility to infectious diseases, most likely due to their genetic differences. The objective of this study was to detect the differentially expressed miRNAs and mRNAs in RSS and normal chickens. By integrating miRNA with mRNA expression profiling, potential molecular mechanisms involved in RSS could be further explored. Twenty-two known miRNAs and 1,159 genes were differentially expressed in RSS chickens compared with normal chickens (P < 0.05). qPCR validation results displayed similar patterns. The differentially expressed genes were primarily involved in energy metabolism pathways. The antisense transcripts were extensively expressed in chicken liver albeit with reduced abundance. Dual-luciferase reporter assay indicated that gga-miR-30b/c directly target CARS through binding to its 3′UTR. The miR-30b/c: CARS regulation mainly occurred in liver. In thigh muscle and the hypothalamus, miR-30b/c are expressed at higher levels in RSS chickens compared with normal chickens from 2 to 6 w of age, and notably significant differences are observed at 4 w of age. PMID:26010155

TGF-ß Regulates Enamel Mineralization and Maturation through KLK4 Expression

PubMed Central

Cho, Andrew; Haruyama, Naoto; Hall, Bradford; Danton, Mary Jo S.; Zhang, Lu; Arany, Praveen; Mooney, David J.; Harichane, Yassine; Goldberg, Michel; Gibson, Carolyn W.; Kulkarni, Ashok B.

2013-01-01

Transforming growth factor-ß (TGF-ß) signaling plays an important role in regulating crucial biological processes such as cell proliferation, differentiation, apoptosis, and extracellular matrix remodeling. Many of these processes are also an integral part of amelogenesis. In order to delineate a precise role of TGF-ß signaling during amelogenesis, we developed a transgenic mouse line that harbors bovine amelogenin promoter-driven Cre recombinase, and bred this line with TGF-ß receptor II floxed mice to generate ameloblast-specific TGF-ß receptor II conditional knockout (cKO) mice. Histological analysis of the teeth at postnatal day 7 (P7) showed altered enamel matrix composition in the cKO mice as compared to the floxed mice that had enamel similar to the wild-type mice. The µCT and SEM analyses revealed decreased mineral content in the cKO enamel concomitant with increased attrition and thinner enamel crystallites. Although the mRNA levels remained unaltered, immunostaining revealed increased amelogenin, ameloblastin, and enamelin localization in the cKO enamel at the maturation stage. Interestingly, KLK4 mRNA levels were significantly reduced in the cKO teeth along with a slight increase in MMP-20 levels, suggesting that normal enamel maturation is regulated by TGF-ß signaling through the expression of KLK4. Thus, our study indicates that TGF-ß signaling plays an important role in ameloblast functions and enamel maturation. PMID:24278477

Expression analysis of NOS family and HSP genes during thermal stress in goat ( Capra hircus)

NASA Astrophysics Data System (ADS)

Yadav, Vijay Pratap; Dangi, Satyaveer Singh; Chouhan, Vikrant Singh; Gupta, Mahesh; Dangi, Saroj K.; Singh, Gyanendra; Maurya, Vijay Prakash; Kumar, Puneet; Sarkar, Mihir

2016-03-01

Approximately 50 genes other than heat shock protein (HSP) expression changes during thermal stress. These genes like nitric oxide synthase (NOS) need proper attention and investigation to find out their possible role in the adaptation to thermal stress in animals. So, the present study was undertaken to demonstrate the expressions of inducible form type II NOS (iNOS), endothelial type III NOS (eNOS), constitutively expressed enzyme NOS (cNOS), HSP70, and HSP90 in peripheral blood mononuclear cells (PBMCs) during different seasons in Barbari goats. Real-time polymerase chain reaction, western blot, and immunocytochemistry were applied to investigate messenger RNA (mRNA) expression, protein expression, and immunolocalization of examined factors. The mRNA and protein expressions of iNOS, eNOS, cNOS, HSP70, and HSP90 were significantly higher ( P < 0.05) during peak summer, and iNOS and eNOS expressions were also observed to be significantly higher ( P < 0.05) during peak winter season as compared with moderate season. The iNOS, eNOS, cNOS, HSP70, and HSP90 were mainly localized in plasma membrane and cytoplasm of PBMCs. To conclude, data generated in the present study indicate the possible involvement of the NOS family genes in amelioration of thermal stress so as to maintain cellular integrity and homeostasis in goats.

The clinical application of HPV E6/E7 mRNA testing in triaging women with atypical squamous cells of undetermined significance or low-grade squamous intra-epithelial lesion Pap smear: A meta-analysis.

PubMed

Yang, Li; Zhu, Yuanhang; Bai, Yang; Zhang, Xiaoan; Ren, Chenchen

2017-01-01

The aim is to evaluate the clinical application value and correlation with cervical lesions' progression of human papillomavirus (HPV) E6/E7 mRNA test in women with atypical squamous cells of undetermined significance (ASCUS/borderline) or low-grade squamous intraepithelial lesions (LSILs/mild dyskaryosis) cytological abnormalities. A meta-analysis was conduct by searching China National Knowledge Infrastructure (1979-2016), Wanfang Date (1998-2016), VIP (1989-2016), PubMed (1950-2016), Web of Science (1950-2016) and Elsevier Science Direct (1998-2016), for studies on effect of HPV E6/E7 mRNA detection in women with ASCUS/LSIL/dyskaryosis. Study selection and appraisal were conducted independently by three authors, according to inclusive and exclusive criteria. Then, a meta-analysis was performed using the RevMan4.2 software. The subgroups analysis was conducted according to women's initial HPV DNA test results. Six articles with a total of 1024 subjects were included in the study. It was concluded that a positive HPV E6/E7 mRNA tested result have a higher risk of progressing to CIN2+ in future 2 years than a negative result. The pooled relative risk (RR) is 3.08, (95% confidence interval [CI] = 1.57-6.07, P < 0.05). The same situation was also observed in the subgroup of HPV DNA tested positive group and HPV DNA tested unlimited group. The pooled RR value of the two subgroups was, respectively, 1.98, (95% CI = 1.19-1.19, P < 0.05) and 7.58, (95% CI = 3.64-3.64, P < 0.05). A positive HPV E6/E7 mRNA testing result suggested the women with ASCUS, or LSIL Pap smear was in a truly dangerous position, which is an adverse prognostic factor. It suggested that cervical lesions stay in a progressing status and these women should be referred for colposcopy and strengthen follow-up promptly. Whereas, women with a negative HPV E6/E7 mRNA testing result can increase follow-up interval, by comprehensively considering their situation, thus, avoiding unnecessary colposcopy and reducing the rate of colposcopy and biopsy.

Retinal-specific ATP-binding cassette transporter (ABCR/ABCA4) is expressed at the choroid plexus in rat brain.

PubMed

Bhongsatiern, Jiraganya; Ohtsuki, Sumio; Tachikawa, Masanori; Hori, Satoko; Terasaki, Tetsuya

2005-03-01

ATP-binding cassette (ABC) transporter A4 is a member of the ABC transporter subfamily A which has been reported to be exclusively expressed in the retina. In contrast, a previous report has suggested a possible relationship between ABCA4 and CNS function. The purpose of the present study was to investigate the localization of ABCA4 mRNA and protein in rat brain. In situ hybridization analysis revealed that ABCA4 mRNA was localized in the lateral ventricles. RT-PCR analysis detected ABCA4 mRNA in isolated rat choroid plexus and conditionally immortalized rat choroid plexus epithelial cells (TR-CSFB). Furthermore, ABCA4 protein was also detected in the isolated rat choroid plexus at about 250 kDa by western blot analysis, and its apparent molecular size was reduced by N-glycosidase F treatment. These results suggest that glycosylated ABCA4 protein is expressed in rat choroid plexus epithelial cells. ABCA4 may play a role in the function of the blood-cerebrospinal fluid barrier and affect CSF conditions.

Statistical modelling of subdiffusive dynamics in the cytoplasm of living cells: A FARIMA approach

NASA Astrophysics Data System (ADS)

Burnecki, K.; Muszkieta, M.; Sikora, G.; Weron, A.

2012-04-01

Golding and Cox (Phys. Rev. Lett., 96 (2006) 098102) tracked the motion of individual fluorescently labelled mRNA molecules inside live E. coli cells. They found that in the set of 23 trajectories from 3 different experiments, the automatically recognized motion is subdiffusive and published an intriguing microscopy video. Here, we extract the corresponding time series from this video by image segmentation method and present its detailed statistical analysis. We find that this trajectory was not included in the data set already studied and has different statistical properties. It is best fitted by a fractional autoregressive integrated moving average (FARIMA) process with the normal-inverse Gaussian (NIG) noise and the negative memory. In contrast to earlier studies, this shows that the fractional Brownian motion is not the best model for the dynamics documented in this video.

Slip of grip of a molecular motor on a crowded track: Modeling shift of reading frame of ribosome on RNA template

NASA Astrophysics Data System (ADS)

Mishra, Bhavya; Schütz, Gunter M.; Chowdhury, Debashish

2016-06-01

We develop a stochastic model for the programmed frameshift of ribosomes synthesizing a protein while moving along a mRNA template. Normally the reading frame of a ribosome decodes successive triplets of nucleotides on the mRNA in a step-by-step manner. We focus on the programmed shift of the ribosomal reading frame, forward or backward, by only one nucleotide which results in a fusion protein; it occurs when a ribosome temporarily loses its grip to its mRNA track. Special “slippery” sequences of nucleotides and also downstream secondary structures of the mRNA strand are believed to play key roles in programmed frameshift. Here we explore the role of an hitherto neglected parameter in regulating -1 programmed frameshift. Specifically, we demonstrate that the frameshift frequency can be strongly regulated also by the density of the ribosomes, all of which are engaged in simultaneous translation of the same mRNA, at and around the slippery sequence. Monte Carlo simulations support the analytical predictions obtained from a mean-field analysis of the stochastic dynamics.

Pressure overload stimulated cardiac hypertrophy leads to a rapid decrease in the mRNA for creatine kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boheler, K.; Popovich, B.; Dillmann, W.H.

1987-05-01

Cardiac hypertrophy (CH) leads to a decrease in creatine kinase (CK) enzymatic activity. To determine if the mRNA for CK also decreases with CH, they performed the following studies. Cardiac RNA was isolated from rats subjected to either abdominal aortic stenosis (AS) or sham surgery. Through Northern blot analysis, total cardiac RNA was quantitated with a CK specific /sup 32/P-labelled cDNA clone. At 3 and 8 days post-constriction, the mRNA for CK decreases by 54.6 +/- 7% and 65.3 +/- 18% respectively, whereas the heart weight increases by 19% and 37% relative to controls. Further studies indicate that CK mRNAmore » also decreases by 41.8% in hypothyroid rats (Tx) but decreases by a total of 68.1% in Tx rats subjected to 8 days of AS. Pressure overload stimulated CH leads to a rapid decrease in CK mRNA in normal and Tx rats. This CK mRNA decrease may account for the decreased efficiency of contraction seen in CH.« less

[Research of expression of L-DOPA decarboxylase in laryngeal cancer].

PubMed

Lai, Shisheng; Wan, Zhili

2014-12-01

This study aimed to investigate the expression levels of L-DOPA decarboxylase (DDC) mRNA and protein in laryngeal cancer, and to determine the clinical significance of DDC in diagnosis and prognosis of laryngeal cancer. Total RNA was isolated from 106 tissue samples surgically removed from 53 laryngeal cancer patients. A quantitative real-time polymerase chain reaction (RT-PCR) methodology based on SYBR Green I fluorescent dye was developed for the quantification of mRNA levels. In addition, Western Blot analysis was performed to detect the expression level of DDC protein. DDC mRNA expression in both primary (P= 0. 000) and recurrent (P=0. 033) laryngeal cancer samples downregulated significantly compared with their nonmalignant counterparts. Moreover, expression of DDC mRNA was not associated with age and histologic grade, but the significantly decreased mRNA were correlated with early TMN stage (P=0. 021). Additionally, DDC protein was detected in both cancerous and noncancerous tissues. Expression levels of DDC may play a vital role in the progression of laryngeal cancer, which can be served as a promising biomarker for the future clinical management of laryngeal cancer patients.

Global Phosphoproteomics Identifies a Major Role for AKT and 14-3-3 in Regulating EDC3*

PubMed Central

Larance, Mark; Rowland, Alexander F.; Hoehn, Kyle L.; Humphreys, David T.; Preiss, Thomas; Guilhaus, Michael; James, David E.

2010-01-01

Insulin plays an essential role in metabolic homeostasis in mammals, and many of the underlying biochemical pathways are regulated via the canonical phosphatidylinositol 3-kinase/AKT pathway. To identify novel metabolic actions of insulin, we conducted a quantitative proteomics analysis of insulin-regulated 14-3-3-binding proteins in muscle cells. These studies revealed a novel role for insulin in the post-transcriptional regulation of mRNA expression. EDC3, a component of the mRNA decay and translation repression pathway associated with mRNA processing bodies, was shown to be phosphorylated by AKT downstream of insulin signaling. The major insulin-regulated site was mapped to Ser-161, and phosphorylation at this site led to increased 14-3-3 binding. Functional studies indicated that induction of 14-3-3 binding to EDC3 causes morphological changes in processing body structures, inhibition of microRNA-mediated mRNA post-transcriptional regulation, and alterations in the protein- protein interactions of EDC3. These data highlight an important new arm of the insulin signaling cascade in the regulation of mRNA utilization. PMID:20051463

Decreased TIM-3 mRNA expression in peripheral blood mononuclear cells from nephropathy patients.

PubMed

Cai, X Z; Liu, N; Qiao, Y; Du, S Y; Chen, Y; Chen, D; Yu, S; Jiang, Y

2015-06-12

Increasing evidence shows that TIM-1 and TIM-3 in-fluence chronic autoimmune diseases, and their expression levels in immune cells from nephritic patients are still unknown. Real-time transcription-polymerase chain reaction analysis was used to deter-mine expression levels of TIM-1 and TIM-3 mRNA in peripheral blood mononuclear cells (PBMCs) from 36 patients with minimal change glo-merulopathy (MCG), 65 patients with lupus nephritis (LN), 78 patients with IgA nephropathy (IgAN), 55 patients with membranous nephropa-thy (MN), 22 patients with crescentic glomerulonephritis (CGN), 26 patients with anaphylactoid purpura nephritis (APN), and 63 healthy controls. TIM-3 mRNA expression significantly decreased in PBMCs from nephritic patients (LN, P < 0.0001; MCG, P < 0.0001; MN, P = 0.0031; CGN, P = 0.0464; IgAN, P = 0.0002; APN, P = 0.0392) com-pared with healthy controls. In contrast, there was no significant differ-ence in TIM-1 mRNA expression between the patients and the healthy controls. Our results suggest that insufficient expression of TIM-3 mRNA may be involved in the pathogenesis of nephropathy.

Characterization of rat calcitonin mRNA.

PubMed Central

Amara, S G; David, D N; Rosenfeld, M G; Roos, B A; Evans, R M

1980-01-01

A chimeric plasmic containing cDNA complementary to rat calcitonin mRNA has been constructed. Partial sequence analysis shows that the insert contains a nucleotide sequence encoding the complete amino acid sequence of calcitonin. Two basic amino acids precede and three basic amino acids follow the hormone sequence, suggesting that calcitonin is generated by the proteolytic cleavage of a larger precursor in a manner analogous to that of other small polypeptide hormones. The COOH-terminal proline, known to be amidated in the secreted hormone, is followed by a glycine in the precursor. The cloned calcitonin DNA was used to characterize the expression of calcitonin mRNA. Cytoplasmic mRNAs from calcitonin-producing rat medullary thyroid carcinoma lines and from normal rat thyroid glands contain a single species, 1050 nucleotides long, whch hybridizes to the cloned calcitonin cDNA. The concentration of calcitonin mRNA sequences is greater in those tumors that produce larger amounts of immunoreactive calcitonin. RNAs from other endocrine tissues, including anterior and neurointermediate lobes of rat pituitary, contain no detectable calcitonin mRNA. Images PMID:6933496

Guanylate-binding protein-1 is a potential new therapeutic target for triple-negative breast cancer.

