mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus

PubMed Central

Legendre, Matthieu; Audic, Stéphane; Poirot, Olivier; Hingamp, Pascal; Seltzer, Virginie; Byrne, Deborah; Lartigue, Audrey; Lescot, Magali; Bernadac, Alain; Poulain, Julie; Abergel, Chantal; Claverie, Jean-Michel

2010-01-01

Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic “virion factory,” we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 5′ and 3′ extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus “virophage.” These results—validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)—will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system. PMID:20360389

Deep sequencing of cardiac microRNA-mRNA interactomes in clinical and experimental cardiomyopathy

PubMed Central

Matkovich, Scot J.; Dorn, Gerald W.

2018-01-01

Summary MicroRNAs are a family of short (~21 nucleotide) noncoding RNAs that serve key roles in cellular growth and differentiation and the response of the heart to stress stimuli. As the sequence-specific recognition element of RNA-induced silencing complexes (RISCs), microRNAs bind mRNAs and prevent their translation via mechanisms that may include transcript degradation and/or prevention of ribosome binding. Short microRNA sequences and the ability of microRNAs to bind to mRNA sites having only partial/imperfect sequence complementarity complicates purely computational analyses of microRNA-mRNA interactomes. Furthermore, computational microRNA target prediction programs typically ignore biological context, and therefore the principal determinants of microRNA-mRNA binding: the presence and quantity of each. To address these deficiencies we describe an empirical method, developed via studies of stressed and failing hearts, to determine disease-induced changes in microRNAs, mRNAs, and the mRNAs targeted to the RISC, without cross-linking mRNAs to RISC proteins. Deep sequencing methods are used to determine RNA abundances, delivering unbiased, quantitative RNA data limited only by their annotation in the genome of interest. We describe the laboratory bench steps required to perform these experiments, experimental design strategies to achieve an appropriate number of sequencing reads per biological replicate, and computer-based processing tools and procedures to convert large raw sequencing data files into gene expression measures useful for differential expression analyses. PMID:25836573

Deep sequencing of cardiac microRNA-mRNA interactomes in clinical and experimental cardiomyopathy.

PubMed

Matkovich, Scot J; Dorn, Gerald W

2015-01-01

MicroRNAs are a family of short (~21 nucleotide) noncoding RNAs that serve key roles in cellular growth and differentiation and the response of the heart to stress stimuli. As the sequence-specific recognition element of RNA-induced silencing complexes (RISCs), microRNAs bind mRNAs and prevent their translation via mechanisms that may include transcript degradation and/or prevention of ribosome binding. Short microRNA sequences and the ability of microRNAs to bind to mRNA sites having only partial/imperfect sequence complementarity complicate purely computational analyses of microRNA-mRNA interactomes. Furthermore, computational microRNA target prediction programs typically ignore biological context, and therefore the principal determinants of microRNA-mRNA binding: the presence and quantity of each. To address these deficiencies we describe an empirical method, developed via studies of stressed and failing hearts, to determine disease-induced changes in microRNAs, mRNAs, and the mRNAs targeted to the RISC, without cross-linking mRNAs to RISC proteins. Deep sequencing methods are used to determine RNA abundances, delivering unbiased, quantitative RNA data limited only by their annotation in the genome of interest. We describe the laboratory bench steps required to perform these experiments, experimental design strategies to achieve an appropriate number of sequencing reads per biological replicate, and computer-based processing tools and procedures to convert large raw sequencing data files into gene expression measures useful for differential expression analyses.

Identification and Characterization of a Cis-Encoded Antisense RNA Associated with the Replication Process of Salmonella enterica Serovar Typhi

PubMed Central

Dadzie, Isaac; Xu, Shungao; Ni, Bin; Zhang, Xiaolei; Zhang, Haifang; Sheng, Xiumei; Xu, Huaxi; Huang, Xinxiang

2013-01-01

Antisense RNAs that originate from the complementary strand of protein coding genes are involved in the regulation of gene expression in all domains of life. In bacteria, some of these antisense RNAs are transcriptional noise whiles others play a vital role to adapt the cell to changing environmental conditions. By deep sequencing analysis of transcriptome of Salmonella enterica serovar Typhi, a partial RNA sequence encoded in-cis to the dnaA gene was revealed. Northern blot and RACE analysis confirmed the transcription of this antisense RNA which was expressed mostly in the stationary phase of the bacterial growth and also under iron limitation and osmotic stress. Pulse expression analysis showed that overexpression of the antisense RNA resulted in a significant increase in the mRNA levels of dnaA, which will ultimately enhance their translation. Our findings have revealed that antisense RNA of dnaA is indeed transcribed not merely as a by-product of the cell's transcription machinery but plays a vital role as far as stability of dnaA mRNA is concerned. PMID:23637809

Digital Marine Bioprospecting: Mining New Neurotoxin Drug Candidates from the Transcriptomes of Cold-Water Sea Anemones

PubMed Central

Urbarova, Ilona; Karlsen, Bård Ove; Okkenhaug, Siri; Seternes, Ole Morten; Johansen, Steinar D.; Emblem, Åse

2012-01-01

Marine bioprospecting is the search for new marine bioactive compounds and large-scale screening in extracts represents the traditional approach. Here, we report an alternative complementary protocol, called digital marine bioprospecting, based on deep sequencing of transcriptomes. We sequenced the transcriptomes from the adult polyp stage of two cold-water sea anemones, Bolocera tuediae and Hormathia digitata. We generated approximately 1.1 million quality-filtered sequencing reads by 454 pyrosequencing, which were assembled into approximately 120,000 contigs and 220,000 single reads. Based on annotation and gene ontology analysis we profiled the expressed mRNA transcripts according to known biological processes. As a proof-of-concept we identified polypeptide toxins with a potential blocking activity on sodium and potassium voltage-gated channels from digital transcriptome libraries. PMID:23170083

Designing deep sequencing experiments: detecting structural variation and estimating transcript abundance.

PubMed

Bashir, Ali; Bansal, Vikas; Bafna, Vineet

2010-06-18

Massively parallel DNA sequencing technologies have enabled the sequencing of several individual human genomes. These technologies are also being used in novel ways for mRNA expression profiling, genome-wide discovery of transcription-factor binding sites, small RNA discovery, etc. The multitude of sequencing platforms, each with their unique characteristics, pose a number of design challenges, regarding the technology to be used and the depth of sequencing required for a particular sequencing application. Here we describe a number of analytical and empirical results to address design questions for two applications: detection of structural variations from paired-end sequencing and estimating mRNA transcript abundance. For structural variation, our results provide explicit trade-offs between the detection and resolution of rearrangement breakpoints, and the optimal mix of paired-read insert lengths. Specifically, we prove that optimal detection and resolution of breakpoints is achieved using a mix of exactly two insert library lengths. Furthermore, we derive explicit formulae to determine these insert length combinations, enabling a 15% improvement in breakpoint detection at the same experimental cost. On empirical short read data, these predictions show good concordance with Illumina 200 bp and 2 Kbp insert length libraries. For transcriptome sequencing, we determine the sequencing depth needed to detect rare transcripts from a small pilot study. With only 1 Million reads, we derive corrections that enable almost perfect prediction of the underlying expression probability distribution, and use this to predict the sequencing depth required to detect low expressed genes with greater than 95% probability. Together, our results form a generic framework for many design considerations related to high-throughput sequencing. We provide software tools http://bix.ucsd.edu/projects/NGS-DesignTools to derive platform independent guidelines for designing sequencing experiments (amount of sequencing, choice of insert length, mix of libraries) for novel applications of next generation sequencing.

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.

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

Genome-wide assessment of differential translations with ribosome profiling data.

PubMed

Xiao, Zhengtao; Zou, Qin; Liu, Yu; Yang, Xuerui

2016-04-04

The closely regulated process of mRNA translation is crucial for precise control of protein abundance and quality. Ribosome profiling, a combination of ribosome foot-printing and RNA deep sequencing, has been used in a large variety of studies to quantify genome-wide mRNA translation. Here, we developed Xtail, an analysis pipeline tailored for ribosome profiling data that comprehensively and accurately identifies differentially translated genes in pairwise comparisons. Applied on simulated and real datasets, Xtail exhibits high sensitivity with minimal false-positive rates, outperforming existing methods in the accuracy of quantifying differential translations. With published ribosome profiling datasets, Xtail does not only reveal differentially translated genes that make biological sense, but also uncovers new events of differential translation in human cancer cells on mTOR signalling perturbation and in human primary macrophages on interferon gamma (IFN-γ) treatment. This demonstrates the value of Xtail in providing novel insights into the molecular mechanisms that involve translational dysregulations.

Transcriptome dynamics through alternative polyadenylation in developmental and environmental responses in plants revealed by deep sequencing

PubMed Central

Shen, Yingjia; Venu, R.C.; Nobuta, Kan; Wu, Xiaohui; Notibala, Varun; Demirci, Caghan; Meyers, Blake C.; Wang, Guo-Liang; Ji, Guoli; Li, Qingshun Q.

2011-01-01

Polyadenylation sites mark the ends of mRNA transcripts. Alternative polyadenylation (APA) may alter sequence elements and/or the coding capacity of transcripts, a mechanism that has been demonstrated to regulate gene expression and transcriptome diversity. To study the role of APA in transcriptome dynamics, we analyzed a large-scale data set of RNA “tags” that signify poly(A) sites and expression levels of mRNA. These tags were derived from a wide range of tissues and developmental stages that were mutated or exposed to environmental treatments, and generated using digital gene expression (DGE)–based protocols of the massively parallel signature sequencing (MPSS-DGE) and the Illumina sequencing-by-synthesis (SBS-DGE) sequencing platforms. The data offer a global view of APA and how it contributes to transcriptome dynamics. Upon analysis of these data, we found that ∼60% of Arabidopsis genes have multiple poly(A) sites. Likewise, ∼47% and 82% of rice genes use APA, supported by MPSS-DGE and SBS-DGE tags, respectively. In both species, ∼49%–66% of APA events were mapped upstream of annotated stop codons. Interestingly, 10% of the transcriptomes are made up of APA transcripts that are differentially distributed among developmental stages and in tissues responding to environmental stresses, providing an additional level of transcriptome dynamics. Examples of pollen-specific APA switching and salicylic acid treatment-specific APA clearly demonstrated such dynamics. The significance of these APAs is more evident in the 3034 genes that have conserved APA events between rice and Arabidopsis. PMID:21813626

Genome-wide analyses of long noncoding RNA expression profiles correlated with radioresistance in nasopharyngeal carcinoma via next-generation deep sequencing.

PubMed

Li, Guo; Liu, Yong; Liu, Chao; Su, Zhongwu; Ren, Shuling; Wang, Yunyun; Deng, Tengbo; Huang, Donghai; Tian, Yongquan; Qiu, Yuanzheng

2016-09-06

Radioresistance is one of the major factors limiting the therapeutic efficacy and prognosis of patients with nasopharyngeal carcinoma (NPC). Accumulating evidence has suggested that aberrant expression of long noncoding RNAs (lncRNAs) contributes to cancer progression. Therefore, here we identified lncRNAs associated with radioresistance in NPC. The differential expression profiles of lncRNAs associated with NPC radioresistance were constructed by next-generation deep sequencing by comparing radioresistant NPC cells with their parental cells. LncRNA-related mRNAs were predicted and analyzed using bioinformatics algorithms compared with the mRNA profiles related to radioresistance obtained in our previous study. Several lncRNAs and associated mRNAs were validated in established NPC radioresistant cell models and NPC tissues. By comparison between radioresistant CNE-2-Rs and parental CNE-2 cells by next-generation deep sequencing, a total of 781 known lncRNAs and 2054 novel lncRNAs were annotated. The top five upregulated and downregulated known/novel lncRNAs were detected using quantitative real-time reverse transcription-polymerase chain reaction, and 7/10 known lncRNAs and 3/10 novel lncRNAs were demonstrated to have significant differential expression trends that were the same as those predicted by deep sequencing. From the prediction process, 13 pairs of lncRNAs and their associated genes were acquired, and the prediction trends of three pairs were validated in both radioresistant CNE-2-Rs and 6-10B-Rs cell lines, including lncRNA n373932 and SLITRK5, n409627 and PRSS12, and n386034 and RIMKLB. LncRNA n373932 and its related SLITRK5 showed dramatic expression changes in post-irradiation radioresistant cells and a negative expression correlation in NPC tissues (R = -0.595, p < 0.05). Our study provides an overview of the expression profiles of radioresistant lncRNAs and potentially related mRNAs, which will facilitate future investigations into the function of lncRNAs in NPC radioresistance.

A first insight into the occurrence and expression of functional amoA and accA genes of autotrophic and ammonia-oxidizing bathypelagic Crenarchaeota of Tyrrhenian Sea

NASA Astrophysics Data System (ADS)

Yakimov, Michail M.; Cono, Violetta La; Denaro, Renata

2009-05-01

The autotrophic and ammonia-oxidizing crenarchaeal assemblage at offshore site located in the deep Mediterranean (Tyrrhenian Sea, depth 3000 m) water was studied by PCR amplification of the key functional genes involved in energy (ammonia mono-oxygenase alpha subunit, amoA) and central metabolism (acetyl-CoA carboxylase alpha subunit, accA). Using two recently annotated genomes of marine crenarchaeons, an initial set of primers targeting archaeal accA-like genes was designed. Approximately 300 clones were analyzed, of which 100% of amoA library and almost 70% of accA library were unambiguously related to the corresponding genes from marine Crenarchaeota. Even though the acetyl-CoA carboxylase is phylogenetically not well conserved and the remaining clones were affiliated to various bacterial acetyl-CoA/propionyl-CoA carboxylase genes, the pool of archaeal sequences was applied for development of quantitative PCR analysis of accA-like distribution using TaqMan ® methodolgy. The archaeal accA gene fragments, together with alignable gene fragments from the Sargasso Sea and North Pacific Subtropical Gyre (ALOHA Station) metagenome databases, were analyzed by multiple sequence alignment. Two accA-like sequences, found in ALOHA Station at the depth of 4000 m, formed a deeply branched clade with 64% of all archaeal Tyrrhenian clones. No close relatives for residual 36% of clones, except of those recovered from Eastern Mediterranean, was found, suggesting the existence of a specific lineage of the crenarchaeal accA genes in deep Mediterranean water. Alignment of Mediterranean amoA sequences defined four cosmopolitan phylotypes of Crenarchaeota putative ammonia mono-oxygenase subunit A gene occurring in the water sample from the 3000 m depth. Without exception all phylotypes fell into Deep Marine Group I cluster that contain the vast majority of known sequences recovered from global deep-sea environment. Remarkably, three phylotypes accounted for 91% of all Mediterranean amoA clones and corresponded to the sequences retrieved from the less deep compartments of the world's ocean, most likely reflecting the higher temperature at the depth of the Mediterranean Sea. In order to verify whether these phylotypes might represent important Crenarchaeota in the functioning of the Mediterranean bathypelagic ecosystem, expression of crenarchaeal amoA gene was monitored by direct RNA retrieval and following analysis of amoA-related mRNA transcripts. Surprisingly, all mRNA-derived sequences formed a tight monophyletic group, which fell into large Shallow Marine Group I cluster with sequences retrieved from shallow (up to 200 m) waters, sediments and corals. This group was not detected in DNA-based clone library, obviously, due to an overwhelming dominance of the Deep Marine Group I. The failure to recover the amoA transcripts, related to Deep Marine Group I of Crenarchaeota, was unanticipated and likely resulted from the physiology of these strongly adapted deep-sea organisms. As far as all seawater samples were treated on-board under atmospheric pressure conditions and sunlight, the decompression and/or photoinhibition likely affected their metabolic activity, followed by the strong decay of gene expression.

Identifying MicroRNAs and Transcript Targets in Jatropha Seeds

PubMed Central

Galli, Vanessa; Guzman, Frank; de Oliveira, Luiz F. V.; Loss-Morais, Guilherme; Körbes, Ana P.; Silva, Sérgio D. A.; Margis-Pinheiro, Márcia M. A. N.; Margis, Rogério

2014-01-01

MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received significant attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library of mature seeds and three mRNA libraries from three different seed development stages were generated by deep sequencing to identify and characterize the miRNAs and pre-miRNAs of J. curcas. Computational analysis was used for the identification of 180 conserved miRNAs and 41 precursors (pre-miRNAs) as well as 16 novel pre-miRNAs. The predicted miRNA target genes are involved in a broad range of physiological functions, including cellular structure, nuclear function, translation, transport, hormone synthesis, defense, and lipid metabolism. Some pre-miRNA and miRNA targets vary in abundance between the three stages of seed development. A search for sequences that produce siRNA was performed, and the results indicated that J. curcas siRNAs play a role in nuclear functions, transport, catalytic processes and disease resistance. This study presents the first large scale identification of J. curcas miRNAs and their targets in mature seeds based on deep sequencing, and it contributes to a functional understanding of these miRNAs. PMID:24551031

Deep RNA sequencing reveals dynamic regulation of myocardial noncoding RNAs in failing human heart and remodeling with mechanical circulatory support.

PubMed

Yang, Kai-Chien; Yamada, Kathryn A; Patel, Akshar Y; Topkara, Veli K; George, Isaac; Cheema, Faisal H; Ewald, Gregory A; Mann, Douglas L; Nerbonne, Jeanne M

2014-03-04

Microarrays have been used extensively to profile transcriptome remodeling in failing human heart, although the genomic coverage provided is limited and fails to provide a detailed picture of the myocardial transcriptome landscape. Here, we describe sequencing-based transcriptome profiling, providing comprehensive analysis of myocardial mRNA, microRNA (miRNA), and long noncoding RNA (lncRNA) expression in failing human heart before and after mechanical support with a left ventricular (LV) assist device (LVAD). Deep sequencing of RNA isolated from paired nonischemic (NICM; n=8) and ischemic (ICM; n=8) human failing LV samples collected before and after LVAD and from nonfailing human LV (n=8) was conducted. These analyses revealed high abundance of mRNA (37%) and lncRNA (71%) of mitochondrial origin. miRNASeq revealed 160 and 147 differentially expressed miRNAs in ICM and NICM, respectively, compared with nonfailing LV. Among these, only 2 (ICM) and 5 (NICM) miRNAs are normalized with LVAD. RNASeq detected 18 480, including 113 novel, lncRNAs in human LV. Among the 679 (ICM) and 570 (NICM) lncRNAs differentially expressed with heart failure, ≈10% are improved or normalized with LVAD. In addition, the expression signature of lncRNAs, but not miRNAs or mRNAs, distinguishes ICM from NICM. Further analysis suggests that cis-gene regulation represents a major mechanism of action of human cardiac lncRNAs. The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.

How Messenger RNA and Nascent Chain Sequences Regulate Translation Elongation.

PubMed

Choi, Junhong; Grosely, Rosslyn; Prabhakar, Arjun; Lapointe, Christopher P; Wang, Jinfan; Puglisi, Joseph D

2018-06-20

Translation elongation is a highly coordinated, multistep, multifactor process that ensures accurate and efficient addition of amino acids to a growing nascent-peptide chain encoded in the sequence of translated messenger RNA (mRNA). Although translation elongation is heavily regulated by external factors, there is clear evidence that mRNA and nascent-peptide sequences control elongation dynamics, determining both the sequence and structure of synthesized proteins. Advances in methods have driven experiments that revealed the basic mechanisms of elongation as well as the mechanisms of regulation by mRNA and nascent-peptide sequences. In this review, we highlight how mRNA and nascent-peptide elements manipulate the translation machinery to alter the dynamics and pathway of elongation.

Deep sequencing of small RNA repertoires in mice reveals metabolic disorders-associated hepatic miRNAs.

PubMed

Liang, Tingming; Liu, Chang; Ye, Zhenchao

2013-01-01

Obesity and associated metabolic disorders contribute importantly to the metabolic syndrome. On the other hand, microRNAs (miRNAs) are a class of small non-coding RNAs that repress target gene expression by inducing mRNA degradation and/or translation repression. Dysregulation of specific miRNAs in obesity may influence energy metabolism and cause insulin resistance, which leads to dyslipidemia, steatosis hepatis and type 2 diabetes. In the present study, we comprehensively analyzed and validated dysregulated miRNAs in ob/ob mouse liver, as well as miRNA groups based on miRNA gene cluster and gene family by using deep sequencing miRNA datasets. We found that over 13.8% of the total analyzed miRNAs were dysregulated, of which 37 miRNA species showed significantly differential expression. Further RT-qPCR analysis in some selected miRNAs validated the similar expression patterns observed in deep sequencing. Interestingly, we found that miRNA gene cluster and family always showed consistent dysregulation patterns in ob/ob mouse liver, although they had various enrichment levels. Functional enrichment analysis revealed the versatile physiological roles (over six signal pathways and five human diseases) of these miRNAs. Biological studies indicated that overexpression of miR-126 or inhibition of miR-24 in AML-12 cells attenuated free fatty acids-induced fat accumulation. Taken together, our data strongly suggest that obesity and metabolic disturbance are tightly associated with functional miRNAs. We also identified hepatic miRNA candidates serving as potential biomarkers for the diagnose of the metabolic syndrome.

Deep mRNA Sequencing of the Tritonia diomedea Brain Transcriptome Provides Access to Gene Homologues for Neuronal Excitability, Synaptic Transmission and Peptidergic Signalling

PubMed Central

Senatore, Adriano; Edirisinghe, Neranjan; Katz, Paul S.

2015-01-01

Background The sea slug Tritonia diomedea (Mollusca, Gastropoda, Nudibranchia), has a simple and highly accessible nervous system, making it useful for studying neuronal and synaptic mechanisms underlying behavior. Although many important contributions have been made using Tritonia, until now, a lack of genetic information has impeded exploration at the molecular level. Results We performed Illumina sequencing of central nervous system mRNAs from Tritonia, generating 133.1 million 100 base pair, paired-end reads. De novo reconstruction of the RNA-Seq data yielded a total of 185,546 contigs, which partitioned into 123,154 non-redundant gene clusters (unigenes). BLAST comparison with RefSeq and Swiss-Prot protein databases, as well as mRNA data from other invertebrates (gastropod molluscs: Aplysia californica, Lymnaea stagnalis and Biomphalaria glabrata; cnidarian: Nematostella vectensis) revealed that up to 76,292 unigenes in the Tritonia transcriptome have putative homologues in other databases, 18,246 of which are below a more stringent E-value cut-off of 1x10-6. In silico prediction of secreted proteins from the Tritonia transcriptome shotgun assembly (TSA) produced a database of 579 unique sequences of secreted proteins, which also exhibited markedly higher expression levels compared to other genes in the TSA. Conclusions Our efforts greatly expand the availability of gene sequences available for Tritonia diomedea. We were able to extract full length protein sequences for most queried genes, including those involved in electrical excitability, synaptic vesicle release and neurotransmission, thus confirming that the transcriptome will serve as a useful tool for probing the molecular correlates of behavior in this species. We also generated a neurosecretome database that will serve as a useful tool for probing peptidergic signalling systems in the Tritonia brain. PMID:25719197

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

HYBRIDIZATION PROPERTIES OF DNA SEQUENCES DIRECTING THE SYNTHESIS OF MESSENGER RNA AND HETEROGENEOUS NUCLEAR RNA

PubMed Central

Greenberg, Jay R.; Perry, Robert P.

1971-01-01

The relationship of the DNA sequences from which polyribosomal messenger RNA (mRNA) and heterogeneous nuclear RNA (NRNA) of mouse L cells are transcribed was investigated by means of hybridization kinetics and thermal denaturation of the hybrids. Hybridization was performed in formamide solutions at DNA excess. Under these conditions most of the hybridizing mRNA and NRNA react at values of Dot (DNA concentration multiplied by time) expected for RNA transcribed from the nonrepeated or rarely repeated fraction of the genome. However, a fraction of both mRNA and NRNA hybridize at values of Dot about 10,000 times lower, and therefore must be transcribed from highly redundant DNA sequences. The fraction of NRNA hybridizing to highly repeated sequences is about 1.7 times greater than the corresponding fraction of mRNA. The hybrids formed by the rapidly reacting fractions of both NRNA and mRNA melt over a narrow temperature range with a midpoint about 11°C below that of native L cell DNA. This indicates that these hybrids consist of partially complementary sequences with approximately 11% mismatching of bases. Hybrids formed by the slowly reacting fraction of NRNA melt within 4°–6°C of native DNA, indicating very little, if any, mismatching of bases. Hybrids of the slowly reacting components of mRNA, formed under conditions of sufficiently low RNA input, have a high thermal stability, similar to that observed for hybrids of the slowly reacting NRNA component. However, when higher inputs of mRNA are used, hybrids are formed which have a strikingly lower thermal stability. This observation can be explained by assuming that there is sufficient similarity among the relatively rare DNA sequences coding for mRNA so that under hybridization conditions, in which these DNA sequences are not truly in excess, reversible hybrids exhibiting a considerable amount of mispairing are formed. The fact that a comparable phenomenon has not been observed for NRNA may mean that there is less similarity among the relatively rare DNA sequences coding for NRNA than there is among the rare sequences coding for mRNA. PMID:4999767

Matrin 3 binds and stabilizes mRNA.

PubMed

Salton, Maayan; Elkon, Ran; Borodina, Tatiana; Davydov, Aleksey; Yaspo, Marie-Laure; Halperin, Eran; Shiloh, Yosef

2011-01-01

Matrin 3 (MATR3) is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM), whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq) identified several small noncoding RNA species. Using microarray analysis to explore MATR3's role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts.

Matrin 3 Binds and Stabilizes mRNA

PubMed Central

Salton, Maayan; Elkon, Ran; Borodina, Tatiana; Davydov, Aleksey; Yaspo, Marie-Laure; Halperin, Eran; Shiloh, Yosef

2011-01-01

Matrin 3 (MATR3) is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM), whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq) identified several small noncoding RNA species. Using microarray analysis to explore MATR3′s role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts. PMID:21858232

Ribosome profiling reveals the what, when, where and how of protein synthesis.

PubMed

Brar, Gloria A; Weissman, Jonathan S

2015-11-01

Ribosome profiling, which involves the deep sequencing of ribosome-protected mRNA fragments, is a powerful tool for globally monitoring translation in vivo. The method has facilitated discovery of the regulation of gene expression underlying diverse and complex biological processes, of important aspects of the mechanism of protein synthesis, and even of new proteins, by providing a systematic approach for experimental annotation of coding regions. Here, we introduce the methodology of ribosome profiling and discuss examples in which this approach has been a key factor in guiding biological discovery, including its prominent role in identifying thousands of novel translated short open reading frames and alternative translation products.

Deep sequencing and proteomic analysis of the microRNA-induced silencing complex in human red blood cells.

PubMed

Azzouzi, Imane; Moest, Hansjoerg; Wollscheid, Bernd; Schmugge, Markus; Eekels, Julia J M; Speer, Oliver

2015-05-01

During maturation, erythropoietic cells extrude their nuclei but retain their ability to respond to oxidant stress by tightly regulating protein translation. Several studies have reported microRNA-mediated regulation of translation during terminal stages of erythropoiesis, even after enucleation. In the present study, we performed a detailed examination of the endogenous microRNA machinery in human red blood cells using a combination of deep sequencing analysis of microRNAs and proteomic analysis of the microRNA-induced silencing complex. Among the 197 different microRNAs detected, miR-451a was the most abundant, representing more than 60% of all read sequences. In addition, miR-451a and its known target, 14-3-3ζ mRNA, were bound to the microRNA-induced silencing complex, implying their direct interaction in red blood cells. The proteomic characterization of endogenous Argonaute 2-associated microRNA-induced silencing complex revealed 26 cofactor candidates. Among these cofactors, we identified several RNA-binding proteins, as well as motor proteins and vesicular trafficking proteins. Our results demonstrate that red blood cells contain complex microRNA machinery, which might enable immature red blood cells to control protein translation independent of de novo nuclei information. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Uncovering microRNA-mediated response to SO2 stress in Arabidopsis thaliana by deep sequencing.

PubMed

Li, Lihong; Xue, Meizhao; Yi, Huilan

2016-10-05

Sulfur dioxide (SO2) is a major air pollutant and has significant impacts on plants. MicroRNAs (miRNAs) are a class of gene expression regulators that play important roles in response to environmental stresses. In this study, deep sequencing was used for genome-wide identification of miRNAs and their expression profiles in response to SO2 stress in Arabidopsis thaliana shoots. A total of 27 conserved miRNAs and 5 novel miRNAs were found to be differentially expressed under SO2 stress. qRT-PCR analysis showed mostly negative correlation between miRNA accumulation and target gene mRNA abundance, suggesting regulatory roles of these miRNAs during SO2 exposure. The target genes of SO2-responsive miRNAs encode transcription factors and proteins that regulate auxin signaling and stress response, and the miRNAs-mediated suppression of these genes could improve plant resistance to SO2 stress. Promoter sequence analysis of genes encoding SO2-responsive miRNAs showed that stress-responsive and phytohormone-related cis-regulatory elements occurred frequently, providing additional evidence of the involvement of miRNAs in adaption to SO2 stress. This study represents a comprehensive expression profiling of SO2-responsive miRNAs in Arabidopsis and broads our perspective on the ubiquitous regulatory roles of miRNAs under stress conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Multihormonal induction of hepatic α2u-globulin mRNA as measured by hybridization to complementary DNA

PubMed Central

Kurtz, David T.; Feigelson, Philip

1977-01-01

A procedure is presented for the preparation of a 3H-labeled complementary DNA (cDNA) specific for the mRNA coding for α2u-globulin, a male rat liver protein under multihormonal control that represents approximately 1% of hepatic protein synthesis. Rat liver polysomes are incubated with monospecific rabbit antiserum to α2u-globulin, which binds to the nascent α2u-globulin chains on the polysomes. These antibody-polysome complexes are then adsorbed to goat antiserum to rabbit IgG that is covalently linked to p-aminobenzylcellulose. mRNA preparations are thus obtained that contain 30-40% α2u-globulin mRNA. A labeled cDNA is made to this α2u-globulin-enriched mRNA preparation by using RNA-dependent DNA polymerase (reverse transcriptase). To remove the non-α2u-globulin sequences, this cDNA preparation is hybridized to an RNA concentration × incubation time (R0t) of 1000 mol of ribonucleotide per liter × sec with female rat liver mRNA, which, though it shares the vast majority of mRNA sequences with male liver, contains no α2u-globulin mRNA sequences. The cDNA remaining single-stranded is isolated by hydroxylapatite chromatography and is shown to be specific for α2u-globulin mRNA by several criteria. Good correlation was found in all endocrine states studied between the hepatic level of α2u-globulin, the level of functional α2u-globulin mRNA as assayed in a wheat germ cell-free translational system, and the level of α2u-globulin mRNA sequences as measured by hybridization to the α2u-globulin cDNA. Thus, the hormonal control of hepatic α2u-globulin synthesis by sex steroids and thyroid hormone occurs through modulation of the cellular level of α2u-globulin mRNA sequences, presumably by hormonal control of transcriptive synthesis. PMID:73184

Genome-wide assessment of differential translations with ribosome profiling data

PubMed Central

Xiao, Zhengtao; Zou, Qin; Liu, Yu; Yang, Xuerui

2016-01-01

The closely regulated process of mRNA translation is crucial for precise control of protein abundance and quality. Ribosome profiling, a combination of ribosome foot-printing and RNA deep sequencing, has been used in a large variety of studies to quantify genome-wide mRNA translation. Here, we developed Xtail, an analysis pipeline tailored for ribosome profiling data that comprehensively and accurately identifies differentially translated genes in pairwise comparisons. Applied on simulated and real datasets, Xtail exhibits high sensitivity with minimal false-positive rates, outperforming existing methods in the accuracy of quantifying differential translations. With published ribosome profiling datasets, Xtail does not only reveal differentially translated genes that make biological sense, but also uncovers new events of differential translation in human cancer cells on mTOR signalling perturbation and in human primary macrophages on interferon gamma (IFN-γ) treatment. This demonstrates the value of Xtail in providing novel insights into the molecular mechanisms that involve translational dysregulations. PMID:27041671

The maize stripe virus major noncapsid protein messenger RNA transcripts contain heterogeneous leader sequences at their 5' termini.