PubMed

Quintero, Melissa; Adamoski, Douglas; Reis, Larissa Menezes Dos; Ascenção, Carolline Fernanda Rodrigues; Oliveira, Krishina Ratna Sousa de; Gonçalves, Kaliandra de Almeida; Dias, Marília Meira; Carazzolle, Marcelo Falsarella; Dias, Sandra Martha Gomes

2017-11-07

Triple-negative breast cancer (TNBC) is characterized by a lack of estrogen and progesterone receptor expression (ESR and PGR, respectively) and an absence of human epithelial growth factor receptor (ERBB2) amplification. Approximately 15-20% of breast malignancies are TNBC. Patients with TNBC often have an unfavorable prognosis. In addition, TNBC represents an important clinical challenge since it does not respond to hormone therapy. In this work, we integrated high-throughput mRNA sequencing (RNA-Seq) data from normal and tumor tissues (obtained from The Cancer Genome Atlas, TCGA) and cell lines obtained through in-house sequencing or available from the Gene Expression Omnibus (GEO) to generate a unified list of differentially expressed (DE) genes. Methylome and proteomic data were integrated to our analysis to give further support to our findings. Genes that were overexpressed in TNBC were then curated to retain new potentially druggable targets based on in silico analysis. Knocking-down was used to assess gene importance for TNBC cell proliferation. Our pipeline analysis generated a list of 243 potential new targets for treating TNBC. We finally demonstrated that knock-down of Guanylate-Binding Protein 1 (GBP1 ), one of the candidate genes, selectively affected the growth of TNBC cell lines. Moreover, we showed that GBP1 expression was controlled by epidermal growth factor receptor (EGFR) in breast cancer cell lines. We propose that GBP1 is a new potential druggable therapeutic target for treating TNBC with enhanced EGFR expression.

Where we stand, where we are moving: Surveying computational techniques for identifying miRNA genes and uncovering their regulatory role.

PubMed

Kleftogiannis, Dimitrios; Korfiati, Aigli; Theofilatos, Konstantinos; Likothanassis, Spiros; Tsakalidis, Athanasios; Mavroudi, Seferina

2013-06-01

Traditional biology was forced to restate some of its principles when the microRNA (miRNA) genes and their regulatory role were firstly discovered. Typically, miRNAs are small non-coding RNA molecules which have the ability to bind to the 3'untraslated region (UTR) of their mRNA target genes for cleavage or translational repression. Existing experimental techniques for their identification and the prediction of the target genes share some important limitations such as low coverage, time consuming experiments and high cost reagents. Hence, many computational methods have been proposed for these tasks to overcome these limitations. Recently, many researchers emphasized on the development of computational approaches to predict the participation of miRNA genes in regulatory networks and to analyze their transcription mechanisms. All these approaches have certain advantages and disadvantages which are going to be described in the present survey. Our work is differentiated from existing review papers by updating the methodologies list and emphasizing on the computational issues that arise from the miRNA data analysis. Furthermore, in the present survey, the various miRNA data analysis steps are treated as an integrated procedure whose aims and scope is to uncover the regulatory role and mechanisms of the miRNA genes. This integrated view of the miRNA data analysis steps may be extremely useful for all researchers even if they work on just a single step. Copyright © 2013 Elsevier Inc. All rights reserved.

General theory for integrated analysis of growth, gene, and protein expression in biofilms.

PubMed

Zhang, Tianyu; Pabst, Breana; Klapper, Isaac; Stewart, Philip S

2013-01-01

A theory for analysis and prediction of spatial and temporal patterns of gene and protein expression within microbial biofilms is derived. The theory integrates phenomena of solute reaction and diffusion, microbial growth, mRNA or protein synthesis, biomass advection, and gene transcript or protein turnover. Case studies illustrate the capacity of the theory to simulate heterogeneous spatial patterns and predict microbial activities in biofilms that are qualitatively different from those of planktonic cells. Specific scenarios analyzed include an inducible GFP or fluorescent protein reporter, a denitrification gene repressed by oxygen, an acid stress response gene, and a quorum sensing circuit. It is shown that the patterns of activity revealed by inducible stable fluorescent proteins or reporter unstable proteins overestimate the region of activity. This is due to advective spreading and finite protein turnover rates. In the cases of a gene induced by either limitation for a metabolic substrate or accumulation of a metabolic product, maximal expression is predicted in an internal stratum of the biofilm. A quorum sensing system that includes an oxygen-responsive negative regulator exhibits behavior that is distinct from any stage of a batch planktonic culture. Though here the analyses have been limited to simultaneous interactions of up to two substrates and two genes, the framework applies to arbitrarily large networks of genes and metabolites. Extension of reaction-diffusion modeling in biofilms to the analysis of individual genes and gene networks is an important advance that dovetails with the growing toolkit of molecular and genetic experimental techniques.

Significance of aquaporins’ expression in the prognosis of gastric cancer

PubMed Central

Thapa, Saroj; Chetry, Mandika; Huang, Kaiyu; Peng, Yangpei; Wang, Jinsheng; Wang, Jiaoni; Zhou, Yingying; Shen, Yigen; Xue, Yangjing; Ji, Kangting

2018-01-01

Gastric carcinoma is one of the most lethal malignancy at present with leading cause of cancer-related deaths worldwide. Aquaporins (AQPs) are a family of small, integral membrane proteins, which have been evidenced to play a crucial role in cell migration and proliferation of different cancer cells including gastric cancers. However, the aberrant expression of specific AQPs and its correlation to detect predictive and prognostic significance in gastric cancer remains elusive. In the present study, we comprehensively explored immunohistochemistry based map of protein expression profiles in normal tissues, cancer and cell lines from publicly available Human Protein Atlas (HPA) database. Moreover, to improve our understanding of general gastric biology and guide to find novel predictive prognostic gastric cancer biomarker, we also retrieved ‘The Kaplan–Meier plotter’ (KM plotter) online database with specific AQPs mRNA to overall survival (OS) in different clinicopathological features. We revealed that ubiquitous expression of AQPs protein can be effective tools to generate gastric cancer biomarker. Furthermore, high level AQP3, AQP9, and AQP11 mRNA expression were correlated with better OS in all gastric patients, whereas AQP0, AQP1, AQP4, AQP5, AQP6, AQP8, and AQP10 mRNA expression were associated with poor OS. With regard to the clinicopathological features including Laurens classification, clinical stage, human epidermal growth factor receptor 2 (HER2) status, and different treatment strategy, we could illustrate significant role of individual AQP mRNA expression in the prognosis of gastric cancer patients. Thus, our results indicated that AQP’s protein and mRNA expression in gastric cancer patients provide effective role to predict prognosis and act as an essential agent to therapeutic strategy. PMID:29678898

Inner nuclear envelope protein SUN1 plays a prominent role in mammalian mRNA export.

PubMed

Li, Ping; Noegel, Angelika A

2015-11-16

Nuclear export of messenger ribonucleoproteins (mRNPs) through the nuclear pore complex (NPC) can be roughly classified into two forms: bulk and specific export, involving an nuclear RNA export factor 1 (NXF1)-dependent pathway and chromosome region maintenance 1 (CRM1)-dependent pathway, respectively. SUN proteins constitute the inner nuclear envelope component of the l I: nker of N: ucleoskeleton and C: ytoskeleton (LINC) complex. Here, we show that mammalian cells require SUN1 for efficient nuclear mRNP export. The results indicate that both SUN1 and SUN2 interact with heterogeneous nuclear ribonucleoprotein (hnRNP) F/H and hnRNP K/J. SUN1 depletion inhibits the mRNP export, with accumulations of both hnRNPs and poly(A)+RNA in the nucleus. Leptomycin B treatment indicates that SUN1 functions in mammalian mRNA export involving the NXF1-dependent pathway. SUN1 mediates mRNA export through its association with mRNP complexes via a direct interaction with NXF1. Additionally, SUN1 associates with the NPC through a direct interaction with Nup153, a nuclear pore component involved in mRNA export. Taken together, our results reveal that the inner nuclear envelope protein SUN1 has additional functions aside from being a central component of the LINC complex and that it is an integral component of the mammalian mRNA export pathway suggesting a model whereby SUN1 recruits NXF1-containing mRNP onto the nuclear envelope and hands it over to Nup153. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Modeling corticosteroid effects in a rat model of rheumatoid arthritis II: mechanistic pharmacodynamic model for dexamethasone effects in Lewis rats with collagen-induced arthritis.

PubMed

Earp, Justin C; Dubois, Debra C; Molano, Diana S; Pyszczynski, Nancy A; Almon, Richard R; Jusko, William J

2008-08-01

A mechanism-based model for pharmacodynamic effects of dexamethasone (DEX) was incorporated into our model for arthritis disease progression in the rat to aid in identification of the primary factors responsible for edema and bone loss. Collagen-induced arthritis was produced in male Lewis rats after injection of type II porcine collagen. DEX was given subcutaneously in single doses of 0.225 or 2.25 mg/kg or 7-day multiple doses of 0.045 or 0.225 mg/kg at 21 days postdisease induction. Effects on disease progression were measured by paw swelling, bone mineral density (BMD), body weights, plasma corticosterone (CST), and tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and glucocorticoid receptor (GR) mRNA expression in paw tissue. Lumbar and femur BMD was determined by PIXImus II dual-energy X-ray absorptiometry. Plasma CST was assayed by high-performance liquid chromatography. Cytokine and GR mRNA were assayed by quantitative real-time polymerase chain reaction. Indirect response models, drug interaction models, transduction processes, and the fifth-generation model of corticosteroid dynamics were integrated and applied using S-ADAPT software to describe how dexamethasone binding to GR can regulate diverse processes. Cytokine mRNA, GR mRNA, plasma CST, and paw edema were suppressed after DEX administration. TNF-alpha mRNA expression and BMD seemed to increase immediately after dosing but were ultimately reduced. Model parameters indicated that IL-6 and IL-1beta were most sensitive to inhibition by DEX. TNF-alpha seemed to primarily influence edema, whereas IL-6 contributed the most to bone loss. Lower doses of corticosteroids may be sufficient to suppress the cytokines most relevant to bone erosion.

Small RNA-based prediction of hybrid performance in maize.

PubMed

Seifert, Felix; Thiemann, Alexander; Schrag, Tobias A; Rybka, Dominika; Melchinger, Albrecht E; Frisch, Matthias; Scholten, Stefan

2018-05-21

Small RNA (sRNA) sequences are known to have a broad impact on gene regulation by various mechanisms. Their performance for the prediction of hybrid traits has not yet been analyzed. Our objective was to analyze the relation of parental sRNA expression with the performance of their hybrids, to develop a sRNA-based prediction approach, and to compare it to more common SNP and mRNA transcript based predictions using a factorial mating scheme of a maize hybrid breeding program. Correlation of genomic differences and messenger RNA (mRNA) or sRNA expression differences between parental lines with hybrid performance of their hybrids revealed that sRNAs showed an inverse relationship in contrast to the other two data types. We associated differences for SNPs, mRNA and sRNA expression between parental inbred lines with the performance of their hybrid combinations and developed two prediction approaches using distance measures based on associated markers. Cross-validations revealed parental differences in sRNA expression to be strong predictors for hybrid performance for grain yield in maize, comparable to genomic and mRNA data. The integration of both positively and negatively associated markers in the prediction approaches enhanced the prediction accurary. The associated sRNAs belong predominantly to the canonical size classes of 22- and 24-nt that show specific genomic mapping characteristics. Expression profiles of sRNA are a promising alternative to SNPs or mRNA expression profiles for hybrid prediction, especially for plant species without reference genome or transcriptome information. The characteristics of the sRNAs we identified suggest that association studies based on breeding populations facilitate the identification of sRNAs involved in hybrid performance.

Expression of transforming growth factor alpha and epidermal growth factor receptor messenger RNA in neoplastic and nonneoplastic human kidney tissue.

PubMed

Mydlo, J H; Michaeli, J; Cordon-Cardo, C; Goldenberg, A S; Heston, W D; Fair, W R

1989-06-15

Using Northern blot analysis, we have demonstrated that mRNA for transforming growth factor alpha (TGF-alpha) was expressed in five malignant kidney tissue specimens but was not detected in their autologous nonneoplastic homologues. In addition, the expression of epidermal growth factor (EGF) receptor mRNA in these malignant tissues was 2- to 3-fold greater than in nontransformed tissues. In two cases examined using immunohistochemistry, we were able to correlate the increased expression of the mRNA with an increase in protein expression. Since TGF-alpha is known to bind to the EGF receptor, the finding of an increased expression of both TGF-alpha and EGF receptor mRNA in kidney tumor tissue suggests that interaction between TGF-alpha and the EGF receptor may play a role in promoting transformation and/or proliferation of kidney neoplasms, perhaps by an autocrine mechanism.

An Analysis of the Sensitivity of Proteogenomic Mapping of Somatic Mutations and Novel Splicing Events in Cancer.

PubMed

Ruggles, Kelly V; Tang, Zuojian; Wang, Xuya; Grover, Himanshu; Askenazi, Manor; Teubl, Jennifer; Cao, Song; McLellan, Michael D; Clauser, Karl R; Tabb, David L; Mertins, Philipp; Slebos, Robbert; Erdmann-Gilmore, Petra; Li, Shunqiang; Gunawardena, Harsha P; Xie, Ling; Liu, Tao; Zhou, Jian-Ying; Sun, Shisheng; Hoadley, Katherine A; Perou, Charles M; Chen, Xian; Davies, Sherri R; Maher, Christopher A; Kinsinger, Christopher R; Rodland, Karen D; Zhang, Hui; Zhang, Zhen; Ding, Li; Townsend, R Reid; Rodriguez, Henry; Chan, Daniel; Smith, Richard D; Liebler, Daniel C; Carr, Steven A; Payne, Samuel; Ellis, Matthew J; Fenyő, David

2016-03-01

Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations, and splice variants identified in cancer cells are translated. Herein, we apply a proteogenomic data integration tool (QUILTS) to illustrate protein variant discovery using whole genome, whole transcriptome, and global proteome datasets generated from a pair of luminal and basal-like breast-cancer-patient-derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS sample process replicates defined here as an independent tandem MS experiment using identical sample material. Despite analysis of over 30 sample process replicates, only about 10% of SNVs (somatic and germline) detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNVs without a detectable mRNA transcript were also observed, suggesting that transcriptome coverage was incomplete (∼80%). In contrast to germline variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than in the luminal tumor, raising the possibility of differential translation or protein degradation effects. In conclusion, this large-scale proteogenomic integration allowed us to determine the degree to which mutations are translated and identify gaps in sequence coverage, thereby benchmarking current technology and progress toward whole cancer proteome and transcriptome analysis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Exploring Valid Reference Genes for Quantitative Real-time PCR Analysis in Plutella xylostella (Lepidoptera: Plutellidae)

PubMed Central

Fu, Wei; Xie, Wen; Zhang, Zhuo; Wang, Shaoli; Wu, Qingjun; Liu, Yong; Zhou, Xiaomao; Zhou, Xuguo; Zhang, Youjun

2013-01-01

Abstract: Quantitative real-time PCR (qRT-PCR), a primary tool in gene expression analysis, requires an appropriate normalization strategy to control for variation among samples. The best option is to compare the mRNA level of a target gene with that of reference gene(s) whose expression level is stable across various experimental conditions. In this study, expression profiles of eight candidate reference genes from the diamondback moth, Plutella xylostella, were evaluated under diverse experimental conditions. RefFinder, a web-based analysis tool, integrates four major computational programs including geNorm, Normfinder, BestKeeper, and the comparative ΔCt method to comprehensively rank the tested candidate genes. Elongation factor 1 (EF1) was the most suited reference gene for the biotic factors (development stage, tissue, and strain). In contrast, although appropriate reference gene(s) do exist for several abiotic factors (temperature, photoperiod, insecticide, and mechanical injury), we were not able to identify a single universal reference gene. Nevertheless, a suite of candidate reference genes were specifically recommended for selected experimental conditions. Our finding is the first step toward establishing a standardized qRT-PCR analysis of this agriculturally important insect pest. PMID:23983612

Assessment of potential biomarkers, metallothionein and vitellogenin mRNA expressions in various chemically exposed benthic Chironomus riparius larvae

NASA Astrophysics Data System (ADS)

Park, Kiyun; Kwak, Inn-Sil

2012-12-01

The objective of this study was conducted to identify the possibility of using Chironomus metallothionein (MT) and vitellogenin (VTG) as biomarkers of stress caused by endocrinedisrupting chemicals (EDCs), heavy metals, herbicides and veterinary antibiotics. We characterized the MT and VTG cDNA in Chironomus riparius and evaluated their mRNA expression profiles following exposure to different environmental pollutants. The gene expression analysis showed that the MT mRNA levels increased significantly after long-term exposure to cadmium (Cd), copper (Cu), Lead (Pb), di(2-ethylhexyl) phthalate (DEHP), and 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, the VTG mRNA expression increased significantly in C. riparius larvae exposed to BPA, NP, DEHP, Cd, 2,4-D and fenbendazole. Evaluation of the long-term effects of environmental pollutants revealed up regulation of Chironomus MT mRNA in response to DEHP exposure among EDCs, and the level of the VTG mRNA was increased significantly following treatment with Cd and herbicide 2,4-D at all concentrations in a dose-dependent manner. These results indicate that VTG could be used as a potential biomarker of herbicide and Cd as well as EDCs, while MT was a potential biomarker of heavy metals such as Cd, Cu, and Pb in aquatic environments.

Effect of Supplemental Trace Minerals on Hsp-70 mRNA Expression in Commercial Broiler Chicken.

PubMed

Rajkumar, U; Vinoth, A; Reddy, E Pradeep Kumar; Shanmugam, M; Rao, S V Rama

2018-01-02

The effects of supplementing the organic forms of selenium (Se), chromium (Cr), and zinc (Zn) on Hsp-70 mRNA expression and body weight in broiler chickens were evaluated. 200 chicks were equally distributed into stainless steel battery brooders at the rate of 5 birds per pen and reared under heat stress condition up to 42 nd day. The chicks were fed with three experimental diets supplemented with organic forms of Se (0.30 mg/kg), Cr (2 mg/kg), and Zn (40 mg/kg) during the starter and finisher phases and a control diet without any supplementation. On the 21st and 42nd day, 20 birds from each period were sacrificed and samples were collected for analysis. Organic Se, Cr, and Zn supplementation significantly (P < 0.05) reduced the expression of Hsp-70 mRNA levels. The Hsp-70 mRNA expression levels were significantly (P < 0.05) different between the tissues studied with spleen having the lowest expression level. Hsp-70 mRNA expression level was not affected by age of the birds. The study concluded that organic trace mineral (oTM) supplementation resulted in low Hsp-70 mRNA expression, indicating reduced heat stress in broilers.

Hamp1 mRNA and plasma hepcidin levels are influenced by sex and strain but do not predict tissue iron levels in inbred mice.

PubMed

McLachlan, Stela; Page, Kathryn E; Lee, Seung-Min; Loguinov, Alex; Valore, Erika; Hui, Simon T; Jung, Grace; Zhou, Jie; Lusis, Aldons J; Fuqua, Brie; Ganz, Tomas; Nemeth, Elizabeta; Vulpe, Chris D

2017-11-01

Iron homeostasis is tightly regulated, and the peptide hormone hepcidin is considered to be a principal regulator of iron metabolism. Previous studies in a limited number of mouse strains found equivocal sex- and strain-dependent differences in mRNA and serum levels of hepcidin and reported conflicting data on the relationship between hepcidin ( Hamp1 ) mRNA levels and iron status. Our aim was to clarify the relationships between strain, sex, and hepcidin expression by examining multiple tissues and the effects of different dietary conditions in multiple inbred strains. Two studies were done: first, Hamp1 mRNA, liver iron, and plasma diferric transferrin levels were measured in 14 inbred strains on a control diet; and second, Hamp1 mRNA and plasma hepcidin levels in both sexes and iron levels in the heart, kidneys, liver, pancreas, and spleen in males were measured in nine inbred/recombinant inbred strains raised on an iron-sufficient or high-iron diet. Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). However, liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice fed iron-sufficient or high-iron diets, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males. We also measured plasma erythroferrone, performed RNA-sequencing analysis of liver samples from six inbred strains fed the iron-sufficient, low-iron, or high-iron diets, and explored differences in gene expression between the strains with the highest and lowest hepcidin levels. NEW & NOTEWORTHY Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). Liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males. Copyright © 2017 the American Physiological Society.

β-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies.

PubMed

Kraan, Claudine M; Cornish, Kim M; Bui, Quang M; Li, Xin; Slater, Howard R; Godler, David E

2018-01-01

Relationships between Fragile X Mental Retardation 1 (FMR1) mRNA levels in blood and intragenic FMR1 CGG triplet expansions support the pathogenic role of RNA gain of function toxicity in premutation (PM: 55-199 CGGs) related disorders. Real-time PCR (RT-PCR) studies reporting these findings normalised FMR1 mRNA level to a single internal control gene called β-glucuronidase (GUS). This study evaluated FMR1 mRNA-CGG correlations in 33 PM and 33 age- and IQ-matched control females using three normalisation strategies in peripheral blood mononuclear cells (PBMCs): (i) GUS as a single internal control; (ii) the mean of GUS, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA); and (iii) the mean of EIF4A2 and SDHA (with no contribution from GUS). GUS mRNA levels normalised to the mean of EIF4A2 and SDHA mRNA levels and EIF4A2/SDHA ratio were also evaluated. FMR1mRNA level normalised to the mean of EIF4A2 and SDHA mRNA levels, with no contribution from GUS, showed the most significant correlation with CGG size and the greatest difference between PM and control groups (p = 10-11). Only 15% of FMR1 mRNA PM results exceeded the maximum control value when normalised to GUS, compared with over 42% when normalised to the mean of EIF4A2 and SDHA mRNA levels. Neither GUS mRNA level normalised to the mean RNA levels of EIF4A2 and SDHA, nor to the EIF4A2/SDHA ratio were correlated with CGG size. However, greater variability in GUS mRNA levels were observed for both PM and control females across the full range of CGG repeat as compared to the EIF4A2/SDHA ratio. In conclusion, normalisation with multiple control genes, excluding GUS, can improve assessment of the biological significance of FMR1 mRNA-CGG size relationships.

A PCR-aided transcript titration assay (PATTY) to measure topoisomerase I gene expression in human tumor specimens.

PubMed

Meersma, G J; Bakker, M; Groen, H J; Van der Zee, A G; Jensen, P B; Giaccone, G; De Vries, E G; Smit, E F

1999-01-01

Topoisomerase I (topo I) inhibitors are promising anticancer agents with demonstrated activity against a wide range of solid tumors. Quantitative information on topol mRNA levels in tumor biopsies may predict response to topo I inhibitors. A polymerase chain reaction aided transcript titration assay (PATTY) was developed to allow quantitation of topol mRNA in small samples. Concentrations of topol mRNA in total RNA samples were estimated by RT-PCR analysis in a human small cell lung cancer (SCLC) cell line (GLC,) and its topotecan (GL2C/SK and F) and camptothecin (GL2C/Campt) resistant sublines, human non-small cell lung cancer (NSCLC) and ovarian carcinoma samples. Topol PATTY showed a decreased topo I mRNA level in GLC2/SK and F (4.5 pg/100 ng total RNA) and GLC,/Campt (2.2 pg/100 ng total RNA), respectively, compared to the parent cell line GLC2 (5.4 pg/100 ng total RNA). Topol protein levels as measured by Western blotting were compatible with topol mRNA levels. Median (range) topol mRNA levels were 3.23 (2.33-5.10) pg/100 ng total RNA in resected NSCLC specimen (n = 6), and 2.03 (0.54-0.95) pg/100 ng total RNA in resected ovarian cancer specimen (n = 6). We conclude that topol PATTY is a new assay that quantitates topol mRNA levels in cell lines and small tumor samples.

Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism

PubMed Central

Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K.; Lehtonen, Jukka Y.A.

2016-01-01

As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3′-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. PMID:26681690

Comprehensive Identification of mRNA-Binding Proteins of Leishmania donovani by Interactome Capture.

PubMed

Nandan, Devki; Thomas, Sneha A; Nguyen, Anne; Moon, Kyung-Mee; Foster, Leonard J; Reiner, Neil E

2017-01-01

Leishmania are unicellular eukaryotes responsible for leishmaniasis in humans. Like other trypanosomatids, leishmania regulate protein coding gene expression almost exclusively at the post-transcriptional level with the help of RNA binding proteins (RBPs). Due to the presence of polycystronic transcription units, leishmania do not regulate RNA polymerase II-dependent transcription initiation. Recent evidence suggests that the main control points in gene expression are mRNA degradation and translation. Protein-RNA interactions are involved in every aspect of RNA biology, such as mRNA splicing, polyadenylation, localization, degradation, and translation. A detailed picture of these interactions would likely prove to be highly informative in understanding leishmania biology and virulence. We developed a strategy involving covalent UV cross-linking of RBPs to mRNA in vivo, followed by interactome capture using oligo(dT) magnetic beads to define comprehensively the mRNA interactome of growing L. donovani amastigotes. The protein mass spectrometry analysis of captured proteins identified 79 mRNA interacting proteins which withstood very stringent washing conditions. Strikingly, we found that 49 of these mRNA interacting proteins had no orthologs or homologs in the human genome. Consequently, these may represent high quality candidates for selective drug targeting leading to novel therapeutics. These results show that this unbiased, systematic strategy has the promise to be applicable to study the mRNA interactome during various biological settings such as metabolic changes, stress (low pH environment, oxidative stress and nutrient deprivation) or drug treatment.

NMD3 regulates both mRNA and rRNA nuclear export in African trypanosomes via an XPOI-linked pathway

PubMed Central

Bühlmann, Melanie; Walrad, Pegine; Rico, Eva; Ivens, Alasdair; Capewell, Paul; Naguleswaran, Arunasalam; Roditi, Isabel; Matthews, Keith R.

2015-01-01

Trypanosomes mostly regulate gene expression through post-transcriptional mechanisms, particularly mRNA stability. However, much mRNA degradation is cytoplasmic such that mRNA nuclear export must represent an important level of regulation. Ribosomal RNAs must also be exported from the nucleus and the trypanosome orthologue of NMD3 has been confirmed to be involved in rRNA processing and export, matching its function in other organisms. Surprisingly, we found that TbNMD3 depletion also generates mRNA accumulation of procyclin-associated genes (PAGs), these being co-transcribed by RNA polymerase I with the procyclin surface antigen genes expressed on trypanosome insect forms. By whole transcriptome RNA-seq analysis of TbNMD3-depleted cells we confirm the regulation of the PAG transcripts by TbNMD3 and using reporter constructs reveal that PAG1 regulation is mediated by its 5′UTR. Dissection of the mechanism of regulation demonstrates that it is not dependent upon translational inhibition mediated by TbNMD3 depletion nor enhanced transcription. However, depletion of the nuclear export factors XPO1 or MEX67 recapitulates the effects of TbNMD3 depletion on PAG mRNAs and mRNAs accumulated in the nucleus of TbNMD3-depleted cells. These results invoke a novel RNA regulatory mechanism involving the NMD3-dependent nuclear export of mRNA cargos, suggesting a shared platform for mRNA and rRNA export. PMID:25873624

Inactivation of MSH3 by promoter methylation correlates with primary tumor stage in nasopharyngeal carcinoma

PubMed Central

Ni, Haifeng; Jiang, Bo; Zhou, Zhen; Yuan, Xiaoyang; Cao, Xiaolin; Huang, Guangwu; Li, Yong

2017-01-01

The aim of this study was to investigate the inactivation of the MutS homolog human 3 (MSH3) gene by promoter methylation in nasopharyngeal carcinoma (NPC). Methylation-specific PCR, semi-quantitative reverse transcription PCR and immunohistochemical analysis were used to detect methylation and the mRNA and protein expression levels of MSH3 in 54 cases of NPC tissues and 16 cases of normal nasopharyngeal epithelial (NNE) tissues. The association between promoter methylation and mRNA expression, and the mRNA and protein expression of the gene and clinical factors was analyzed. The promoter methylation of MSH3 was detected in 50% (27/54) of the primary tumors, but not in the 16 NNE tissues. The mRNA and protein expression levels were significantly decreased in the 54 cases of human NPC as compared to the 16 NNE tissues (P<0.05). The MSH3-methylated cases exhibited significantly lower mRNA and protein expression levels than the unmethylated cases (P<0.05). The MSH3 mRNA and protein expression levels were significantly associated with the variable T stage (P<0.05); however, they did not correlate with the age and sex of the patients, or with the N stage, TNM classification or histopathological subtype (P>0.05). On the whole, MSH3 was frequently inactivated by promoter methylation and its mRNA and protein expression correlated with the primary tumor stage in NPC. PMID:28656302

Molecular cloning, characterization, and expression analysis of a heat shock protein (HSP) 70 gene from Paphia undulata.

PubMed

Wu, Xiangwei; Tan, Jing; Cai, Mingyi; Liu, Xiande

2014-06-15

In this study, a full-length HSP70 cDNA from Paphia undulata was cloned using reverse transcriptase polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends (RACE). The full-length cDNA is 2,351 bp, consisting of a 5'-untranslated region (UTR) of 83 bp, a 3'-UTR of 315 bp, and an open reading frame (ORF) of 1,953 bp. This cDNA encodes 650 amino acids with an estimated molecular weight of 71.3 kDa and an isoelectric point of 5.51. Based on the amino acid sequence analysis and phylogenetic analysis, this HSP70 gene was identified as a member of the cytoplasmic HSP70 family, being the constitutive expression, and it was designated as PuHSC70. The distribution of PuHSC70 mRNA in the mantle, digestive gland, adductor muscle, gonad, gill, heart, and hemocytes suggested that PuHSC70 is ubiquitously expressed. The mRNA levels of PuHSC70 under high temperature and high salinity stresses were analyzed by real-time PCR. Under high temperature stress of 32°C, PuHSC70 mRNA in the mantle, digestive gland, gill, and heart was significantly up-regulated at 1h and 2h, and it was then progressively down-regulated. In the adductor muscle, the level of PuHSC70 mRNA gradually increased throughout the study period; the mRNA levels in the gonad and hemocytes increased significantly at 4h and 8h (P<0.05) and then decreased at 8h and 14 h, respectively, however they increased again afterwards, reaching the highest levels at 50h. Under high salinity (32 ‰) stress, the mRNA levels of PuHSC70 in the mantle and gonad were increased significantly only at 24h and 48 h (P<0.05), and at the rest of the study period they were slightly elevated. Compared with the pretreatment level, the levels of expression in the digestive gland and gill were unchanged or reduced throughout the study period. The levels of PuHSC70 mRNA in the adductor muscle, hemocytes, and heart were significantly increased, reaching a maximum at 24h, and then they gradually decreased; moreover, in the heart, the mRNA expression recovered to the pretreatment level at 50h; while in the adductor muscle and hemocytes, the expression level remained higher than that of the control. The cloning and expression analyses of PuHSC70 provide theoretical basis to further study the mechanism of physiological response to thermal and high salinity stresses. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel in vitro bovine cartilage punch model for assessing the regeneration of focal cartilage defects with biocompatible bacterial nanocellulose.