PubMed

Huiet, L; Feldstein, P A; Tsai, J H; Falk, B W

1993-12-01

Primer extension analyses and a PCR-based cloning strategy were used to identify and characterize 5' nucleotide sequences on the maize stripe virus (MStV) RNA4 mRNA transcripts encoding the major noncapsid protein (NCP). Direct RNA sequence analysis by primer extension showed that the NCP mRNA transcripts had 10-15 nucleotides beyond the 5' terminus of the MStV RNA4 nucleotide sequence. MStV genomic RNAs isolated from ribonucleoprotein particles (RNPs) lacked the additional 5' nucleotides. cDNA clones representing the 5' region of the mRNA transcripts were constructed, and the nucleotide sequences of the 5' regions were determined for 16 clones. Each was found to have a distinct 10-15 nucleotide sequence immediately 5' of the MStV RNA4 sequence. Eleven of 16 clones had the correct MStV RNA4 5' nucleotide sequence, while five showed minor variations at or near the 5' most MStV RNA4 nucleotide. These characteristics show strong similarities to other viral mRNA transcripts which are synthesized by cap snatching.

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.

Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing.

PubMed

Liu, Zhen; Lu, Zongyang; Yang, Guang; Huang, Shisheng; Li, Guanglei; Feng, Songjie; Liu, Yajing; Li, Jianan; Yu, Wenxia; Zhang, Yu; Chen, Jia; Sun, Qiang; Huang, Xingxu

2018-06-14

A recently developed adenine base editor (ABE) efficiently converts A to G and is potentially useful for clinical applications. However, its precision and efficiency in vivo remains to be addressed. Here we achieve A-to-G conversion in vivo at frequencies up to 100% by microinjection of ABE mRNA together with sgRNAs. We then generate mouse models harboring clinically relevant mutations at Ar and Hoxd13, which recapitulates respective clinical defects. Furthermore, we achieve both C-to-T and A-to-G base editing by using a combination of ABE and SaBE3, thus creating mouse model harboring multiple mutations. We also demonstrate the specificity of ABE by deep sequencing and whole-genome sequencing (WGS). Taken together, ABE is highly efficient and precise in vivo, making it feasible to model and potentially cure relevant genetic diseases.

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

Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

PubMed Central

Thess, Andreas; Grund, Stefanie; Mui, Barbara L; Hope, Michael J; Baumhof, Patrick; Fotin-Mleczek, Mariola; Schlake, Thomas

2015-01-01

Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies. PMID:26050989

Evidence for a Complex Class of Nonadenylated mRNA in Drosophila

PubMed Central

Zimmerman, J. Lynn; Fouts, David L.; Manning, Jerry E.

1980-01-01

The amount, by mass, of poly(A+) mRNA present in the polyribosomes of third-instar larvae of Drosophila melanogaster, and the relative contribution of the poly(A+) mRNA to the sequence complexity of total polysomal RNA, has been determined. Selective removal of poly(A+) mRNA from total polysomal RNA by use of either oligo-dT-cellulose, or poly(U)-sepharose affinity chromatography, revealed that only 0.15% of the mass of the polysomal RNA was present as poly(A+) mRNA. The present study shows that this RNA hybridized at saturation with 3.3% of the single-copy DNA in the Drosophila genome. After correction for asymmetric transcription and reactability of the DNA, 7.4% of the single-copy DNA in the Drosophila genome is represented in larval poly(A+) mRNA. This corresponds to 6.73 x 106 nucleotides of mRNA coding sequences, or approximately 5,384 diverse RNA sequences of average size 1,250 nucleotides. However, total polysomal RNA hybridizes at saturation to 10.9% of the single-copy DNA sequences. After correcting this value for asymmetric transcription and tracer DNA reactability, 24% of the single-copy DNA in Drosophila is represented in total polysomal RNA. This corresponds to 2.18 x 107 nucleotides of RNA coding sequences or 17,440 diverse RNA molecules of size 1,250 nucleotides. This value is 3.2 times greater than that observed for poly(A+) mRNA, and indicates that ≃69% of the polysomal RNA sequence complexity is contributed by nonadenylated RNA. Furthermore, if the number of different structural genes represented in total polysomal RNA is ≃1.7 x 104, then the number of genes expressed in third-instar larvae exceeds the number of chromomeres in Drosophila by about a factor of three. This numerology indicates that the number of chromomeres observed in polytene chromosomes does not reflect the number of structural gene sequences in the Drosophila genome. PMID:6777246

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

Using complementary DNA from MyoD-transduced fibroblasts to sequence large muscle genes.

PubMed

Waddell, Leigh B; Monnier, Nicole; Cooper, Sandra T; North, Kathryn N; Clarke, Nigel F

2011-08-01

Large muscle genes are often sequenced using complementary DNA (cDNA) made from muscle messenger RNA (mRNA) to reduce the cost and workload associated with sequencing from genomic DNA. Two potential barriers are the availability of a frozen muscle biopsy, and difficulties in detecting nonsense mutations due to nonsense-mediated mRNA decay (NMD). We present patient examples showing that use of MyoD-transduced fibroblasts as a source of muscle-specific mRNA overcomes these potential difficulties in sequencing large muscle-related genes. Copyright © 2011 Wiley Periodicals, Inc.

A Bioinformatics-Based Alternative mRNA Splicing Code that May Explain Some Disease Mutations Is Conserved in Animals.

PubMed

Qu, Wen; Cingolani, Pablo; Zeeberg, Barry R; Ruden, Douglas M

2017-01-01

Deep sequencing of cDNAs made from spliced mRNAs indicates that most coding genes in many animals and plants have pre-mRNA transcripts that are alternatively spliced. In pre-mRNAs, in addition to invariant exons that are present in almost all mature mRNA products, there are at least 6 additional types of exons, such as exons from alternative promoters or with alternative polyA sites, mutually exclusive exons, skipped exons, or exons with alternative 5' or 3' splice sites. Our bioinformatics-based hypothesis is that, in analogy to the genetic code, there is an "alternative-splicing code" in introns and flanking exon sequences, analogous to the genetic code, that directs alternative splicing of many of the 36 types of introns. In humans, we identified 42 different consensus sequences that are each present in at least 100 human introns. 37 of the 42 top consensus sequences are significantly enriched or depleted in at least one of the 36 types of introns. We further supported our hypothesis by showing that 96 out of 96 analyzed human disease mutations that affect RNA splicing, and change alternative splicing from one class to another, can be partially explained by a mutation altering a consensus sequence from one type of intron to that of another type of intron. Some of the alternative splicing consensus sequences, and presumably their small-RNA or protein targets, are evolutionarily conserved from 50 plant to animal species. We also noticed the set of introns within a gene usually share the same splicing codes, thus arguing that one sub-type of splicesosome might process all (or most) of the introns in a given gene. Our work sheds new light on a possible mechanism for generating the tremendous diversity in protein structure by alternative splicing of pre-mRNAs.

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.

Genome-wide mapping of alternative splicing in Arabidopsis thaliana

PubMed Central

Filichkin, Sergei A.; Priest, Henry D.; Givan, Scott A.; Shen, Rongkun; Bryant, Douglas W.; Fox, Samuel E.; Wong, Weng-Keen; Mockler, Todd C.

2010-01-01

Alternative splicing can enhance transcriptome plasticity and proteome diversity. In plants, alternative splicing can be manifested at different developmental stages, and is frequently associated with specific tissue types or environmental conditions such as abiotic stress. We mapped the Arabidopsis transcriptome at single-base resolution using the Illumina platform for ultrahigh-throughput RNA sequencing (RNA-seq). Deep transcriptome sequencing confirmed a majority of annotated introns and identified thousands of novel alternatively spliced mRNA isoforms. Our analysis suggests that at least ∼42% of intron-containing genes in Arabidopsis are alternatively spliced; this is significantly higher than previous estimates based on cDNA/expressed sequence tag sequencing. Random validation confirmed that novel splice isoforms empirically predicted by RNA-seq can be detected in vivo. Novel introns detected by RNA-seq were substantially enriched in nonconsensus terminal dinucleotide splice signals. Alternative isoforms with premature termination codons (PTCs) comprised the majority of alternatively spliced transcripts. Using an example of an essential circadian clock gene, we show that intron retention can generate relatively abundant PTC+ isoforms and that this specific event is highly conserved among diverse plant species. Alternatively spliced PTC+ isoforms can be potentially targeted for degradation by the nonsense mediated mRNA decay (NMD) surveillance machinery or regulate the level of functional transcripts by the mechanism of regulated unproductive splicing and translation (RUST). We demonstrate that the relative ratios of the PTC+ and reference isoforms for several key regulatory genes can be considerably shifted under abiotic stress treatments. Taken together, our results suggest that like in animals, NMD and RUST may be widespread in plants and may play important roles in regulating gene expression. PMID:19858364

Transcription elongation rate has a tissue-specific impact on alternative cleavage and polyadenylation in Drosophila melanogaster.

PubMed

Liu, Xiaochuan; Freitas, Jaime; Zheng, Dinghai; Oliveira, Marta S; Hoque, Mainul; Martins, Torcato; Henriques, Telmo; Tian, Bin; Moreira, Alexandra

2017-12-01

Alternative polyadenylation (APA) is a mechanism that generates multiple mRNA isoforms with different 3'UTRs and/or coding sequences from a single gene. Here, using 3' region extraction and deep sequencing (3'READS), we have systematically mapped cleavage and polyadenylation sites (PASs) in Drosophila melanogaster , expanding the total repertoire of PASs previously identified for the species, especially those located in A-rich genomic sequences. Cis -element analysis revealed distinct sequence motifs around fly PASs when compared to mammalian ones, including the greater enrichment of upstream UAUA elements and the less prominent presence of downstream UGUG elements. We found that over 75% of mRNA genes in Drosophila melanogaster undergo APA. The head tissue tends to use distal PASs when compared to the body, leading to preferential expression of APA isoforms with long 3'UTRs as well as with distal terminal exons. The distance between the APA sites and intron location of PAS are important parameters for APA difference between body and head, suggesting distinct PAS selection contexts. APA analysis of the RpII215 C4 mutant strain, which harbors a mutant RNA polymerase II (RNAPII) with a slower elongation rate, revealed that a 50% decrease in transcriptional elongation rate leads to a mild trend of more usage of proximal, weaker PASs, both in 3'UTRs and in introns, consistent with the "first come, first served" model of APA regulation. However, this trend was not observed in the head, suggesting a different regulatory context in neuronal cells. Together, our data expand the PAS collection for Drosophila melanogaster and reveal a tissue-specific effect of APA regulation by RNAPII elongation rate. © 2017 Liu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

The conserved CAAGAAAGA spacer sequence is an essential element for the formation of 3' termini of the sea urchin H3 histone mRNA by RNA processing.

PubMed Central

Georgiev, O; Birnstiel, M L

1985-01-01

Analysis of cDNA sequences obtained from the small nuclear RNA U7 has previously suggested specific contacts, by base pairing, between the conserved stem-loop structure and CAAGAAAGA sequence of the histone pre-mRNA and the 5'-terminal sequence of the U7 RNA during RNA processing. In order to test some aspects of the model we have created a series of linker scan, deletion and insertion mutants of the 3' terminus of a sea urchin H3 histone gene and have injected mutant DNAs or in vitro synthesized precursors into frog oocyte nuclei for interpretation. We find that, in addition to the stem-loop structure of the mRNA, the CAAGAAAGA spacer transcript within the histone pre-mRNA is required absolutely for RNA processing, as predicted from our model. Spacer sequences immediately downstream of the CAAGAAAGA motif are not complementary to U7 RNA. Nevertheless, they are necessary for obtaining a maximal rate of RNA processing, as is the ACCA sequence coding for the 3' terminus of the mature mRNA. An increase of distance between the mRNA palindrome and the CAAGAAAGA by as little as six nucleotides abolishes all processing. It may, therefore, be useful to regard both these sequence motifs as part of one and the same RNA processing signal with narrowly defined topologies. Interestingly, U7 RNA-dependent 3' processing of histone pre-mRNA can occur in RNA injection experiments only when the in vitro synthesized pre-mRNA contains sequence extensions well beyond the region of sequence complementarities to the U7 RNA. In addition to directing 3' processing the terminal mRNA sequences may have a role in histone mRNA stabilization in the cytoplasmic compartment. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2410259

Characterisation of myosin heavy chain gene variants in the fast and slow muscle fibres of gammarid amphipods.

PubMed

Whiteley, N M; Magnay, J L; McCleary, S J; Nia, S Khazraee; El Haj, A J; Rock, J

2010-10-01

Recent molecular work has revealed a large diversity of myosin heavy chain (MyHC) gene variants in the abdominal musculature of gammarid amphipods. An unusual truncated MyHC transcript from the loop 1 region (Variant A(3)) was consistently observed in multiple species and populations. The current study aimed to determine whether this MyHC variant is specific to a particular muscle fibre type, as a change in net charge to the loop 1 region of Variant A(3) could be functionally significant. The localisation of different fibre types within the abdominal musculature of several gammarid species revealed that the deep flexor and extensor muscles are fast-twitch muscle fibres. The dorsal superficial muscles were identified as slow fibres and the muscles extrinsic to the pleopods were identified as intermediate fibres. Amplification of loop 1 region mRNA from isolated superficial extensor and deep flexor muscles, and subsequent liquid chromatography and sequence analysis revealed that Variant A(3) was the primary MyHC variant in slow muscles, and the conserved A(1) sequence was the primary variant in fast muscles. The specific role of Variant A(3) in the slow muscles remains to be investigated. 2010 Elsevier Inc. All rights reserved.

Translation of vph mRNA in Streptomyces lividans and Escherichia coli after removal of the 5' untranslated leader.

PubMed

Wu, C J; Janssen, G R

1996-10-01

The Streptomyces vinaceus viomycin phosphotransferase (vph) mRNA contains an untranslated leader with a conventional Shine-Dalgarno homology. The vph leader was removed by ligation of the vph coding sequence to the transcriptional start site of a Streptomyces or an Escherichia coli promoter, such that transcription would initiate at the first position of the vph start codon. Analysis of mRNA demonstrated that transcription initiated primarily at the A of the vph AUG translational start codon in both Streptomyces lividans and E. coli; cells expressing the unleadered vph mRNA were resistant to viomycin indicating that the Shine-Dalgarno sequence, or other features contained within the leader, was not necessary for vph translation. Addition of four nucleotides (5'-AUGC-3') onto the 5' end of the unleadered vph mRNA resulted in translation initiation from the vph start codon and the AUG triplet contained within the added sequence. Translational fusions of vph sequence to a Tn5 neo reporter gene indicated that the first 16 codons of vph coding sequence were sufficient to specify the translational start site and reading frame for expression of neomycin resistance in both E. coli and S. lividans.

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

Dwell-Time Distribution, Long Pausing and Arrest of Single-Ribosome Translation through the mRNA Duplex.

PubMed

Xie, Ping

2015-10-09

Proteins in the cell are synthesized by a ribosome translating the genetic information encoded on the single-stranded messenger RNA (mRNA). It has been shown that the ribosome can also translate through the duplex region of the mRNA by unwinding the duplex. Here, based on our proposed model of the ribosome translation through the mRNA duplex we study theoretically the distribution of dwell times of the ribosome translation through the mRNA duplex under the effect of a pulling force externally applied to the ends of the mRNA to unzip the duplex. We provide quantitative explanations of the available single molecule experimental data on the distribution of dwell times with both short and long durations, on rescuing of the long paused ribosomes by raising the pulling force to unzip the duplex, on translational arrests induced by the mRNA duplex and Shine-Dalgarno(SD)-like sequence in the mRNA. The functional consequences of the pauses or arrests caused by the mRNA duplex and the SD sequence are discussed and compared with those obtained from other types of pausing, such as those induced by "hungry" codons or interactions of specific sequences in the nascent chain with the ribosomal exit tunnel.

Dwell-Time Distribution, Long Pausing and Arrest of Single-Ribosome Translation through the mRNA Duplex

PubMed Central

Xie, Ping

2015-01-01

Proteins in the cell are synthesized by a ribosome translating the genetic information encoded on the single-stranded messenger RNA (mRNA). It has been shown that the ribosome can also translate through the duplex region of the mRNA by unwinding the duplex. Here, based on our proposed model of the ribosome translation through the mRNA duplex we study theoretically the distribution of dwell times of the ribosome translation through the mRNA duplex under the effect of a pulling force externally applied to the ends of the mRNA to unzip the duplex. We provide quantitative explanations of the available single molecule experimental data on the distribution of dwell times with both short and long durations, on rescuing of the long paused ribosomes by raising the pulling force to unzip the duplex, on translational arrests induced by the mRNA duplex and Shine-Dalgarno(SD)-like sequence in the mRNA. The functional consequences of the pauses or arrests caused by the mRNA duplex and the SD sequence are discussed and compared with those obtained from other types of pausing, such as those induced by “hungry” codons or interactions of specific sequences in the nascent chain with the ribosomal exit tunnel. PMID:26473825

Nuclease footprint analyses of the interactions between RNase P ribozyme and a model mRNA substrate.

PubMed Central

Trang, P; Hsu, A W; Liu, F

1999-01-01

RNase P ribozyme cleaves an RNA helix substrate which resembles the acceptor stem and T-stem structures of its natural tRNA substrate. By linking the ribozyme covalently to a sequence (guide sequence) complementary to a target RNA, the catalytic RNA can be converted into a sequence-specific ribozyme, M1GS RNA. We have previously shown that M1GS RNA can efficiently cleave the mRNA sequence encoding thymidine kinase (TK) of herpes simplex virus 1. In this study, a footprint procedure using different nucleases was carried out to map the regions of a M1GS ribozyme that potentially interact with the TK mRNA substrate. The ribozyme regions that are protected from nuclease degradation in the presence of the TK mRNA substrate include those that interact with the acceptor stem and T-stem, the 3' terminal CCA sequence and the cleavage site of a tRNA substrate. However, some of the protected regions (e.g. P13 and P14) are unique and not among those protected in the presence of a tRNA substrate. Identification of the regions that interact with a mRNA substrate will allow us to study how M1GS RNA recognizes a mRNA substrate and facilitate the development of mRNA-cleaving ribozymes for gene-targeting applications. PMID:10556315

Monitoring the Spatiotemporal Activities of miRNAs in Small Animal Models Using Molecular Imaging Modalities

PubMed Central

Baril, Patrick; Ezzine, Safia; Pichon, Chantal

2015-01-01

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by binding mRNA targets via sequence complementary inducing translational repression and/or mRNA degradation. A current challenge in the field of miRNA biology is to understand the functionality of miRNAs under physiopathological conditions. Recent evidence indicates that miRNA expression is more complex than simple regulation at the transcriptional level. MiRNAs undergo complex post-transcriptional regulations such miRNA processing, editing, accumulation and re-cycling within P-bodies. They are dynamically regulated and have a well-orchestrated spatiotemporal localization pattern. Real-time and spatio-temporal analyses of miRNA expression are difficult to evaluate and often underestimated. Therefore, important information connecting miRNA expression and function can be lost. Conventional miRNA profiling methods such as Northern blot, real-time PCR, microarray, in situ hybridization and deep sequencing continue to contribute to our knowledge of miRNA biology. However, these methods can seldom shed light on the spatiotemporal organization and function of miRNAs in real-time. Non-invasive molecular imaging methods have the potential to address these issues and are thus attracting increasing attention. This paper reviews the state-of-the-art of methods used to detect miRNAs and discusses their contribution in the emerging field of miRNA biology and therapy. PMID:25749473

Monitoring the spatiotemporal activities of miRNAs in small animal models using molecular imaging modalities.

PubMed

Baril, Patrick; Ezzine, Safia; Pichon, Chantal

2015-03-04

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by binding mRNA targets via sequence complementary inducing translational repression and/or mRNA degradation. A current challenge in the field of miRNA biology is to understand the functionality of miRNAs under physiopathological conditions. Recent evidence indicates that miRNA expression is more complex than simple regulation at the transcriptional level. MiRNAs undergo complex post-transcriptional regulations such miRNA processing, editing, accumulation and re-cycling within P-bodies. They are dynamically regulated and have a well-orchestrated spatiotemporal localization pattern. Real-time and spatio-temporal analyses of miRNA expression are difficult to evaluate and often underestimated. Therefore, important information connecting miRNA expression and function can be lost. Conventional miRNA profiling methods such as Northern blot, real-time PCR, microarray, in situ hybridization and deep sequencing continue to contribute to our knowledge of miRNA biology. However, these methods can seldom shed light on the spatiotemporal organization and function of miRNAs in real-time. Non-invasive molecular imaging methods have the potential to address these issues and are thus attracting increasing attention. This paper reviews the state-of-the-art of methods used to detect miRNAs and discusses their contribution in the emerging field of miRNA biology and therapy.

Deep sequencing of evolving pathogen populations: applications, errors, and bioinformatic solutions

PubMed Central

2014-01-01

Deep sequencing harnesses the high throughput nature of next generation sequencing technologies to generate population samples, treating information contained in individual reads as meaningful. Here, we review applications of deep sequencing to pathogen evolution. Pioneering deep sequencing studies from the virology literature are discussed, such as whole genome Roche-454 sequencing analyses of the dynamics of the rapidly mutating pathogens hepatitis C virus and HIV. Extension of the deep sequencing approach to bacterial populations is then discussed, including the impacts of emerging sequencing technologies. While it is clear that deep sequencing has unprecedented potential for assessing the genetic structure and evolutionary history of pathogen populations, bioinformatic challenges remain. We summarise current approaches to overcoming these challenges, in particular methods for detecting low frequency variants in the context of sequencing error and reconstructing individual haplotypes from short reads. PMID:24428920

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.

Protein structure and the sequential structure of mRNA: alpha-helix and beta-sheet signals at the nucleotide level.

PubMed

Brunak, S; Engelbrecht, J

1996-06-01

A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed. We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting protein. The degeneracy of the genetic code allows for a biased selection of codons which may control the translational rate of the ribosome, and may thus in vivo have a catalyzing effect on the folding of the polypeptide chain. A complete search for GenBank nucleotide sequences coding for structural entries in the Brookhaven Protein Data Bank produced 719 protein chains with matching mRNA sequence, amino acid sequence, and secondary structure assignment. By neural network analysis, we found strong signals in mRNA sequence regions surrounding helices and sheets. These signals do not originate from the clustering of rare codons, but from the similarity of codons coding for very abundant amino acid residues at the N- and C-termini of helices and sheets. No correlation between the positioning of rare codons and the location of structural units was found. The mRNA signals were also compared with conserved nucleotide features of 16S-like ribosomal RNA sequences and related to mechanisms for maintaining the correct reading frame by the ribosome.

GWIPS-viz: development of a ribo-seq genome browser

PubMed Central

Michel, Audrey M.; Fox, Gearoid; M. Kiran, Anmol; De Bo, Christof; O’Connor, Patrick B. F.; Heaphy, Stephen M.; Mullan, James P. A.; Donohue, Claire A.; Higgins, Desmond G.; Baranov, Pavel V.

2014-01-01

We describe the development of GWIPS-viz (http://gwips.ucc.ie), an online genome browser for viewing ribosome profiling data. Ribosome profiling (ribo-seq) is a recently developed technique that provides genome-wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of ribosome-protected messenger RNA (mRNA) fragments, which allows the ribosome density along all mRNA transcripts present in the cell to be quantified. Since its inception, ribo-seq has been carried out in a number of eukaryotic and prokaryotic organisms. Owing to the increasing interest in ribo-seq, there is a pertinent demand for a dedicated ribo-seq genome browser. GWIPS-viz is based on The University of California Santa Cruz (UCSC) Genome Browser. Ribo-seq tracks, coupled with mRNA-seq tracks, are currently available for several genomes: human, mouse, zebrafish, nematode, yeast, bacteria (Escherichia coli K12, Bacillus subtilis), human cytomegalovirus and bacteriophage lambda. Our objective is to continue incorporating published ribo-seq data sets so that the wider community can readily view ribosome profiling information from multiple studies without the need to carry out computational processing. PMID:24185699

Optimization of preservation and storage time of sponge tissues to obtain quality mRNA for next-generation sequencing.

PubMed

Riesgo, Ana; Pérez-Porro, Alicia R; Carmona, Susana; Leys, Sally P; Giribet, Gonzalo

2012-03-01

Transcriptome sequencing with next-generation sequencing technologies has the potential for addressing many long-standing questions about the biology of sponges. Transcriptome sequence quality depends on good cDNA libraries, which requires high-quality mRNA. Standard protocols for preserving and isolating mRNA often require optimization for unusual tissue types. Our aim was assessing the efficiency of two preservation modes, (i) flash freezing with liquid nitrogen (LN₂) and (ii) immersion in RNAlater, for the recovery of high-quality mRNA from sponge tissues. We also tested whether the long-term storage of samples at -80 °C affects the quantity and quality of mRNA. We extracted mRNA from nine sponge species and analysed the quantity and quality (A260/230 and A260/280 ratios) of mRNA according to preservation method, storage time, and taxonomy. The quantity and quality of mRNA depended significantly on the preservation method used (LN₂) outperforming RNAlater), the sponge species, and the interaction between them. When the preservation was analysed in combination with either storage time or species, the quantity and A260/230 ratio were both significantly higher for LN₂-preserved samples. Interestingly, individual comparisons for each preservation method over time indicated that both methods performed equally efficiently during the first month, but RNAlater lost efficiency in storage times longer than 2 months compared with flash-frozen samples. In summary, we find that for long-term preservation of samples, flash freezing is the preferred method. If LN₂ is not available, RNAlater can be used, but mRNA extraction during the first month of storage is advised. © 2011 Blackwell Publishing Ltd.

Synthesis and biological activity of artificial mRNA prepared with novel phosphorylating reagents

PubMed Central

Nagata, Seigo; Hamasaki, Tomohiro; Uetake, Koichi; Masuda, Hirofumi; Takagaki, Kazuchika; Oka, Natsuhisa; Wada, Takeshi; Ohgi, Tadaaki; Yano, Junichi

2010-01-01

Though medicines that target mRNA are under active investigation, there has been little or no effort to develop mRNA itself as a medicine. Here, we report the synthesis of a 130-nt mRNA sequence encoding a 33-amino-acid peptide that includes the sequence of glucagon-like peptide-1, a peptide that stimulates glucose-dependent insulin secretion from the pancreas. The synthesis method used, which had previously been developed in our laboratory, was based on the use of 2-cyanoethoxymethyl as the 2′-hydroxy protecting group. We also developed novel, highly reactive phosphotriester pyrophosphorylating reagents to pyrophosphorylate the 5′-end of the 130-mer RNA in preparation for capping. We completed the synthesis of the artificial mRNA by the enzymatic addition of a 5′-cap and a 3′-poly(A) tail to the pyrophosphorylated 130-mer and showed that the resulting mRNA supported protein synthesis in a cell-free system and in whole cells. As far as we know, this is the first time that mRNA has been prepared from a chemically synthesized RNA sequence. As well as providing a research tool for the intracellular expression of peptides, the technology described here may be used for the production of mRNA for medical applications. PMID:20660478

Large-scale identification and comparative analysis of miRNA expression profile in the respiratory tree of the sea cucumber Apostichopus japonicus during aestivation.

PubMed

Chen, Muyan; Storey, Kenneth B

2014-02-01

The sea cucumber Apostichopus japonicus withstands high water temperatures in the summer by suppressing its metabolic rate and entering a state of aestivation. We hypothesized that changes in the expression of miRNAs could provide important post-transcriptional regulation of gene expression during hypometabolism via control over mRNA translation. The present study analyzed profiles of miRNA expression in the sea cucumber respiratory tree using Solexa deep sequencing technology. We identified 279 sea cucumber miRNAs, including 15 novel miRNAs specific to sea cucumber. Animals sampled during deep aestivation (DA; after at least 15 days of continuous torpor) were compared with animals from a non-aestivation (NA) state (animals that had passed through aestivation and returned to an active state). We identified 30 differentially expressed miRNAs ([RPM (reads per million) >10, |FC| (|fold change|)≥1, FDR (false discovery rate)<0.01]) during aestivation, which were validated by two other miRNA profiling methods: miRNA microarray and real-time PCR. Among the most prominent miRNA species, miR-124, miR-124-3p, miR-79, miR-9 and miR-2010 were significantly over-expressed during deep aestivation compared with non-aestivation animals, suggesting that these miRNAs may play important roles in metabolic rate suppression during aestivation. High-throughput sequencing data and microarray data have been submitted to the GEO database with accession number: 16902695. Copyright © 2014 Elsevier B.V. All rights reserved.

mRNA stability in mammalian cells.

PubMed Central

Ross, J

1995-01-01

This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end. PMID:7565413

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

PubMed

Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell W; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay A A

2014-10-01

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.