PubMed

Pretzel, David; Linss, Stefanie; Ahrem, Hannes; Endres, Michaela; Kaps, Christian; Klemm, Dieter; Kinne, Raimund W

2013-01-01

Current therapies for articular cartilage defects fail to achieve qualitatively sufficient tissue regeneration, possibly because of a mismatch between the speed of cartilage rebuilding and the resorption of degradable implant polymers. The present study focused on the self-healing capacity of resident cartilage cells in conjunction with cell-free and biocompatible (but non-resorbable) bacterial nanocellulose (BNC). This was tested in a novel in vitro bovine cartilage punch model. Standardized bovine cartilage discs with a central defect filled with BNC were cultured for up to eight weeks with/without stimulation with transforming growth factor-β1 (TGF-β1. Cartilage formation and integrity were analyzed by histology, immunohistochemistry and electron microscopy. Content, release and neosynthesis of the matrix molecules proteoglycan/aggrecan, collagen II and collagen I were also quantified. Finally, gene expression of these molecules was profiled in resident chondrocytes and chondrocytes migrated onto the cartilage surface or the implant material. Non-stimulated and especially TGF-β1-stimulated cartilage discs displayed a preserved structural and functional integrity of the chondrocytes and surrounding matrix, remained vital in long-term culture (eight weeks) without signs of degeneration and showed substantial synthesis of cartilage-specific molecules at the protein and mRNA level. Whereas mobilization of chondrocytes from the matrix onto the surface of cartilage and implant was pivotal for successful seeding of cell-free BNC, chondrocytes did not immigrate into the central BNC area, possibly due to the relatively small diameter of its pores (2 to 5 μm). Chondrocytes on the BNC surface showed signs of successful redifferentiation over time, including increase of aggrecan/collagen type II mRNA, decrease of collagen type I mRNA and initial deposition of proteoglycan and collagen type II in long-term high-density pellet cultures. Although TGF-β1 stimulation showed protective effects on matrix integrity, effects on other parameters were limited. The present bovine cartilage punch model represents a robust, reproducible and highly suitable tool for the long-term culture of cartilage, maintaining matrix integrity and homoeostasis. As an alternative to animal studies, this model may closely reflect early stages of cartilage regeneration, allowing the evaluation of promising biomaterials with/without chondrogenic factors.

Metabolic syndrome alters expression of insulin signaling-related genes in swine mesenchymal stem cells.

PubMed

Conley, Sabena M; Zhu, Xiang-Yang; Eirin, Alfonso; Tang, Hui; Lerman, Amir; van Wijnen, Andre J; Lerman, Lilach O

2018-02-20

Metabolic syndrome (MetS) is associated with insulin resistance (IR) and impaired glucose metabolism in muscle, fat, and other cells, and may induce inflammation and vascular remodeling. Endogenous reparative systems, including adipose tissue-derived mesenchymal stem/stromal cells (MSC), are responsible for repair of damaged tissue. MSC have also been proposed as an exogenous therapeutic intervention in patients with cardiovascular and chronic kidney disease (CKD). The feasibility of using autologous cells depends on their integrity, but whether in MetS IR involves adipose tissue-derived MSC remains unknown. The aim of this study was to examine the expression of mRNA involved in insulin signaling in MSC from subjects with MetS. Domestic pigs consumed a lean or obese diet (n=6 each) for 16weeks. MSC were collected from subcutaneous abdominal fat and analyzed using high-throughput RNA-sequencing for expression of genes involved in insulin signaling. Expression profiles for enriched (fold change>1.4, p<0.05) and suppressed (fold change<0.7, p<0.05) mRNAs in MetS pigs were functionally interpreted by gene ontology analysis. The most prominently upregulated and downregulated mRNAs were further probed. We identified in MetS-MSC 168 up-regulated and 51 down-regulated mRNAs related to insulin signaling. Enriched mRNAs were implicated in biological pathways including hepatic glucose metabolism, adipocyte differentiation, and transcription regulation, and down-regulated mRNAs in intracellular calcium signaling and cleaving peptides. Functional analysis suggested that overall these alterations could increase IR. MetS alters mRNA expression related to insulin signaling in adipose tissue-derived MSC. These observations mandate caution during administration of autologous MSC in subjects with MetS. Copyright © 2017. Published by Elsevier B.V.

Predicting miRNA targets for head and neck squamous cell carcinoma using an ensemble method.

PubMed

Gao, Hong; Jin, Hui; Li, Guijun

2018-01-01

This study aimed to uncover potential microRNA (miRNA) targets in head and neck squamous cell carcinoma (HNSCC) using an ensemble method which combined 3 different methods: Pearson's correlation coefficient (PCC), Lasso and a causal inference method (i.e., intervention calculus when the directed acyclic graph (DAG) is absent [IDA]), based on Borda count election. The Borda count election method was used to integrate the top 100 predicted targets of each miRNA generated by individual methods. Afterwards, to validate the performance ability of our method, we checked the TarBase v6.0, miRecords v2013, miRWalk v2.0 and miRTarBase v4.5 databases to validate predictions for miRNAs. Pathway enrichment analysis of target genes in the top 1,000 miRNA-messenger RNA (mRNA) interactions was conducted to focus on significant KEGG pathways. Finally, we extracted target genes based on occurrence frequency ≥3. Based on an absolute value of PCC >0.7, we found 33 miRNAs and 288 mRNAs for further analysis. We extracted 10 target genes with predicted frequencies not less than 3. The target gene MYO5C possessed the highest frequency, which was predicted by 7 different miRNAs. Significantly, a total of 8 pathways were identified; the pathways of cytokine-cytokine receptor interaction and chemokine signaling pathway were the most significant. We successfully predicted target genes and pathways for HNSCC relying on miRNA expression data, mRNA expression profile, an ensemble method and pathway information. Our results may offer new information for the diagnosis and estimation of the prognosis of HNSCC.

Chronic gastrointestinal inflammation induces anxiety-like behavior and alters central nervous system biochemistry in mice.

PubMed

Bercik, Premysl; Verdu, Elena F; Foster, Jane A; Macri, Joseph; Potter, Murray; Huang, Xiaxing; Malinowski, Paul; Jackson, Wendy; Blennerhassett, Patricia; Neufeld, Karen A; Lu, Jun; Khan, Waliul I; Corthesy-Theulaz, Irene; Cherbut, Christine; Bergonzelli, Gabriela E; Collins, Stephen M

2010-12-01

Clinical and preclinical studies have associated gastrointestinal inflammation and infection with altered behavior. We investigated whether chronic gut inflammation alters behavior and brain biochemistry and examined underlying mechanisms. AKR mice were infected with the noninvasive parasite Trichuris muris and given etanercept, budesonide, or specific probiotics. Subdiaphragmatic vagotomy was performed in a subgroup of mice before infection. Gastrointestinal inflammation was assessed by histology and quantification of myeloperoxidase activity. Serum proteins were measured by proteomic analysis, circulating cytokines were measured by fluorescence activated cell sorting array, and serum tryptophan and kynurenine were measured by liquid chromatography. Behavior was assessed using light/dark preference and step-down tests. In situ hybridization was used to assess brain-derived neurotrophic factor (BDNF) expression in the brain. T muris caused mild to moderate colonic inflammation and anxiety-like behavior that was associated with decreased hippocampal BDNF messenger RNA (mRNA). Circulating tumor necrosis factor-α and interferon-γ, as well as the kynurenine and kynurenine/tryptophan ratio, were increased. Proteomic analysis showed altered levels of several proteins related to inflammation and neural function. Administration of etanercept, and to a lesser degree of budesonide, normalized behavior, reduced cytokine and kynurenine levels, but did not influence BDNF expression. The probiotic Bifidobacterium longum normalized behavior and BDNF mRNA but did not affect cytokine or kynurenine levels. Anxiety-like behavior was present in infected mice after vagotomy. Chronic gastrointestinal inflammation induces anxiety-like behavior and alters central nervous system biochemistry, which can be normalized by inflammation-dependent and -independent mechanisms, neither of which requires the integrity of the vagus nerve. Copyright © 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.

Global miRNA expression and correlation with mRNA levels in primary human bone cells

PubMed Central

Laxman, Navya; Rubin, Carl-Johan; Mallmin, Hans; Nilsson, Olle; Pastinen, Tomi; Grundberg, Elin; Kindmark, Andreas

2015-01-01

MicroRNAs (miRNAs) are important post-transcriptional regulators that have recently introduced an additional level of intricacy to our understanding of gene regulation. The aim of this study was to investigate miRNA–mRNA interactions that may be relevant for bone metabolism by assessing correlations and interindividual variability in miRNA levels as well as global correlations between miRNA and mRNA levels in a large cohort of primary human osteoblasts (HOBs) obtained during orthopedic surgery in otherwise healthy individuals. We identified differential expression (DE) of 24 miRNAs, and found 9 miRNAs exhibiting DE between males and females. We identified hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b and their target genes as important modulators of bone metabolism. Further, we used an integrated analysis of global miRNA–mRNA correlations, mRNA-expression profiling, DE, bioinformatics analysis, and functional studies to identify novel target genes for miRNAs with the potential to regulate osteoblast differentiation and extracellular matrix production. Functional studies by overexpression and knockdown of miRNAs showed that, the differentially expressed miRNAs hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b target genes highly relevant to bone metabolism, e.g., collagen, type I, α1 (COL1A1), osteonectin (SPARC), Runt-related transcription factor 2 (RUNX2), osteocalcin (BGLAP), and frizzled-related protein (FRZB). These miRNAs orchestrate the activities of key regulators of osteoblast differentiation and extracellular matrix proteins by their convergent action on target genes and pathways to control the skeletal gene expression. PMID:26078267

Mechanisms of fibrosis in acute liver failure.

PubMed

He, Yingli; Jin, Li; Wang, Jing; Yan, Zhi; Chen, Tianyan; Zhao, Yingren

2015-07-01

Acute liver failure (ALF) is a condition with high mortality and morbidity. Fibrosis in chronic liver disease was extensively researched, whereas fibrosis and underlying mechanism in acute liver failure remains unclear. Hepatitis B virus related ALF patients were recruited to investigate if there was ongoing fibrosis by liver histology and liver stiffness measurement(LSM) analysis as well as fibrosis markers assay. Sera HMGB1 were kinetically detected in progression and remission stage of ALF. Hepatic stellate cell(HSC) activation by HMGB1 was explored by testing mRNA and protein level of α-SMA and collagen 1a1 by using qPCR and western blot. Autophagy induction by HMGB1 was explored by LC3-II conversion, autophagy flux and fluorescence. Firstly, ongoing fibrosis in progression stage of ALF was confirmed by histological analysis, LS measurement as well as fibrosis markers detection. HSC activation and autophagy induction in explanted liver tissue also revealed. Next, kinetic monitoring sera HMGB1 revealed elevated HMGB1 in progression stage of ALF vs HBsAg carrier, and drop back to base level in remission stage. Thirdly, rHMGB1 dose dependently activated HSCs, as indicated by increased mRNA and proteins level in α-SMA and collagen 1a1. Moreover, autophagy was induced in HSC treated with rHMGB1, as illustrated by increased LC3 lipidation, elevated autophagy flux and GFP-LC3 puncta. Acute liver failure is accompanied by ongoing fibrosis, HSC activation and autophagy induction. Increased HMGB1 activates HSC via autophagy induction. Those findings integrate HMGB1, HSCs activation, autophagy into a common framework that underlies the fibrosis in ALF. © 2014 The Authors. Liver International Published by John Wiley & Sons Ltd.

The flow dependency of Tie2 expression in endotoxemia.

PubMed

Kurniati, Neng F; Jongman, Rianne M; vom Hagen, Franziska; Spokes, Katherine C; Moser, Jill; Regan, Erzsébet Ravasz; Krenning, Guido; Moonen, Jan-Renier A J; Harmsen, Martin C; Struys, Michel M R F; Hammes, Hans-Peter; Zijlstra, Jan G; Aird, William C; Heeringa, Peter; Molema, Grietje; van Meurs, Matijs

2013-07-01

Tie2 is predominantly expressed by endothelial cells and is involved in vascular integrity control during sepsis. Changes in Tie2 expression during sepsis development may contribute to microvascular dysfunction. Understanding the kinetics and molecular basis of these changes may assist in the development of therapeutic intervention to counteract microvascular dysfunction. To investigate the molecular mechanisms underlying the changes in Tie2 expression upon lipopolysaccharide (LPS) challenge. Studies were performed in LPS and pro-inflammatory cytokine challenged mice as well as in mice subjected to hemorrhagic shock, primary endothelial cells were used for in vitro experiments in static and flow conditions. Eight hours after LPS challenge, Tie2 mRNA loss was observed in all major organs, while loss of Tie2 protein was predominantly observed in lungs and kidneys, in the capillaries. A similar loss could be induced by secondary cytokines TNF-α and IL-1β. Ang2 protein administration did not affect Tie2 protein expression nor was Tie2 protein rescued in LPS-challenged Ang2-deficient mice, excluding a major role for Ang2 in Tie2 down regulation. In vitro, endothelial loss of Tie2 was observed upon lowering of shear stress, not upon LPS and TNF-α stimulation, suggesting that inflammation related haemodynamic changes play a major role in loss of Tie2 in vivo, as also hemorrhagic shock induced Tie2 mRNA loss. In vitro, this loss was partially counteracted by pre-incubation with a pharmacologically NF-кB inhibitor (BAY11-7082), an effect further substantiated in vivo by pre-treatment of mice with the NF-кB inhibitor prior to the inflammatory challenge. Microvascular bed specific loss of Tie2 mRNA and protein in vivo upon LPS, TNFα, IL-1β challenge, as well as in response to hemorrhagic shock, is likely an indirect effect caused by a change in endothelial shear stress. This loss of Tie2 mRNA, but not Tie2 protein, induced by TNFα exposure was shown to be controlled by NF-кB signaling. Drugs aiming at restoring vascular integrity in sepsis could focus on preventing the Tie2 loss.

Retinoic acid stimulates interstitial collagenase messenger ribonucleic acid in osteosarcoma cells

NASA Technical Reports Server (NTRS)

Connolly, T. J.; Clohisy, J. C.; Shilt, J. S.; Bergman, K. D.; Partridge, N. C.; Quinn, C. O.

1994-01-01

The rat osteoblastic osteosarcoma cell line UMR 106-01 secretes interstitial collagenase in response to retinoic acid (RA). The present study demonstrates by Northern blot analysis that RA causes an increase in collagenase messenger RNA (mRNA) at 6 h, which is maximal at 24 h (20.5 times basal) and declines toward basal level by 72 h. This stimulation is dose dependent, with a maximal response at 5 x 10(-7) M RA. Nuclear run-on assays show a greater than 20-fold increase in the rate of collagenase mRNA transcription between 12-24 h after RA treatment. Cycloheximide blocks RA stimulation of collagenase mRNA, demonstrating the need for de novo protein synthesis. RA not only causes an increase in collagenase secretion, but is known to decrease collagen synthesis in UMR 106-01 cells. In this study, the increase in collagenase mRNA is accompanied by a concomitant decrease in the level of alpha 1(I) procollagen mRNA, which is maximal at 24 h (70% decrease), with a return to near-control levels by 72 h. Nuclear run-on assays demonstrated that the decrease in alpha 1 (I) procollagen expression does not have a statistically significant transcriptional component. RA did not statistically decrease the stability of alpha 1 (I) procollagen mRNA (calculated t1/2 = 8.06 +/- 0.30 and 9.01 +/- 0.62 h in the presence and absence of RA, respectively). However, transcription and stability together probably contribute to the major decrease in stable alpha 1 (I) procollagen mRNA observed. Cycloheximide treatment inhibits basal level alpha 1 (I) procollagen mRNA accumulation, demonstrating the need for on-going protein synthesis to maintain basal expression of this gene.

Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood.

PubMed

Souza, K de Picoli; Nunes, M T

2014-08-01

Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g) and triiodothyronine (0.5-50 µg/100 g) for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P) days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold). Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60%) ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold), and decreased heart rate (5%), fast muscle myoglobin mRNA (30%) and body weight (20%) in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30%) and body weight (14%). These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood.

mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma.

PubMed

Bache, Matthias; Rot, Swetlana; Keßler, Jacqueline; Güttler, Antje; Wichmann, Henri; Greither, Thomas; Wach, Sven; Taubert, Helge; Söling, Ariane; Bilkenroth, Udo; Kappler, Matthias; Vordermark, Dirk

2015-06-01

The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme.

Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood

PubMed Central

de Picoli Souza, K.; Nunes, M.T.

2014-01-01

Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g) and triiodothyronine (0.5-50 µg/100 g) for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P) days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold). Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60%) ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold), and decreased heart rate (5%), fast muscle myoglobin mRNA (30%) and body weight (20%) in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30%) and body weight (14%). These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood. PMID:25098716

Self-sampling with HPV mRNA analyses from vagina and urine compared with cervical samples.

PubMed

Asciutto, Katrin Christine; Ernstson, Avalon; Forslund, Ola; Borgfeldt, Christer

2018-04-01

In order to increase coverage in the organized cervical screening program, self-sampling with HPV analyses has been suggested. The aim was to compare human papillomavirus (HPV) mRNA detection in vaginal and urine self-collected samples with clinician-taken cervical samples and the corresponding clinician-taken histological specimens. Self-collected vaginal, urine and clinician-taken cervical samples were analyzed from 209 women with the Aptima mRNA assay (Hologic Inc, MA, USA). Cervical cytology, colposcopy, biopsy and/or the loop electrosurgical excision procedure (LEEP) were performed in every examination. The sensitivity of the HPV mRNA test in detecting high-grade squamous intraepithelial lesions (HSIL)/adenocarcinoma in situ (AIS)/cancer cases was as follows: for the vaginal self-samples 85.5% (95% CI; 75.0-92.8), the urinary samples 44.8% (95% CI; 32.6-57.4), and for routine cytology 81.7% (95% CI; 70.7-89.9). For the clinician-taken cervical HPV samples the sensitivity of the HPV mRNA test in detecting HSIL/AIS/cancer was 100.0% (95% CI; 94.9-100.0). The specificity of the HPV mRNA was similar for the clinician-taken cervical HPV samples and the self-samples: 49.0% vs. 48.1%. The urinary HPV samples had a specificity of 61.9% and cytology had a specificity of 93.3%. The sensitivity of the Aptima HPV mRNA test in detecting HSIL/AIS/cancer from vaginal self-samples was similar to that of routine cytology. The Aptima HPV mRNA vaginal self-sampling analysis may serve as a complement in screening programs. Copyright © 2018 Elsevier B.V. All rights reserved.

Comparative effects of low-level laser therapy pre- and post-injury on mRNA expression of MyoD, myogenin, and IL-6 during the skeletal muscle repair.

PubMed

Alves, Agnelo Neves; Ribeiro, Beatriz Guimarães; Fernandes, Kristianne Porta Santos; Souza, Nadhia Helena Costa; Rocha, Lília Alves; Nunes, Fabio Daumas; Bussadori, Sandra Kalil; Mesquita-Ferrari, Raquel Agnelli

2016-05-01

This study analyzed the effect of pre-injury and post-injury irradiation with low-level laser therapy (LLLT) on the mRNA expression of myogenic regulatory factors and interleukin 6 (IL-6) during the skeletal muscle repair. Male rats were divided into six groups: control group, sham group, LLLT group, injury group; pre-injury LLLT group, and post-injury LLLT group. LLLT was performed with a diode laser (wavelength 780 nm; output power 40 mW' and total energy 3.2 J). Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly onto the belly of the tibialis anterior (TA) muscle. After euthanasia, the TA muscle was removed for the isolation of total RNA and analysis of MyoD, myogenin, and IL-6 using real-time quantitative PCR. Significant increases were found in the expression of MyoD mRNA at 3 and 7 days as well as the expression of myogenin mRNA at 14 days in the post-injury LLLT group in comparison to injury group. A significant reduction was found in the expression of IL-6 mRNA at 3 and 7 days in the pre-injury LLLT and post-injury LLLT groups. A significant increase in IL-6 mRNA was found at 14 days in the post-injury LLLT group in comparison to the injury group. LLLT administered following muscle injury modulates the mRNA expression of MyoD and myogenin. Moreover, the both forms of LLLT administration were able to modulate the mRNA expression of IL-6 during the muscle repair process.

RT-PCR amplification of RNA extracted from formalin-fixed, paraffin-embedded oral cancer sections: analysis of p53 pathway.

PubMed

Tachibana, Masatsugu; Shinagawa, Yasuhiro; Kawamata, Hitoshi; Omotehara, Fumie; Horiuchi, Hideki; Ohkura, Yasuo; Kubota, Keiichi; Imai, Yutaka; Fujibayashi, Takashi; Fujimori, Takahiro

2003-01-01

We present a new approach towards the detection of the mRNAs in formalin-fixed, paraffin-embedded samples using a reverse transcriptase (RT)-polymerase chain reaction (PCR). The total RNAs were extracted from 10-micron-thick sections and were reverse-transcribed, then the RT-products were subjected to PCR amplification of GAPDH mRNA for screening the mRNA degradation. Next, nested PCR was performed for examining the expression of p53-related genes, p21WAF1, MDM2, p33ING1 and p14ARF. GAPDH mRNA expression was detectable in 12 out of 21 oral squamous cell carcinoma (SCC) samples. p21WAF1 mRNA expression was detectable in 5 out of 12 SCC samples, MDM2 mRNA expression was detectable in 5 our of 12 SCC samples and p33ING1 mRNA expression was detectable in 6 out of 12 SCC samples. However, the expression of p14ARF mRNA was not detectable in any of the samples. Seven out of 12 oral SCC samples showed abnormal nuclear accumulation of p53 protein by immunohistochemical staining, whereas 5 out of 12 oral SCCs showed negative staining for p53 protein. Of of p33ING1 mRNA. One of these was a verrucous carcinoma in which the p53 gene products might be inactivated by the oncoprotein E6 of human papilloma virus. Thus, the p53 tumor suppressor pathway was disrupted in most oral SCCs at the cellular levels, due to either an abnormality in p53 itself or loss of expression of p53 regulatory factors. This method would assist in making diagnosis, determining therapeutic strategy and predicting the prognosis of various cancers including oral SCCs.

Quantitative Single-Cell mRNA Analysis in Hydrogel Beads.

PubMed

Rakszewska, Agata; Stolper, Rosa J; Kolasa, Anna B; Piruska, Aigars; Huck, Wilhelm T S

2016-06-01

In recent years, technologies capable of analyzing single cells have emerged that are transforming many fields of biological research. Herein we report how DNA-functionalized hydrogel beads can serve as a matrix to capture mRNA from lysed single cells. mRNA quantification free of pre-amplification bias is ensured by using padlock probes and rolling circle amplification followed by hybridization with fluorescent probes. The number of transcripts in individual cells is assessed by simply counting fluorescent dots inside gel beads. The method extends the potential of existing techniques and provides a general platform for capturing molecules of interest from single cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Polysome Fractionation to Analyze mRNA Distribution Profiles.

PubMed

Panda, Amaresh C; Martindale, Jennifer L; Gorospe, Myriam

2017-02-05

Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al. , 2014a and 2014b; Abdelmohsen et al. , 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ribosomes with a given mRNA. It provides valuable information about the translational status of that mRNA, depending on the number of ribosomes with which they are associated, and identifies mRNAs that are not translated (Panda et al. , 2016). mRNAs associated with many ribosomes form large polysomes that are predicted to be actively translated, while mRNAs associated with few or no ribosomes are expected to be translated poorly if at all. In sum, polysome fractionation analysis allows the direct determination of translation efficiencies at the level of the whole transcriptome as well as individual mRNAs.

Isolation and characterization of Xenopus laevis homologs of the mouse inv gene and functional analysis of the conserved calmodulin binding sites.

PubMed

Yasuhiko, Yukuto; Shiokawa, Koichiro; Mochizuki, Toshio; Asashima, Makoto; Yokoyama, Takahiko

2006-04-01

The homozygous inv (inversion of embryonic turning) mouse mutant shows situs inversus and polycystic kidney disease, both of which result from the lack of the inv gene. Previously, we suggested that inv may be important for the left-right axis formation, not only in mice but also in Xenopus, and that calmodulin regulates this inv protein function. Here, we isolated and characterized two Xenopus laevis homologs (Xinv-1 and Xinv-2) of the mouse inv gene, and performed functional analysis of the conserved IQ motifs that interact with calmodulin. Xinv-1 expresses early in development in the same manner as mouse inv does. Unexpectedly, a full-length Xenopus inv mRNA did not randomize cardiac orientation when injected into Xenopus embryos, which is different from mouse inv mRNA. Contrary to mouse inv mRNA, Xenopus inv mRNA with mutated IQ randomized cardiac orientation. The present study indicates that calmodulin binding sites (IQ motifs) are crucial in controlling the biological activity of both mouse and Xenopus inv proteins. Although mouse and Xenopus inv genes have a quite similar structure, the interaction with calmodulin and IQ motifs of Xenopus inv and mouse inv proteins may regulate their function in different ways.

Nondestructive cryomicro-CT imaging enables structural and molecular analysis of human lung tissue.

PubMed

Vasilescu, Dragoş M; Phillion, André B; Tanabe, Naoya; Kinose, Daisuke; Paige, David F; Kantrowitz, Jacob J; Liu, Gang; Liu, Hanqiao; Fishbane, Nick; Verleden, Stijn E; Vanaudenaerde, Bart M; Lenburg, Marc; Stevenson, Christopher S; Spira, Avrum; Cooper, Joel D; Hackett, Tillie-Louise; Hogg, James C

2017-01-01

Micro-computed tomography (CT) enables three-dimensional (3D) imaging of complex soft tissue structures, but current protocols used to achieve this goal preclude cellular and molecular phenotyping of the tissue. Here we describe a radiolucent cryostage that permits micro-CT imaging of unfixed frozen human lung samples at an isotropic voxel size of (11 µm) 3 under conditions where the sample is maintained frozen at -30°C during imaging. The cryostage was tested for thermal stability to maintain samples frozen up to 8 h. This report describes the methods used to choose the materials required for cryostage construction and demonstrates that whole genome mRNA integrity and expression are not compromised by exposure to micro-CT radiation and that the tissue can be used for immunohistochemistry. The new cryostage provides a novel method enabling integration of 3D tissue structure with cellular and molecular analysis to facilitate the identification of molecular determinants of disease. The described micro-CT cryostage provides a novel way to study the three-dimensional lung structure preserved without the effects of fixatives while enabling subsequent studies of the cellular matrix composition and gene expression. This approach will, for the first time, enable researchers to study structural changes of lung tissues that occur with disease and correlate them with changes in gene or protein signatures. Copyright © 2017 the American Physiological Society.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruggles, Kelly V.; Tang, Zuojian; Wang, Xuya

Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations and splice variants identified in cancer cells are translated. Herein we therefore describe a proteogenomic data integration tool (QUILTS) and illustrate its application to whole genome, transcriptome and global MS peptide sequence datasets generated from a pair of luminal and basal-like breast cancer patient derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS process replicates. Despite over thirty sample replicates, only about 10% of all SNV (somatic andmore » germline) were detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNV without a detectable mRNA transcript were also observed demonstrating the transcriptome coverage was also incomplete (~80%). In contrast to germ-line variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than the luminal tumor raising the possibility of differential translation or protein degradation effects. In conclusion, the QUILTS program integrates DNA, RNA and peptide sequencing to assess the degree to which somatic mutations are translated and therefore biologically active. By identifying gaps in sequence coverage QUILTS benchmarks current technology and assesses progress towards whole cancer proteome and transcriptome analysis.« less

Mitomycin C induces apoptosis in cultured corneal fibroblasts derived from type II granular corneal dystrophy corneas.

PubMed

Kim, Tae-im; Choi, Seung-il; Lee, Hyung Keun; Cho, Young Jae; Kim, Eung Kweon

2008-06-30

The present study investigated the effect of mitomycin C (MMC) on cell viability, apoptosis, and transforming growth factor beta-induced protein (TGFBIp) expression in cultured normal corneal fibroblasts and heterozygote or homozygote granular corneal dystrophy type II (GCD II) corneal fibroblasts. Keratocytes were obtained from normal cornea or from heterozygote or homozygote GCD II patients after lamellar or penetrating keratoplasty. To measure cell viability, corneal fibroblasts were incubated with 0.02% MMC for 3 h, 6 h, and 24 h or with 0%, 0.01%, 0.02%, and 0.04% MMC for 24 h and then tested using lactate dehydrogenase (LDH) and 3-[4,5-demethylthiazol-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assays. To measure apoptosis, cells were analyzed by FACS analysis and annexin V staining. Bcl-xL, Bax, and TGFBI mRNA expression was measured using reverse transcription polymerase chain reaction (RT-PCR) assays. Cellular and media levels of TGFBIp protein were measured by immunoblotting. MTT and LDH assays showed that MMC reduced cell viability in all three cell types in a dose-dependent and time-dependent manner (p<0.05). FACS analysis and annexin V staining showed that MMC caused apoptosis with GCD II homozygote cells being most affected. RT-PCR analysis showed that MMC decreased Bcl-xL mRNA expression and increased Bax mRNA expression in all cell types. RT-PCR and immunoblotting analysis showed that MMC reduced TGFBI mRNA levels and cellular and media TGFBIp protein levels in all cell types. MMC induced apoptosis, and the effects of MMC were greatest in GCD II homozygote cells. MMC also reduced the production of TGFBIp in all three types of corneal fibroblasts. These findings may explain the additional therapeutic effect of MMC in GCD II patients.

RNA analysis of inner ear cells from formalin fixed paraffin embedded (FFPE) archival human temporal bone section using laser microdissection--a technical report.

PubMed

Kimura, Yurika; Kubo, Sachiho; Koda, Hiroko; Shigemoto, Kazuhiro; Sawabe, Motoji; Kitamura, Ken

2013-08-01

Molecular analysis using archival human inner ear specimens is challenging because of the anatomical complexity, long-term fixation, and decalcification. However, this method may provide great benefit for elucidation of otological diseases. Here, we extracted mRNA for RT-PCR from tissues dissected from archival FFPE human inner ears by laser microdissection. Three human temporal bones obtained at autopsy were fixed in formalin, decalcified by EDTA, and embedded in paraffin. The samples were isolated into spiral ligaments, outer hair cells, spiral ganglion cells, and stria vascularis by laser microdissection. RNA was extracted and heat-treated in 10 mM citrate buffer to remove the formalin-derived modification. To identify the sites where COCH and SLC26A5 mRNA were expressed, semi-nested RT-PCR was performed. We also examined how long COCH mRNA could be amplified by semi-nested RT-PCR in archival temporal bone. COCH was expressed in the spiral ligament and stria vascularis. However, SLC26A5 was expressed only in outer hair cells. The maximum base length of COCH mRNA amplified by RT-PCR was 98 bp in 1 case and 123 bp in 2 cases. We detected COCH and SLC26A5 mRNA in specific structures and cells of the inner ear from archival human temporal bone. Our innovative method using laser microdissection and semi-nested RT-PCR should advance future RNA study of human inner ear diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular Basis of the Increase in Invertase Activity Elicited by Gravistimulation of Oat-Shoot Pulvini

NASA Technical Reports Server (NTRS)

Wu, Liu-Lai; Song, Il; Kim, Donghern; Kaufman, Peter B.