Staufen1 senses overall transcript secondary structure to regulate translation

PubMed Central

Ricci, Emiliano P; Kucukural, Alper; Cenik, Can; Mercier, Blandine C; Singh, Guramrit; Heyer, Erin E; Ashar-Patel, Ami; Peng, Lingtao; Moore, Melissa J

2015-01-01

Human Staufen1 (Stau1) is a double-stranded RNA (dsRNA)-binding protein implicated in multiple post-transcriptional gene-regulatory processes. Here we combined RNA immunoprecipitation in tandem (RIPiT) with RNase footprinting, formaldehyde cross-linking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1-binding sites transcriptome wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3′ untranslated regions (UTRs) or in ‘strongly distal’ 3′ UTRs. Stau1 also interacts with actively translating ribosomes and with mRNA coding sequences (CDSs) and 3′ UTRs in proportion to their GC content and propensity to form internal secondary structure. On mRNAs with high CDS GC content, higher Stau1 levels lead to greater ribosome densities, thus suggesting a general role for Stau1 in modulating translation elongation through structured CDS regions. Our results also indicate that Stau1 regulates translation of transcription-regulatory proteins. PMID:24336223

AthMethPre: a web server for the prediction and query of mRNA m6A sites in Arabidopsis thaliana.

PubMed

Xiang, Shunian; Yan, Zhangming; Liu, Ke; Zhang, Yaou; Sun, Zhirong

2016-10-18

N 6 -Methyladenosine (m 6 A) is the most prevalent and abundant modification in mRNA that has been linked to many key biological processes. High-throughput experiments have generated m 6 A-peaks across the transcriptome of A. thaliana, but the specific methylated sites were not assigned, which impedes the understanding of m 6 A functions in plants. Therefore, computational prediction of mRNA m 6 A sites becomes emergently important. Here, we present a method to predict the m 6 A sites for A. thaliana mRNA sequence(s). To predict the m 6 A sites of an mRNA sequence, we employed the support vector machine to build a classifier using the features of the positional flanking nucleotide sequence and position-independent k-mer nucleotide spectrum. Our method achieved good performance and was applied to a web server to provide service for the prediction of A. thaliana m 6 A sites. The server also provides a comprehensive database of predicted transcriptome-wide m 6 A sites and curated m 6 A-seq peaks from the literature for query and visualization. The AthMethPre web server is the first web server that provides a user-friendly tool for the prediction and query of A. thaliana mRNA m 6 A sites, which is freely accessible for public use at .

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

Deep sequencing shows low-level oncogenic hepatitis B virus variants persists post-liver transplant despite potent anti-HBV prophylaxis.

PubMed

Lau, K C K; Osiowy, C; Giles, E; Lusina, B; van Marle, G; Burak, K W; Coffin, C S

2018-06-01

Recent studies suggest that withdrawal of hepatitis B immune globulin (HBIG) and nucleos(t)ide analogues (NA) prophylaxis may be considered in HBV surface antigen (HBsAg)-negative liver transplant (LT) recipients with a low risk of disease recurrence. However, the frequency of occult HBV infection (OBI) and HBV variants after LT in the current era of potent NA therapy is unknown. Twelve LT recipients on prophylaxis were tested in matched plasma and peripheral blood mononuclear cells (PBMCs) for HBV quasispecies by in-house nested PCR and next-generation sequencing of amplicons. HBV covalently closed circular DNA (cccDNA) was detected in Hirt DNA isolated from PBMCs with cccDNA-specific primers and confirmed by nucleic acid hybridization and Sanger sequencing. HBV mRNA in PBMC was detected with reverse-transcriptase nested PCR. In LT recipients on immunosuppressive therapy (10/12 male; median age 57.5 [IQR: 39.8-66.5]; median follow-up post-LT 60 months; 6 pre-LT hepatocellular carcinoma [HCC]), 9 were HBsAg-. HBV DNA was detected in all plasma and PBMC tested; cccDNA and/or mRNA was detected in the PBMC of 10/12 patients. Significant HBV quasispecies diversity (ie 143-2212 nonredundant HBV species) was noted in both sites, and single nucleotide polymorphisms associated with cirrhosis and HCC were detected at varying frequencies. In conclusion, OBI and HBV variants associated with severe liver disease persist in LT recipients on prophylaxis. Although HBV control and cccDNA transcriptional silencing may occur despite immunosuppression, complete virological eradication does not occur in LT recipients with a history of HBV-related end-stage liver disease. © 2018 John Wiley & Sons Ltd.

Molecular evolution of adiponectin in Carnivora and its mRNA expression in relation to hepatic lipidosis.

PubMed

Nieminen, Petteri; Rouvinen-Watt, Kirsti; Kapiainen, Suvi; Harris, Lora; Mustonen, Anne-Mari

2010-09-15

Adiponectin is a novel adipocyte-derived hormone with low circulating concentrations and/or mRNA expression in obesity and non-alcoholic fatty liver disease (NAFLD). The adiponectin mRNA of several Carnivora species was sequenced to enable further gene expression studies in this clade with potential experimental species to examine the connections of hypoadiponectinemia to hepatic lipidosis. In addition, adiponectin mRNA expression was studied in the retroperitoneal fat of the American mink (Neovison vison), as hepatic lipidosis with close similarities to NAFLD can be rapidly induced to the species by fasting. The mRNA expression was determined after overnight-7d of food deprivation and 28d of re-feeding and correlated to the liver fat %. The homologies between the determined carnivoran mRNA sequences and that of the domestic dog were 92.2-99.1%. As the mRNA expression was not affected by short-term fasting and did not correlate with the liver fat %, there seems to be no clear connection between adiponectin and the development of lipidosis in the American mink. In the future, the obtained sequences can be utilized in further studies of adiponectin expression in comparative endocrinology. Copyright (c) 2010 Elsevier Inc. All rights reserved.

DeepBase: annotation and discovery of microRNAs and other noncoding RNAs from deep-sequencing data.

PubMed

Yang, Jian-Hua; Qu, Liang-Hu

2012-01-01

Recent advances in high-throughput deep-sequencing technology have produced large numbers of short and long RNA sequences and enabled the detection and profiling of known and novel microRNAs (miRNAs) and other noncoding RNAs (ncRNAs) at unprecedented sensitivity and depth. In this chapter, we describe the use of deepBase, a database that we have developed to integrate all public deep-sequencing data and to facilitate the comprehensive annotation and discovery of miRNAs and other ncRNAs from these data. deepBase provides an integrative, interactive, and versatile web graphical interface to evaluate miRBase-annotated miRNA genes and other known ncRNAs, explores the expression patterns of miRNAs and other ncRNAs, and discovers novel miRNAs and other ncRNAs from deep-sequencing data. deepBase also provides a deepView genome browser to comparatively analyze these data at multiple levels. deepBase is available at http://deepbase.sysu.edu.cn/.

Nucleotide sequences of bovine alpha S1- and kappa-casein cDNAs.

PubMed Central

Stewart, A F; Willis, I M; Mackinlay, A G

1984-01-01

The nucleotide sequences corresponding to bovine alpha S1- and kappa-casein mRNAs are presented. An unusual alpha S1-casein cDNA has been characterised whose 5' end commences upstream from its putative TATA box. The alpha S1-casein mRNA is compared to rat alpha-casein mRNA and two components of divergence are identified. Firstly, the two sequences have diverged at a high point mutation rate and the rate of amino acid replacement by this mechanism is at least as great as the rate of divergence of any other part of the mRNAs. Secondly, the protein coding sequence has been subjected to several insertion/deletion events, one of which may be an example of exon shuffling . The kappa-casein mRNA sequence verifies the proposition that it has arisen from a different ancestral gene to the other caseins. Images PMID:6328443

Making sense of deep sequencing

PubMed Central

Goldman, D.; Domschke, K.

2016-01-01

This review, the first of an occasional series, tries to make sense of the concepts and uses of deep sequencing of polynucleic acids (DNA and RNA). Deep sequencing, synonymous with next-generation sequencing, high-throughput sequencing and massively parallel sequencing, includes whole genome sequencing but is more often and diversely applied to specific parts of the genome captured in different ways, for example the highly expressed portion of the genome known as the exome and portions of the genome that are epigenetically marked either by DNA methylation, the binding of proteins including histones, or that are in different configurations and thus more or less accessible to enzymes that cleave DNA. Deep sequencing of RNA (RNASeq) reverse-transcribed to complementary DNA is invaluable for measuring RNA expression and detecting changes in RNA structure. Important concepts in deep sequencing include the length and depth of sequence reads, mapping and assembly of reads, sequencing error, haplotypes, and the propensity of deep sequencing, as with other types of ‘big data’, to generate large numbers of errors, requiring monitoring for methodologic biases and strategies for replication and validation. Deep sequencing yields a unique genetic fingerprint that can be used to identify a person, and a trove of predictors of genetic medical diseases. Deep sequencing to identify epigenetic events including changes in DNA methylation and RNA expression can reveal the history and impact of environmental exposures. Because of the power of sequencing to identify and deliver biomedically significant information about a person and their blood relatives, it creates ethical dilemmas and practical challenges in research and clinical care, for example the decision and procedures to report incidental findings that will increasingly and frequently be discovered. PMID:24925306

Strategies for Protein Overproduction in Escherichia coli.

ERIC Educational Resources Information Center

Mott, John E.

1984-01-01

Examines heterologous expression in Escherichia coli and the role of regulatory sequences which control gene expression at transcription resulting in abundant production of messenger RNA and regulatory sequences in mRNA which promote efficient translation. Also examines the role of E. coli cells in stabilizing mRNA and protein that is…

Application of sorting and next generation sequencing to study 5΄-UTR influence on translation efficiency in Escherichia coli

PubMed Central

Evfratov, Sergey A.; Osterman, Ilya A.; Komarova, Ekaterina S.; Pogorelskaya, Alexandra M.; Rubtsova, Maria P.; Zatsepin, Timofei S.; Semashko, Tatiana A.; Kostryukova, Elena S.; Mironov, Andrey A.; Burnaev, Evgeny; Krymova, Ekaterina; Gelfand, Mikhail S.; Govorun, Vadim M.; Bogdanov, Alexey A.; Dontsova, Olga A.

2017-01-01

Abstract Yield of protein per translated mRNA may vary by four orders of magnitude. Many studies analyzed the influence of mRNA features on the translation yield. However, a detailed understanding of how mRNA sequence determines its propensity to be translated is still missing. Here, we constructed a set of reporter plasmid libraries encoding CER fluorescent protein preceded by randomized 5΄ untranslated regions (5΄-UTR) and Red fluorescent protein (RFP) used as an internal control. Each library was transformed into Escherchia coli cells, separated by efficiency of CER mRNA translation by a cell sorter and subjected to next generation sequencing. We tested efficiency of translation of the CER gene preceded by each of 48 natural 5΄-UTR sequences and introduced random and designed mutations into natural and artificially selected 5΄-UTRs. Several distinct properties could be ascribed to a group of 5΄-UTRs most efficient in translation. In addition to known ones, several previously unrecognized features that contribute to the translation enhancement were found, such as low proportion of cytidine residues, multiple SD sequences and AG repeats. The latter could be identified as translation enhancer, albeit less efficient than SD sequence in several natural 5΄-UTRs. PMID:27899632

Deep sequencing and genome-wide analysis reveals the expansion of MicroRNA genes in the gall midge Mayetiola destructor

PubMed Central

2013-01-01

Background MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in regulating post transcriptional gene expression. Gall midges encompass a large group of insects that are of economic importance and also possess fascinating biological traits. The gall midge Mayetiola destructor, commonly known as the Hessian fly, is a destructive pest of wheat and model organism for studying gall midge biology and insect – host plant interactions. Results In this study, we systematically analyzed miRNAs from the Hessian fly. Deep-sequencing a Hessian fly larval transcriptome led to the identification of 89 miRNA species that are either identical or very similar to known miRNAs from other insects, and 184 novel miRNAs that have not been reported from other species. A genome-wide search through a draft Hessian fly genome sequence identified a total of 611 putative miRNA-encoding genes based on sequence similarity and the existence of a stem-loop structure for miRNA precursors. Analysis of the 611 putative genes revealed a striking feature: the dramatic expansion of several miRNA gene families. The largest family contained 91 genes that encoded 20 different miRNAs. Microarray analyses revealed the expression of miRNA genes was strictly regulated during Hessian fly larval development and abundance of many miRNA genes were affected by host genotypes. Conclusion The identification of a large number of miRNAs for the first time from a gall midge provides a foundation for further studies of miRNA functions in gall midge biology and behavior. The dramatic expansion of identical or similar miRNAs provides a unique system to study functional relations among miRNA iso-genes as well as changes in sequence specificity due to small changes in miRNAs and in their mRNA targets. These results may also facilitate the identification of miRNA genes for potential pest control through transgenic approaches. PMID:23496979

Curcumin may serve an anticancer role in human osteosarcoma cell line U-2 OS by targeting ITPR1.

PubMed

Luo, Zhanpeng; Li, Dawei; Luo, Xiaobo; Li, Litao; Gu, Suxi; Yu, Long; Ma, Yuanzheng

2018-04-01

The present study aimed to determine the mechanisms of action of curcumin in osteosarcoma. Human osteosarcoma U-2 OS cells was purchased from the Cell Bank of the Chinese Academy of Sciences. RNA sequencing analysis was performed for 2 curcumin-treated samples and 2 control samples using Illumina deep sequencing technology. The differentially expressed genes were identified using Cufflink software. Enrichment and protein-protein interaction network analyses were performed separately using cluster Profiler package and Cytoscape software to identify key genes. Then, the mRNA levels of key genes were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in U-2 OS cells. Finally, cell apoptosis, proliferation, migration and invasion arrays were performed. In total, 201 DEGs were identified in the curcumin-treated group. EEF1A1 (degree=88), ATF7IP, HIF1A, SMAD7, CLTC, MCM10, ITPR1, ADAM15, WWP2 and ATP5C1, which were enriched in 'biological process', exhibited higher degrees than other genes in the PPI network. RT-qPCR demonstrated that treatment with curcumin was able to significantly increase the levels of CLTC and ITPR1 mRNA in curcumin-treated cells compared with control. In addition, targeting ITPR1 with curcumin significantly promoted apoptosis and suppressed proliferation, migration and invasion. Targeting ITPR1 via curcumin may serve an anticancer role by mediating apoptosis, proliferation, migration and invasion in U-2 OS cells.

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

cAMP-Mediated Stimulation of Tyrosine Hydroxylase mRNA Translation Is Mediated by Polypyrimidine-Rich Sequences within Its 3′-Untranslated Region and Poly(C)-Binding Protein 2

PubMed Central

Xu, Lu; Sterling, Carol R.

2009-01-01

Tyrosine hydroxylase (TH) plays a critical role in maintaining the appropriate concentrations of catecholamine neurotransmitters in brain and periphery, particularly during long-term stress, long-term drug treatment, or neurodegenerative diseases. Its expression is controlled by both transcriptional and post-transcriptional mechanisms. In a previous report, we showed that treatment of rat midbrain slice explant cultures or mouse MN9D cells with cAMP analog or forskolin leads to induction of TH protein without concomitant induction of TH mRNA. We further showed that cAMP activates mechanisms that regulate TH mRNA translation via cis-acting sequences within its 3′-untranslated region (UTR). In the present report, we extend these studies to show that MN9D cytoplasmic proteins bind to the same TH mRNA 3′-UTR domain that is required for the cAMP response. RNase T1 mapping demonstrates binding of proteins to a 27-nucleotide polypyrimidine-rich sequence within this domain. A specific mutation within the polypyrimidine-rich sequence inhibits protein binding and cAMP-mediated translational activation. UV-cross-linking studies identify a ∼44-kDa protein as a major TH mRNA 3′-UTR binding factor, and cAMP induces the 40- to 42-kDa poly(C)-binding protein-2 (PCBP2) in MN9D cells. We show that PCBP2 binds to the TH mRNA 3′-UTR domain that participates in the cAMP response. Overexpression of PCBP2 induces TH protein without concomitant induction of TH mRNA. These results support a model in which cAMP induces PCBP2, leading to increased interaction with its cognate polypyrimidine binding site in the TH mRNA 3′-UTR. This increased interaction presumably plays a role in the activation of TH mRNA translation by cAMP in dopaminergic neurons. PMID:19620256

De novo peptide sequencing by deep learning

PubMed Central

Tran, Ngoc Hieu; Zhang, Xianglilan; Xin, Lei; Shan, Baozhen; Li, Ming

2017-01-01

De novo peptide sequencing from tandem MS data is the key technology in proteomics for the characterization of proteins, especially for new sequences, such as mAbs. In this study, we propose a deep neural network model, DeepNovo, for de novo peptide sequencing. DeepNovo architecture combines recent advances in convolutional neural networks and recurrent neural networks to learn features of tandem mass spectra, fragment ions, and sequence patterns of peptides. The networks are further integrated with local dynamic programming to solve the complex optimization task of de novo sequencing. We evaluated the method on a wide variety of species and found that DeepNovo considerably outperformed state of the art methods, achieving 7.7–22.9% higher accuracy at the amino acid level and 38.1–64.0% higher accuracy at the peptide level. We further used DeepNovo to automatically reconstruct the complete sequences of antibody light and heavy chains of mouse, achieving 97.5–100% coverage and 97.2–99.5% accuracy, without assisting databases. Moreover, DeepNovo is retrainable to adapt to any sources of data and provides a complete end-to-end training and prediction solution to the de novo sequencing problem. Not only does our study extend the deep learning revolution to a new field, but it also shows an innovative approach in solving optimization problems by using deep learning and dynamic programming. PMID:28720701

DeepMirTar: a deep-learning approach for predicting human miRNA targets.

PubMed

Wen, Ming; Cong, Peisheng; Zhang, Zhimin; Lu, Hongmei; Li, Tonghua

2018-06-01

MicroRNAs (miRNAs) are small noncoding RNAs that function in RNA silencing and post-transcriptional regulation of gene expression by targeting messenger RNAs (mRNAs). Because the underlying mechanisms associated with miRNA binding to mRNA are not fully understood, a major challenge of miRNA studies involves the identification of miRNA-target sites on mRNA. In silico prediction of miRNA-target sites can expedite costly and time-consuming experimental work by providing the most promising miRNA-target-site candidates. In this study, we reported the design and implementation of DeepMirTar, a deep-learning-based approach for accurately predicting human miRNA targets at the site level. The predicted miRNA-target sites are those having canonical or non-canonical seed, and features, including high-level expert-designed, low-level expert-designed, and raw-data-level, were used to represent the miRNA-target site. Comparison with other state-of-the-art machine-learning methods and existing miRNA-target-prediction tools indicated that DeepMirTar improved overall predictive performance. DeepMirTar is freely available at https://github.com/Bjoux2/DeepMirTar_SdA. lith@tongji.edu.cn, hongmeilu@csu.edu.cn. Supplementary data are available at Bioinformatics online.

Analysis of Ribosome Stalling and Translation Elongation Dynamics by Deep Learning.

PubMed

Zhang, Sai; Hu, Hailin; Zhou, Jingtian; He, Xuan; Jiang, Tao; Zeng, Jianyang

2017-09-27

Ribosome stalling is manifested by the local accumulation of ribosomes at specific codon positions of mRNAs. Here, we present ROSE, a deep learning framework to analyze high-throughput ribosome profiling data and estimate the probability of a ribosome stalling event occurring at each genomic location. Extensive validation tests on independent data demonstrated that ROSE possessed higher prediction accuracy than conventional prediction models, with an increase in the area under the receiver operating characteristic curve by up to 18.4%. In addition, genome-wide statistical analyses showed that ROSE predictions can be well correlated with diverse putative regulatory factors of ribosome stalling. Moreover, the genome-wide ribosome stalling landscapes of both human and yeast computed by ROSE recovered the functional interplays between ribosome stalling and cotranslational events in protein biogenesis, including protein targeting by the signal recognition particles and protein secondary structure formation. Overall, our study provides a novel method to complement the ribosome profiling techniques and further decipher the complex regulatory mechanisms underlying translation elongation dynamics encoded in the mRNA sequence. Copyright © 2017 Elsevier Inc. All rights reserved.

Genomic organization and sequence of the Gus-s/sup a/ allele of the murine. beta. -glucuronidase gene

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

Funkenstein, B.; Leary, S.L.; Stein, J.C.

1988-03-01

The Gus-s/sup ..cap alpha../ allele of the mouse ..beta..-glucuronidase gene exhibits a high degree of inducibility by androgens due to its linkage with the Gus-r/sup ..cap alpha../ regulatory locus. The authors isolated Gus-s/sup ..cap alpha../ on a 28-kilobase pair fragment of mouse chromosome 5 and found that it contains 12 exons and 11 intervening sequences spanning 14 kilobase pairs of this genomic segment. The mRNA cap site was identified by ribonuclease protection and primer extension analyses which revealed an unusually short 5' noncoding sequence of 12 nucleotides. Proximal regulatory sequences in the 5'-flanking DNA and the complete sequence of themore » Gus-s/sup ..cap alpha../ mRNA transcript were also determined. Comparison of the amino acid sequence determined from the Gus-s/sup ..cap alpha../ nucleotide sequence with that of human ..beta..-glucuronidase indicated that the two human mRNA species differ due to alternate splicing of an exon homologous to exon 6 of the mouse gene.« less

Short intronic repeat sequences facilitate circular RNA production

PubMed Central

Liang, Dongming

2014-01-01

Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery “backsplices” and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3′ end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA. PMID:25281217

A role for exon sequences in alternative splicing of the human fibronectin gene.

PubMed Central

Mardon, H J; Sebastio, G; Baralle, F E

1987-01-01

Exon EDIIIA of the fibronectin (Fn) gene is alternatively spliced via pathways which either skip or include the whole exon in the messenger RNA (mRNA). We have investigated the role of EDIIIA exon sequences in the human Fn gene in determining alternative splicing of this exon during transient expression of alpha globin/Fn minigene hybrids in HeLa cells. We demonstrate that a DNA sequence of 81bp within the central region of exon EDIIIA is required for alternative splicing during processing of the primary transcript to generate both EDIIIA+ and EDIIIA- mRNA's. Furthermore, alternative splicing of EDIIIA only occurs when this sequence is present in the correct orientation since when it is in antisense orientation splicing always occurs via exon-skipping generating EDIIIA- mRNA. Images PMID:3671064

Single-cell mRNA cytometry via sequence-specific nanoparticle clustering and trapping

NASA Astrophysics Data System (ADS)

Labib, Mahmoud; Mohamadi, Reza M.; Poudineh, Mahla; Ahmed, Sharif U.; Ivanov, Ivaylo; Huang, Ching-Lung; Moosavi, Maral; Sargent, Edward H.; Kelley, Shana O.

2018-05-01

Cell-to-cell variation in gene expression creates a need for techniques that can characterize expression at the level of individual cells. This is particularly true for rare circulating tumour cells, in which subtyping and drug resistance are of intense interest. Here we describe a method for cell analysis—single-cell mRNA cytometry—that enables the isolation of rare cells from whole blood as a function of target mRNA sequences. This approach uses two classes of magnetic particles that are labelled to selectively hybridize with different regions of the target mRNA. Hybridization leads to the formation of large magnetic clusters that remain localized within the cells of interest, thereby enabling the cells to be magnetically separated. Targeting specific intracellular mRNAs enablescirculating tumour cells to be distinguished from normal haematopoietic cells. No polymerase chain reaction amplification is required to determine RNA expression levels and genotype at the single-cell level, and minimal cell manipulation is required. To demonstrate this approach we use single-cell mRNA cytometry to detect clinically important sequences in prostate cancer specimens.

Exploring fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing

NASA Astrophysics Data System (ADS)

Zhang, Xiao-Yong; Wang, Guang-Hua; Xu, Xin-Ya; Nong, Xu-Hua; Wang, Jie; Amin, Muhammad; Qi, Shu-Hua

2016-10-01

The present study investigated the fungal diversity in four different deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing of the nuclear ribosomal internal transcribed spacer-1 (ITS1). A total of 40,297 fungal ITS1 sequences clustered into 420 operational taxonomic units (OTUs) with 97% sequence similarity and 170 taxa were recovered from these sediments. Most ITS1 sequences (78%) belonged to the phylum Ascomycota, followed by Basidiomycota (17.3%), Zygomycota (1.5%) and Chytridiomycota (0.8%), and a small proportion (2.4%) belonged to unassigned fungal phyla. Compared with previous studies on fungal diversity of sediments from deep-sea environments by culture-dependent approach and clone library analysis, the present result suggested that Illumina sequencing had been dramatically accelerating the discovery of fungal community of deep-sea sediments. Furthermore, our results revealed that Sordariomycetes was the most diverse and abundant fungal class in this study, challenging the traditional view that the diversity of Sordariomycetes phylotypes was low in the deep-sea environments. In addition, more than 12 taxa accounted for 21.5% sequences were found to be rarely reported as deep-sea fungi, suggesting the deep-sea sediments from Okinawa Trough harbored a plethora of different fungal communities compared with other deep-sea environments. To our knowledge, this study is the first exploration of the fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing.

The VP35 and VP40 proteins of filoviruses. Homology between Marburg and Ebola viruses.

PubMed

Bukreyev, A A; Volchkov, V E; Blinov, V M; Netesov, S V

1993-05-03

The fragments of genomic RNA sequences of Marburg (MBG) and Ebola (EBO) viruses are reported. These fragments were found to encode the VP35 and VP40 proteins. The canonic sequences were revealed before and after each open reading frame. It is suggested that these sequences are mRNA extremities and at the same time the regulatory elements for mRNA transcription. Homology between the MBG and EBO proteins was discovered.

Replacement of the yeast TRP4 3' untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail.

PubMed Central

Düvel, Katrin; Valerius, Oliver; Mangus, David A; Jacobson, Allan; Braus, Gerhard H

2002-01-01

The mRNA poly(A) tail serves different purposes, including the facilitation of nuclear export, mRNA stabilization, efficient translation, and, finally, specific degradation. The posttranscriptional addition of a poly(A) tail depends on sequence motifs in the 3' untranslated region (3' UTR) of the mRNA and a complex trans-acting protein machinery. In this study, we have replaced the 3' UTR of the yeast TRP4 gene with sequences encoding a hammerhead ribozyme that efficiently cleaves itself in vivo. Expression of the TRP4-ribozyme allele resulted in the accumulation of a nonpolyadenylated mRNA. Cells expressing the TRP4-ribozyme mRNA showed a reduced growth rate due to a reduction in Trp4p enzyme activity. The reduction in enzyme activity was not caused by inefficient mRNA export from the nucleus or mRNA destabilization. Rather, analyses of mRNA association with polyribosomes indicate that translation of the ribozyme-containing mRNA is impaired. This translational defect allows sufficient synthesis of Trp4p to support growth of trp4 cells, but is, nevertheless, of such magnitude as to activate the general control network of amino acid biosynthesis. PMID:12003493

Cellular microRNA miR-10a-5p inhibits replication of porcine reproductive and respiratory syndrome virus by targeting the host factor signal recognition particle 14.

PubMed

Zhao, Guangwei; Hou, Jianye; Xu, Gaoxiao; Xiang, Aoqi; Kang, Yanmei; Yan, Yunhuan; Zhang, Xiaobin; Yang, Gongshe; Xiao, Shuqi; Sun, Shiduo

2017-04-01

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viruses affecting the swine industry worldwide. MicroRNAs have recently been demonstrated to play vital roles in virus-host interactions. Our previous research on small RNA deep sequencing showed that the expression level of miR-10a increased during the viral life cycle. The present study sought to determine the function of miR-10a and its molecular mechanism during PRRSV infection. In the current study, the result of PRRSV infection inducing miR-10a expression was validated by quantitative reverse transcriptase PCR. Overexpression of miR-10a-5p using its mimics markedly reduced the expression level of intracellular PRRSV ORF7 mRNA and N protein. Simultaneously, overexpression of miR-10a-5p also significantly decreased the expression level of extracellular viral RNA and virus titres in the supernatants. These results demonstrated that miR-10a-5p could suppress the replication of PRRSV. A direct interaction between miR-10a-5p and signal recognition particle 14 (SRP14) was confirmed using bioinformatic prediction and experimental verification. miR-10a-5p could directly target the 3'UTR of pig SRP14 mRNA in a sequence-specific manner and decrease SRP14 expression through translational repression but not mRNA degradation. Further, knockdown of SRP14 by small interfering RNA also inhibits the replication of PRRSV. Collectively, these results suggested that miR-10a-5p inhibits PRRSV replication through suppression of SRP14 expression, which not only provides new insights into virus-host interactions during PRRSV infection but also suggests potential new antiviral strategies against PRRSV infection.

Expression and activity levels of chymase in mast cells of burn wound tissues increase during the healing process in a hamster model.

PubMed

Dong, Xianglin; Xu, Tao; Ma, Shaolin; Wen, Hao

2015-06-01

The present study aimed to investigate the changes in the expression levels and activity of mast cell chymase in the process of burn wound healing in a hamster model of deep second-degree burn. The hamster model was established by exposing a ~3 cm diameter area of bare skin to hot water (75°C) for 0, 6, 8, 10 or 12 sec. Tissue specimens were collected 24 h after burning and histological analysis revealed that hot water contact for 12 sec was required to produce a deep second-degree burn. Quantitative polymerase chain reaction and a radioimmunoassay were used to the determine changes in chymase mRNA expression levels and activity. The mRNA expression levels and activity of chymase were increased in the burn wound tissues when compared with the normal skin. However, no statistically significant differences were observed in mast cell chymase activity amongst the various post-burn stages. Chymase mRNA expression levels peaked at day 1 post-burn, subsequently decreasing at days 3 and 7 post-burn and finally increasing again at day 14 post-burn. In summary, a hamster model of deep second-degree burn can be created by bringing the skin into contact with water at 75°C for 12 sec. Furthermore, the mRNA expression levels and activity of chymase in the burn wound tissues increased when compared with those in normal skin tissues.

Expression and activity levels of chymase in mast cells of burn wound tissues increase during the healing process in a hamster model

PubMed Central

DONG, XIANGLIN; XU, TAO; MA, SHAOLIN; WEN, HAO

2015-01-01

The present study aimed to investigate the changes in the expression levels and activity of mast cell chymase in the process of burn wound healing in a hamster model of deep second-degree burn. The hamster model was established by exposing a ~3 cm diameter area of bare skin to hot water (75°C) for 0, 6, 8, 10 or 12 sec. Tissue specimens were collected 24 h after burning and histological analysis revealed that hot water contact for 12 sec was required to produce a deep second-degree burn. Quantitative polymerase chain reaction and a radioimmunoassay were used to the determine changes in chymase mRNA expression levels and activity. The mRNA expression levels and activity of chymase were increased in the burn wound tissues when compared with the normal skin. However, no statistically significant differences were observed in mast cell chymase activity amongst the various post-burn stages. Chymase mRNA expression levels peaked at day 1 post-burn, subsequently decreasing at days 3 and 7 post-burn and finally increasing again at day 14 post-burn. In summary, a hamster model of deep second-degree burn can be created by bringing the skin into contact with water at 75°C for 12 sec. Furthermore, the mRNA expression levels and activity of chymase in the burn wound tissues increased when compared with those in normal skin tissues. PMID:26136958

Prenatal Exposure to Bisphenol A Disrupts Naturally Occurring Bimodal DNA Methylation at Proximal Promoter of fggy, an Obesity-Relevant Gene Encoding a Carbohydrate Kinase, in Gonadal White Adipose Tissues of CD-1 Mice.