1993-01-01

An asymmetric (top vs. bottom) increase in invertase activity is elicited by gravistimulation in oatshoot pulvini starting within 3h after treatment. In order to analyze the regulation of invertase gene expression in this system, we examined the effect of gravistimulation on invertase mRNA induction. Total RNA and poly(A)(+)RNA, isolated from oat pulvini, and two oligonucleotide primers, corresponding to two conserved amino-acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the Polymerase Chain Reaction (PCR). A partial-length cDNA (550 base pairs) was obtained and characterized. There was a 52 % deduced amino-acid sequence homology to that of carrot beta-fructosi- dase and a 48 % homology to that of tomato invertase. Northern blot analysis showed that there was an obvious transient accumulation of invertase mRNA elicited by gravistimulation of oat pulvini. The mRNA was rapidly induced to a maximum level at 1h following gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher (five-fold) than that in the top half of the pulvinus tissue. The induction of invertase mRNA was consistent with the transient enhancement of invertase activity during the graviresponse of the pulvinus. These data indicate that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional and/or translational levels. Southern blot analysis showed that there were four genomic DNA fragments hybridized to the invertase cDNA. This suggests that an invertase gene family may exist in oat plants.

Human L-DOPA decarboxylase mRNA is a target of miR-145: A prediction to validation workflow.

PubMed

Papadopoulos, Emmanuel I; Fragoulis, Emmanuel G; Scorilas, Andreas

2015-01-10

l-DOPA decarboxylase (DDC) is a multiply-regulated gene which encodes the enzyme that catalyzes the biosynthesis of dopamine in humans. MicroRNAs comprise a novel class of endogenously transcribed small RNAs that can post-transcriptionally regulate the expression of various genes. Given that the mechanism of microRNA target recognition remains elusive, several genes, including DDC, have not yet been identified as microRNA targets. Nevertheless, a number of specifically designed bioinformatic algorithms provide candidate miRNAs for almost every gene, but still their results exhibit moderate accuracy and should be experimentally validated. Motivated by the above, we herein sought to discover a microRNA that regulates DDC expression. By using the current algorithms according to bibliographic recommendations we found that miR-145 could be predicted with high specificity as a candidate regulatory microRNA for DDC expression. Thus, a validation experiment followed by firstly transfecting an appropriate cell culture system with a synthetic miR-145 sequence and sequentially assessing the mRNA and protein levels of DDC via real-time PCR and Western blotting, respectively. Our analysis revealed that miR-145 had no significant impact on protein levels of DDC but managed to dramatically downregulate its mRNA expression. Overall, the experimental and bioinformatic analysis conducted herein indicate that miR-145 has the ability to regulate DDC mRNA expression and potentially this occurs by recognizing its mRNA as a target. Copyright © 2014. Published by Elsevier B.V.

A complex structure in the mRNA of Tf1 is recognized and cleaved to generate the primer of reverse transcription.

PubMed

Lin, J H; Levin, H L

1997-01-15

All retroviruses and LTR-containing retrotransposons are thought to require specific tRNA molecules to serve as primers of reverse transcription. An exception is the LTR-containing retrotransposon Tf1, isolated from Schizosaccharomyces pombe. Instead of requiring a tRNA, the reverse transcriptase of Tf1 uses the first 11 bases of the Tf1 transcript as the primer for reverse transcription. The primer is generated by a cleavage that occurs between bases 11 and 12 of the Tf1 mRNA. Sequence analysis of the 5' untranslated region of the Tf1 mRNA resulted in the identification of a region with the potential to form an RNA structure of 89 bases that included the primer binding site and the first 11 bases of the Tf1 mRNA. Systematic mutagenesis of this region revealed 34 single-point mutants in the structure that resulted in reduced transposition activity. The defects in transposition correlated with reduced level of Tf1 reverse transcripts as determined by DNA blot analysis. Evidence that the RNA structure did form in vivo included the result that strains with second site mutations that restored complementarity resulted in increased levels of reverse transcripts and Tf1 transposition. The majority of the mutants defective for reverse transcription were unable to cleave the Tf1 mRNA between bases 11 and 12. These data indicate that formation of an extensive RNA structure was required for the cleavage reaction that generated the primer for Tf1 reverse transcription.

Noise analysis of genome-scale protein synthesis using a discrete computational model of translation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Racle, Julien; Hatzimanikatis, Vassily, E-mail: vassily.hatzimanikatis@epfl.ch; Swiss Institute of Bioinformatics

2015-07-28

Noise in genetic networks has been the subject of extensive experimental and computational studies. However, very few of these studies have considered noise properties using mechanistic models that account for the discrete movement of ribosomes and RNA polymerases along their corresponding templates (messenger RNA (mRNA) and DNA). The large size of these systems, which scales with the number of genes, mRNA copies, codons per mRNA, and ribosomes, is responsible for some of the challenges. Additionally, one should be able to describe the dynamics of ribosome exchange between the free ribosome pool and those bound to mRNAs, as well as howmore » mRNA species compete for ribosomes. We developed an efficient algorithm for stochastic simulations that addresses these issues and used it to study the contribution and trade-offs of noise to translation properties (rates, time delays, and rate-limiting steps). The algorithm scales linearly with the number of mRNA copies, which allowed us to study the importance of genome-scale competition between mRNAs for the same ribosomes. We determined that noise is minimized under conditions maximizing the specific synthesis rate. Moreover, sensitivity analysis of the stochastic system revealed the importance of the elongation rate in the resultant noise, whereas the translation initiation rate constant was more closely related to the average protein synthesis rate. We observed significant differences between our results and the noise properties of the most commonly used translation models. Overall, our studies demonstrate that the use of full mechanistic models is essential for the study of noise in translation and transcription.« less

Comparative Analysis of mRNA Isoform Expression in Cardiac Hypertrophy and Development Reveals Multiple Post-Transcriptional Regulatory Modules

PubMed Central

Park, Ji Yeon; Li, Wencheng; Zheng, Dinghai; Zhai, Peiyong; Zhao, Yun; Matsuda, Takahisa; Vatner, Stephen F.; Sadoshima, Junichi; Tian, Bin

2011-01-01

Cardiac hypertrophy is enlargement of the heart in response to physiological or pathological stimuli, chiefly involving growth of myocytes in size rather than in number. Previous studies have shown that the expression pattern of a group of genes in hypertrophied heart induced by pressure overload resembles that at the embryonic stage of heart development, a phenomenon known as activation of the “fetal gene program”. Here, using a genome-wide approach we systematically defined genes and pathways regulated in short- and long-term cardiac hypertrophy conditions using mice with transverse aortic constriction (TAC), and compared them with those regulated at different stages of embryonic and postnatal development. In addition, exon-level analysis revealed widespread mRNA isoform changes during cardiac hypertrophy resulting from alternative usage of terminal or internal exons, some of which are also developmentally regulated and may be attributable to decreased expression of Fox-1 protein in cardiac hypertrophy. Genes with functions in certain pathways, such as cell adhesion and cell morphology, are more likely to be regulated by alternative splicing. Moreover, we found 3′UTRs of mRNAs were generally shortened through alternative cleavage and polyadenylation in hypertrophy, and microRNA target genes were generally de-repressed, suggesting coordinated mechanisms to increase mRNA stability and protein production during hypertrophy. Taken together, our results comprehensively delineated gene and mRNA isoform regulation events in cardiac hypertrophy and revealed their relations to those in development, and suggested that modulation of mRNA isoform expression plays an importance role in heart remodeling under pressure overload. PMID:21799842

Poly A tail length analysis of in vitro transcribed mRNA by LC-MS.

PubMed

Beverly, Michael; Hagen, Caitlin; Slack, Olga

2018-02-01

The 3'-polyadenosine (poly A) tail of in vitro transcribed (IVT) mRNA was studied using liquid chromatography coupled to mass spectrometry (LC-MS). Poly A tails were cleaved from the mRNA using ribonuclease T1 followed by isolation with dT magnetic beads. Extracted tails were then analyzed by LC-MS which provided tail length information at single-nucleotide resolution. A 2100-nt mRNA with plasmid-encoded poly A tail lengths of either 27, 64, 100, or 117 nucleotides was used for these studies as enzymatically added poly A tails showed significant length heterogeneity. The number of As observed in the tails closely matched Sanger sequencing results of the DNA template, and even minor plasmid populations with sequence variations were detected. When the plasmid sequence contained a discreet number of poly As in the tail, analysis revealed a distribution that included tails longer than the encoded tail lengths. These observations were consistent with transcriptional slippage of T7 RNAP taking place within a poly A sequence. The type of RNAP did not alter the observed tail distribution, and comparison of T3, T7, and SP6 showed all three RNAPs produced equivalent tail length distributions. The addition of a sequence at the 3' end of the poly A tail did, however, produce narrower tail length distributions which supports a previously described model of slippage where the 3' end can be locked in place by having a G or C after the poly nucleotide region. Graphical abstract Determination of mRNA poly A tail length using magnetic beads and LC-MS.

Identification and Functional Analysis of the First Aquaporin from Striped Stem Borer, Chilo suppressalis

PubMed Central

Lu, Ming-Xing; Pan, Dan-Dan; Xu, Jing; Liu, Yang; Wang, Gui-Rong; Du, Yu-Zhou

2018-01-01

Aquaporins are integral membrane proteins some of which form high capacity water-selective channels, promoting water permeation across cell membranes. In this study, we isolated the aquaporin transcript (CsDrip1) of Chilo suppressalis, one of the important rice pests. CsDrip1 included two variants, CsDrip1_v1 and CsDrip1_v2. Although CsDrip1_v2 sequence (>409 bp) was longer than CsDrip1_v1, they possessed the same open reading frame (ORF). Protein structure and topology of CsDrip1 was analyzed using a predicted model, and the results demonstrated the conserved properties of insect water-specific aquaporins, including 6 transmembrane domains, 2 NPA motifs, ar/R constriction region (Phe69, His194, Ser203, and Arg209) and the C-terminal peptide sequence ending in “SYDF.” Our data revealed that the Xenopus oocytes expressing CsDrip1 indicated CsDrip1 could transport water instead of glycerol, trehalose and urea. Further, the transcript of CsDrip1 expressed ubiquitously but differentially in different tissues or organs and developmental stages of C. suppressalis. CsDrip1 mRNA exhibited the highest level of expression within hindgut and the third instar larvae. Regardless of pupae and adults, there were significantly different expression levels of CsDrip1 gene between male and female. Different from at low temperature, the transcript of CsDrip1 in larvae exposed to high temperature was increased significantly. Moreover, the mRNA levels of CsDrip1 in the third instar larvae, the fifth instar larvae, pupae (male and female), and adults (male and female) under different humidities were investigated. However, the mRNA levels of CsDrip1 of only female and male adults were changed remarkably. In conclusions, CsDrip1 plays important roles in maintaining water homeostasis in this important rice pest. PMID:29467668

Cellular RNA binding proteins NS1-BP and hnRNP K regulate influenza A virus RNA splicing.

PubMed

Tsai, Pei-Ling; Chiou, Ni-Ting; Kuss, Sharon; García-Sastre, Adolfo; Lynch, Kristen W; Fontoura, Beatriz M A

2013-01-01

Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA3, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.

WNT2B2 mRNA, up-regulated in primary gastric cancer, is a positive regulator of the WNT- beta-catenin-TCF signaling pathway.

PubMed

Katoh, M; Kirikoshi, H; Terasaki, H; Shiokawa, K

2001-12-21

Genetic alterations of WNT signaling molecules lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway. We have previously cloned and characterized WNT2B/WNT13 gene on human chromosome 1p13, which is homologous to proto-oncogene WNT2 on human chromosome 7q31. WNT2B1 and WNT2B2 mRNAs, generated from the WNT2B gene due to alternative splicing of the alternative promoter type, encode almost identical polypeptides with divergence in the N-terminal region. WNT2B2 mRNA rather than WNT2B1 mRNA is preferentially expressed in NT2 cells with the potential of neuronal differentiation. Here, we describe our investigations of expression of WNT2B mRNAs in various types of human primary cancer. Matched tumor/normal expression array analysis revealed that WNT2B mRNAs were significantly up-regulated in 2 of 8 cases of primary gastric cancer. WNT2B2 mRNA rather than WNT2B1 mRNA was found to be preferentially up-regulated in a case of primary gastric cancer (signet ring cell carcinoma). Function of WNT2B1 mRNA and that of WNT2B2 mRNA were investigated by using Xenopus axis duplication assay. Injection of synthetic WNT2B1 mRNA into the ventral marginal zone of fertilized Xenopus eggs at the 4-cell stage did not induce axis duplication. In contrast, ventral injection of synthetic WNT2B2 mRNA induced axis duplication in 90% of embryos (complete axis duplication, 24%). These results strongly suggest that WNT2B2 up-regulation in some cases of gastric cancer might lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway.

Differential global gene expression in red and white skeletal muscle

NASA Technical Reports Server (NTRS)

Campbell, W. G.; Gordon, S. E.; Carlson, C. J.; Pattison, J. S.; Hamilton, M. T.; Booth, F. W.

2001-01-01

The differences in gene expression among the fiber types of skeletal muscle have long fascinated scientists, but for the most part, previous experiments have only reported differences of one or two genes at a time. The evolving technology of global mRNA expression analysis was employed to determine the potential differential expression of approximately 3,000 mRNAs between the white quad (white muscle) and the red soleus muscle (mixed red muscle) of female ICR mice (30-35 g). Microarray analysis identified 49 mRNA sequences that were differentially expressed between white and mixed red skeletal muscle, including newly identified differential expressions between muscle types. For example, the current findings increase the number of known, differentially expressed mRNAs for transcription factors/coregulators by nine and signaling proteins by three. The expanding knowledge of the diversity of mRNA expression between white and mixed red muscle suggests that there could be quite a complex regulation of phenotype between muscles of different fiber types.

Public data mining plus domestic experimental study defined involvement of the old-yet-uncharacterized gene matrix-remodeling associated 7 (MXRA7) in physiopathology of the eye.

PubMed

Jia, Changkai; Zhang, Feng; Zhu, Ying; Qi, Xia; Wang, Yiqiang

2017-10-20

Matrix-remodeling associated 7 (MXRA7) gene was first reported in 2002 and named so for its co-expression with several genes known to relate with matrix-remodeling. However, not any studies had been intentionally performed to characterize this gene. We started defining the functions of MXRA7 by integrating bioinformatics analysis and experimental study. Data mining of MXRA7 expression in BioGPS, Gene Expression Omnibus and EurExpress platforms highlighted high level expression of Mxra7 in murine ocular tissues. Real-time PCR was employed to measure Mxra7 mRNA in tissues of adult C57BL/6 mice and demonstrated that Mxra7 was preferentially expressed at higher level in retina, corneas and lens than in other tissues. Then the inflammatory corneal neovascularization (CorNV) model and fungal corneal infections were induced in Balb/c mice, and mRNA levels of Mxra7 as well as several matrix-remodeling related genes (Mmp3, Mmp13, Ecm1, Timp1) were monitored with RT-PCR. The results demonstrated a time-dependent Mxra7 under-expression pattern (U-shape curve along timeline), while all other matrix-remodeling related genes manifested an opposite changes pattern (dome-shape curve). When limited data from BioGPS concerning human MXRA7 gene expression in human tissues were looked at, it was found that ocular tissue was also the one expressing highest level of MXRA7. To conclude, integrative assay of MXRA7 gene expression in public databank as well as domestic animal models revealed a selective high expression MXRA7 in murine and human ocular tissues, and its change patterns in two corneal disease models implied that MXRA7 might play a role in pathological processes or diseases involving injury, neovascularization and would healing. Copyright © 2017 Elsevier B.V. All rights reserved.

Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine.

PubMed

O'Sullivan, Finbarr; Keenan, Joanne; Aherne, Sinead; O'Neill, Fiona; Clarke, Colin; Henry, Michael; Meleady, Paula; Breen, Laura; Barron, Niall; Clynes, Martin; Horgan, Karina; Doolan, Padraig; Murphy, Richard

2017-11-07

To identify miRNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine. Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for mRNA, miRNA and proteomic profiling. mRNA microarray profiling analysis was carried out using the Affymetrix GeneChip Human Gene 1.0 ST array. miRNA microarray profiling analysis was carried out using the Affymetrix Genechip miRNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear ion trap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets (miRNA, proteomics, mRNA) was conducted in the R software environment using the Euclidean distance measure and Ward's clustering algorithm. The prediction of miRNA and oppositely correlated protein/mRNA interactions was performed using TargetScan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE miRNA, protein and mRNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database. Differential expression (DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 miRNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the miRNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology (GO) analysis of the DE mRNA identified cell adhesion, migration and ECM organization, cellular lipid and cholesterol metabolic processes, small molecule transport and a range of responses to external stimuli, while similar analysis of the DE protein list identified gene expression/transcription, epigenetic mechanisms, DNA replication, differentiation and translation ontology categories. The DE protein and gene lists were found to share 15 biological processes including for example epithelial cell differentiation [ P value ≤ 1.81613E-08 (protein list); P ≤ 0.000434311 (gene list)] and actin filament bundle assembly [ P value ≤ 0.001582797 (protein list); P ≤ 0.002733714 (gene list)]. Analysis was conducted on the three data streams acquired in parallel to identify targets undergoing potential miRNA translational repression identified 34 proteins, whose respective mRNAs were detected but no change in expression was observed. Of these 34 proteins, 27 proteins downregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 19 unique anti-correlated/upregulated microRNAs and 7 proteins upregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 15 unique anti-correlated/downregulated microRNAs. This first study providing "tri-omics" analysis of the principal intestinal cell line models Caco-2 and HT-29 has identified 34 proteins potentially undergoing miRNA translational repression.

Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine

PubMed Central

O’Sullivan, Finbarr; Keenan, Joanne; Aherne, Sinead; O’Neill, Fiona; Clarke, Colin; Henry, Michael; Meleady, Paula; Breen, Laura; Barron, Niall; Clynes, Martin; Horgan, Karina; Doolan, Padraig; Murphy, Richard

2017-01-01

AIM To identify miRNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine. METHODS Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for mRNA, miRNA and proteomic profiling. mRNA microarray profiling analysis was carried out using the Affymetrix GeneChip Human Gene 1.0 ST array. miRNA microarray profiling analysis was carried out using the Affymetrix Genechip miRNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear ion trap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets (miRNA, proteomics, mRNA) was conducted in the R software environment using the Euclidean distance measure and Ward’s clustering algorithm. The prediction of miRNA and oppositely correlated protein/mRNA interactions was performed using TargetScan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE miRNA, protein and mRNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database. RESULTS Differential expression (DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 miRNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the miRNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology (GO) analysis of the DE mRNA identified cell adhesion, migration and ECM organization, cellular lipid and cholesterol metabolic processes, small molecule transport and a range of responses to external stimuli, while similar analysis of the DE protein list identified gene expression/transcription, epigenetic mechanisms, DNA replication, differentiation and translation ontology categories. The DE protein and gene lists were found to share 15 biological processes including for example epithelial cell differentiation [P value ≤ 1.81613E-08 (protein list); P ≤ 0.000434311 (gene list)] and actin filament bundle assembly [P value ≤ 0.001582797 (protein list); P ≤ 0.002733714 (gene list)]. Analysis was conducted on the three data streams acquired in parallel to identify targets undergoing potential miRNA translational repression identified 34 proteins, whose respective mRNAs were detected but no change in expression was observed. Of these 34 proteins, 27 proteins downregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 19 unique anti-correlated/upregulated microRNAs and 7 proteins upregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 15 unique anti-correlated/downregulated microRNAs. CONCLUSION This first study providing “tri-omics” analysis of the principal intestinal cell line models Caco-2 and HT-29 has identified 34 proteins potentially undergoing miRNA translational repression. PMID:29151691

Feminization and masculinization of western mosquitofish (Gambusia affinis) observed in rivers impacted by municipal wastewaters

PubMed Central

Huang, Guo-Yong; Liu, You-Sheng; Chen, Xiao-Wen; Liang, Yan-Qiu; Liu, Shuang-Shuang; Yang, Yuan-Yuan; Hu, Li-Xin; Shi, Wen-Jun; Tian, Fei; Zhao, Jian-Liang; Chen, Jun; Ying, Guang-Guo

2016-01-01

Municipal wastewaters have been known to contain various estrogens and androgens. Little is known about the joint action of these chemicals from wastewaters on fishes in the aquatic environment. The objectives of this study were to investigate the estrogenic and/or androgenic effects in wild mosquitofish (Gambusia affinis) of two effluent-impacted rivers in South China by determining morphological changes and hepatic mRNA expression levels of relevant genes such as vitellogenin (Vtg), estrogen receptor (ERα) and androgen receptors (ARα and ARβ), and to assess the linkages of those morphological changes and hepatic mRNA expression levels to the chemical concentrations measured by in vitro bioassays and chemical analysis. The results showed a significant induction of Vtg and ERα mRNA in the livers of the males and a gonopodium-like anal fin in the females collected at the majority of sites. Redundancy analysis and Pearson correlation analysis showed that the chemical concentrations obtained by in vitro bioassays and chemical analysis had significant correlations with some of the endpoints for the estrogenic and/or androgenic effects in mosquitofish. The findings from this study indicate that the estrogens and androgens present in the two rivers could cause the observed estrogenic and androgenic effects in mosquitofish. PMID:26876344

Bridging the gap between high-throughput genetic and transcriptional data reveals cellular pathways responding to alpha-synuclein toxicity

PubMed Central

Yeger-Lotem, Esti; Riva, Laura; Su, Linhui Julie; Gitler, Aaron D.; Cashikar, Anil; King, Oliver D.; Auluck, Pavan K.; Geddie, Melissa L.; Valastyan, Julie S.; Karger, David R.; Lindquist, Susan; Fraenkel, Ernest

2009-01-01

Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens, yet the functional roles of the genes identified by these assays often remain enigmatic. By comparing the results of these two assays across various cellular responses, we found that they are consistently distinct. Moreover, genetic screens tend to identify response regulators, while mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We harnessed this approach to reveal cellular pathways related to alpha-synuclein, a small lipid-binding protein implicated in several neurodegenerative disorders including Parkinson disease. For this we screened an established yeast model for alpha-synuclein toxicity to identify genes that when overexpressed alter cellular survival. Application of our algorithm to these data and data from mRNA profiling provided functional explanations for many of these genes and revealed novel relations between alpha-synuclein toxicity and basic cellular pathways. PMID:19234470

IMP3 Stabilization of WNT5B mRNA Facilitates TAZ Activation in Breast Cancer.

PubMed

Samanta, Sanjoy; Guru, Santosh; Elaimy, Ameer L; Amante, John J; Ou, Jianhong; Yu, Jun; Zhu, Lihua J; Mercurio, Arthur M

2018-05-29

Insulin-like growth factor-2 mRNA-binding protein 3 (IMP3) is an oncofetal protein associated with many aggressive cancers and implicated in the function of breast cancer stem cells (CSCs). The mechanisms involved, however, are poorly understood. We observed that IMP3 facilitates the activation of TAZ, a transcriptional co-activator of Hippo signaling that is necessary for the function of breast CSCs. The mechanism by which IMP3 activates TAZ involves both mRNA stability and transcriptional regulation. IMP3 stabilizes the mRNA of an alternative WNT ligand (WNT5B) indirectly by repressing miR145-5p, which targets WNT5B, resulting in TAZ activation by alternative WNT signaling. IMP3 also facilitates the transcription of SLUG, which is necessary for TAZ nuclear localization and activation, by a mechanism that is also mediated by WNT5B. These results demonstrate that TAZ can be regulated by an mRNA-binding protein and that this regulation involves the integration of Hippo and alternative WNT-signaling pathways. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Small RNAome profiling from human skeletal muscle: novel miRNAs and their targets associated with cancer cachexia.

PubMed

Narasimhan, Ashok; Ghosh, Sunita; Stretch, Cynthia; Greiner, Russell; Bathe, Oliver F; Baracos, Vickie; Damaraju, Sambasivarao

2017-06-01

MicroRNAs (miRs) are small non-coding RNAs that regulate gene (mRNA) expression. Although the pathological role of miRs have been studied in muscle wasting conditions such as myotonic and muscular dystrophy, their roles in cancer cachexia (CC) are still emerging. The objectives are (i) to profile human skeletal muscle expressed miRs; (ii) to identify differentially expressed (DE) miRs between cachectic and non-cachectic cancer patients; (iii) to identify mRNA targets for the DE miRs to gain mechanistic insights; and (iv) to investigate if miRs show potential prognostic and predictive value. Study subjects were classified based on the international consensus diagnostic criteria for CC. Forty-two cancer patients were included, of which 22 were cachectic cases and 20 were non-cachectic cancer controls. Total RNA isolated from muscle biopsies were subjected to next-generation sequencing. A total of 777 miRs were profiled, and 82 miRs with read counts of ≥5 in 80% of samples were retained for analysis. We identified eight DE miRs (up-regulated, fold change of ≥1.4 at P < 0.05). A total of 191 potential mRNA targets were identified for the DE miRs using previously described human skeletal muscle mRNA expression data (n = 90), and a majority of them were also confirmed in an independent mRNA transcriptome dataset. Ingenuity pathway analysis identified pathways related to myogenesis and inflammation. qRT-PCR analysis of representative miRs showed similar direction of effect (P < 0.05), as observed in next-generation sequencing. The identified miRs also showed prognostic and predictive value. In all, we identified eight novel miRs associated with CC. © 2017 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.

Identification of tumor-restricted antigens NY-BR-1, SCP-1, and a new cancer/testis-like antigen NW-BR-3 by serological screening of a testicular library with breast cancer serum.

PubMed

Jäger, Dirk; Unkelbach, Marc; Frei, Claudia; Bert, Florian; Scanlan, Matthew J; Jäger, Elke; Old, Lloyd J; Chen, Yao-Tseng; Knuth, Alexander

2002-06-28

Serological analysis of recombinant cDNA expression libraries (SEREX) has led to the identification of several categories of new tumor antigens. We analyzed a testicular cDNA expression library with serum obtained from a breast cancer patient and isolated 13 genes designated NW-BR-1 through NW-BR-13. Of these, 3 showed tumor-restricted expression (NW-BR-1, -2 and -3), the others being expressed ubiquitously. NW-BR-3, representing 9 of 24 primary clones, showed tissue-restricted mRNA expression, being expressed in normal testis but not in 15 other normal tissues tested by Northern blotting. RT-PCR analysis showed strong NW-BR-3 expression in normal testis, weak expression in brain, kidney, trachea, uterus and normal prostate, and was negative in liver, heart, lung, colon, small intestine, bone marrow, breast, thymus, muscle, spleen, and stomach. NW-BR-3 mRNA expression was found in different tumor tissues and tumor cell lines by RT-PCR, thus showing a 'cancer/testis' (CT)-like mRNA expression pattern. NW-BR-3 shares 71% nucleotide and amino acid homology to a mouse gene cloned from mouse testicular tissue. Based on the mRNA expression pattern, NW-BR-3 represents a new candidate target gene for cancer immunotherapy. NW-BR-1 and NW-BR-2 also showed tumor-restricted mRNA expression. NW-BR-1 is a partial clone of the breast differentiation antigen NY-BR-1 previously identified by SEREX. NY-BR-1 is expressed in normal breast, testis and 80% of breast cancers. NW-BR-2 is identical to the CT antigen SCP-1, initially isolated by SEREX analysis of renal cancer. This study provides further evidence that SEREX is a powerful tool to identify new tumor antigens potentially relevant for immunotherapy approaches.

Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations

PubMed Central

Laudenbach, Beatrice Theres; Martínez-Montero, Saúl; Cencic, Regina; Habjan, Matthias; Pichlmair, Andreas; Damha, Masad J.; Pelletier, Jerry; Nagar, Bhushan

2017-01-01

IFIT1 (IFN-induced protein with tetratricopeptide repeats-1) is an effector of the host innate immune antiviral response that prevents propagation of virus infection by selectively inhibiting translation of viral mRNA. It relies on its ability to compete with the translation initiation factor eIF4F to specifically recognize foreign capped mRNAs, while remaining inactive against host mRNAs marked by ribose 2′-O methylation at the first cap-proximal nucleotide (N1). We report here several crystal structures of RNA-bound human IFIT1, including a 1.6-Å complex with capped RNA. IFIT1 forms a water-filled, positively charged RNA-binding tunnel with a separate hydrophobic extension that unexpectedly engages the cap in multiple conformations (syn and anti) giving rise to a relatively plastic and nonspecific mode of binding, in stark contrast to eIF4E. Cap-proximal nucleotides encircled by the tunnel provide affinity to compete with eIF4F while allowing IFIT1 to select against N1 methylated mRNA. Gel-shift binding assays confirm that N1 methylation interferes with IFIT1 binding, but in an RNA-dependent manner, whereas translation assays reveal that N1 methylation alone is not sufficient to prevent mRNA recognition at high IFIT1 concentrations. Structural and functional analysis show that 2′-O methylation at N2, another abundant mRNA modification, is also detrimental for RNA binding, thus revealing a potentially synergistic role for it in self- versus nonself-mRNA discernment. Finally, structure-guided mutational analysis confirms the importance of RNA binding for IFIT1 restriction of a human coronavirus mutant lacking viral N1 methylation. Our structural and biochemical analysis sheds new light on the molecular basis for IFIT1 translational inhibition of capped viral RNA. PMID:28251928

Genome‐Wide MicroRNA and Gene Analysis of Mesenchymal Stem Cell Chondrogenesis Identifies an Essential Role and Multiple Targets for miR‐140‐5p

PubMed Central

Tselepi, Maria; Gómez, Rodolfo; Woods, Steven; Hui, Wang; Smith, Graham R.; Shanley, Daryl P.; Clark, Ian M.; Young, David A.

2015-01-01

Abstract microRNAs (miRNAs) are abundantly expressed in development where they are critical determinants of cell differentiation and phenotype. Accordingly miRNAs are essential for normal skeletal development and chondrogenesis in particular. However, the question of which miRNAs are specific to the chondrocyte phenotype has not been fully addressed. Using microarray analysis of miRNA expression during mesenchymal stem cell chondrogenic differentiation and detailed examination of the role of essential differentiation factors, such as SOX9, TGF‐β, and the cell condensation phase, we characterize the repertoire of specific miRNAs involved in chondrocyte development, highlighting in particular miR‐140 and miR‐455. Further with the use of mRNA microarray data we integrate miRNA expression and mRNA expression during chondrogenesis to underline the particular importance of miR‐140, especially the ‐5p strand. We provide a detailed identification and validation of direct targets of miR‐140‐5p in both chondrogenesis and adult chondrocytes with the use of microarray and 3′UTR analysis. This emphasizes the diverse array of targets and pathways regulated by miR‐140‐5p. We are also able to confirm previous experimentally identified targets but, additionally, identify a novel positive regulation of the Wnt signaling pathway by miR‐140‐5p. Wnt signaling has a complex role in chondrogenesis and skeletal development and these findings illustrate a previously unidentified role for miR‐140‐5p in regulation of Wnt signaling in these processes. Together these developments further highlight the role of miRNAs during chondrogenesis to improve our understanding of chondrocyte development and guide cartilage tissue engineering. Stem Cells 2015;33:3266–3280 PMID:26175215

Proteins S100A8 and S100A9 are potential biomarkers for renal cell carcinoma in the early stages: results from a proteomic study integrated with bioinformatics analysis.