PubMed

Taylor, Julia A; Shioda, Keiko; Mitsunaga, Shino; Yawata, Shiomi; Angle, Brittany M; Nagel, Susan C; Vom Saal, Frederick S; Shioda, Toshi

2018-02-01

Exposure of mammalian fetuses to endocrine disruptors can increase the risk of adult-onset diseases. We previously showed that exposure of mouse fetuses to bisphenol A (BPA) caused adult-onset obesity. To examine roles of epigenetic changes in this delayed toxicity, we determined the effects of fetal mouse exposure to BPA on genome-wide DNA methylation and messenger RNA (mRNA) expression in gonadal white adipose tissues (WATs) by deep sequencing, bisulfite pyrosequencing, and real-time quantitative polymerase chain reaction. Pregnant CD-1 mice (F0) were dosed daily with 0, 5, or 500 μg/kg/d BPA during gestational days 9 to 18, and the weaned F1 animals were fed ad libitum with standard chow until they were euthanized at 19 weeks old. In the vehicle-exposed F1 animals, fggy promoter showed a clear bimodal pattern of very strong (55% to 95%) or very weak (5% to 30%) DNA methylation occurring at nearly equal incidence with no intermediate strength. Promoter hypermethylation completely suppressed mRNA expression. BPA exposure eliminated this naturally occurring dichotomy, shifting fggy promoter toward the hypomethylation state to release transcriptional suppression. The strength of Fggy mRNA expression significantly correlated with increased whole body weight and gonadal fat weight of males but not females. Bioinformatics studies showed that expression of Fggy mRNA is stronger in mouse WATs than in brown adipose tissues and enhanced in gonadal fat by diet-induced obesity. These observations suggest that prenatal exposure to BPA may disrupt the physiological bimodal nature of epigenetic regulation of fggy in mouse WATs, possibly contributing to the adult-onset obesity phenotype. Copyright © 2018 Endocrine Society.

Identification of tissue-specific, abiotic stress-responsive gene expression patterns in wine grape (Vitis vinifera L.) based on curation and mining of large-scale EST data sets

PubMed Central

2011-01-01

Background Abiotic stresses, such as water deficit and soil salinity, result in changes in physiology, nutrient use, and vegetative growth in vines, and ultimately, yield and flavor in berries of wine grape, Vitis vinifera L. Large-scale expressed sequence tags (ESTs) were generated, curated, and analyzed to identify major genetic determinants responsible for stress-adaptive responses. Although roots serve as the first site of perception and/or injury for many types of abiotic stress, EST sequencing in root tissues of wine grape exposed to abiotic stresses has been extremely limited to date. To overcome this limitation, large-scale EST sequencing was conducted from root tissues exposed to multiple abiotic stresses. Results A total of 62,236 expressed sequence tags (ESTs) were generated from leaf, berry, and root tissues from vines subjected to abiotic stresses and compared with 32,286 ESTs sequenced from 20 public cDNA libraries. Curation to correct annotation errors, clustering and assembly of the berry and leaf ESTs with currently available V. vinifera full-length transcripts and ESTs yielded a total of 13,278 unique sequences, with 2302 singletons and 10,976 mapped to V. vinifera gene models. Of these, 739 transcripts were found to have significant differential expression in stressed leaves and berries including 250 genes not described previously as being abiotic stress responsive. In a second analysis of 16,452 ESTs from a normalized root cDNA library derived from roots exposed to multiple, short-term, abiotic stresses, 135 genes with root-enriched expression patterns were identified on the basis of their relative EST abundance in roots relative to other tissues. Conclusions The large-scale analysis of relative EST frequency counts among a diverse collection of 23 different cDNA libraries from leaf, berry, and root tissues of wine grape exposed to a variety of abiotic stress conditions revealed distinct, tissue-specific expression patterns, previously unrecognized stress-induced genes, and many novel genes with root-enriched mRNA expression for improving our understanding of root biology and manipulation of rootstock traits in wine grape. mRNA abundance estimates based on EST library-enriched expression patterns showed only modest correlations between microarray and quantitative, real-time reverse transcription-polymerase chain reaction (qRT-PCR) methods highlighting the need for deep-sequencing expression profiling methods. PMID:21592389

Toxins of Prokaryotic Toxin-Antitoxin Systems with Sequence-Specific Endoribonuclease Activity

PubMed Central

Masuda, Hisako; Inouye, Masayori

2017-01-01

Protein translation is the most common target of toxin-antitoxin system (TA) toxins. Sequence-specific endoribonucleases digest RNA in a sequence-specific manner, thereby blocking translation. While past studies mainly focused on the digestion of mRNA, recent analysis revealed that toxins can also digest tRNA, rRNA and tmRNA. Purified toxins can digest single-stranded portions of RNA containing recognition sequences in the absence of ribosome in vitro. However, increasing evidence suggests that in vivo digestion may occur in association with ribosomes. Despite the prevalence of recognition sequences in many mRNA, preferential digestion seems to occur at specific positions within mRNA and also in certain reading frames. In this review, a variety of tools utilized to study the nuclease activities of toxins over the past 15 years will be reviewed. A recent adaptation of an RNA-seq-based technique to analyze entire sets of cellular RNA will be introduced with an emphasis on its strength in identifying novel targets and redefining recognition sequences. The differences in biochemical properties and postulated physiological roles will also be discussed. PMID:28420090

Modulation of c-fms proto-oncogene in an ovarian carcinoma cell line by a hammerhead ribozyme.

PubMed Central

Yokoyama, Y.; Morishita, S.; Takahashi, Y.; Hashimoto, M.; Tamaya, T.

1997-01-01

Co-expression of macrophage colony-stimulating factor (M-CSF) and its receptor (c-fms) is often found in ovarian epithelial carcinoma, suggesting the existence of autocrine regulation of cell growth by M-CSF. To block this autocrine loop, we have developed hammerhead ribozymes against c-fms mRNA. As target sites of the ribozyme, we chose the GUC sequence in codon 18 and codon 27 of c-fms mRNA. Two kinds of ribozymes were able to cleave an artificial c-fms RNA substrate in a cell-free system, although the ribozyme against codon 18 was much more efficient than that against codon 27. We next constructed an expression vector carrying a ribozyme sequence that targeted the GUC sequence in codon 18 of c-fms mRNA. It was introduced into TYK-nu cells that expressed M-CSF and its receptor. Its transfectant showed a reduced growth potential. The expression levels of c-fms protein and mRNA in the transfectant were clearly decreased with the expression of ribozyme RNA compared with that of an untransfected control or a transfectant with the vector without the ribozyme sequence. These results suggest that the ribozyme against GUC in codon 18 of c-fms mRNA is a promising tool for blocking the autocrine loop of M-CSF in ovarian epithelial carcinoma. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:9376277

Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis.

PubMed

Spangler, Jacob B; Feltus, Frank Alex

2013-01-01

Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression.

Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis

PubMed Central

Spangler, Jacob B.; Feltus, Frank Alex

2013-01-01

Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression. PMID:23675377

Molecular characterization, mRNA expression of prolactin receptor (PRLR) gene during pregnancy, nonpregnancy in the yak (Bos grunniens).

PubMed

Zi, Xiang-Dong; Chen, Da-Wen; Wang, Hong-Mei

2012-02-01

Prolactin (PRL) plays central roles in a wide range of body functions in mammals, and the actions are mediated by the specific cell surface receptor, the prolactin receptor (PRLR). To better understand the role of PRL in the yak (Bos grunniens), in the present study, we first cloned yak PRLR cDNA, and compared its mRNA expression in several tissues with cattle (Bos taurus). By reverse transcriptase-polymerase chain reaction (RT-PCR) strategy, we obtained full-length of yak PRLR cDNA sequence comprised of an open reading frame of 1746bp encoding a 581 amino acid protein, and contained a signal sequence and a transmembrane region. The intracellular domain had two pairs of cysteine residues and a WSXWS motif. The cytoplasmic domain comprised 323 residues and contained box 1 sequence. The yak PRLR shared 66.0-98.5% protein sequence identity with mammalian homologs. Real-time PCR analysis revealed that PRLR mRNA was higher in mammary tissue than in ovary and endometrium (P<0.01). During pregnancy, the ovary and mammary PRLR mRNA expression increased by 33- and 2.9-fold in yak, respectively, and increased by 46- and 3.8-fold in cattle, respectively. PRLR mRNA expression was higher (P<0.05) in mammary tissue and ovary of pregnant cow than that of pregnant yak. It is proposed that the increased ovarian and mammary PRLR mRNA expression during pregnancy may be associated with corpus luteum function for maintenance of pregnancy and mammary development for subsequent lactation. Copyright © 2011 Elsevier Inc. All rights reserved.

Quantitative phenotyping via deep barcode sequencing.

PubMed

Smith, Andrew M; Heisler, Lawrence E; Mellor, Joseph; Kaper, Fiona; Thompson, Michael J; Chee, Mark; Roth, Frederick P; Giaever, Guri; Nislow, Corey

2009-10-01

Next-generation DNA sequencing technologies have revolutionized diverse genomics applications, including de novo genome sequencing, SNP detection, chromatin immunoprecipitation, and transcriptome analysis. Here we apply deep sequencing to genome-scale fitness profiling to evaluate yeast strain collections in parallel. This method, Barcode analysis by Sequencing, or "Bar-seq," outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex chemogenomic assay, Bar-seq quantitatively identifies drug targets, with performance superior to the benchmark microarray assay. We also show that Bar-seq is well-suited for a multiplex format. We completely re-sequenced and re-annotated the yeast deletion collection using deep sequencing, found that approximately 20% of the barcodes and common priming sequences varied from expectation, and used this revised list of barcode sequences to improve data quality. Together, this new assay and analysis routine provide a deep-sequencing-based toolkit for identifying gene-environment interactions on a genome-wide scale.

Deep Sequencing to Identify the Causes of Viral Encephalitis

PubMed Central

Chan, Benjamin K.; Wilson, Theodore; Fischer, Kael F.; Kriesel, John D.

2014-01-01

Deep sequencing allows for a rapid, accurate characterization of microbial DNA and RNA sequences in many types of samples. Deep sequencing (also called next generation sequencing or NGS) is being developed to assist with the diagnosis of a wide variety of infectious diseases. In this study, seven frozen brain samples from deceased subjects with recent encephalitis were investigated. RNA from each sample was extracted, randomly reverse transcribed and sequenced. The sequence analysis was performed in a blinded fashion and confirmed with pathogen-specific PCR. This analysis successfully identified measles virus sequences in two brain samples and herpes simplex virus type-1 sequences in three brain samples. No pathogen was identified in the other two brain specimens. These results were concordant with pathogen-specific PCR and partially concordant with prior neuropathological examinations, demonstrating that deep sequencing can accurately identify viral infections in frozen brain tissue. PMID:24699691

Vesicular monoamine transporter-1 (VMAT-1) mRNA and immunoreactive proteins in mouse brain.

PubMed

Ashe, Karen M; Chiu, Wan-Ling; Khalifa, Ahmed M; Nicolas, Antoine N; Brown, Bonnie L; De Martino, Randall R; Alexander, Clayton P; Waggener, Christopher T; Fischer-Stenger, Krista; Stewart, Jennifer K

2011-01-01

Vesicular monoamine transporter 1 (VMAT-1) mRNA and protein were examined (1) to determine whether adult mouse brain expresses full-length VMAT-1 mRNA that can be translated to functional transporter protein and (2) to compare immunoreactive VMAT-1 proteins in brain and adrenal. VMAT-1 mRNA was detected in mouse brain with RT-PCR. The cDNA was sequenced, cloned into an expression vector, transfected into COS-1 cells, and cell protein was assayed for VMAT-1 activity. Immunoreactive proteins were examined on western blots probed with four different antibodies to VMAT-1. Sequencing confirmed identity of the entire coding sequences of VMAT-1 cDNA from mouse medulla oblongata/pons and adrenal to a Gen-Bank reference sequence. Transfection of the brain cDNA into COS-1 cells resulted in transporter activity that was blocked by the VMAT inhibitor reserpine and a proton ionophore, but not by tetrabenazine, which has a high affinity for VMAT-2. Antibodies to either the C- or N- terminus of VMAT-1 detected two proteins (73 and 55 kD) in transfected COS-1 cells. The C-terminal antibodies detected both proteins in extracts of mouse medulla/pons, cortex, hypothalamus, and cerebellum but only the 73 kD protein and higher molecular weight immunoreactive proteins in mouse adrenal and rat PC12 cells, which are positive controls for rodent VMAT-1. These findings demonstrate that a functional VMAT-1 mRNA coding sequence is expressed in mouse brain and suggest processing of VMAT-1 protein differs in mouse adrenal and brain.

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.

Local upregulation of transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 ion channels in rectosigmoid deep infiltrating endometriosis

PubMed Central

Bohonyi, Noémi; Pohóczky, Krisztina; Szalontai, Bálint; Perkecz, Anikó; Kovács, Krisztina; Kajtár, Béla; Orbán, Lajos; Varga, Tamás; Szegedi, Sarolta; Bódis, József; Koppán, Miklós

2017-01-01

Transient Receptor Potential Vanilloid 1 (TRPV1) and Transient Receptor Potential Ankyrin 1 (TRPA1) expressed mainly by primary sensory neurons function as major nociceptive integrators. They are also present on the rat endometrium in an oestrogen-regulated manner. TRPV1 is upregulated in peritoneal and ovarian endometriosis patients, but there is no information about TRPA1 and their pathophysiological significances. In this study, patients undergoing laparoscopic surgery were investigated: severe dysmenorrhoea due to rectosigmoid deep infiltrating endometriosis (n = 15), uterine fibroid-induced moderate dysmenorrhoea (n = 7) and tubal infertility with no pain (n = 6). TRPA1 and TRPV1 mRNA and protein expressions were determined by quantitative polymerase chain reaction and semi-quantitative immunohistochemistry from the endometrium samples taken by curettage. Results were correlated with the clinical characteristics including pain intensity. TRPA1 and TRPV1 receptors were expressed in the healthy human endometrium at mRNA and protein levels. Sparse, scattered cytoplasmic TRPA1 and TRPV1 immunopositivities were found in the stroma and epithelial layers. We detected upregulated mRNA levels in deep infiltrating endometriosis lesions, and TRPV1 gene expression was also elevated in autocontrol endometrium of deep infiltrating endometriosis patients. Histological scoring revealed significant TRPA1 and TRPV1 difference between deep infiltrating endometriosis stroma and epithelium, and in deep infiltrating endometriosis epithelium compared to control samples. Besides, we measured elevated stromal TRPV1 immunopositivity in deep infiltrating endometriosis. Stromal TRPA1 and TRPV1 immunoreactivities strongly correlated with dysmenorrhoea severity, as well TRPV1 expression on ectopic epithelial cells and macrophages with dyspareunia. Epithelial TRPA1 and stromal TRPV1 immunopositivity also positively correlated with dyschezia severity. We provide the first evidence for the presence of non-neuronal TRPA1 receptor in the healthy human endometrium and confirm the expression of TRPV1 channels. Their upregulations in rectosigmoid deep infiltrating endometriosis lesions and correlations with pain intensity suggest potential roles in pathophysiological mechanisms of the disease. PMID:28478727

Local upregulation of transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 ion channels in rectosigmoid deep infiltrating endometriosis.

PubMed

Bohonyi, Noémi; Pohóczky, Krisztina; Szalontai, Bálint; Perkecz, Anikó; Kovács, Krisztina; Kajtár, Béla; Orbán, Lajos; Varga, Tamás; Szegedi, Sarolta; Bódis, József; Helyes, Zsuzsanna; Koppán, Miklós

2017-01-01

Transient Receptor Potential Vanilloid 1 (TRPV1) and Transient Receptor Potential Ankyrin 1 (TRPA1) expressed mainly by primary sensory neurons function as major nociceptive integrators. They are also present on the rat endometrium in an oestrogen-regulated manner. TRPV1 is upregulated in peritoneal and ovarian endometriosis patients, but there is no information about TRPA1 and their pathophysiological significances. In this study, patients undergoing laparoscopic surgery were investigated: severe dysmenorrhoea due to rectosigmoid deep infiltrating endometriosis ( n = 15), uterine fibroid-induced moderate dysmenorrhoea ( n = 7) and tubal infertility with no pain ( n = 6). TRPA1 and TRPV1 mRNA and protein expressions were determined by quantitative polymerase chain reaction and semi-quantitative immunohistochemistry from the endometrium samples taken by curettage. Results were correlated with the clinical characteristics including pain intensity. TRPA1 and TRPV1 receptors were expressed in the healthy human endometrium at mRNA and protein levels. Sparse, scattered cytoplasmic TRPA1 and TRPV1 immunopositivities were found in the stroma and epithelial layers. We detected upregulated mRNA levels in deep infiltrating endometriosis lesions, and TRPV1 gene expression was also elevated in autocontrol endometrium of deep infiltrating endometriosis patients. Histological scoring revealed significant TRPA1 and TRPV1 difference between deep infiltrating endometriosis stroma and epithelium, and in deep infiltrating endometriosis epithelium compared to control samples. Besides, we measured elevated stromal TRPV1 immunopositivity in deep infiltrating endometriosis. Stromal TRPA1 and TRPV1 immunoreactivities strongly correlated with dysmenorrhoea severity, as well TRPV1 expression on ectopic epithelial cells and macrophages with dyspareunia. Epithelial TRPA1 and stromal TRPV1 immunopositivity also positively correlated with dyschezia severity. We provide the first evidence for the presence of non-neuronal TRPA1 receptor in the healthy human endometrium and confirm the expression of TRPV1 channels. Their upregulations in rectosigmoid deep infiltrating endometriosis lesions and correlations with pain intensity suggest potential roles in pathophysiological mechanisms of the disease.

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.

MicroRNA-200c Modulates the Expression of MUC4 and MUC16 by Directly Targeting Their Coding Sequences in Human Pancreatic Cancer

PubMed Central

Radhakrishnan, Prakash; Mohr, Ashley M.; Grandgenett, Paul M.; Steele, Maria M.; Batra, Surinder K.; Hollingsworth, Michael A.

2013-01-01

Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer. PMID:24204560

MicroRNA-200c modulates the expression of MUC4 and MUC16 by directly targeting their coding sequences in human pancreatic cancer.

PubMed

Radhakrishnan, Prakash; Mohr, Ashley M; Grandgenett, Paul M; Steele, Maria M; Batra, Surinder K; Hollingsworth, Michael A

2013-01-01

Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer.

Identification and Characterization of a Cis Antisense RNA of the rpoH Gene of Salmonella enterica Serovar Typhi.

PubMed

Xiong, Changyan; Li, Xuejiao; Liu, Juanli; Zhao, Xin; Xu, Shungao; Huang, Xinxiang

2018-01-01

Antisense RNAs from complementary strands of protein coding genes regulate the expression of genes involved in many cellular processes. Using deep sequencing analysis of the Salmonella enterica serovar Typhi ( S. Typhi) transcriptome, a novel antisense RNA encoded on the strand complementary to the rpoH gene was revealed. In this study, the molecular features of this antisense RNA were assessed using northern blotting and rapid amplification of cDNA ends. The 3,508 nt sequence of RNA was identified as the antisense RNA of the rpoH gene and was named ArpH. ArpH was found to attenuate the invasion of HeLa cells by S. Typhi by regulating the expression of SPI-1 genes. In an rpoH mutant strain, the invasive capacity of S. Typhi was increased, whereas overexpression of ArpH positively regulates rpoH mRNA levels. Results of this study suggest that the cis -encoded antisense RNA ArpH is likely to affect the invasive capacity of S. Typhi by regulating the expression of rpoH .

A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

PubMed

Nestorowa, Sonia; Hamey, Fiona K; Pijuan Sala, Blanca; Diamanti, Evangelia; Shepherd, Mairi; Laurenti, Elisa; Wilson, Nicola K; Kent, David G; Göttgens, Berthold

2016-08-25

Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice. © 2016 by The American Society of Hematology.

Stabilization and cytoskeletal-association of LDL receptor mRNA are mediated by distinct domains in its 3' untranslated region.

PubMed

Wilson, G M; Vasa, M Z; Deeley, R G

1998-05-01

The mRNA encoding the human low density lipoprotein (LDL) receptor is transiently stabilized after phorbol ester treatment of HepG2 cells and has been shown to associate with components of the cytoskeleton in this cell line (G. M. Wilson, E. A. Roberts, and R. G. Deeley, J. Lipid Res. 1997. 38: 437-446). Using an episomal expression system, fragments of the 3' untranslated region (3'UTR) of LDL receptor mRNA were transcribed in fusion with the coding region of beta-globin mRNA in HepG2 cells. Analyses of the decay kinetics of these beta-globin-LDL receptor fusion mRNA deletion mutants showed that sequences in the proximal 3'UTR of LDL receptor mRNA including several AU-rich elements (AREs) were sufficient to confer short constitutive mRNA half-life in the heterologous system. Stabilization of LDL receptor mRNA in the presence of PMA required sequences in the distal 3'UTR, at or near three Alu-like repetitive elements. Furthermore, the 3'UTR of LDL receptor mRNA conferred cytoskeletal association on the otherwise unassociated beta-globin mRNA, by a mechanism involving at least two distinct RNA elements. Comparisons of decay kinetics and subcellular localization of endogenous LDL receptor mRNA and beta-globin-LDL receptor mRNA fusions in HepG2 cells have demonstrated that several cis-acting elements in the receptor 3'UTR contribute to post-transcriptional regulation of receptor expression, and provide further support for involvement of the cytoskeleton in the regulation of LDL receptor mRNA turnover.

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.

The katG mRNA of Mycobacterium tuberculosis and Mycobacterium smegmatis is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation

PubMed Central

Sala, Claudia; Forti, Francesca; Magnoni, Francesca; Ghisotti, Daniela

2008-01-01

Background In Mycobacterium tuberculosis and in Mycobacterium smegmatis the furA-katG loci, encoding the FurA regulatory protein and the KatG catalase-peroxidase, are highly conserved. In M. tuberculosis furA-katG constitute a single operon, whereas in M. smegmatis a single mRNA covering both genes could not be found. In both species, specific 5' ends have been identified: the first one, located upstream of the furA gene, corresponds to transcription initiation from the furA promoter; the second one is the katG mRNA 5' end, located in the terminal part of furA. Results In this work we demonstrate by in vitro transcription and by RNA polymerase Chromatin immunoprecipitation that no promoter is present in the M. smegmatis region covering the latter 5' end, suggesting that it is produced by specific processing of longer transcripts. Several DNA fragments of M. tuberculosis and M. smegmatis were inserted in a plasmid between the sigA promoter and the lacZ reporter gene, and expression of the reporter gene was measured. A polypurine sequence, located four bp upstream of the katG translation start codon, increased beta-galactosidase activity and stabilized the lacZ transcript. Mutagenesis of this sequence led to destabilization of the mRNA. Analysis of constructs, in which the polypurine sequence of M. smegmatis was followed by an increasing number of katG codons, demonstrated that mRNA stability requires translation of at least 20 amino acids. In order to define the requirements for the 5' processing of the katG transcript, we created several mutations in this region and analyzed the 5' ends of the transcripts: the distance from the polypurine sequence does not seem to influence the processing, neither the sequence around the cutting point. Only mutations which create a double stranded region around the processing site prevented RNA processing. Conclusion This is the first reported case in mycobacteria, in which both a polypurine sequence and translation initiation are shown to contribute to mRNA stability. The furA-katG mRNA is transcribed from the furA promoter and immediately processed; this processing is prevented by a double stranded RNA at the cutting site, suggesting that the endoribonuclease responsible for the cleavage cuts single stranded RNA. PMID:18394163

Quantitative phenotyping via deep barcode sequencing

PubMed Central

Smith, Andrew M.; Heisler, Lawrence E.; Mellor, Joseph; Kaper, Fiona; Thompson, Michael J.; Chee, Mark; Roth, Frederick P.; Giaever, Guri; Nislow, Corey

2009-01-01

Next-generation DNA sequencing technologies have revolutionized diverse genomics applications, including de novo genome sequencing, SNP detection, chromatin immunoprecipitation, and transcriptome analysis. Here we apply deep sequencing to genome-scale fitness profiling to evaluate yeast strain collections in parallel. This method, Barcode analysis by Sequencing, or “Bar-seq,” outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex chemogenomic assay, Bar-seq quantitatively identifies drug targets, with performance superior to the benchmark microarray assay. We also show that Bar-seq is well-suited for a multiplex format. We completely re-sequenced and re-annotated the yeast deletion collection using deep sequencing, found that ∼20% of the barcodes and common priming sequences varied from expectation, and used this revised list of barcode sequences to improve data quality. Together, this new assay and analysis routine provide a deep-sequencing-based toolkit for identifying gene–environment interactions on a genome-wide scale. PMID:19622793

External Guide Sequences Targeting the aac(6′)-Ib mRNA Induce Inhibition of Amikacin Resistance▿

PubMed Central

Bistué, Alfonso J. C. Soler; Ha, Hongphuc; Sarno, Renee; Don, Michelle; Zorreguieta, Angeles; Tolmasky, Marcelo E.

2007-01-01

The dissemination of AAC(6′)-I-type acetyltransferases have rendered amikacin and other aminoglycosides all but useless in some parts of the world. Antisense technologies could be an alternative to extend the life of these antibiotics. External guide sequences are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. Thirteen-nucleotide external guide sequences complementary to locations within five regions accessible for interaction with antisense oligonucleotides in the mRNA that encodes AAC(6′)-Ib were analyzed. While small variations in the location targeted by different external guide sequences resulted in big changes in efficiency of binding to native aac(6′)-Ib mRNA, most of them induced high levels of RNase P-mediated cleavage in vitro. Recombinant plasmids coding for selected external guide sequences were introduced into Escherichia coli harboring aac(6′)-Ib, and the transformant strains were tested to determine their resistance to amikacin. The two external guide sequences that showed the strongest binding efficiency to the mRNA in vitro, EGSC3 and EGSA2, interfered with expression of the resistance phenotype at different degrees. Growth curve experiments showed that E. coli cells harboring a plasmid coding for EGSC3, the external guide sequence with the highest mRNA binding affinity in vitro, did not grow for at least 300 min in the presence of 15 μg of amikacin/ml. EGSA2, which had a lower mRNA-binding affinity in vitro than EGSC3, inhibited the expression of amikacin resistance at a lesser level; growth of E. coli harboring a plasmid coding for EGSA2, in the presence of 15 μg of amikacin/ml was undetectable for 200 min but reached an optical density at 600 nm of 0.5 after 5 h of incubation. Our results indicate that the use of external guide sequences could be a viable strategy to preserve the efficacy of amikacin. PMID:17387154

Sequence and structure determinants of Drosophila Hsp70 mRNA translation: 5'UTR secondary structure specifically inhibits heat shock protein mRNA translation.

PubMed Central

Hess, M A; Duncan, R F

1996-01-01

Preferential translation of Drosophila heat shock protein 70 (Hsp70) mRNA requires only the 5'-untranslated region (5'-UTR). The sequence of this region suggests that it has relatively little secondary structure, which may facilitate efficient protein synthesis initiation. To determine whether minimal 5'-UTR secondary structure is required for preferential translation during heat shock, the effect of introducing stem-loops into the Hsp70 mRNA 5'-UTR was measured. Stem-loops of -11 kcal/mol abolished translation during heat shock, but did not reduce translation in non-heat shocked cells. A -22 kcal/mol stem-loop was required to comparably inhibit translation during growth at normal temperatures. To investigate whether specific sequence elements are also required for efficient preferential translation, deletion and mutation analyses were conducted in a truncated Hsp70 5'-UTR containing only the cap-proximal and AUG-proximal segments. Linker-scanner mutations in the cap-proximal segment (+1 to +37) did not impair translation. Re-ordering the segments reduced mRNA translational efficiency by 50%. Deleting the AUG-proximal segment severely inhibited translation. A 5-extension of the full-length leader specifically impaired heat shock translation. These results indicate that heat shock reduces the capacity to unwind 5-UTR secondary structure, allowing only mRNAs with minimal 5'-UTR secondary structure to be efficiently translated. A function for specific sequences is also suggested. PMID:8710519

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.

DNA sequence analysis of simian virus 40 mutants with deletions mapping in the leader region of the late viral mRNA's: mutants with deletions similar in size and position exhibit varied phenotypes.

PubMed

Barkan, A; Mertz, J E

1981-02-01

The nucleotide sequences of 10 viable yet partially defective deletion mutants of simian virus 40 were determined. The deletions mapped within, and, in many cases, 5' to, the predominant leader sequence of the late viral mRNA's. They ranged from 74 to 187 nucleotide pairs in length. Six of the mutants had lost the sequence that corresponds to the "cap" site (5' terminus) of the most abundant class of 16S mRNA's. One of these mutants had a deletion that extended 103 nucleotide pairs into the region preceding this primary cap site and, therefore, was missing many secondary cap sites as well. A seventh mutant lacked the entire major 16S leader sequence except for the first six nucleotides at its 5' end and the last nine at its 3' end. Although these mutants differed in the size and position of their deletions, we were unable to discover any simple correlations between their growth characteristics and their DNA sequences. This finding indicates that the secondary structures of the RNA transcripts may play a more important role than the exact nucleotide sequence of the RNAs in determining how they function within the cell.

Bacillus subtilis MazF-bs (EndoA) is a UACAU-specific mRNA interferase.

PubMed

Park, Jung-Ho; Yamaguchi, Yoshihiro; Inouye, Masayori

2011-08-04

MazF is an mRNA interferase which cleaves mRNAs at a specific sequence. Here, we show that in contrast to MazF-ec from Escherichia coli, which specifically cleaves ACA sequences, MazF-bs from Bacillus subtilis is an mRNA interferase that specifically cleaves a five-base sequence, UACAU. MazF homologues widely prevailing in Gram-positive bacteria were found to be highly homologous to MazF-bs, suggesting that they may also have similar cleavage specificity. This cleavage site is over-represented in the B. subtilis genes associated with biosynthesis of secondary metabolites, suggesting that MazF-bs may be involved in the regulation of the production of secondary metabolites. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Discovery radiomics via evolutionary deep radiomic sequencer discovery for pathologically proven lung cancer detection.

PubMed

Shafiee, Mohammad Javad; Chung, Audrey G; Khalvati, Farzad; Haider, Masoom A; Wong, Alexander

2017-10-01

While lung cancer is the second most diagnosed form of cancer in men and women, a sufficiently early diagnosis can be pivotal in patient survival rates. Imaging-based, or radiomics-driven, detection methods have been developed to aid diagnosticians, but largely rely on hand-crafted features that may not fully encapsulate the differences between cancerous and healthy tissue. Recently, the concept of discovery radiomics was introduced, where custom abstract features are discovered from readily available imaging data. We propose an evolutionary deep radiomic sequencer discovery approach based on evolutionary deep intelligence. Motivated by patient privacy concerns and the idea of operational artificial intelligence, the evolutionary deep radiomic sequencer discovery approach organically evolves increasingly more efficient deep radiomic sequencers that produce significantly more compact yet similarly descriptive radiomic sequences over multiple generations. As a result, this framework improves operational efficiency and enables diagnosis to be run locally at the radiologist's computer while maintaining detection accuracy. We evaluated the evolved deep radiomic sequencer (EDRS) discovered via the proposed evolutionary deep radiomic sequencer discovery framework against state-of-the-art radiomics-driven and discovery radiomics methods using clinical lung CT data with pathologically proven diagnostic data from the LIDC-IDRI dataset. The EDRS shows improved sensitivity (93.42%), specificity (82.39%), and diagnostic accuracy (88.78%) relative to previous radiomics approaches.

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.

Short intronic repeat sequences facilitate circular RNA production.

PubMed

Liang, Dongming; Wilusz, Jeremy E

2014-10-15

Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery "backsplices" and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼ 30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3' end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA. © 2014 Liang and Wilusz; Published by Cold Spring Harbor Laboratory Press.

Unravelling the complexity of microRNA-mediated gene regulation in black pepper (Piper nigrum L.) using high-throughput small RNA profiling.

PubMed

Asha, Srinivasan; Sreekumar, Sweda; Soniya, E V

2016-01-01

Analysis of high-throughput small RNA deep sequencing data, in combination with black pepper transcriptome sequences revealed microRNA-mediated gene regulation in black pepper ( Piper nigrum L.). Black pepper is an important spice crop and its berries are used worldwide as a natural food additive that contributes unique flavour to foods. In the present study to characterize microRNAs from black pepper, we generated a small RNA library from black pepper leaf and sequenced it by Illumina high-throughput sequencing technology. MicroRNAs belonging to a total of 303 conserved miRNA families were identified from the sRNAome data. Subsequent analysis from recently sequenced black pepper transcriptome confirmed precursor sequences of 50 conserved miRNAs and four potential novel miRNA candidates. Stem-loop qRT-PCR experiments demonstrated differential expression of eight conserved miRNAs in black pepper. Computational analysis of targets of the miRNAs showed 223 potential black pepper unigene targets that encode diverse transcription factors and enzymes involved in plant development, disease resistance, metabolic and signalling pathways. RLM-RACE experiments further mapped miRNA-mediated cleavage at five of the mRNA targets. In addition, miRNA isoforms corresponding to 18 miRNA families were also identified from black pepper. This study presents the first large-scale identification of microRNAs from black pepper and provides the foundation for the future studies of miRNA-mediated gene regulation of stress responses and diverse metabolic processes in black pepper.

COL1A1 transgene expression in stably transfected osteoblastic cells. Relative contributions of first intron, 3'-flanking sequences, and sequences derived from the body of the human COL1A1 minigene

NASA Technical Reports Server (NTRS)

Breault, D. T.; Lichtler, A. C.; Rowe, D. W.

1997-01-01

Collagen reporter gene constructs have be used to identify cell-specific sequences needed for transcriptional activation. The elements required for endogenous levels of COL1A1 expression, however, have not been elucidated. The human COL1A1 minigene is expressed at high levels and likely harbors sequence elements required for endogenous levels of activity. Using stably transfected osteoblastic Py1a cells, we studied a series of constructs (pOBColCAT) designed to characterize further the elements required for high level of expression. pOBColCAT, which contains the COL1A1 first intron, was expressed at 50-100-fold higher levels than ColCAT 3.6, which lacks the first intron. This difference is best explained by improved mRNA processing rather than a transcriptional effect. Furthermore, variation in activity observed with the intron deletion constructs is best explained by altered mRNA splicing. Two major regions of the human COL1A1 minigene, the 3'-flanking sequences and the minigene body, were introduced into pOBColCAT to assess both transcriptional enhancing activity and the effect on mRNA stability. Analysis of the minigene body, which includes the first five exons and introns fused with the terminal six introns and exons, revealed an orientation-independent 5-fold increase in CAT activity. In contrast the 3'-flanking sequences gave rise to a modest 61% increase in CAT activity. Neither region increased the mRNA half-life of the parent construct, suggesting that CAT-specific mRNA instability elements may serve as dominant negative regulators of stability. This study suggests that other sites within the body of the COL1A1 minigene are important for high expression, e.g. during periods of rapid extracellular matrix production.

Individual microRNAs (miRNAs) display distinct mRNA targeting "rules".

PubMed

Wang, Wang-Xia; Wilfred, Bernard R; Xie, Kevin; Jennings, Mary H; Hu, Yanling Hu; Stromberg, Arnold J; Nelson, Peter T

2010-01-01

MicroRNAs (miRNAs) guide Argonaute (AGO)-containing microribonucleoprotein (miRNP) complexes to target mRNAs.It has been assumed that miRNAs behave similarly to each other with regard to mRNA target recognition. The usual assumptions, which are based on prior studies, are that miRNAs target preferentially sequences in the 3'UTR of mRNAs,guided by the 5' "seed" portion of the miRNAs. Here we isolated AGO- and miRNA-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with anti-AGO antibody. Cells were transfected with miR-107, miR-124,miR-128, miR-320, or a negative control miRNA. Co-IPed RNAs were subjected to downstream high-density Affymetrix Human Gene 1.0 ST microarray analyses using an assay we validated previously-a "RIP-Chip" experimental design. RIP-Chip data provided a list of mRNAs recruited into the AGO-miRNP in correlation to each miRNA. These experimentally identified miRNA targets were analyzed for complementary six nucleotide "seed" sequences within the transfected miRNAs. We found that miR-124 targets tended to have sequences in the 3'UTR that would be recognized by the 5' seed of miR-124, as described in previous studies. By contrast, miR-107 targets tended to have 'seed' sequences in the mRNA open reading frame, but not the 3' UTR. Further, mRNA targets of miR-128 and miR-320 are less enriched for 6-mer seed sequences in comparison to miR-107 and miR-124. In sum, our data support the importance of the 5' seed in determining binding characteristics for some miRNAs; however, the "binding rules" are complex, and individual miRNAs can have distinct sequence determinants that lead to mRNA targeting.

The Nucleotide Sequence and Spliced pol mRNA Levels of the Nonprimate Spumavirus Bovine Foamy Virus

PubMed Central

Holzschu, Donald L.; Delaney, Mari A.; Renshaw, Randall W.; Casey, James W.

1998-01-01

We have determined the complete nucleotide sequence of a replication-competent clone of bovine foamy virus (BFV) and have quantitated the amount of splice pol mRNA processed early in infection. The 544-amino-acid Gag protein precursor has little sequence similarity with its primate foamy virus homologs, but the putative nucleocapsid (NC) protein, like the primate NCs, contains the three glycine-arginine-rich regions that are postulated to bind genomic RNA during virion assembly. The BFV gag and pol open reading frames overlap, with pro and pol in the same translational frame. As with the human foamy virus (HFV) and feline foamy virus, we have detected a spliced pol mRNA by PCR. Quantitatively, this mRNA approximates the level of full-length genomic RNA early in infection. The integrase (IN) domain of reverse transcriptase does not contain the canonical HH-CC zinc finger motif present in all characterized retroviral INs, but it does contain a nearby histidine residue that could conceivably participate as a member of the zinc finger. The env gene encodes a protein that is over 40% identical in sequence to the HFV Env. By comparison, the Gag precursor of BFV is predicted to be only 28% identical to the HFV protein. PMID:9499074

Detection of canine cytokine gene expression by reverse transcription-polymerase chain reaction.

PubMed

Pinelli, E; van der Kaaij, S Y; Slappendel, R; Fragio, C; Ruitenberg, E J; Bernadina, W; Rutten, V P

1999-08-02

Further characterization of the canine immune system will greatly benefit from the availability of tools to detect canine cytokines. Our interest concerns the study on the role of cytokines in canine visceral leishmaniasis. For this purpose, we have designed specific primers using previously published sequences for the detection of canine IL-2, IFN-gamma and IL10 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). For IL-4, we have cloned and sequenced this cytokine gene, and developed canine-specific primers. To control for sample-to-sample variation in the quantity of mRNA and variation in the RT and PCR reactions, the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a housekeeping gene, were determined in parallel. Primers to amplify G3PDH were designed from consensus sequences obtained from the Genbank database. The mRNA levels of the cytokines mentioned here were detected from ConA-stimulated peripheral mononuclear cells derived from Leishmania-infected dogs. A different pattern of cytokine production among infected animals was found.

Molecular characterization of a phloem-specific gene encoding the filament protein, phloem protein 1 (PP1), from Cucurbita maxima.

PubMed

Clark, A M; Jacobsen, K R; Bostwick, D E; Dannenhoffer, J M; Skaggs, M I; Thompson, G A

1997-07-01

Sieve elements in the phloem of most angiosperms contain proteinaceous filaments and aggregates called P-protein. In the genus Cucurbita, these filaments are composed of two major proteins: PP1, the phloem filament protein, and PP2, the phloem lactin. The gene encoding the phloem filament protein in pumpkin (Cucurbita maxima Duch.) has been isolated and characterized. Nucleotide sequence analysis of the reconstructed gene gPP1 revealed a continuous 2430 bp protein coding sequence, with no introns, encoding an 809 amino acid polypeptide. The deduced polypeptide had characteristics of PP1 and contained a 15 amino acid sequence determined by N-terminal peptide sequence analysis of PP1. The sequence of PP1 was highly repetitive with four 200 amino acid sequence domains containing structural motifs in common with cysteine proteinase inhibitors. Expression of the PP1 gene was detected in roots, hypocotyls, cotyledons, stems, and leaves of pumpkin plants. PP1 and its mRNA accumulated in pumpkin hypocotyls during the period of rapid hypocotyl elongation after which mRNA levels declined, while protein levels remained elevated. PP1 was immunolocalized in slime plugs and P-protein bodies in sieve elements of the phloem. Occasionally, PP1 was detected in companion cells. PP1 mRNA was localized by in situ hybridization in companion cells at early stages of vascular differentiation. The developmental accumulation and localization of PP1 and its mRNA paralleled the phloem lactin, further suggesting an interaction between these phloem-specific proteins.

Predicting RNA-protein binding sites and motifs through combining local and global deep convolutional neural networks.

PubMed

Pan, Xiaoyong; Shen, Hong-Bin

2018-05-02

RNA-binding proteins (RBPs) take over 5∼10% of the eukaryotic proteome and play key roles in many biological processes, e.g. gene regulation. Experimental detection of RBP binding sites is still time-intensive and high-costly. Instead, computational prediction of the RBP binding sites using pattern learned from existing annotation knowledge is a fast approach. From the biological point of view, the local structure context derived from local sequences will be recognized by specific RBPs. However, in computational modeling using deep learning, to our best knowledge, only global representations of entire RNA sequences are employed. So far, the local sequence information is ignored in the deep model construction process. In this study, we present a computational method iDeepE to predict RNA-protein binding sites from RNA sequences by combining global and local convolutional neural networks (CNNs). For the global CNN, we pad the RNA sequences into the same length. For the local CNN, we split a RNA sequence into multiple overlapping fixed-length subsequences, where each subsequence is a signal channel of the whole sequence. Next, we train deep CNNs for multiple subsequences and the padded sequences to learn high-level features, respectively. Finally, the outputs from local and global CNNs are combined to improve the prediction. iDeepE demonstrates a better performance over state-of-the-art methods on two large-scale datasets derived from CLIP-seq. We also find that the local CNN run 1.8 times faster than the global CNN with comparable performance when using GPUs. Our results show that iDeepE has captured experimentally verified binding motifs. https://github.com/xypan1232/iDeepE. xypan172436@gmail.com or hbshen@sjtu.edu.cn. Supplementary data are available at Bioinformatics online.

Cloning of a cDNA encoding 1-aminocyclopropane-1-carboxylate synthase and expression of its mRNA in ripening apple fruit.

PubMed

Dong, J G; Kim, W T; Yip, W K; Thompson, G A; Li, L; Bennett, A B; Yang, S F

1991-08-01

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) purified from apple (Malus sylvestris Mill.) fruit was subjected to trypsin digestion. Following separation by reversed-phase high-pressure liquid chromatography, ten tryptic peptides were sequenced. Based on the sequences of three tryptic peptides, three sets of mixed oligonucleotide probes were synthesized and used to screen a plasmid cDNA library prepared from poly(A)(+) RNA of ripe apple fruit. A 1.5-kb (kilobase) cDNA clone which hybridized to all three probes were isolated. The clone contained an open reading frame of 1214 base pairs (bp) encoding a sequence of 404 amino acids. While the polyadenine tail at the 3'-end was intact, it lacked a portion of sequence at the 5'-end. Using the RNA-based polymerase chain reaction, an additional sequence of 148 bp was obtained at the 5'-end. Thus, 1362 bp were sequenced and they encode 454 amino acids. The deduced amino-acid sequence contained peptide sequences corresponding to all ten tryptic fragments, confirming the identity of the cDNA clone. Comparison of the deduced amino-acid sequence between ACC synthase from apple fruit and those from tomato (Lycopersicon esculentum Mill.) and winter squash (Cucurbita maxima Duch.) fruits demonstrated the presence of seven highly conserved regions, including the previously identified region for the active site. The size of the translation product of ACC-synthase mRNA was similar to that of the mature protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that apple ACC-synthase undergoes only minor, if any, post-translational proteolytic processing. Analysis of ACC-synthase mRNA by in-vitro translation-immunoprecipitation, and by Northern blotting indicates that the ACC-synthase mRNA was undetectable in unripe fruit, but was accumulated massively during the ripening proccess. These data demonstrate that the expression of the ACC-synthase gene is developmentally regulated.

High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling

PubMed Central

Jones, Joshua D.; Chung, Betty Y.-W.; Siddell, Stuart G.; Brierley, Ian

2016-01-01

Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus. PMID:26919232

Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting

PubMed Central

Piazza, Carol Lyn; Smith, Dorie

2018-01-01

Group II introns are mobile ribozymes that are rare in bacterial genomes, often cohabiting with various mobile elements, and seldom interrupting housekeeping genes. What accounts for this distribution has not been well understood. Here, we demonstrate that Ll.LtrB, the group II intron residing in a relaxase gene on a conjugative plasmid from Lactococcus lactis, inhibits its host gene expression and restrains the naturally cohabiting mobile element from conjugative horizontal transfer. We show that reduction in gene expression is mainly at the mRNA level, and results from the interaction between exon-binding sequences (EBSs) in the intron and intron-binding sequences (IBSs) in the mRNA. The spliced intron targets the relaxase mRNA and reopens ligated exons, causing major mRNA loss. Taken together, this study provides an explanation for the distribution and paucity of group II introns in bacteria, and suggests a potential force for those introns to evolve into spliceosomal introns. PMID:29905149

Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules

PubMed Central

Gifford, Lida K.; Opalinska, Joanna B.; Jordan, David; Pattanayak, Vikram; Greenham, Paul; Kalota, Anna; Robbins, Michelle; Vernovsky, Kathy; Rodriguez, Lesbeth C.; Do, Bao T.; Lu, Ponzy; Gewirtz, Alan M.

2005-01-01

We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20–30 base oligodeoxynucleotides with 5–6 bp complementary ends to which a 5′ fluorophore and 3′ quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem–loop of the SQRM suggests that SQRM be made to target natural stem–loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells. PMID:15718294

Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting.

PubMed

Qu, Guosheng; Piazza, Carol Lyn; Smith, Dorie; Belfort, Marlene

2018-06-15

Group II introns are mobile ribozymes that are rare in bacterial genomes, often cohabiting with various mobile elements, and seldom interrupting housekeeping genes. What accounts for this distribution has not been well understood. Here, we demonstrate that Ll.LtrB, the group II intron residing in a relaxase gene on a conjugative plasmid from Lactococcus lactis , inhibits its host gene expression and restrains the naturally cohabiting mobile element from conjugative horizontal transfer. We show that reduction in gene expression is mainly at the mRNA level, and results from the interaction between exon-binding sequences (EBSs) in the intron and intron-binding sequences (IBSs) in the mRNA. The spliced intron targets the relaxase mRNA and reopens ligated exons, causing major mRNA loss. Taken together, this study provides an explanation for the distribution and paucity of group II introns in bacteria, and suggests a potential force for those introns to evolve into spliceosomal introns. © 2018, Qu et al.

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

DeepText2GO: Improving large-scale protein function prediction with deep semantic text representation.

PubMed

You, Ronghui; Huang, Xiaodi; Zhu, Shanfeng

2018-06-06

As of April 2018, UniProtKB has collected more than 115 million protein sequences. Less than 0.15% of these proteins, however, have been associated with experimental GO annotations. As such, the use of automatic protein function prediction (AFP) to reduce this huge gap becomes increasingly important. The previous studies conclude that sequence homology based methods are highly effective in AFP. In addition, mining motif, domain, and functional information from protein sequences has been found very helpful for AFP. Other than sequences, alternative information sources such as text, however, may be useful for AFP as well. Instead of using BOW (bag of words) representation in traditional text-based AFP, we propose a new method called DeepText2GO that relies on deep semantic text representation, together with different kinds of available protein information such as sequence homology, families, domains, and motifs, to improve large-scale AFP. Furthermore, DeepText2GO integrates text-based methods with sequence-based ones by means of a consensus approach. Extensive experiments on the benchmark dataset extracted from UniProt/SwissProt have demonstrated that DeepText2GO significantly outperformed both text-based and sequence-based methods, validating its superiority. Copyright © 2018 Elsevier Inc. All rights reserved.

An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5′ UTRs and its implications for eukaryotic gene translation regulation

PubMed Central

Pánek, Josef; Kolář, Michal; Vohradský, Jiří; Shivaya Valášek, Leoš

2013-01-01

There are several key mechanisms regulating eukaryotic gene expression at the level of protein synthesis. Interestingly, the least explored mechanisms of translational control are those that involve the translating ribosome per se, mediated for example via predicted interactions between the ribosomal RNAs (rRNAs) and mRNAs. Here, we took advantage of robustly growing large-scale data sets of mRNA sequences for numerous organisms, solved ribosomal structures and computational power to computationally explore the mRNA–rRNA complementarity that is statistically significant across the species. Our predictions reveal highly specific sequence complementarity of 18S rRNA sequences with mRNA 5′ untranslated regions (UTRs) forming a well-defined 3D pattern on the rRNA sequence of the 40S subunit. Broader evolutionary conservation of this pattern may imply that 5′ UTRs of eukaryotic mRNAs, which have already emerged from the mRNA-binding channel, may contact several complementary spots on 18S rRNA situated near the exit of the mRNA binding channel and on the middle-to-lower body of the solvent-exposed 40S ribosome including its left foot. We discuss physiological significance of this structurally conserved pattern and, in the context of previously published experimental results, propose that it modulates scanning of the 40S subunit through 5′ UTRs of mRNAs. PMID:23804757

Both coding exons of the c-myc gene contribute to its posttranscriptional regulation in the quiescent liver and regenerating liver and after protein synthesis inhibition.

PubMed Central

Lavenu, A; Pistoi, S; Pournin, S; Babinet, C; Morello, D

1995-01-01

In vivo, the steady-state level of c-myc mRNA is mainly controlled by posttranscriptional mechanisms. Using a panel of transgenic mice in which various versions of the human c-myc proto-oncogene were under the control of major histocompatibility complex H-2Kb class I regulatory sequences, we have shown that the 5' and the 3' noncoding sequences are dispensable for obtaining a regulated expression of the transgene in adult quiescent tissues, at the start of liver regeneration, and after inhibition of protein synthesis. These results indicated that the coding sequences were sufficient to ensure a regulated c-myc expression. In the present study, we have pursued this analysis with transgenes containing one or the other of the two c-myc coding exons either alone or in association with the c-myc 3' untranslated region. We demonstrate that each of the exons contains determinants which control c-myc mRNA expression. Moreover, we show that in the liver, c-myc exon 2 sequences are able to down-regulate an otherwise stable H-2K mRNA when embedded within it and to induce its transient accumulation after cycloheximide treatment and soon after liver ablation. Finally, the use of transgenes with different coding capacities has allowed us to postulate that the primary mRNA sequence itself and not c-Myc peptides is an important component of c-myc posttranscriptional regulation. PMID:7623834

LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs.

PubMed

Hong, Sungki; Freeberg, Mallory A; Han, Ting; Kamath, Avani; Yao, Yao; Fukuda, Tomoko; Suzuki, Tsukasa; Kim, John K; Inoki, Ken

2017-06-26

The RNA binding protein, LARP1, has been proposed to function downstream of mTORC1 to regulate the translation of 5'TOP mRNAs such as those encoding ribosome proteins (RP). However, the roles of LARP1 in the translation of 5'TOP mRNAs are controversial and its regulatory roles in mTORC1-mediated translation remain unclear. Here we show that LARP1 is a direct substrate of mTORC1 and Akt/S6K1. Deep sequencing of LARP1-bound mRNAs reveal that non-phosphorylated LARP1 interacts with both 5' and 3'UTRs of RP mRNAs and inhibits their translation. Importantly, phosphorylation of LARP1 by mTORC1 and Akt/S6K1 dissociates it from 5'UTRs and relieves its inhibitory activity on RP mRNA translation. Concomitantly, phosphorylated LARP1 scaffolds mTORC1 on the 3'UTRs of translationally-competent RP mRNAs to facilitate mTORC1-dependent induction of translation initiation. Thus, in response to cellular mTOR activity, LARP1 serves as a phosphorylation-sensitive molecular switch for turning off or on RP mRNA translation and subsequent ribosome biogenesis.

Insights into Deep-Sea Sediment Fungal Communities from the East Indian Ocean Using Targeted Environmental Sequencing Combined with Traditional Cultivation

PubMed Central

Zhang, Xiao-yong; Tang, Gui-ling; Xu, Xin-ya; Nong, Xu-hua; Qi, Shu-Hua

2014-01-01

The fungal diversity in deep-sea environments has recently gained an increasing amount attention. Our knowledge and understanding of the true fungal diversity and the role it plays in deep-sea environments, however, is still limited. We investigated the fungal community structure in five sediments from a depth of ∼4000 m in the East India Ocean using a combination of targeted environmental sequencing and traditional cultivation. This approach resulted in the recovery of a total of 45 fungal operational taxonomic units (OTUs) and 20 culturable fungal phylotypes. This finding indicates that there is a great amount of fungal diversity in the deep-sea sediments collected in the East Indian Ocean. Three fungal OTUs and one culturable phylotype demonstrated high divergence (89%–97%) from the existing sequences in the GenBank. Moreover, 44.4% fungal OTUs and 30% culturable fungal phylotypes are new reports for deep-sea sediments. These results suggest that the deep-sea sediments from the East India Ocean can serve as habitats for new fungal communities compared with other deep-sea environments. In addition, different fungal community could be detected when using targeted environmental sequencing compared with traditional cultivation in this study, which suggests that a combination of targeted environmental sequencing and traditional cultivation will generate a more diverse fungal community in deep-sea environments than using either targeted environmental sequencing or traditional cultivation alone. This study is the first to report new insights into the fungal communities in deep-sea sediments from the East Indian Ocean, which increases our knowledge and understanding of the fungal diversity in deep-sea environments. PMID:25272044

Long-depth imaging of specific gene expressions in whole-mount mouse embryos with single-photon excitation confocal fluorescence microscopy and FISH.

PubMed

Palmes-Saloma, C; Saloma, C

2000-07-01

Long-depth imaging of specific gene expression in the midgestation whole-mount mouse embryo (WME) is demonstrated with single-photon excitation (1PE) confocal fluorescence microscopy and fluorescence in situ hybridization. Expression domains of Pax-6 mRNA transcripts were labeled with an in situ hybridization probe that is a RNA sequence complementary to the cloned gene fragment and were rendered visible using two fluorochrome-conjugated antibodies that fluoresce at peak wavelengths of lambda(F) = 0.525 microm and lambda(F) = 0. 580 microm, respectively. Distributions of Pax-6 mRNA domains as deep as 1000 microm in the day 9.5 WME were imaged with a long-working-distance (13.6 mm) objective lens (magnification 5x). The scattering problem posed by the optically thick WME sample is alleviated by careful control of the detector pinhole size and the application of simple but fast postdetection image enhancement techniques, such as space and wavelength averaging to produce high-quality fluorescence images. A three-dimensional reconstruction that clearly shows the Pax-6 mRNA expression domains in the forebrain, diencephalon, optic cup, and spinal cord of the day 9.5 WME is obtained. The advantages of 1PE confocal fluorescence imaging over two-photon excitation fluorescence imaging are discussed for the case of long-depth imaging in highly scattering media. Imaging in midgestation WMEs at optical depths of more than 350 microm has not yet been realized with two-photon fluorescence excitation. Copyright 2000 Academic Press.

Disruption of the Axonal Trafficking of Tyrosine Hydroxylase mRNA Impairs Catecholamine Biosynthesis in the Axons of Sympathetic Neurons

PubMed Central

Gioio, Anthony E.

2017-01-01

Abstract Tyrosine hydroxylase (TH) is the enzyme that catalyzes the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. In a previous communication, evidence was provided that TH mRNA is trafficked to the axon, where it is locally translated. In addition, a 50-bp sequence element in the 3′untranslated region (3’UTR) of TH mRNA was identified that directs TH mRNA to distal axons (i.e., zip-code). In the present study, the hypothesis was tested that local translation of TH plays an important role in the biosynthesis of the catecholamine neurotransmitters in the axon and/or presynaptic nerve terminal. Toward this end, a targeted deletion of the axonal transport sequence element was developed, using the lentiviral delivery of the CRISPR/Cas9 system, and two guide RNA (gRNA) sequences flanking the 50-bp cis-acting regulatory element in rat superior cervical ganglion (SCG) neurons. Deletion of the axonal transport element reduced TH mRNA levels in the distal axons and reduced the axonal protein levels of TH and TH activity as measured by phosphorylation of SER40 in SCG neurons. Moreover, deletion of the zip-code diminished the axonal levels of dopamine (DA) and norepinephrine (NE). Conversely, the local translation of exogenous TH mRNA in the distal axon enhanced TH levels and activity, and elevated axonal NE levels. Taken together, these results provide direct evidence to support the hypothesis that TH mRNA trafficking and local synthesis of TH play an important role in the synthesis of catecholamines in the axon and presynaptic terminal. PMID:28630892

Disruption of the Axonal Trafficking of Tyrosine Hydroxylase mRNA Impairs Catecholamine Biosynthesis in the Axons of Sympathetic Neurons.

PubMed

Aschrafi, Armaz; Gioio, Anthony E; Dong, Lijin; Kaplan, Barry B

2017-01-01

Tyrosine hydroxylase (TH) is the enzyme that catalyzes the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. In a previous communication, evidence was provided that TH mRNA is trafficked to the axon, where it is locally translated. In addition, a 50-bp sequence element in the 3'untranslated region (3'UTR) of TH mRNA was identified that directs TH mRNA to distal axons (i.e., zip-code). In the present study, the hypothesis was tested that local translation of TH plays an important role in the biosynthesis of the catecholamine neurotransmitters in the axon and/or presynaptic nerve terminal. Toward this end, a targeted deletion of the axonal transport sequence element was developed, using the lentiviral delivery of the CRISPR/Cas9 system, and two guide RNA (gRNA) sequences flanking the 50-bp cis- acting regulatory element in rat superior cervical ganglion (SCG) neurons. Deletion of the axonal transport element reduced TH mRNA levels in the distal axons and reduced the axonal protein levels of TH and TH activity as measured by phosphorylation of SER40 in SCG neurons. Moreover, deletion of the zip-code diminished the axonal levels of dopamine (DA) and norepinephrine (NE). Conversely, the local translation of exogenous TH mRNA in the distal axon enhanced TH levels and activity, and elevated axonal NE levels. Taken together, these results provide direct evidence to support the hypothesis that TH mRNA trafficking and local synthesis of TH play an important role in the synthesis of catecholamines in the axon and presynaptic terminal.

Expression of myosin heavy chain isoforms mRNA transcripts in the temporalis muscle of common chimpanzees (Pan troglodytes).

PubMed

Ciurana, Neus; Artells, Rosa; Muñoz, Carmen; Arias-Martorell, Júlia; Bello-Hellegouarch, Gaëlle; Casado, Aroa; Cuesta, Elisabeth; Pérez-Pérez, Alejandro; Pastor, Juan Francisco; Potau, Josep Maria

2017-11-01

The common chimpanzee (Pan troglodytes) is the primate that is phylogenetically most closely related to humans (Homo sapiens). In order to shed light on the anatomy and function of the temporalis muscle in the chimpanzee, we have analyzed the expression patterns of the mRNA transcripts of the myosin heavy chain (MyHC) isoforms in different parts of the muscle. We dissected the superficial, deep and sphenomandibularis portions of the temporalis muscle in five adult P. troglodytes and quantified the expression of the mRNA transcripts of the MyHC isoforms in each portion using real-time quantitative polymerase chain reaction. We observed significant differences in the patterns of expression of the mRNA transcripts of the MyHC-IIM isoform between the sphenomandibularis portion and the anterior superficial temporalis (33.6% vs 47.0%; P=0.032) and between the sphenomandibularis portion and the anterior deep temporalis (33.6% vs 43.0; P=0.016). We also observed non-significant differences between the patterns of expression in the anterior and posterior superficial temporalis. The differential expression patterns of the mRNA transcripts of the MyHC isoforms in the temporalis muscle in P. troglodytes may be related to the functional differences that have been observed in electromyographic studies in other species of primates. Our findings can be applicable to the fields of comparative anatomy, evolutionary anatomy, and anthropology. Copyright © 2017 Elsevier GmbH. All rights reserved.