PubMed

Zhang, Limin; Jiang, Haowen; Xu, Gang; Wen, Hui; Gu, Bin; Liu, Jun; Mao, Shanghua; Na, Rong; Jing, Yan; Ding, Qiang; Zhang, Yuanfang

2015-06-01

In order to investigate the two members of the EF‑hand Ca2+ binding protein S100 family, S100A8 and S100A9, in renal cell carcinoma (RCC), serum samples were collected from patients with RCC, transitional cell carcinoma in the kidney, benign renal masses and normal controls. The samples were analyzed by isobaric tags for relative and absolute quantification technology to identify the differential expression of S100A8 and S100A9 in the respective groups. Hierarchical clustering analysis was then conducted for the samples and the relevant selected gene. The cross‑platform analysis for the external validation was performed by means of The Cancer Genome Atlas database, containing the gene/microRNA expression pattern and clinical information of patients with RCC. Immunohistochemical staining was used to verify the expression of S100A8 and S100A9 in the four groups. As a result, serum and mRNA expression levels of S100A8 and S100A9 were found to be upregulated in patients with RCC compared with the other three groups, which was consistent with the result of the upregulated expression of mRNA levels in RCC tissue. The overexpression of S100A8 and S100A9 in cancer cells was also confirmed by immunohistochemistry. In addition, bioinformatics revealed that let‑7, a microRNA formerly identified as an inhibiting factor of RCC was downregulated in RCC, which contrasted with S100A8. It was also complementary to the sequence at the 3' untranslated region terminal of S100A8. Therefore, indicating that S100A8 and S100A9 may serve as biomarkers for the detection of RCC.

miRge - A Multiplexed Method of Processing Small RNA-Seq Data to Determine MicroRNA Entropy

PubMed Central

Myers, Jason R.; Gupta, Simone; Weng, Lien-Chun; Ashton, John M.; Cornish, Toby C.; Pandey, Akhilesh; Halushka, Marc K.

2015-01-01

Small RNA RNA-seq for microRNAs (miRNAs) is a rapidly developing field where opportunities still exist to create better bioinformatics tools to process these large datasets and generate new, useful analyses. We built miRge to be a fast, smart small RNA-seq solution to process samples in a highly multiplexed fashion. miRge employs a Bayesian alignment approach, whereby reads are sequentially aligned against customized mature miRNA, hairpin miRNA, noncoding RNA and mRNA sequence libraries. miRNAs are summarized at the level of raw reads in addition to reads per million (RPM). Reads for all other RNA species (tRNA, rRNA, snoRNA, mRNA) are provided, which is useful for identifying potential contaminants and optimizing small RNA purification strategies. miRge was designed to optimally identify miRNA isomiRs and employs an entropy based statistical measurement to identify differential production of isomiRs. This allowed us to identify decreasing entropy in isomiRs as stem cells mature into retinal pigment epithelial cells. Conversely, we show that pancreatic tumor miRNAs have similar entropy to matched normal pancreatic tissues. In a head-to-head comparison with other miRNA analysis tools (miRExpress 2.0, sRNAbench, omiRAs, miRDeep2, Chimira, UEA small RNA Workbench), miRge was faster (4 to 32-fold) and was among the top-two methods in maximally aligning miRNAs reads per sample. Moreover, miRge has no inherent limits to its multiplexing. miRge was capable of simultaneously analyzing 100 small RNA-Seq samples in 52 minutes, providing an integrated analysis of miRNA expression across all samples. As miRge was designed for analysis of single as well as multiple samples, miRge is an ideal tool for high and low-throughput users. miRge is freely available at http://atlas.pathology.jhu.edu/baras/miRge.html. PMID:26571139

Integrated microRNA and gene expression profiling reveals the crucial miRNAs in curcumin anti-lung cancer cell invasion.

PubMed

Zhan, Jian-Wei; Jiao, De-Min; Wang, Yi; Song, Jia; Wu, Jin-Hong; Wu, Li-Jun; Chen, Qing-Yong; Ma, Sheng-Lin

2017-09-01

Curcumin (diferuloylmethane) has chemopreventive and therapeutic properties against many types of tumors, both in vitro and in vivo. Previous reports have shown that curcumin exhibits anti-invasive activities, but the mechanisms remain largely unclear. In this study, both microRNA (miRNA) and messenger RNA (mRNA) expression profiles were used to characterize the anti-metastasis mechanisms of curcumin in human non-small cell lung cancer A549 cell line. Microarray analysis revealed that 36 miRNAs were differentially expressed between the curcumin-treated and control groups. miR-330-5p exhibited maximum upregulation, while miR-25-5p exhibited maximum downregulation in the curcumin treatment group. mRNA expression profiles and functional analysis indicated that 226 differentially expressed mRNAs belonged to different functional categories. Significant pathway analysis showed that mitogen-activated protein kinase, transforming growth factor-β, and Wnt signaling pathways were significantly downregulated. At the same time, axon guidance, glioma, and ErbB tyrosine kinase receptor signaling pathways were significantly upregulated. We constructed a miRNA gene network that contributed to the curcumin inhibition of metastasis in lung cancer cells. let-7a-3p, miR-1262, miR-499a-5p, miR-1276, miR-331-5p, and miR-330-5p were identified as key microRNA regulators in the network. Finally, using miR-330-5p as an example, we confirmed the role of miR-330-5p in mediating the anti-migration effect of curcumin, suggesting the importance of miRNAs in the regulation of curcumin biological activity. Our findings provide new insights into the anti-metastasis mechanism of curcumin in lung cancer. © 2017 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

Identification of miRNA-Mediated Core Gene Module for Glioma Patient Prediction by Integrating High-Throughput miRNA, mRNA Expression and Pathway Structure

PubMed Central

Han, Junwei; Shang, Desi; Zhang, Yunpeng; Zhang, Wei; Yao, Qianlan; Han, Lei; Xu, Yanjun; Yan, Wei; Bao, Zhaoshi; You, Gan; Jiang, Tao; Kang, Chunsheng; Li, Xia

2014-01-01

The prognosis of glioma patients is usually poor, especially in patients with glioblastoma (World Health Organization (WHO) grade IV). The regulatory functions of microRNA (miRNA) on genes have important implications in glioma cell survival. However, there are not many studies that have investigated glioma survival by integrating miRNAs and genes while also considering pathway structure. In this study, we performed sample-matched miRNA and mRNA expression profilings to systematically analyze glioma patient survival. During this analytical process, we developed pathway-based random walk to identify a glioma core miRNA-gene module, simultaneously considering pathway structure information and multi-level involvement of miRNAs and genes. The core miRNA-gene module we identified was comprised of four apparent sub-modules; all four sub-modules displayed a significant correlation with patient survival in the testing set (P-values≤0.001). Notably, one sub-module that consisted of 6 miRNAs and 26 genes also correlated with survival time in the high-grade subgroup (WHO grade III and IV), P-value = 0.0062. Furthermore, the 26-gene expression signature from this sub-module had robust predictive power in four independent, publicly available glioma datasets. Our findings suggested that the expression signatures, which were identified by integration of miRNA and gene level, were closely associated with overall survival among the glioma patients with various grades. PMID:24809850

Structure of the gene encoding VGF, a nervous system-specific mRNA that is rapidly and selectively induced by nerve growth factor in PC12 cells.

PubMed

Salton, S R; Fischberg, D J; Dong, K W

1991-05-01

Nerve growth factor (NGF) plays a critical role in the development and survival of neurons in the peripheral nervous system. Following treatment with NGF but not epidermal growth factor, rat pheochromocytoma (PC12) cells undergo neural differentiation. We have cloned a nervous system-specific mRNA, NGF33.1, that is rapidly and relatively selectively induced by treatment of PC12 cells with NGF and basic fibroblast growth factor in comparison with epidermal growth factor. Analysis of the nucleic acid and predicted amino acid sequences of the NGF33.1 cDNA clone suggested that this clone corresponded to the NGF-inducible mRNA called VGF (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985; R. Possenti, J. D. Eldridge, B. M. Paterson, A. Grasso, and A. Levi, EMBO J. 8:2217-2223, 1989). We have used the NGF33.1 cDNA clone to isolate and characterize the VGF gene, and in this paper we report the complete sequence of the VGF gene, including 853 bases of 5' flank revealed TATAA and CCAAT elements, several GC boxes, and a consensus cyclic AMP response element-binding protein binding site. The VGF promoter contains sequences homologous to other NGF-inducible, neuronal promoters. We further show that VGF mRNA is induced in PC12 cells to a greater extent by depolarization and by phorbol-12-myristate-13-acetate treatment than by 8-bromo-cyclic AMP treatment. By Northern (RNA) and RNase protection analysis, VGF mRNA is detectable in embryonic and postnatal central and peripheral nervous tissues but not in a number of nonneural tissues. In the cascade of events which ultimately leads to the neural differentiation of NGF-treated PC12 cells, the VGF gene encodes the most rapidly and selectively regulated, nervous-system specific mRNA yet identified.

Exosomal particles secreted by prostate cancer cells are potent mRNA and protein vehicles for the interference of tumor and tumor environment.

PubMed

Rauschenberger, Lisa; Staar, Doreen; Thom, Kathleen; Scharf, Christian; Venz, Simone; Homuth, Georg; Schlüter, Rabea; Brandenburg, Lars-Ove; Ziegler, Patrick; Zimmermann, Uwe; Weitschies, Werner; Völker, Uwe; Lendeckel, Uwe; Walther, Reinhard; Burchardt, Martin; Stope, Matthias B

2016-03-01

Remodeling of the tumor environment and the modulation of tumor associated non-malignant cells are essential events in tumor progression. Exosomes are small membranous vesicles of 50-150 nm in diameter, which are secreted into the extracellular space and supposedly serve as vehicles for signal and effector molecules to modulate adjacent target cells. We characterized the mRNA and protein composition as well as cellular functions of prostate cancer cell-derived exosomes. Exosomes were prepared from prostate cancer cell culture supernatant by ultracentrifugation and subsequently characterized by dynamic light scattering and electron microscopy. Exosomal mRNA and protein composition were analyzed by DNA microarrays and gel electrophoresis coupled with mass spectrometry. Physiological effects of exosomes were studied by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release cell assays. Using a SILAC approach, putative uptake of exosomal human proteins in canine cells and canine de novo synthesis of proteins specified by exosome-transferred human mRNA was analyzed in MDCK cells via mass spectrometry. Preparations of exosomes revealed typical cup shaped particles of 150 nm in diameter. Analysis of mRNA and protein composition of exosomes exhibited a wide range of mRNA and protein species. Interestingly, the packaging of at least small proteins into exosomes was apparently unspecific, as shown with the example of two model proteins. In cell culture incubation experiments exosomal preparations of prostate cancer cells caused anti-proliferative effects. MS analysis revealed the uptake of exosomal human proteins into canine cells after 6 hr of incubation. The results reveal a distinct exosomal functionality in the modulation of the prostatic tumor adjacent environment. The multitude of translocated factors implies the induction of numerous effects in tumor-associated target cells, including impact on cellular growth. © 2015 Wiley Periodicals, Inc.

Stability of the Osmoregulated Promoter-Derived proP mRNA Is Posttranscriptionally Regulated by RNase III in Escherichia coli

PubMed Central

Lim, Boram

2015-01-01

ABSTRACT The enzymatic activity of Escherichia coli endo-RNase III determines the stability of a subgroup of mRNA species, including bdm, betT, and proU, whose protein products are associated with the cellular response to osmotic stress. Here, we report that the stability of proP mRNA, which encodes a transporter of osmoprotectants, is controlled by RNase III in response to osmotic stress. We observed that steady-state levels of proP mRNA and ProP protein are inversely correlated with cellular RNase III activity and, in turn, affect the proline uptake capacity of the cell. In vitro and in vivo analyses of proP mRNA revealed RNase III cleavage sites in a stem-loop within the 5′ untranslated region present only in proP mRNA species synthesized from the osmoregulated P1 promoter. Introduction of nucleotide substitutions in the cleavage site identified inhibited the ribonucleolytic activity of RNase III on proP mRNA, increasing the steady-state levels and half-life of the mRNA. In addition, decreased RNase III activity coincided with a significant increase in both the half-life and abundance of proP mRNA under hyperosmotic stress conditions. Analysis of the RNA bound to RNase III via in vivo cross-linking and immunoprecipitation indicated that this phenomenon is related to the decreased RNA binding capacity of RNase III. Our findings suggest the existence of an RNase III-mediated osmoregulatory network that rapidly balances the expression levels of factors associated with the cellular response to osmotic stress in E. coli. IMPORTANCE Our results demonstrate that RNase III activity on proP mRNA degradation is downregulated in Escherichia coli cells under osmotic stress. In addition, we show that the downregulation of RNase III activity is associated with decreased RNA binding capacity of RNase III under hyperosmotic conditions. In particular, our findings demonstrate a link between osmotic stress and RNase III activity, underscoring the growing importance of posttranscriptional regulation in modulating rapid physiological adjustment to environmental changes. PMID:25645556

Localization and expression of messenger RNAs for the peroxisome proliferator-activated receptors in ovarian tissue from naturally cycling and pseudopregnant rats.

PubMed

Komar, Carolyn M; Curry, Thomas E

2002-05-01

Structural and functional development of the corpus luteum (CL) involves tissue remodeling, angiogenesis, lipid metabolism, and steroid production. The peroxisome proliferator-activated receptors (PPARs) have been shown to play a role in these as well as in a multitude of other cellular processes. To examine the expression of mRNA corresponding to the PPAR family members (alpha, delta, and gamma) in luteal tissue, ovaries were collected from gonadotropin-treated, immature rats on Days 1, 4, 8, and 14 of pseudopregnancy and from adult, cycling animals on each day of the estrous cycle. Ovaries were processed for in situ hybridization or RNA isolation for analysis by RNase protection assay. The expression of PPARgamma mRNA was abundant in granulosa cells of developing follicles during both pseudopregnancy and the estrous cycle and was low to undetectable in CL from pseudopregnant rats. However, luteal tissue in cycling animals, especially CL remaining from previous cycles, had high levels of PPARgamma mRNA. The PPARalpha mRNA was localized mainly in the theca and stroma, and PPARdelta mRNA was expressed throughout the ovary. Levels of mRNA for PPARgamma decreased between Days 1 and 4 of pseudopregnancy, and PPARalpha mRNA levels were lower on the day of estrus compared to pro- and metestrus (P < 0.05). The PPARdelta mRNA levels remained steady throughout the estrous cycle and pseudopregnancy. These data illustrate a difference in the luteal expression of mRNA for PPARgamma between the adult, cycling rat and the immature, gonadotropin-treated rat. This differential pattern of expression may be related to the difference in timing of the preovulatory prolactin surge, because the gonadotropin-primed animals would not experience a prolactin surge coincident with the LH surge, as occurs in adult, cycling animals. Additionally, the expression pattern of PPARdelta mRNA indicates that it may be involved in cellular functions involved with maintaining basal ovarian function, whereas PPARalpha may play a role in lipid metabolism in the theca and stroma.