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.

Natural antisense transcript-targeted regulation of inducible nitric oxide synthase mRNA levels.

PubMed

Yoshigai, Emi; Hara, Takafumi; Araki, Yoshiro; Tanaka, Yoshito; Oishi, Masaharu; Tokuhara, Katsuji; Kaibori, Masaki; Okumura, Tadayoshi; Kwon, A-Hon; Nishizawa, Mikio

2013-04-01

Natural antisense transcripts (asRNAs) are frequently transcribed from mammalian genes. Recently, we found that non-coding asRNAs are transcribed from the 3' untranslated region (3'UTR) of the rat and mouse genes encoding inducible nitric oxide synthase (iNOS), which catalyzes the production of the inflammatory mediator nitric oxide. The iNOS asRNA stabilizes iNOS mRNA by interacting with the mRNA 3'UTR. Furthermore, single-stranded 'sense' oligonucleotides corresponding to the iNOS mRNA sequence were found to reduce iNOS mRNA levels by interfering with mRNA-asRNA interactions in rat hepatocytes. This method was named natural antisense transcript-targeted regulation (NATRE) technology. In this study, we detected human iNOS asRNA expressed in hepatocarcinoma and colon carcinoma tissues. The human iNOS asRNA harbored a sequence complementary to an evolutionarily conserved region of the iNOS mRNA 3'UTR. When introduced into hepatocytes, iNOS sense oligonucleotides that were modified by substitution with partial phosphorothioate bonds and locked nucleic acids or 2'-O-methyl nucleic acids greatly reduced levels of iNOS mRNA and iNOS protein. Moreover, sense oligonucleotides and short interfering RNAs decreased iNOS mRNA to comparable levels. These results suggest that NATRE technology using iNOS sense oligonucleotides could potentially be used to treat human inflammatory diseases and cancers by reducing iNOS mRNA levels. Copyright © 2013 Elsevier Inc. All rights reserved.

Transcriptomic investigation of meat tenderness in two Italian cattle breeds.

PubMed

Bongiorni, S; Gruber, C E M; Bueno, S; Chillemi, G; Ferrè, F; Failla, S; Moioli, B; Valentini, A

2016-06-01

Our objectives for this study were to understand the biological basis of meat tenderness and to provide an overview of the gene expression profiles related to meat quality as a tool for selection. Through deep mRNA sequencing, we analyzed gene expression in muscle tissues of two Italian cattle breeds: Maremmana and Chianina. We uncovered several differentially expressed genes that encode for proteins belonging to a family of tripartite motif proteins, which are involved in growth, cell differentiation and apoptosis, such as TRIM45, or play an essential role in regulating skeletal muscle differentiation and the regeneration of adult skeletal muscle, such as TRIM32. Other differentially expressed genes (SCN2B, SLC9A7 and KCNK3) emphasize the involvement of potassium-sodium pumps in tender meat. By mapping splice junctions in RNA-Seq reads, we found significant differences in gene isoform expression levels. The PRKAG3 gene, which is involved in the regulation of energy metabolism, showed four isoforms that were differentially expressed. This distinct pattern of PRKAG3 gene expression could indicate impaired glycogen storage in skeletal muscle, and consequently, this gene very likely has a role in the tenderization process. Furthermore, with this deep RNA-sequencing, we captured a high number of expressed SNPs, for example, we found 1462 homozygous SNPs showing the alternative allele with a 100% frequency when comparing tender and tough meat. SNPs were then classified into categories by their position and also by their effect on gene coding (174 non-synonymous polymorphisms) based on the available UMD_3.1 annotations. © 2016 Stichting International Foundation for Animal Genetics.

RNActive® Technology: Generation and Testing of Stable and Immunogenic mRNA Vaccines.

PubMed

Rauch, Susanne; Lutz, Johannes; Kowalczyk, Aleksandra; Schlake, Thomas; Heidenreich, Regina

2017-01-01

Developing effective mRNA vaccines poses certain challenges concerning mRNA stability and ability to induce sufficient immune stimulation and requires a specific panel of techniques for production and testing. Here, we describe the production of stabilized mRNA with enhanced immunogenicity, generated using conventional nucleotides only, by introducing changes to the mRNA sequence and by complexation with the nucleotide-binding peptide protamine (RNActive® technology). Methods described here include the synthesis, purification, and protamine complexation of mRNA vaccines as well as a comprehensive panel of in vitro and in vivo methods for evaluation of vaccine quality and immunogenicity.

Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq

PubMed Central

Ode, Hirotaka; Matsuda, Masakazu; Matsuoka, Kazuhiro; Hachiya, Atsuko; Hattori, Junko; Kito, Yumiko; Yokomaku, Yoshiyuki; Iwatani, Yasumasa; Sugiura, Wataru

2015-01-01

Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of aHIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only < 1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients' samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome. PMID:26617593

The Drosophila Tis11 protein and its effects on mRNA expression in flies.

PubMed

Choi, Youn-Jeong; Lai, Wi S; Fedic, Robert; Stumpo, Deborah J; Huang, Weichun; Li, Leping; Perera, Lalith; Brewer, Brandy Y; Wilson, Gerald M; Mason, James M; Blackshear, Perry J

2014-12-19

Members of the mammalian tristetraprolin family of CCCH tandem zinc finger proteins can bind to certain AU-rich elements (AREs) in mRNAs, leading to their deadenylation and destabilization. Mammals express three or four members of this family, but Drosophila melanogaster and other insects appear to contain a single gene, Tis11. We found that recombinant Drosophila Tis11 protein could bind to ARE-containing RNA oligonucleotides with low nanomolar affinity. Remarkably, co-expression in mammalian cells with "target" RNAs demonstrated that Tis11 could promote destabilization of ARE-containing mRNAs and that this was partially dependent on a conserved C-terminal sequence resembling the mammalian NOT1 binding domain. Drosophila Tis11 promoted both deadenylation and decay of a target transcript in this heterologous cell system. We used chromosome deletion/duplication and P element insertion to produce two types of Tis11 deficiency in adult flies, both of which were viable and fertile. To address the hypothesis that Tis11 deficiency would lead to the abnormal accumulation of potential target transcripts, we analyzed gene expression in adult flies by deep mRNA sequencing. We identified 69 transcripts from 56 genes that were significantly up-regulated more than 1.5-fold in both types of Tis11-deficient flies. Ten of the up-regulated transcripts encoded probable proteases, but many other functional classes of proteins were represented. Many of the up-regulated transcripts contained potential binding sites for tristetraprolin family member proteins that were conserved in other Drosophila species. Tis11 is thus an ARE-binding, mRNA-destabilizing protein that may play a role in post-transcriptional gene expression in Drosophila and other insects. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The Drosophila Tis11 Protein and Its Effects on mRNA Expression in Flies*

PubMed Central

Choi, Youn-Jeong; Lai, Wi S.; Fedic, Robert; Stumpo, Deborah J.; Huang, Weichun; Li, Leping; Perera, Lalith; Brewer, Brandy Y.; Wilson, Gerald M.; Mason, James M.; Blackshear, Perry J.

2014-01-01

Members of the mammalian tristetraprolin family of CCCH tandem zinc finger proteins can bind to certain AU-rich elements (AREs) in mRNAs, leading to their deadenylation and destabilization. Mammals express three or four members of this family, but Drosophila melanogaster and other insects appear to contain a single gene, Tis11. We found that recombinant Drosophila Tis11 protein could bind to ARE-containing RNA oligonucleotides with low nanomolar affinity. Remarkably, co-expression in mammalian cells with “target” RNAs demonstrated that Tis11 could promote destabilization of ARE-containing mRNAs and that this was partially dependent on a conserved C-terminal sequence resembling the mammalian NOT1 binding domain. Drosophila Tis11 promoted both deadenylation and decay of a target transcript in this heterologous cell system. We used chromosome deletion/duplication and P element insertion to produce two types of Tis11 deficiency in adult flies, both of which were viable and fertile. To address the hypothesis that Tis11 deficiency would lead to the abnormal accumulation of potential target transcripts, we analyzed gene expression in adult flies by deep mRNA sequencing. We identified 69 transcripts from 56 genes that were significantly up-regulated more than 1.5-fold in both types of Tis11-deficient flies. Ten of the up-regulated transcripts encoded probable proteases, but many other functional classes of proteins were represented. Many of the up-regulated transcripts contained potential binding sites for tristetraprolin family member proteins that were conserved in other Drosophila species. Tis11 is thus an ARE-binding, mRNA-destabilizing protein that may play a role in post-transcriptional gene expression in Drosophila and other insects. PMID:25342740

Virus Identification in Unknown Tropical Febrile Illness Cases Using Deep Sequencing

PubMed Central

Balmaseda, Angel; Harris, Eva; DeRisi, Joseph L.

2012-01-01

Dengue virus is an emerging infectious agent that infects an estimated 50–100 million people annually worldwide, yet current diagnostic practices cannot detect an etiologic pathogen in ∼40% of dengue-like illnesses. Metagenomic approaches to pathogen detection, such as viral microarrays and deep sequencing, are promising tools to address emerging and non-diagnosable disease challenges. In this study, we used the Virochip microarray and deep sequencing to characterize the spectrum of viruses present in human sera from 123 Nicaraguan patients presenting with dengue-like symptoms but testing negative for dengue virus. We utilized a barcoding strategy to simultaneously deep sequence multiple serum specimens, generating on average over 1 million reads per sample. We then implemented a stepwise bioinformatic filtering pipeline to remove the majority of human and low-quality sequences to improve the speed and accuracy of subsequent unbiased database searches. By deep sequencing, we were able to detect virus sequence in 37% (45/123) of previously negative cases. These included 13 cases with Human Herpesvirus 6 sequences. Other samples contained sequences with similarity to sequences from viruses in the Herpesviridae, Flaviviridae, Circoviridae, Anelloviridae, Asfarviridae, and Parvoviridae families. In some cases, the putative viral sequences were virtually identical to known viruses, and in others they diverged, suggesting that they may derive from novel viruses. These results demonstrate the utility of unbiased metagenomic approaches in the detection of known and divergent viruses in the study of tropical febrile illness. PMID:22347512

Molecular analysis of two phytohemagglutinin genes and their expression in Phaseolus vulgaris cv. Pinto, a lectin-deficient cultivar of the bean.

PubMed

Voelker, T A; Staswick, P; Chrispeels, M J

1986-12-01

Phytohemagglutinin (PHA), the seed lectin of the common bean, Phaseolus vulgaris, is encoded by two highly homologous, tandemly linked genes, dlec1 and dlec2, which are coordinately expressed at high levels in developing cotyledons. Their respective transcripts translate into closely related polypeptides, PHA-E and PHA-L, constituents of the tetrameric lectin which accumulates at high levels in developing seeds. In the bean cultivar Pinto UI111, PHA-E is not detectable, and PHA-L accumulates at very reduced levels. To investigate the cause of the Pinto phenotype, we cloned and sequenced the two PHA genes of Pinto, called Pdlec1 and Pdlec2, and determined the abundance of their respective mRNAs in developing cotyledons. Both genes are more than 90% homologous to the normal PHA genes found in other cultivars. Pdlec1 carries a 1-bp frameshift mutation close to the 5' end of its coding sequence. Only very truncated polypeptides could be made from its mRNA. The gene Pdlec2 encodes a polypeptide, which resembles PHA-L and its predicted amino acid sequence agrees with the available Pinto PHA amino acid sequence data. Analysis of the mRNA of developing cotyledons revealed that the Pdlec1 message is reduced 600-fold, and Pdlec2 mRNA is reduced 20-fold with respect to mRNA levels in normal cultivars. A comparison of the sequences which are upstream from the coding sequence shows that Pdlec2 has a 100-bp deletion compared to the other genes (dlec1, dlec2 and Pdlec1). This deletion which contains a large tandem repeat may be responsible for the low level of expression of Pdlec2. The very low expression of Pdlec1 is as yet unexplained.

RNA editing: trypanosomes rewrite the genetic code.

PubMed

Stuart, K

1998-01-01

The understanding of how genetic information is stored and expressed has advanced considerably since the "central dogma" asserted that genetic information flows from the nucleotide sequence of DNA to that of messenger RNA (mRNA) which in turn specifies the amino acid sequence of a protein. It was found that genetic information can be stored as RNA (e.g. in RNA viruses) and can flow from RNA to DNA by reverse transcriptase enzyme activity. In addition, some genes contain introns, nucleotide sequences that are removed from their RNA (by RNA splicing) and thus are not represented in the resultant protein. Furthermore, alternative splicing was found to produce variant proteins from a single gene. More recently, the study of trypanosome parasites revealed an unexpected and indeed counter-intuitive genetic complexity. Genetic information for a single protein can be dispersed among several (DNA) genes in these organisms. One of these genes specifies an encrypted precursor mRNA that is converted to a functional mRNA by a process called RNA editing that inserts and deletes uridylate nucleotides. The sequence of the edited mRNA is specified by multiple small RNAs, named guide RNAs, (gRNAs) each of which is encoded in a separate gene. Thus, edited mRNA sequences are assembled from multiple genes by the transfer of information from one type of RNA to another. The existence of editing was surprising but has stimulated the discovery of other types of RNA editing. The Stuart laboratory has been exploring RNA editing in trypanosomes from the time of its discovery. They found dramatic differences between the mitochondrial gene sequences and those of the corresponding mRNAs, which indicated editing by the insertion and deletion of uridylates. Some editing was modest; simply eliminating shifts in sequence register of minimally extending the protein coding sequence. However, editing of many mRNAs was startingly extensive. The RNA sequence was essentially entirely remodeled with its sequence more the result of editing than the gene sequence. The identities of genes for such extensively edited RNA were not recognizable from the DNA sequence but they were readily identifiable from the edited mRNA sequence. Thus, despite the complex and extensive editing the resultant mRNA sequence is precise. Characterization of partially edited RNAs indicated that editing proceeds in the direction opposite to that used to specify the protein which reflects the use of the gRNAs. The numerous gRNAs that are used for editing are encoded in the DNA molecules whose role was previously a mystery. Using information gained in our earlier studies, the Stuart group developed an in vitro system that reproduces the fundamental process of editing in order to resolve the mechanism by which it occurs. They determined that editing entails a series of enzymatic steps rather than the mechanism used in RNA splicing. They also showed that chimeric gRNA-mRNA molecules are aberrant by-products of editing rather than intermediates in the process as had been proposed. Additional studies are exploring precisely how the number of added and deleted uridylates is specified by the gRNA. The Stuart laboratory showed that editing is performed by an aggregation of enzymes that catalyze the separate steps of editing. It also developed a method to purify this multimolecule complex that contains several, perhaps tens of, proteins. This will allow the study of its composition and the functions of its component parts. Indeed, the gene for one component has been identified and its detailed characterization begun. These studies are developing tools to explore related processes. An early finding in the lab was that the various mRNAs are differentially edited during the life cycle of the parasite. The pattern of this editing indicates that editing serves to regulate the alternation between two modes of energy generation. This regulation is coordinated with other events that are occurring during the life c

Familial retinoblastoma due to intronic LINE-1 insertion causes aberrant and noncanonical mRNA splicing of the RB1 gene.

PubMed

Rodríguez-Martín, Carlos; Cidre, Florencia; Fernández-Teijeiro, Ana; Gómez-Mariano, Gema; de la Vega, Leticia; Ramos, Patricia; Zaballos, Ángel; Monzón, Sara; Alonso, Javier

2016-05-01

Retinoblastoma (RB, MIM 180200) is the paradigm of hereditary cancer. Individuals harboring a constitutional mutation in one allele of the RB1 gene have a high predisposition to develop RB. Here, we present the first case of familial RB caused by a de novo insertion of a full-length long interspersed element-1 (LINE-1) into intron 14 of the RB1 gene that caused a highly heterogeneous splicing pattern of RB1 mRNA. LINE-1 insertion was inferred by mRNA studies and full-length sequenced by massive parallel sequencing. Some of the aberrant mRNAs were produced by noncanonical acceptor splice sites, a new finding that up to date has not been described to occur upon LINE-1 retrotransposition. Our results clearly show that RNA-based strategies have the potential to detect disease-causing transposon insertions. It also confirms that the incorporation of new genetic approaches, such as massive parallel sequencing, contributes to characterize at the sequence level these unique and exceptional genetic alterations.

Isolation of a complementary DNA clone for thyroid microsomal antigen. Homology with the gene for thyroid peroxidase.

PubMed Central

Seto, P; Hirayu, H; Magnusson, R P; Gestautas, J; Portmann, L; DeGroot, L J; Rapoport, B

1987-01-01

The thyroid microsomal antigen (MSA) in autoimmune thyroid disease is a protein of approximately 107 kD. We screened a human thyroid cDNA library constructed in the expression vector lambda gt11 with anti-107-kD monoclonal antibodies. Of five clones obtained, the recombinant beta-galactosidase fusion protein from one clone (PM-5) was confirmed to react with the monoclonal antiserum. The complementary DNA (cDNA) insert from PM-5 (0.8 kb) was used as a probe on Northern blot analysis to estimate the size of the mRNA coding for the MSA. The 2.9-kb messenger RNA (mRNA) species observed was the same size as that coding for human thyroid peroxidase (TPO). The probe did not bind to human liver mRNA, indicating the thyroid-specific nature of the PM-5-related mRNA. The nucleotide sequence of PM-5 (842 bp) was determined and consisted of a single open reading frame. Comparison of the nucleotide sequence of PM-5 with that presently available for pig TPO indicates 84% homology. In conclusion, a cDNA clone representing part of the microsomal antigen has been isolated. Sequence homology with porcine TPO, as well as identity in the size of the mRNA species for both the microsomal antigen and TPO, indicate that the microsomal antigen is, at least in part, TPO. Images PMID:3654979

Two divergent endo-beta-1,4-glucanase genes exhibit overlapping expression in ripening fruit and abscising flowers.

PubMed Central

Lashbrook, C C; Gonzalez-Bosch, C; Bennett, A B

1994-01-01

Two structurally divergent endo-beta-1,4-glucanase (EGase) cDNAs were cloned from tomato. Although both cDNAs (Cel1 and Cel2) encode potentially glycosylated, basic proteins of 51 to 53 kD and possess multiple amino acid domains conserved in both plant and microbial EGases, Cel1 and Cel2 exhibit only 50% amino acid identity at the overall sequence level. Amino acid sequence comparisons to other plant EGases indicate that tomato Cel1 is most similar to bean abscission zone EGase (68%), whereas Cel2 exhibits greatest sequence identity to avocado fruit EGase (57%). Sequence comparisons suggest the presence of at least two structurally divergent EGase families in plants. Unlike ripening avocado fruit and bean abscission zones in which a single EGase mRNA predominates, EGase expression in tomato reflects the overlapping accumulation of both Cel1 and Cel2 transcripts in ripening fruit and in plant organs undergoing cell separation. Cel1 mRNA contributes significantly to total EGase mRNA accumulation within plant organs undergoing cell separation (abscission zones and mature anthers), whereas Cel2 mRNA is most abundant in ripening fruit. The overlapping expression of divergent EGase genes within a single species may suggest that multiple activities are required for the cooperative disassembly of cell wall components during fruit ripening, floral abscission, and anther dehiscence. PMID:7994180

Inhibition of herpes simplex virus 1 gene expression and replication by RNase P-associated external guide sequences.

PubMed

Liu, Jin; Shao, Luyao; Trang, Phong; Yang, Zhu; Reeves, Michael; Sun, Xu; Vu, Gia-Phong; Wang, Yu; Li, Hongjian; Zheng, Congyi; Lu, Sangwei; Liu, Fenyong

2016-06-09

An external guide sequence (EGS) is a RNA sequence which can interact with a target mRNA to form a tertiary structure like a pre-tRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, to degrade target mRNA. Previously, an in vitro selection procedure has been used by us to engineer new EGSs that are more robust in inducing human RNase P to cleave their targeted mRNAs. In this study, we constructed EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. The EGS variant induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS generated from a natural pre-tRNA. A decrease of about 97% and 75% in the level of ICP4 gene expression and an inhibition of about 7,000- and 500-fold in viral growth were observed in HSV infected cells expressing the variant and the pre-tRNA-derived EGS, respectively. This study shows that engineered EGSs can inhibit HSV-1 gene expression and viral growth. Furthermore, these results demonstrate the potential for engineered EGS RNAs to be developed and used as anti-HSV therapeutics.

Inhibition of herpes simplex virus 1 gene expression and replication by RNase P-associated external guide sequences

PubMed Central

Liu, Jin; Shao, Luyao; Trang, Phong; Yang, Zhu; Reeves, Michael; Sun, Xu; Vu, Gia-Phong; Wang, Yu; Li, Hongjian; Zheng, Congyi; Lu, Sangwei; Liu, Fenyong

2016-01-01

An external guide sequence (EGS) is a RNA sequence which can interact with a target mRNA to form a tertiary structure like a pre-tRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, to degrade target mRNA. Previously, an in vitro selection procedure has been used by us to engineer new EGSs that are more robust in inducing human RNase P to cleave their targeted mRNAs. In this study, we constructed EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. The EGS variant induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS generated from a natural pre-tRNA. A decrease of about 97% and 75% in the level of ICP4 gene expression and an inhibition of about 7,000- and 500-fold in viral growth were observed in HSV infected cells expressing the variant and the pre-tRNA-derived EGS, respectively. This study shows that engineered EGSs can inhibit HSV-1 gene expression and viral growth. Furthermore, these results demonstrate the potential for engineered EGS RNAs to be developed and used as anti-HSV therapeutics. PMID:27279482

Distant sequences determine 5′ end formation of cox3 transcripts in Arabidopsis thaliana ecotype C24

PubMed Central

Forner, Joachim; Weber, Bärbel; Wiethölter, Caterina; Meyer, Rhonda C.; Binder, Stefan

2005-01-01

The genomic environments and the transcripts of the mitochondrial cox3 gene are investigated in three Arabidopsis thaliana ecotypes. While the proximate 5′ sequences up to nucleotide position −584, the coding regions and the 3′ flanking regions are identical in Columbia (Col), C24 and Landsberg erecta (Ler), genomic variation is detected in regions further upstream. In the mitochondrial DNA of Col, a 1790 bp fragment flanked by a nonanucleotide direct repeat is present beyond position −584 with respect to the ATG. While in Ler only part of this insertion is conserved, this sequence is completely absent in C24, except for a single copy of the nonanucleotide direct repeat. Northern hybridization reveals identical major transcripts in the three ecotypes, but identifies an additional abundant 60 nt larger mRNA species in C24. The extremities of the most abundant mRNA species are identical in the three ecotypes. In C24, an extra major 5′ end is abundant. This terminus and the other major 5′ ends are located in identical sequence regions. Inspection of Atcox3 transcripts in C24/Col hybrids revealed a female inheritance of the mRNA species with the extra 5′ terminus. Thus, a mitochondrially encoded factor determines the generation of an extra 5′ mRNA end. PMID:16107557

Protein functional features are reflected in the patterns of mRNA translation speed.

PubMed

López, Daniel; Pazos, Florencio

2015-07-09

The degeneracy of the genetic code makes it possible for the same amino acid string to be coded by different messenger RNA (mRNA) sequences. These "synonymous mRNAs" may differ largely in a number of aspects related to their overall translational efficiency, such as secondary structure content and availability of the encoded transfer RNAs (tRNAs). Consequently, they may render different yields of the translated polypeptides. These mRNA features related to translation efficiency are also playing a role locally, resulting in a non-uniform translation speed along the mRNA, which has been previously related to some protein structural features and also used to explain some dramatic effects of "silent" single-nucleotide-polymorphisms (SNPs). In this work we perform the first large scale analysis of the relationship between three experimental proxies of mRNA local translation efficiency and the local features of the corresponding encoded proteins. We found that a number of protein functional and structural features are reflected in the patterns of ribosome occupancy, secondary structure and tRNA availability along the mRNA. One or more of these proxies of translation speed have distinctive patterns around the mRNA regions coding for certain protein local features. In some cases the three patterns follow a similar trend. We also show specific examples where these patterns of translation speed point to the protein's important structural and functional features. This support the idea that the genome not only codes the protein functional features as sequences of amino acids, but also as subtle patterns of mRNA properties which, probably through local effects on the translation speed, have some consequence on the final polypeptide. These results open the possibility of predicting a protein's functional regions based on a single genomic sequence, and have implications for heterologous protein expression and fine-tuning protein function.

Exon 2-mediated c-myc mRNA decay in vivo is independent of its translation.

PubMed Central

Pistoi, S; Roland, J; Babinet, C; Morello, D

1996-01-01

We have previously shown that the steady-state level of c-myc mRNA in vivo is primarily controlled by posttranscriptional regulatory mechanisms. To identify the sequences involved in this process, we constructed a series of H-2/myc transgenic lines in which various regions of the human c-MYC gene were placed under the control of the quasi-ubiquitous H-2K class I regulatory sequences. We demonstrated that the presence of one of the two coding exons, exon 2 or exon 3, is sufficient to confer a level of expression of transgene mRNA similar to that of endogenous c-myc in various adult tissues as well as after partial hepatectomy or after protein synthesis inhibition. We now focus on the molecular mechanisms involved in modulation of expression of mRNAs containing c-myc exon 2 sequences, with special emphasis on the coupling between translation and c-myc mRNA turnover. We have undertaken an analysis of expression, both at the mRNA level and at the protein level, of new transgenic constructs in which the translation is impaired either by disruption of the initiation codon or by addition of stop codons upstream of exon 2. Our results show that the translation of c-myc exon 2 is not required for regulated expression of the transgene in the different situations analyzed, and therefore they indicate that the mRNA destabilizing function of exon 2 is independent of translation by ribosomes. Our investigations also reveal that, in the thymus, some H-2/myc transgenes express high levels of mRNA but low levels of protein. Besides the fact that these results suggest the existence of tissue-specific mechanisms that control c-myc translatability in vivo, they also bring another indication of the uncoupling of c-myc mRNA translation and degradation. PMID:8756668

Next-generation sequencing facilitates quantitative analysis of wild-type and Nrl−/− retinal transcriptomes

PubMed Central

Brooks, Matthew J.; Rajasimha, Harsha K.; Roger, Jerome E.

2011-01-01

Purpose Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis. Methods Retinal mRNA profiles of 21-day-old wild-type (WT) and neural retina leucine zipper knockout (Nrl−/−) mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl−/− retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. PMID:22162623

High-Throughput Mapping of Single-Neuron Projections by Sequencing of Barcoded RNA.

PubMed

Kebschull, Justus M; Garcia da Silva, Pedro; Reid, Ashlan P; Peikon, Ian D; Albeanu, Dinu F; Zador, Anthony M

2016-09-07

Neurons transmit information to distant brain regions via long-range axonal projections. In the mouse, area-to-area connections have only been systematically mapped using bulk labeling techniques, which obscure the diverse projections of intermingled single neurons. Here we describe MAPseq (Multiplexed Analysis of Projections by Sequencing), a technique that can map the projections of thousands or even millions of single neurons by labeling large sets of neurons with random RNA sequences ("barcodes"). Axons are filled with barcode mRNA, each putative projection area is dissected, and the barcode mRNA is extracted and sequenced. Applying MAPseq to the locus coeruleus (LC), we find that individual LC neurons have preferred cortical targets. By recasting neuroanatomy, which is traditionally viewed as a problem of microscopy, as a problem of sequencing, MAPseq harnesses advances in sequencing technology to permit high-throughput interrogation of brain circuits. Copyright © 2016 Elsevier Inc. All rights reserved.

Genetic analysis of tumorigenesis: XXXII. Localization of constitutionally amplified KRAS sequences to Chinese hamster chromosomes X and Y by in situ hybridization.

PubMed

Stenman, G; Anisowicz, A; Sager, R

1988-11-01

The KRAS gene is constitutionally amplified in the Chinese hamster. We have mapped the amplified sequences by in situ hybridization to two major sites on the X and Y chromosomes, Xq4 and Yp2. No autosomal site was detected despite a search under relaxed hybridization conditions. KRAS DNA is amplified about 50-fold compared to a human cell line known to have a diploid number of KRAS sequences, whereas mRNA expression is 5- to 10-fold lower than in normal human cells. While mRNA expression levels do not necessarily parallel gene copy number, the low expression level strongly suggests that the amplified sequences are transcriptionally silent. It is suggested that the amplified sequences arose from the original KRAS gene on chromosome 8 and that the KRAS sequences on the Y chromosome arose by X-Y recombination.

deepTools: a flexible platform for exploring deep-sequencing data.

PubMed

Ramírez, Fidel; Dündar, Friederike; Diehl, Sarah; Grüning, Björn A; Manke, Thomas

2014-07-01

We present a Galaxy based web server for processing and visualizing deeply sequenced data. The web server's core functionality consists of a suite of newly developed tools, called deepTools, that enable users with little bioinformatic background to explore the results of their sequencing experiments in a standardized setting. Users can upload pre-processed files with continuous data in standard formats and generate heatmaps and summary plots in a straight-forward, yet highly customizable manner. In addition, we offer several tools for the analysis of files containing aligned reads and enable efficient and reproducible generation of normalized coverage files. As a modular and open-source platform, deepTools can easily be expanded and customized to future demands and developments. The deepTools webserver is freely available at http://deeptools.ie-freiburg.mpg.de and is accompanied by extensive documentation and tutorials aimed at conveying the principles of deep-sequencing data analysis. The web server can be used without registration. deepTools can be installed locally either stand-alone or as part of Galaxy. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Single Molecule Force Measurement for Protein Synthesis on the Ribosome

NASA Astrophysics Data System (ADS)

Uemura, Sotaro

2008-04-01

The ribosome is a molecular machine that translates the genetic code described on the messenger RNA (mRNA) into an amino acid sequence through repetitive cycles of transfer RNA (tRNA) selection, peptide bond formation and translocation. Although the detailed interactions between the translation components have been revealed by extensive structural and biochemical studies, it is not known how the precise regulation of macromolecular movements required at each stage of translation is achieved. Here we demonstrate an optical tweezer assay to measure the rupture force between a single ribosome complex and mRNA. The rupture force was compared between ribosome complexes assembled on an mRNA with and without a strong Shine-Dalgarno (SD) sequence. The removal of the SD sequence significantly reduced the rupture force, indicating that the SD interactions contribute significantly to the stability of the ribosomal complex on the mRNA in a pre-peptidyl transfer state. In contrast, the post-peptidyl transfer state weakened the rupture force as compared to the complex in a pre-peptidyl transfer state and it was the same for both the SD-containing and SD-deficient mRNAs. The results suggest that formation of the first peptide bond destabilizes the SD interaction, resulting in the weakening of the force with which the ribosome grips an mRNA. This might be an important requirement to facilitate movement of the ribosome along mRNA during the first translocation step. In this article, we discuss about the above new results including the introduction of the ribosome translation mechanism and the optical tweezer method.

Isolates of viral hemorrhagic septicemia virus from North America and Europe can be detected and distinguished by DNA probes

USGS Publications Warehouse

Batts, W.N.; Arakawa, C.K.; Bernard, J.; Winton, J.R.

1993-01-01

Biotinylated DNA probes were constructed to hybndize with speclfic sequences within the messenger RNA (mRNA) of the nucleoprotein (N) gene of vlral hemorrhagic septicemia virus (VHSV) reference strains from Europe (07-71) and North Arnenca (Makah) Probes were synthesized that were complementary to (1) a 29-nucleotide sequence near the center of the N gene conlmon to both the 07-71 and Makah reference strains of the virus (2) a unique 28- nucleotide sequence that followed the open readng frame of the Makah N gene mRNA most of which was absent In the 07-71 strain, and (3) a 22-nucleobde sequence wthin the 07-71 N gene that had 6 nllsmatches \

Codon influence on protein expression in E. coli correlates with mRNA levels

PubMed Central

Boël, Grégory; Wong, Kam-Ho; Su, Min; Luff, Jon; Valecha, Mayank; Everett, John K.; Acton, Thomas B.; Xiao, Rong; Montelione, Gaetano T.; Aalberts, Daniel P.; Hunt, John F.

2016-01-01

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyze the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli. PMID:26760206

Definition of the complete Schistosoma mansoni hemoglobinase mRNA sequence and gene expression in developing parasites.

PubMed

el Meanawy, M A; Aji, T; Phillips, N F; Davis, R E; Salata, R A; Malhotra, I; McClain, D; Aikawa, M; Davis, A H

1990-07-01

Schistosoma mansoni uses a variety of proteases termed hemoglobinases to obtain nutrition from host globin. Previous reports have characterized cDNAs encoding 1 of these enzymes. However, these sequences did not define the primary structures of the mRNA and protein. The complete sequence of the 1390 base mRNA has now been determined. It encodes a 50 kDa primary translation product. In vitro translations coupled with immunoprecipitations and Western blots of parasite lysates allowed visualization of the 50 kDa form. Production of the 31 kDa mature hemoglobinase from the 50 kDa species involves removal of both NH2 and COOH terminal residues from the primary translation product. Expression of hemoglobinase mRNA and protein was examined during larval parasite development. Low levels were observed in young schistosomula. After 6-9 days in culture, high hemoglobinase levels were seen which correlated with the onset of red blood cell feeding. Immunoelectron microscopy was employed to examine hemoglobinase location and function. In adult worms the enzyme was associated with the gut lumen and gut epithelium. In cercariae, the protease was observed in the head gland, suggesting new roles for the protease.

Identification of microRNAs involved in lipid biosynthesis and seed size in developing sea buckthorn seeds using high-throughput sequencing.

PubMed

Ding, Jian; Ruan, Chengjiang; Guan, Ying; Krishna, Priti

2018-03-05

Sea buckthorn is a plant of medicinal and nutritional importance owing in part to the high levels of essential fatty acids, linoleic (up to 42%) and α-linolenic (up to 39%) acids in the seed oil. Sea buckthorn can produce seeds either via the sexual pathway or by apomixis. The seed development and maturation programs are critically dependent on miRNAs. To understand miRNA-mediated regulation of sea buckthorn seed development, eight small RNA libraries were constructed for deep sequencing from developing seeds of a low oil content line 'SJ1' and a high oil content line 'XE3'. High-throughput sequencing identified 137 known miRNA from 27 families and 264 novel miRNAs. The potential targets of the identified miRNAs were predicted based on sequence homology. Nineteen (four known and 15 novel) and 22 (six known and 16 novel) miRNAs were found to be involved in lipid biosynthesis and seed size, respectively. An integrated analysis of mRNA and miRNA transcriptome and qRT-PCR identified some key miRNAs and their targets (miR164d-ARF2, miR168b-Δ9D, novelmiRNA-108-ACC, novelmiRNA-23-GPD1, novelmiRNA-58-DGAT1, and novelmiRNA-191-DGAT2) potentially involved in seed size and lipid biosynthesis of sea buckthorn seed. These results indicate the potential importance of miRNAs in regulating lipid biosynthesis and seed size in sea buckthorn.

FATHEAD MINNOW VITELLOGENIN: CDNA SEQUENCE AND MRNA AND PROTEIN EXPRESSION AFTER 17 BETA-ESTRADIOL TREATMENT

EPA Science Inventory

In the present study, a sensitive ribonuclease protection assay (RPA) for VTG mRNA was developed for the fathead minnow (Pimephales promelas), a species proposed for routine endocrine-disrupting chemical (EDC) screening.

PreTIS: A Tool to Predict Non-canonical 5’ UTR Translational Initiation Sites in Human and Mouse

PubMed Central

Reuter, Kerstin; Helms, Volkhard

2016-01-01

Translation of mRNA sequences into proteins typically starts at an AUG triplet. In rare cases, translation may also start at alternative non–AUG codons located in the annotated 5’ UTR which leads to an increased regulatory complexity. Since ribosome profiling detects translational start sites at the nucleotide level, the properties of these start sites can then be used for the statistical evaluation of functional open reading frames. We developed a linear regression approach to predict in–frame and out–of–frame translational start sites within the 5’ UTR from mRNA sequence information together with their translation initiation confidence. Predicted start codons comprise AUG as well as near–cognate codons. The underlying datasets are based on published translational start sites for human HEK293 and mouse embryonic stem cells that were derived by the original authors from ribosome profiling data. The average prediction accuracy of true vs. false start sites for HEK293 cells was 80%. When applied to mouse mRNA sequences, the same model predicted translation initiation sites observed in mouse ES cells with an accuracy of 76%. Moreover, we illustrate the effect of in silico mutations in the flanking sequence context of a start site on the predicted initiation confidence. Our new webservice PreTIS visualizes alternative start sites and their respective ORFs and predicts their ability to initiate translation. Solely, the mRNA sequence is required as input. PreTIS is accessible at http://service.bioinformatik.uni-saarland.de/pretis. PMID:27768687

Rapid amplification of 5' complementary DNA ends (5' RACE).

PubMed

2005-08-01

This method is used to extend partial cDNA clones by amplifying the 5' sequences of the corresponding mRNAs 1-3. The technique requires knowledge of only a small region of sequence within the partial cDNA clone. During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence; the second primer required for PCR is complementary to a general feature of the target-in the case of 5' RACE, to a homopolymeric tail added (via terminal transferase) to the 3' termini of cDNAs transcribed from a preparation of mRNA. This synthetic tail provides a primer-binding site upstream of the unknown 5' sequence of the target mRNA. The products of the amplification reaction are cloned into a plasmid vector for sequencing and subsequent manipulation.

Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.

PubMed

Bernsen, M R; Dijkman, H B; de Vries, E; Figdor, C G; Ruiter, D J; Adema, G J; van Muijen, G N

1998-10-01

Molecular analysis of small tissue samples has become increasingly important in biomedical studies. Using a laser dissection microscope and modified nucleic acid isolation protocols, we demonstrate that multiple mRNA as well as DNA sequences can be identified from a single-cell sample. In addition, we show that the specificity of procurement of tissue samples is not compromised by smear contamination resulting from scraping of the microtome knife during sectioning of lesions. The procedures described herein thus allow for efficient RT-PCR or PCR analysis of multiple nucleic acid sequences from small tissue samples obtained by laser-assisted microdissection.

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.

Transcriptional and Posttranscriptional Control of Phaseolin and Phytohemagglutinin Gene Expression in Developing Cotyledons of Phaseolus vulgaris.

PubMed

Chappell, J; Chrispeels, M J

1986-05-01

The expression of phaseolin and phytohemagglutinin (PHA) in the developing cotyledons of a normal (Greensleeves) and a PHA-deficient (Pinto 111) cultivar of Phaseolus vulgaris was investigated. Phaseolin mRNA translational activity and abundance were present at similar levels in both cultivars. In contrast, PHA mRNA translational activity and abundance in Pinto 111 were less than 1% of the levels measured in Greensleeves. Using nuclear runoff assays, the transcription rate of phaseolin gene sequences was similar in both cultivars. The transcription rate of PHA gene sequences in Pinto 111 was only 20% of that measured in Greensleeves. Comparison of the transcription rates with the relative mRNA amounts measured in RNA blot hybridizations indicated that the normally expressed storage protein gene mRNAs were very stable with half-lives greater than several days. Because a low level of PHA gene transcription in Pinto 111 was measurable but no PHA mRNA accumulated, these results suggest that the PHA deficiency in Pinto 111 is due to a reduced transcription rate and possibly an instability of the mRNA.

miR-328 Functions as an RNA Decoy to Modulate hnRNP E2 Regulation of mRNA Translation in Leukemic Blasts

PubMed Central

Eiring, Anna M.; Harb, Jason G.; Neviani, Paolo; Garton, Christopher; Oaks, Joshua J.; Spizzo, Riccardo; Liu, Shujun; Schwind, Sebastian; Santhanam, Ramasamy; Hickey, Christopher J.; Becker, Heiko; Chandler, Jason C.; Andino, Raul; Cortes, Jorge; Hokland, Peter; Huettner, Claudia S.; Bhatia, Ravi; Roy, Denis C.; Liebhaber, Stephen A.; Caligiuri, Michael A.; Marcucci, Guido; Garzon, Ramiro; Croce, Carlo M.; Calin, George A.; Perrotti, Danilo

2010-01-01

SUMMARY MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA’s seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins. PMID:20211135

An Updated Collection of Sequence Barcoded Temperature-Sensitive Alleles of Yeast Essential Genes

PubMed Central

Kofoed, Megan; Milbury, Karissa L.; Chiang, Jennifer H.; Sinha, Sunita; Ben-Aroya, Shay; Giaever, Guri; Nislow, Corey; Hieter, Philip; Stirling, Peter C.

2015-01-01

Systematic analyses of essential gene function using mutant collections in Saccharomyces cerevisiae have been conducted using collections of heterozygous diploids, promoter shut-off alleles, through alleles with destabilized mRNA, destabilized protein, or bearing mutations that lead to a temperature-sensitive (ts) phenotype. We previously described a method for construction of barcoded ts alleles in a systematic fashion. Here we report the completion of this collection of alleles covering 600 essential yeast genes. This resource covers a larger gene repertoire than previous collections and provides a complementary set of strains suitable for single gene and genomic analyses. We use deep sequencing to characterize the amino acid changes leading to the ts phenotype in half of the alleles. We also use high-throughput approaches to describe the relative ts behavior of the alleles. Finally, we demonstrate the experimental usefulness of the collection in a high-content, functional genomic screen for ts alleles that increase spontaneous P-body formation. By increasing the number of alleles and improving the annotation, this ts collection will serve as a community resource for probing new aspects of biology for essential yeast genes. PMID:26175450

An Updated Collection of Sequence Barcoded Temperature-Sensitive Alleles of Yeast Essential Genes.

PubMed

Kofoed, Megan; Milbury, Karissa L; Chiang, Jennifer H; Sinha, Sunita; Ben-Aroya, Shay; Giaever, Guri; Nislow, Corey; Hieter, Philip; Stirling, Peter C

2015-07-14

Systematic analyses of essential gene function using mutant collections in Saccharomyces cerevisiae have been conducted using collections of heterozygous diploids, promoter shut-off alleles, through alleles with destabilized mRNA, destabilized protein, or bearing mutations that lead to a temperature-sensitive (ts) phenotype. We previously described a method for construction of barcoded ts alleles in a systematic fashion. Here we report the completion of this collection of alleles covering 600 essential yeast genes. This resource covers a larger gene repertoire than previous collections and provides a complementary set of strains suitable for single gene and genomic analyses. We use deep sequencing to characterize the amino acid changes leading to the ts phenotype in half of the alleles. We also use high-throughput approaches to describe the relative ts behavior of the alleles. Finally, we demonstrate the experimental usefulness of the collection in a high-content, functional genomic screen for ts alleles that increase spontaneous P-body formation. By increasing the number of alleles and improving the annotation, this ts collection will serve as a community resource for probing new aspects of biology for essential yeast genes. Copyright © 2015 Kofoed et al.

Human ribosomal protein L37 has motifs predicting serine/threonine phosphorylation and a zinc-finger domain.

PubMed

Barnard, G F; Staniunas, R J; Puder, M; Steele, G D; Chen, L B

1994-08-02

Ribosomal protein L37 mRNA is overexpressed in colon cancer. The nucleotide sequences of human L37 from several tumor and normal, colon and liver cDNA sources were determined to be identical. L37 mRNA was approximately 375 nucleotides long encoding 97 amino acids with M(r) = 11,070, pI = 12.6, multiple potential serine/threonine phosphorylation sites and a zinc-finger domain. The human sequence is compared to other species.

A HuD-ZBP1 ribonucleoprotein complex localizes GAP-43 mRNA into axons through its 3′ untranslated region AU-rich regulatory element

PubMed Central

Yoo, Soonmoon; Kim, Hak Hee; Kim, Paul; Donnelly, Christopher J.; Kalinski, Ashley L.; Vuppalanchi, Deepika; Park, Michael; Lee, Seung Joon; Merianda, Tanuja T.; Perrone-Bizzozero, Nora I.; Twiss, Jeffery L.

2013-01-01

Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β-actin and GAP-43 mRNAs. β-actin 3′UTR has a defined element for interaction with ZBP1, but GAP-43 mRNA shows no homology to this RNA sequence. Here, we show that an AU-rich element (ARE) in GAP-43’s 3′UTR is necessary and sufficient for its axonal localization. Axonal GAP-43 mRNA levels increase after in vivo injury, and GAP-43 mRNA shows an increased half-life in regenerating axons. GAP-43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 coimmunoprecipitate in an RNA-dependent fashion. Reporter mRNA with the GAP-43 ARE competes with endogenous β-actin mRNA for axonal localization and decreases axon length and branching similar to the β-actin 3′UTR competing with endogenous GAP-43 mRNA. Conversely, overexpressing GAP-43 coding sequence with it’s 3′UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP-43’s 3′UTR. PMID:23586486

Presence and Expression of Microbial Genes Regulating Soil Nitrogen Dynamics Along the Tanana River Successional Sequence

NASA Astrophysics Data System (ADS)

Boone, R. D.; Rogers, S. L.

2004-12-01

We report on work to assess the functional gene sequences for soil microbiota that control nitrogen cycle pathways along the successional sequence (willow, alder, poplar, white spruce, black spruce) on the Tanana River floodplain, Interior Alaska. Microbial DNA and mRNA were extracted from soils (0-10 cm depth) for amoA (ammonium monooxygenase), nifH (nitrogenase reductase), napA (nitrate reductase), and nirS and nirK (nitrite reductase) genes. Gene presence was determined by amplification of a conserved sequence of each gene employing sequence specific oligonucleotide primers and Polymerase Chain Reaction (PCR). Expression of the genes was measured via nested reverse transcriptase PCR amplification of the extracted mRNA. Amplified PCR products were visualized on agarose electrophoresis gels. All five successional stages show evidence for the presence and expression of microbial genes that regulate N fixation (free-living), nitrification, and nitrate reduction. We detected (1) nifH, napA, and nirK presence and amoA expression (mRNA production) for all five successional stages and (2) nirS and amoA presence and nifH, nirK, and napA expression for early successional stages (willow, alder, poplar). The results highlight that the existing body of previous process-level work has not sufficiently considered the microbial potential for a nitrate economy and free-living N fixation along the complete floodplain successional sequence.

Low-Latency Telerobotic Sample Return and Biomolecular Sequencing for Deep Space Gateway

NASA Astrophysics Data System (ADS)

Lupisella, M.; Bleacher, J.; Lewis, R.; Dworkin, J.; Wright, M.; Burton, A.; Rubins, K.; Wallace, S.; Stahl, S.; John, K.; Archer, D.; Niles, P.; Regberg, A.; Smith, D.; Race, M.; Chiu, C.; Russell, J.; Rampe, E.; Bywaters, K.

2018-02-01

Low-latency telerobotics, crew-assisted sample return, and biomolecular sequencing can be used to acquire and analyze lunar farside and/or Apollo landing site samples. Sequencing can also be used to monitor and study Deep Space Gateway environment and crew health.

Differential effects of simple repeating DNA sequences on gene expression from the SV40 early promoter.

PubMed

Amirhaeri, S; Wohlrab, F; Wells, R D

1995-02-17

The influence of simple repeat sequences, cloned into different positions relative to the SV40 early promoter/enhancer, on the transient expression of the chloramphenicol acetyltransferase (CAT) gene was investigated. Insertion of (G)29.(C)29 in either orientation into the 5'-untranslated region of the CAT gene reduced expression in CV-1 cells 50-100 fold when compared with controls with random sequence inserts. Analysis of CAT-specific mRNA levels demonstrated that the effect was due to a reduction of CAT mRNA production rather than to posttranscriptional events. In contrast, insertion of the same insert in either orientation upstream of the promoter-enhancer or downstream of the gene stimulated gene expression 2-3-fold. These effects could be reversed by cotransfection of a competitor plasmid carrying (G)25.(C)25 sequences. The results suggest that a G.C-binding transcription factor modulates gene expression in this system and that promoter strength can be regulated by providing protein-binding sites in trans. Although constructs containing longer tracts of alternating (C-G), (T-G), or (A-T) sequences inhibited CAT expression when inserted in the 5'-untranslated region of the CAT gene, the amount of CAT mRNA was unaffected. Hence, these inhibitions must be due to posttranscriptional events, presumably at the level of translation. These effects of microsatellite sequences on gene expression are discussed with respect to recent data on related simple repeat sequences which cause several human genetic diseases.

Deep sequencing of small RNA libraries from human prostate epithelial and stromal cells reveal distinct pattern of microRNAs primarily predicted to target growth factors.

PubMed

Singh, Savita; Zheng, Yun; Jagadeeswaran, Guru; Ebron, Jey Sabith; Sikand, Kavleen; Gupta, Sanjay; Sunker, Ramanjulu; Shukla, Girish C

2016-02-28

Complex epithelial and stromal cell interactions are required during the development and progression of prostate cancer. Regulatory small non-coding microRNAs (miRNAs) participate in the spatiotemporal regulation of messenger RNA (mRNA) and regulation of translation affecting a large number of genes involved in prostate carcinogenesis. In this study, through deep-sequencing of size fractionated small RNA libraries we profiled the miRNAs of prostate epithelial (PrEC) and stromal (PrSC) cells. Over 50 million reads were obtained for PrEC in which 860,468 were unique sequences. Similarly, nearly 76 million reads for PrSC were obtained in which over 1 million were unique reads. Expression of many miRNAs of broadly conserved and poorly conserved miRNA families were identified. Sixteen highly expressed miRNAs with significant change in expression in PrSC than PrEC were further analyzed in silico. ConsensusPathDB showed the target genes of these miRNAs were significantly involved in adherence junction, cell adhesion, EGRF, TGF-β and androgen signaling. Let-7 family of tumor-suppressor miRNAs expression was highly pervasive in both, PrEC and PrSC cells. In addition, we have also identified several miRNAs that are unique to PrEC or PrSC cells and their predicted putative targets are a group of transcription factors. This study provides perspective on the miRNA expression in PrEC and PrSC, and reveals a global trend in miRNA interactome. We conclude that the most abundant miRNAs are potential regulators of development and differentiation of the prostate gland by targeting a set of growth factors. Additionally, high level expression of the most members of let-7 family miRNAs suggests their role in the fine tuning of the growth and proliferation of prostate epithelial and stromal cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Identification of 88 regulatory small RNAs in the TIGR4 strain of the human pathogen Streptococcus pneumoniae

PubMed Central

Acebo, Paloma; Martin-Galiano, Antonio J.; Navarro, Sara; Zaballos, Ángel; Amblar, Mónica

2012-01-01

Streptococcus pneumoniae is the main etiological agent of community-acquired pneumonia and a major cause of mortality and morbidity among children and the elderly. Genome sequencing of several pneumococcal strains revealed valuable information about the potential proteins and genetic diversity of this prevalent human pathogen. However, little is known about its transcriptional regulation and its small regulatory noncoding RNAs. In this study, we performed deep sequencing of the S. pneumoniae TIGR4 strain RNome to identify small regulatory RNA candidates expressed in this pathogen. We discovered 1047 potential small RNAs including intragenic, 5′- and/or 3′-overlapping RNAs and 88 small RNAs encoded in intergenic regions. With this approach, we recovered many of the previously identified intergenic small RNAs and identified 68 novel candidates, most of which are conserved in both sequence and genomic context in other S. pneumoniae strains. We confirmed the independent expression of 17 intergenic small RNAs and predicted putative mRNA targets for six of them using bioinformatics tools. Preliminary results suggest that one of these six is a key player in the regulation of competence development. This study is the biggest catalog of small noncoding RNAs reported to date in S. pneumoniae and provides a highly complete view of the small RNA network in this pathogen. PMID:22274957

Massive Analysis of Rice Small RNAs: Mechanistic Implications of Regulated MicroRNAs and Variants for Differential Target RNA Cleavage[W][OA

PubMed Central

Jeong, Dong-Hoon; Park, Sunhee; Zhai, Jixian; Gurazada, Sai Guna Ranjan; De Paoli, Emanuele; Meyers, Blake C.; Green, Pamela J.

2011-01-01

Small RNAs have a variety of important roles in plant development, stress responses, and other processes. They exert their influence by guiding mRNA cleavage, translational repression, and chromatin modification. To identify previously unknown rice (Oryza sativa) microRNAs (miRNAs) and those regulated by environmental stress, 62 small RNA libraries were constructed from rice plants and used for deep sequencing with Illumina technology. The libraries represent several tissues from control plants and plants subjected to different environmental stress treatments. More than 94 million genome-matched reads were obtained, resulting in more than 16 million distinct small RNA sequences. This allowed an evaluation of ~400 annotated miRNAs with current criteria and the finding that among these, ~150 had small interfering RNA–like characteristics. Seventy-six new miRNAs were found, and miRNAs regulated in response to water stress, nutrient stress, or temperature stress were identified. Among the new examples of miRNA regulation were members of the same miRNA family that were differentially regulated in different organs and had distinct sequences Some of these distinct family members result in differential target cleavage and provide new insight about how an agriculturally important rice phenotype could be regulated in the panicle. This high-resolution analysis of rice miRNAs should be relevant to plant miRNAs in general, particularly in the Poaceae. PMID:22158467

Fungal communities from the calcareous deep-sea sediments in the Southwest India Ridge revealed by Illumina sequencing technology.

PubMed

Zhang, Likui; Kang, Manyu; Huang, Yangchao; Yang, Lixiang

2016-05-01

The diversity and ecological significance of bacteria and archaea in deep-sea environments have been thoroughly investigated, but eukaryotic microorganisms in these areas, such as fungi, are poorly understood. To elucidate fungal diversity in calcareous deep-sea sediments in the Southwest India Ridge (SWIR), the internal transcribed spacer (ITS) regions of rRNA genes from two sediment metagenomic DNA samples were amplified and sequenced using the Illumina sequencing platform. The results revealed that 58-63 % and 36-42 % of the ITS sequences (97 % similarity) belonged to Basidiomycota and Ascomycota, respectively. These findings suggest that Basidiomycota and Ascomycota are the predominant fungal phyla in the two samples. We also found that Agaricomycetes, Leotiomycetes, and Pezizomycetes were the major fungal classes in the two samples. At the species level, Thelephoraceae sp. and Phialocephala fortinii were major fungal species in the two samples. Despite the low relative abundance, unidentified fungal sequences were also observed in the two samples. Furthermore, we found that there were slight differences in fungal diversity between the two sediment samples, although both were collected from the SWIR. Thus, our results demonstrate that calcareous deep-sea sediments in the SWIR harbor diverse fungi, which augment the fungal groups in deep-sea sediments. This is the first report of fungal communities in calcareous deep-sea sediments in the SWIR revealed by Illumina sequencing.

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.

Position-specific automated processing of V3 env ultra-deep pyrosequencing data for predicting HIV-1 tropism

PubMed Central

Jeanne, Nicolas; Saliou, Adrien; Carcenac, Romain; Lefebvre, Caroline; Dubois, Martine; Cazabat, Michelle; Nicot, Florence; Loiseau, Claire; Raymond, Stéphanie; Izopet, Jacques; Delobel, Pierre

2015-01-01

HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds. PMID:26585833

Position-specific automated processing of V3 env ultra-deep pyrosequencing data for predicting HIV-1 tropism.

PubMed

Jeanne, Nicolas; Saliou, Adrien; Carcenac, Romain; Lefebvre, Caroline; Dubois, Martine; Cazabat, Michelle; Nicot, Florence; Loiseau, Claire; Raymond, Stéphanie; Izopet, Jacques; Delobel, Pierre

2015-11-20

HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds.

Accurate identification of RNA editing sites from primitive sequence with deep neural networks.

PubMed

Ouyang, Zhangyi; Liu, Feng; Zhao, Chenghui; Ren, Chao; An, Gaole; Mei, Chuan; Bo, Xiaochen; Shu, Wenjie

2018-04-16

RNA editing is a post-transcriptional RNA sequence alteration. Current methods have identified editing sites and facilitated research but require sufficient genomic annotations and prior-knowledge-based filtering steps, resulting in a cumbersome, time-consuming identification process. Moreover, these methods have limited generalizability and applicability in species with insufficient genomic annotations or in conditions of limited prior knowledge. We developed DeepRed, a deep learning-based method that identifies RNA editing from primitive RNA sequences without prior-knowledge-based filtering steps or genomic annotations. DeepRed achieved 98.1% and 97.9% area under the curve (AUC) in training and test sets, respectively. We further validated DeepRed using experimentally verified U87 cell RNA-seq data, achieving 97.9% positive predictive value (PPV). We demonstrated that DeepRed offers better prediction accuracy and computational efficiency than current methods with large-scale, mass RNA-seq data. We used DeepRed to assess the impact of multiple factors on editing identification with RNA-seq data from the Association of Biomolecular Resource Facilities and Sequencing Quality Control projects. We explored developmental RNA editing pattern changes during human early embryogenesis and evolutionary patterns in Drosophila species and the primate lineage using DeepRed. Our work illustrates DeepRed's state-of-the-art performance; it may decipher the hidden principles behind RNA editing, making editing detection convenient and effective.

Sequence and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency

PubMed Central

Sroubek, Jakub; Krishnan, Yamini; McDonald, Thomas V.

2013-01-01

Human ether-á-gogo-related gene (HERG) encodes a potassium channel that is highly susceptible to deleterious mutations resulting in susceptibility to fatal cardiac arrhythmias. Most mutations adversely affect HERG channel assembly and trafficking. Why the channel is so vulnerable to missense mutations is not well understood. Since nothing is known of how mRNA structural elements factor in channel processing, we synthesized a codon-modified HERG cDNA (HERG-CM) where the codons were synonymously changed to reduce GC content, secondary structure, and rare codon usage. HERG-CM produced typical IKr-like currents; however, channel synthesis and processing were markedly different. Translation efficiency was reduced for HERG-CM, as determined by heterologous expression, in vitro translation, and polysomal profiling. Trafficking efficiency to the cell surface was greatly enhanced, as assayed by immunofluorescence, subcellular fractionation, and surface labeling. Chimeras of HERG-NT/CM indicated that trafficking efficiency was largely dependent on 5′ sequences, while translation efficiency involved multiple areas. These results suggest that HERG translation and trafficking rates are independently governed by noncoding information in various regions of the mRNA molecule. Noncoding information embedded within the mRNA may play a role in the pathogenesis of hereditary arrhythmia syndromes and could provide an avenue for targeted therapeutics.—Sroubek, J., Krishnan, Y., McDonald, T V. Sequence- and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency. PMID:23608144

Regulation of cytoplasmic mRNA decay

PubMed Central

Schoenberg, Daniel R.; Maquat, Lynne E.

2012-01-01

Discoveries made over the past 20 years highlight the importance of mRNA decay as a means to modulate gene expression and thereby protein production. Up until recently, studies focused largely on identifying cis-acting sequences that serve as mRNA stability or instability elements, the proteins that bind these elements, how the process of translation influences mRNA decay, and the ribonucleases that catalyze decay. Now, current studies have begun to elucidate how the decay process is regulated. This review examines our current understanding of how mammalian-cell mRNA decay is controlled by different signaling pathways and lays out a framework for future research. PMID:22392217

Engineered external guide sequences are highly effective in inhibiting gene expression and replication of hepatitis B virus in cultured cells.

PubMed

Zhang, Zhigang; Vu, Gia-Phong; Gong, Hao; Xia, Chuan; Chen, Yuan-Chuan; Liu, Fenyong; Wu, Jianguo; Lu, Sangwei

2013-01-01

External guide sequences (EGSs) are RNA molecules that consist of a sequence complementary to a target mRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, for specific degradation of the target mRNA. We have previously used an in vitro selection procedure to generate EGS variants that efficiently induce human RNase P to cleave a target mRNA in vitro. In this study, we constructed EGSs from a variant to target the overlapping region of the S mRNA, pre-S/L mRNA, and pregenomic RNA (pgRNA) of hepatitis B virus (HBV), which are essential for viral replication and infection. The EGS variant was about 50-fold more efficient in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Following Salmonella-mediated gene delivery, the EGSs were expressed in cultured HBV-carrying cells. A reduction of about 97% and 75% in the level of HBV RNAs and proteins and an inhibition of about 6,000- and 130-fold in the levels of capsid-associated HBV DNA were observed in cells treated with Salmonella vectors carrying the expression cassette for the variant and the tRNA-derived EGS, respectively. Our study provides direct evidence that the EGS variant is more effective in blocking HBV gene expression and DNA replication than the tRNA-derived EGS. Furthermore, these results demonstrate the feasibility of developing Salmonella-mediated gene delivery of highly active EGS RNA variants as a novel approach for gene-targeting applications such as anti-HBV therapy.

Designing oligo libraries taking alternative splicing into account

NASA Astrophysics Data System (ADS)

Shoshan, Avi; Grebinskiy, Vladimir; Magen, Avner; Scolnicov, Ariel; Fink, Eyal; Lehavi, David; Wasserman, Alon

2001-06-01

We have designed sequences for DNA microarrays and oligo libraries, taking alternative splicing into account. Alternative splicing is a common phenomenon, occurring in more than 25% of the human genes. In many cases, different splice variants have different functions, are expressed in different tissues or may indicate different stages of disease. When designing sequences for DNA microarrays or oligo libraries, it is very important to take into account the sequence information of all the mRNA transcripts. Therefore, when a gene has more than one transcript (as a result of alternative splicing, alternative promoter sites or alternative poly-adenylation sites), it is very important to take all of them into account in the design. We have used the LEADS transcriptome prediction system to cluster and assemble the human sequences in GenBank and design optimal oligonucleotides for all the human genes with a known mRNA sequence based on the LEADS predictions.

Comparative Genomic and Transcriptomic Characterization of the Toxigenic Marine Dinoflagellate Alexandrium ostenfeldii

PubMed Central

Jaeckisch, Nina; Yang, Ines; Wohlrab, Sylke; Glöckner, Gernot; Kroymann, Juergen; Vogel, Heiko; Cembella, Allan; John, Uwe

2011-01-01

Many dinoflagellate species are notorious for the toxins they produce and ecological and human health consequences associated with harmful algal blooms (HABs). Dinoflagellates are particularly refractory to genomic analysis due to the enormous genome size, lack of knowledge about their DNA composition and structure, and peculiarities of gene regulation, such as spliced leader (SL) trans-splicing and mRNA transposition mechanisms. Alexandrium ostenfeldii is known to produce macrocyclic imine toxins, described as spirolides. We characterized the genome of A. ostenfeldii using a combination of transcriptomic data and random genomic clones for comparison with other dinoflagellates, particularly Alexandrium species. Examination of SL sequences revealed similar features as in other dinoflagellates, including Alexandrium species. SL sequences in decay indicate frequent retro-transposition of mRNA species. This probably contributes to overall genome complexity by generating additional gene copies. Sequencing of several thousand fosmid and bacterial artificial chromosome (BAC) ends yielded a wealth of simple repeats and tandemly repeated longer sequence stretches which we estimated to comprise more than half of the whole genome. Surprisingly, the repeats comprise a very limited set of 79–97 bp sequences; in part the genome is thus a relatively uniform sequence space interrupted by coding sequences. Our genomic sequence survey (GSS) represents the largest genomic data set of a dinoflagellate to date. Alexandrium ostenfeldii is a typical dinoflagellate with respect to its transcriptome and mRNA transposition but demonstrates Alexandrium-like stop codon usage. The large portion of repetitive sequences and the organization within the genome is in agreement with several other studies on dinoflagellates using different approaches. It remains to be determined whether this unusual composition is directly correlated to the exceptionally genome organization of dinoflagellates with a low amount of histones and histone-like proteins. PMID:22164224

Genetic and Molecular Characterization of the Caenorhabditis Elegans Spermatogenesis-Defective Gene Spe-17

PubMed Central

L'Hernault, S. W.; Benian, G. M.; Emmons, R. B.

1993-01-01

Two self-sterile mutations that define the spermatogenesis-defective gene spe-17 have been analyzed. These mutations affect unc-22 and fail to complement each other for both Unc-22 and spermatogenesis defects. Both of these mutations are deficiencies (hcDf1 and hDf13) that affect more than one transcription unit. Genomic DNA adjacent to and including the region deleted by the smaller deficiency (hcDf1) has been sequenced and four mRNAs (including unc-22) have been localized to this sequenced region. The three non unc-22 mRNAs are shown to be sex-specific: a 1.2-kb mRNA that can be detected in sperm-free hermaphrodites and 1.2- and 0.56-kb mRNAs found in males. hDf13 deletes at least 55 kb of chromosome IV, including all of unc-22, both male-specific mRNAs and at least part of the female-specific mRNA. hcDf1, which is approximately 15.6 kb, deletes only the 5' end of unc-22 and the gene that encodes the 0.56-kb male-specific mRNA. The common defect that apparently accounts for the defective sperm in hcDf1 and hDf13 homozygotes is deletion of the spe-17 gene, which encodes the 0.56-kb mRNA. Strains carrying two copies of either deletion are self-fertile when they are transgenic for any of four extrachromosomal array that include spe-17. We have sequenced two spe-17 cDNAs, and the deduced 142 amino acid protein sequence is highly charged and rich in serine and threonine, but shows no significant homology to any previously determined protein sequence. PMID:8349108

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.

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

Genome Analysis Reveals Interplay between 5′UTR Introns and Nuclear mRNA Export for Secretory and Mitochondrial Genes

PubMed Central

Cenik, Can; Chua, Hon Nian; Zhang, Hui; Tarnawsky, Stefan P.; Akef, Abdalla; Derti, Adnan; Tasan, Murat; Moore, Melissa J.; Palazzo, Alexander F.; Roth, Frederick P.

2011-01-01

In higher eukaryotes, messenger RNAs (mRNAs) are exported from the nucleus to the cytoplasm via factors deposited near the 5′ end of the transcript during splicing. The signal sequence coding region (SSCR) can support an alternative mRNA export (ALREX) pathway that does not require splicing. However, most SSCR–containing genes also have introns, so the interplay between these export mechanisms remains unclear. Here we support a model in which the furthest upstream element in a given transcript, be it an intron or an ALREX–promoting SSCR, dictates the mRNA export pathway used. We also experimentally demonstrate that nuclear-encoded mitochondrial genes can use the ALREX pathway. Thus, ALREX can also be supported by nucleotide signals within mitochondrial-targeting sequence coding regions (MSCRs). Finally, we identified and experimentally verified novel motifs associated with the ALREX pathway that are shared by both SSCRs and MSCRs. Our results show strong correlation between 5′ untranslated region (5′UTR) intron presence/absence and sequence features at the beginning of the coding region. They also suggest that genes encoding secretory and mitochondrial proteins share a common regulatory mechanism at the level of mRNA export. PMID:21533221

A deep learning method for lincRNA detection using auto-encoder algorithm.

PubMed

Yu, Ning; Yu, Zeng; Pan, Yi

2017-12-06

RNA sequencing technique (RNA-seq) enables scientists to develop novel data-driven methods for discovering more unidentified lincRNAs. Meantime, knowledge-based technologies are experiencing a potential revolution ignited by the new deep learning methods. By scanning the newly found data set from RNA-seq, scientists have found that: (1) the expression of lincRNAs appears to be regulated, that is, the relevance exists along the DNA sequences; (2) lincRNAs contain some conversed patterns/motifs tethered together by non-conserved regions. The two evidences give the reasoning for adopting knowledge-based deep learning methods in lincRNA detection. Similar to coding region transcription, non-coding regions are split at transcriptional sites. However, regulatory RNAs rather than message RNAs are generated. That is, the transcribed RNAs participate the biological process as regulatory units instead of generating proteins. Identifying these transcriptional regions from non-coding regions is the first step towards lincRNA recognition. The auto-encoder method achieves 100% and 92.4% prediction accuracy on transcription sites over the putative data sets. The experimental results also show the excellent performance of predictive deep neural network on the lincRNA data sets compared with support vector machine and traditional neural network. In addition, it is validated through the newly discovered lincRNA data set and one unreported transcription site is found by feeding the whole annotated sequences through the deep learning machine, which indicates that deep learning method has the extensive ability for lincRNA prediction. The transcriptional sequences of lincRNAs are collected from the annotated human DNA genome data. Subsequently, a two-layer deep neural network is developed for the lincRNA detection, which adopts the auto-encoder algorithm and utilizes different encoding schemes to obtain the best performance over intergenic DNA sequence data. Driven by those newly annotated lincRNA data, deep learning methods based on auto-encoder algorithm can exert their capability in knowledge learning in order to capture the useful features and the information correlation along DNA genome sequences for lincRNA detection. As our knowledge, this is the first application to adopt the deep learning techniques for identifying lincRNA transcription sequences.

Epitranscriptional orchestration of genetic reprogramming is an emergent property of stress-regulated cardiac microRNAs

PubMed Central

Hu, Yuanxin; Matkovich, Scot J.; Hecker, Peter A.; Zhang, Yan; Edwards, John R.; Dorn, Gerald W.

2012-01-01

Cardiac stress responses are driven by an evolutionarily conserved gene expression program comprising dozens of microRNAs and hundreds of mRNAs. Functionalities of different individual microRNAs are being studied, but the overall purpose of interactions between stress-regulated microRNAs and mRNAs and potentially distinct roles for microRNA-mediated epigenetic and conventional transcriptional genetic reprogramming of the stressed heart are unknown. Here we used deep sequencing to interrogate microRNA and mRNA regulation in pressure-overloaded mouse hearts, and performed a genome-wide examination of microRNA–mRNA interactions during early cardiac hypertrophy. Based on abundance and regulatory patterns, cardiac microRNAs were categorized as constitutively expressed housekeeping, regulated homeostatic, or dynamic early stress-responsive microRNAs. Regulation of 62 stress-responsive cardiac microRNAs directly affected levels of only 66 mRNAs, but the global impact of microRNA-mediated epigenetic regulation was amplified by preferential targeting of mRNAs encoding transcription factors, kinases, and phosphatases exerting amplified secondary effects. Thus, an emergent cooperative property of stress-regulated microRNAs is orchestration of transcriptional and posttranslational events that help determine the stress-reactive cardiac phenotype. This global functionality explains how large end-organ effects can be induced through modest individual changes in target mRNA and protein content by microRNAs that sense and respond dynamically to a changing physiological milieu. PMID:23150554

The primary structure of the Saccharomyces cerevisiae gene for 3-phosphoglycerate kinase.

PubMed Central

Hitzeman, R A; Hagie, F E; Hayflick, J S; Chen, C Y; Seeburg, P H; Derynck, R

1982-01-01

The DNA sequence of the gene for the yeast glycolytic enzyme, 3-phosphoglycerate kinase (PGK), has been obtained by sequencing part of a 3.1 kbp HindIII fragment obtained from the yeast genome. The structural gene sequence corresponds to a reading frame of 1251 bp coding for 416 amino acids with no intervening DNA sequences. The amino acid sequence is approximately 65 percent homologous with human and horse PGK protein sequences and is in general agreement with the published protein sequence for yeast PGK. As for other highly expressed structural genes in yeast, the coding sequence is highly codon biased with 95 percent of the amino acids coded for by a select 25 codons (out of 61 possible). Besides structural DNA sequence, 291 bp of 5'-flanking sequence and 286 bp of 3'-flanking sequence were determined. Transcription starts 36 nucleotides upstream from the translational start and stops 86-93 nucleotides downstream from the translational stop. These results suggest a non-polyadenylated mRNA length of 1373 to 1380 nucleotides, which is consistent with the observed length of 1500 nucleotides for polyadenylated PGK mRNA. A sequence TATATATAAA is found at 145 nucleotides upstream from the translational start. This sequence resembles the TATAAA box that is possibly associated with RNA polymerase II binding. Images PMID:6296791

Identification of Small RNAs in Desulfovibrio vulgaris Hildenborough

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

Burns, Andrew; Joachimiak, Marcin; Deutschbauer, Adam

2010-05-17

Desulfovibrio vulgaris is an anaerobic sulfate-reducing bacterium capable of facilitating the removal of toxic metals such as uranium from contaminated sites via reduction. As such, it is essential to understand the intricate regulatory cascades involved in how D. vulgaris and its relatives respond to stressors in such sites. One approach is the identification and analysis of small non-coding RNAs (sRNAs); molecules ranging in size from 20-200 nucleotides that predominantly affect gene regulation by binding to complementary mRNA in an anti-sense fashion and therefore provide an immediate regulatory response. To identify sRNAs in D. vulgaris, a bacterium that does not possessmore » an annotated hfq gene, RNA was pooled from stationary and exponential phases, nitrate exposure, and biofilm conditions. The subsequent RNA was size fractionated, modified, and converted to cDNA for high throughput transcriptomic deep sequencing. A computational approach to identify sRNAs via the alignment of seven separate Desulfovibrio genomes was also performed. From the deep sequencing analysis, 2,296 reads between 20 and 250 nt were identified with expression above genome background. Analysis of those reads limited the number of candidates to ~;;87 intergenic, while ~;;140 appeared to be antisense to annotated open reading frames (ORFs). Further BLAST analysis of the intergenic candidates and other Desulfovibrio genomes indicated that eight candidates were likely portions of ORFs not previously annotated in the D. vulgaris genome. Comparison of the intergenic and antisense data sets to the bioinformatical predicted candidates, resulted in ~;;54 common candidates. Current approaches using Northern analysis and qRT-PCR are being used toverify expression of the candidates and to further develop the role these sRNAs play in D. vulgaris regulation.« less

Neuroglobin of seals and whales: evidence for a divergent role in the diving brain.

PubMed

Schneuer, M; Flachsbarth, S; Czech-Damal, N U; Folkow, L P; Siebert, U; Burmester, T

2012-10-25

Although many physiological adaptations of diving mammals have been reported, little is known about how their brains sustain the high demands for metabolic energy and thus O(2) when submerged. A recent study revealed in the deep-diving hooded seal (Cystophora cristata) a unique shift of the oxidative energy metabolism and neuroglobin, a respiratory protein that is involved in neuronal hypoxia tolerance, from neurons to astrocytes. Here we have investigated neuroglobin in another pinniped species, the harp seal (Pagophilus groenlandicus), and in two cetaceans, the harbor porpoise (Phocoena phocoena) and the minke whale (Balaenoptera acutorostrata). Neuroglobin sequences, expression levels and patterns were compared with those of terrestrial relatives, the ferret (Mustela putorius furo) and the cattle (Bos taurus), respectively. Neuroglobin sequences of whales and seals only differ in two or three amino acids from those of cattle and ferret, and are unlikely to confer functional differences, e.g. in O(2) affinity. Neuroglobin is expressed in the astrocytes also of P. groenlandicus, suggesting that the shift of neuroglobin and oxidative metabolism is a common adaptation in the brains of deep-diving phocid seals. In the cetacean brain neuroglobin resides in neurons, like in terrestrial mammals. However, neuroglobin mRNA expression levels were 4-15 times higher in the brains of harbor porpoises and minke whales than in terrestrial mammals or in seals. Thus neuroglobin appears to play a specific role in diving mammals, but seals and whales have evolved divergent strategies to cope with cerebral hypoxia. The specific function of neuroglobin that conveys hypoxia tolerance may either relate to oxygen supply or protection from reactive oxygen species. The different strategies in seals and whales resulted from a divergent evolution and an independent adaptation to diving. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Molecular identification of differentially regulated genes in the hydrothermal-vent species Bathymodiolus thermophilus and Paralvinella pandorae in response to temperature

PubMed Central

Boutet, Isabelle; Jollivet, Didier; Shillito, Bruce; Moraga, Dario; Tanguy, Arnaud

2009-01-01

Background Hydrothermal vents and cold seeps represent oases of life in the deep-sea environment, but are also characterized by challenging physical and chemical conditions. The effect of temperature fluctuations on vent organisms in their habitat has not been well explored, in particular at a molecular level, most gene expression studies being conducted on coastal marine species. In order to better understand the response of hydrothermal organisms to different temperature regimes, differentially expressed genes (obtained by a subtractive suppression hybridization approach) were identified in the mussel Bathymodiolus thermophilus and the annelid Paralvinella pandorae irlandei to characterize the physiological processes involved when animals are subjected to long term exposure (2 days) at two contrasting temperatures (10° versus 20°C), while maintained at in situ pressures. To avoid a potential effect of pressure, the experimental animals were initially thermally acclimated for 24 hours in a pressurized vessel. Results For each species, we produced two subtractive cDNA libraries (forward and reverse) from sets of deep-sea mussels and annelids exposed together to a thermal challenge under pressure. RNA extracted from the gills, adductor muscle, mantle and foot tissue were used for B. thermophilus. For the annelid model, whole animals (small individuals) were used. For each of the four libraries, we sequenced 200 clones, resulting in 78 and 83 unique sequences in mussels and annelids (about 20% of the sequencing effort), respectively, with only half of them corresponding to known genes. Real-time PCR was used to validate differentially expressed genes identified in the corresponding libraries. Strong expression variations have been observed for some specific genes such as the intracellular hemoglobin, the nidogen protein, and Rab7 in P. pandorae, and the SPARC protein, cyclophilin, foot protein and adhesive plaque protein in B. thermophilus. Conclusion Our results indicate that mussels and worms are not responding in the same way to temperature variations. While the results obtained for the mussel B. thermophilus seem to indicate a metabolic depression (strong decrease in the level of mRNA expression of numerous genes) when temperature increased, the annelid P. pandorae mainly displayed a strong regulation of the mRNA encoding subunits and linkers of respiratory pigments and some proteins involved in membrane structure. In both cases, these regulations seem to be partly due to a possible cellular oxidative stress induced by the simulated thermal environment (10°C to 20°C). This work will serve as a starting point for studying the transcriptomic response of hydrothermal mussels and annelids in future experiments in response to thermal stress at various conditions of duration and temperature challenge. PMID:19439073

X-linked Alport syndrome caused by splicing mutations in COL4A5.

PubMed

Nozu, Kandai; Vorechovsky, Igor; Kaito, Hiroshi; Fu, Xue Jun; Nakanishi, Koichi; Hashimura, Yuya; Hashimoto, Fusako; Kamei, Koichi; Ito, Shuichi; Kaku, Yoshitsugu; Imasawa, Toshiyuki; Ushijima, Katsumi; Shimizu, Junya; Makita, Yoshio; Konomoto, Takao; Yoshikawa, Norishige; Iijima, Kazumoto

2014-11-07

X-linked Alport syndrome is caused by mutations in the COL4A5 gene. Although many COL4A5 mutations have been detected, the mutation detection rate has been unsatisfactory. Some men with X-linked Alport syndrome show a relatively mild phenotype, but molecular basis investigations have rarely been conducted to clarify the underlying mechanism. In total, 152 patients with X-linked Alport syndrome who were suspected of having Alport syndrome through clinical and pathologic investigations and referred to the hospital for mutational analysis between January of 2006 and January of 2013 were genetically diagnosed. Among those patients, 22 patients had suspected splice site mutations. Transcripts are routinely examined when suspected splice site mutations for abnormal transcripts are detected; 11 of them showed expected exon skipping, but others showed aberrant splicing patterns. The mutation detection strategy had two steps: (1) genomic DNA analysis using PCR and direct sequencing and (2) mRNA analysis using RT-PCR to detect RNA processing abnormalities. Six splicing consensus site mutations resulting in aberrant splicing patterns, one exonic mutation leading to exon skipping, and four deep intronic mutations producing cryptic splice site activation were identified. Interestingly, one case produced a cryptic splice site with a single nucleotide substitution in the deep intron that led to intronic exonization containing a stop codon; however, the patient showed a clearly milder phenotype for X-linked Alport syndrome in men with a truncating mutation. mRNA extracted from the kidney showed both normal and abnormal transcripts, with the normal transcript resulting in the milder phenotype. This novel mechanism leads to mild clinical characteristics. This report highlights the importance of analyzing transcripts to enhance the mutation detection rate and provides insight into genotype-phenotype correlations. This approach can clarify the cause of atypically mild phenotypes in X-linked Alport syndrome. Copyright © 2014 by the American Society of Nephrology.

CsrA Participates in a PNPase Autoregulatory Mechanism by Selectively Repressing Translation of pnp Transcripts That Have Been Previously Processed by RNase III and PNPase

PubMed Central

Park, Hongmarn; Yakhnin, Helen; Connolly, Michael; Romeo, Tony

2015-01-01

ABSTRACT Csr is a conserved global regulatory system that represses or activates gene expression posttranscriptionally. CsrA of Escherichia coli is a homodimeric RNA binding protein that regulates transcription elongation, translation initiation, and mRNA stability by binding to the 5′ untranslated leader or initial coding sequence of target transcripts. pnp mRNA, encoding the 3′ to 5′ exoribonuclease polynucleotide phosphorylase (PNPase), was previously identified as a CsrA target by transcriptome sequencing (RNA-seq). Previous studies also showed that RNase III and PNPase participate in a pnp autoregulatory mechanism in which RNase III cleavage of the untranslated leader, followed by PNPase degradation of the resulting 5′ fragment, leads to pnp repression by an undefined translational repression mechanism. Here we demonstrate that CsrA binds to two sites in pnp leader RNA but only after the transcript is fully processed by RNase III and PNPase. In the absence of processing, both of the binding sites are sequestered in an RNA secondary structure, which prevents CsrA binding. The CsrA dimer bridges the upstream high-affinity site to the downstream site that overlaps the pnp Shine-Dalgarno sequence such that bound CsrA causes strong repression of pnp translation. CsrA-mediated translational repression also leads to a small increase in the pnp mRNA decay rate. Although CsrA has been shown to regulate translation and mRNA stability of numerous genes in a variety of organisms, this is the first example in which prior mRNA processing is required for CsrA-mediated regulation. IMPORTANCE CsrA protein represses translation of numerous mRNA targets, typically by binding to multiple sites in the untranslated leader region preceding the coding sequence. We found that CsrA represses translation of pnp by binding to two sites in the pnp leader transcript but only after it is processed by RNase III and PNPase. Processing by these two ribonucleases alters the mRNA secondary structure such that it becomes accessible to the ribosome for translation as well as to CsrA. As one of the CsrA binding sites overlaps the pnp ribosome binding site, bound CsrA prevents ribosome binding. This is the first example in which regulation by CsrA requires prior mRNA processing and should link pnp expression to conditions affecting CsrA activity. PMID:26438818

Molecular analysis of two phytohemagglutinin genes and their expression in Phaseolus vulgaris cv. Pinto, a lectin-deficient cultivar of the bean

PubMed Central

Voelker, Toni A.; Staswick, Paul; Chrispeels, Maarten J.

1986-01-01

Phytohemagglutinin (PHA), the seed lectin of the common bean, Phaseolus vulgaris, is encoded by two highly homologous, tandemly linked genes, dlec1 and dlec2, which are coordinately expressed at high levels in developing cotyledons. Their respective transcripts translate into closely related polypeptides, PHA-E and PHA-L, constituents of the tetrameric lectin which accumulates at high levels in developing seeds. In the bean cultivar Pinto UI111, PHA-E is not detectable, and PHA-L accumulates at very reduced levels. To investigate the cause of the Pinto phenotype, we cloned and sequenced the two PHA genes of Pinto, called Pdlec1 and Pdlec2, and determined the abundance of their respective mRNAs in developing cotyledons. Both genes are more than 90% homologous to the normal PHA genes found in other cultivars. Pdlec1 carries a 1-bp frameshift mutation close to the 5' end of its coding sequence. Only very truncated polypeptides could be made from its mRNA. The gene Pdlec2 encodes a polypeptide, which resembles PHA-L and its predicted amino acid sequence agrees with the available Pinto PHA amino acid sequence data. Analysis of the mRNA of developing cotyledons revealed that the Pdlec1 message is reduced 600-fold, and Pdlec2 mRNA is reduced 20-fold with respect to mRNA levels in normal cultivars. A comparison of the sequences which are upstream from the coding sequence shows that Pdlec2 has a 100-bp deletion compared to the other genes (dlec1, dlec2 and Pdlec1). This deletion which contains a large tandem repeat may be responsible for the low level of expression of Pdlec2. The very low expression of Pdlec1 is as yet unexplained. ImagesFig. 5. PMID:16453730

Population-genomic variation within RNA viruses of the Western honey bee, Apis mellifera, inferred from deep sequencing

USDA-ARS?s Scientific Manuscript database

Deep sequencing of viruses isolated from infected hosts is an efficient way to measure population-genetic variation and can reveal patterns of dispersal and natural selection. In this study, we mined existing Illumina sequence reads to investigate single-nucleotide polymorphisms (SNPs) within two RN...

Deciphering mRNA Sequence Determinants of Protein Production Rate

NASA Astrophysics Data System (ADS)

Szavits-Nossan, Juraj; Ciandrini, Luca; Romano, M. Carmen

2018-03-01

One of the greatest challenges in biophysical models of translation is to identify coding sequence features that affect the rate of translation and therefore the overall protein production in the cell. We propose an analytic method to solve a translation model based on the inhomogeneous totally asymmetric simple exclusion process, which allows us to unveil simple design principles of nucleotide sequences determining protein production rates. Our solution shows an excellent agreement when compared to numerical genome-wide simulations of S. cerevisiae transcript sequences and predicts that the first 10 codons, which is the ribosome footprint length on the mRNA, together with the value of the initiation rate, are the main determinants of protein production rate under physiological conditions. Finally, we interpret the obtained analytic results based on the evolutionary role of the codons' choice for regulating translation rates and ribosome densities.

Transcriptional regulation of ceruloplasmin gene expression during inflammation

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

Gitlin, J.D.

1988-05-05

Mixed sequence oligonucleotides were used to isolate a series of acute-phase human liver cDNA clones corresponding to the serum ..cap alpha../sub 2/-globulin ceruloplasmin. These clones were characterized, sequenced, and used to analyze changes in hepatic ceruloplasmin mRNA content during inflammation. In all species examined, hepatic ceruloplasmin mRNA content increased approximately 6-10-fold over control values within 24 h following the induction of inflammation. The mechanisms leading to this increase in hepatic ceruloplasmin mRNA content were studied following turpentine-induced inflammation in Syrian hamsters. Nuclear run-on assays demonstrated an increase in the relative rate of transcription of the ceruloplasmin gene within 3 hmore » following induction, reaching maximum values by 18 h. Hepatic ceruloplasmin mRNA content increased 2-fold within 12 h following induction, reached maximum values by 24 h, and returned to control within 72 h. In contrast, serum ceruloplasmin concentration did not increase until 36 h, reached maximal levels by 120 h, and remained elevated for the course of the study. These data indicate that inflammation leads to a rapid increase in hepatic ceruloplasmin mRNA content. This increase is largely the result of increased ceruloplasmin gene transcription, but comparison of the relative rate of transcription and mRNA accumulation suggests that changes in ceruloplasmin mRNA turnover are also involved. In addition, translational and/or post-translational mechanisms must account for the observed changes in serum ceruloplasmin concentration seen during inflammation.« less

Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen.

PubMed

Deng, Dawei; Li, Yang; Xue, Jianpeng; Wang, Jie; Ai, Guanhua; Li, Xin; Gu, Yueqing

2015-01-01

Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem-loop-stem oligonucleotide sequences attached by Au-S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.

Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen

PubMed Central

Deng, Dawei; Li, Yang; Xue, Jianpeng; Wang, Jie; Ai, Guanhua; Li, Xin; Gu, Yueqing

2015-01-01

Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem–loop–stem oligonucleotide sequences attached by Au–S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis. PMID:25987838

Ferritin gene transcription is regulated by iron in soybean cell cultures.

PubMed

Lescure, A M; Proudhon, D; Pesey, H; Ragland, M; Theil, E C; Briat, J F

1991-09-15

Iron-regulated ferritin synthesis in animals is dominated by translational control of stored mRNA; iron-induced transcription of ferritin genes, when it occurs, changes the subunit composition of ferritin mRNA and protein and is coupled to translational control. Ferritins in plants and animals have evolved from a common progenitor, based on the similarity of protein sequence; however, sequence divergence occurs in the C termini; structure prediction suggests that plant ferritin has the E-helix, which, in horse ferritin, forms a large channel at the tetrameric interface. In contemporary plants, a transit peptide is encoded by ferritin mRNA to target the protein to plastids. Iron-regulated synthesis of ferritin in plants and animals appears to be very different since the 50- to 60-fold increases of ferritin protein, previously observed to be induced by iron in cultured soybean cells, is accompanied by an equivalent accumulation of hybridizable ferritin mRNA and by increased transcription of ferritin genes. Ferritin mRNA from iron-induced cells and the constitutive ferritin mRNA from soybean hypocotyls are identical. The iron-induced protein is translocated normally to plastids. Differences in animal ferritin structure coincide with the various iron storage functions (reserve for iron proteins and detoxification). In contrast, the constancy of structure of soybean ferritin, iron-induced and constitutive, coupled with the potential for vacuolar storage of excess iron in plants suggest that rapid synthesis of ferritin from a stored ferritin mRNA may not be needed in plants for detoxification of iron.

Advanced colorectal adenoma related gene expression signature may predict prognostic for colorectal cancer patients with adenoma-carcinoma sequence.

PubMed

Li, Bing; Shi, Xiao-Yu; Liao, Dai-Xiang; Cao, Bang-Rong; Luo, Cheng-Hua; Cheng, Shu-Jun

2015-01-01

There are still no absolute parameters predicting progression of adenoma into cancer. The present study aimed to characterize functional differences on the multistep carcinogenetic process from the adenoma-carcinoma sequence. All samples were collected and mRNA expression profiling was performed by using Agilent Microarray high-throughput gene-chip technology. Then, the characteristics of mRNA expression profiles of adenoma-carcinoma sequence were described with bioinformatics software, and we analyzed the relationship between gene expression profiles of adenoma-adenocarcinoma sequence and clinical prognosis of colorectal cancer. The mRNA expressions of adenoma-carcinoma sequence were significantly different between high-grade intraepithelial neoplasia group and adenocarcinoma group. The biological process of gene ontology function enrichment analysis on differentially expressed genes between high-grade intraepithelial neoplasia group and adenocarcinoma group showed that genes enriched in the extracellular structure organization, skeletal system development, biological adhesion and itself regulated growth regulation, with the P value after FDR correction of less than 0.05. In addition, IPR-related protein mainly focused on the insulin-like growth factor binding proteins. The variable trends of gene expression profiles for adenoma-carcinoma sequence were mainly concentrated in high-grade intraepithelial neoplasia and adenocarcinoma. The differentially expressed genes are significantly correlated between high-grade intraepithelial neoplasia group and adenocarcinoma group. Bioinformatics analysis is an effective way to study the gene expression profiles in the adenoma-carcinoma sequence, and may provide an effective tool to involve colorectal cancer research strategy into colorectal adenoma or advanced adenoma.

Geoseq: a tool for dissecting deep-sequencing datasets.

PubMed

Gurtowski, James; Cancio, Anthony; Shah, Hardik; Levovitz, Chaya; George, Ajish; Homann, Robert; Sachidanandam, Ravi

2010-10-12

Datasets generated on deep-sequencing platforms have been deposited in various public repositories such as the Gene Expression Omnibus (GEO), Sequence Read Archive (SRA) hosted by the NCBI, or the DNA Data Bank of Japan (ddbj). Despite being rich data sources, they have not been used much due to the difficulty in locating and analyzing datasets of interest. Geoseq http://geoseq.mssm.edu provides a new method of analyzing short reads from deep sequencing experiments. Instead of mapping the reads to reference genomes or sequences, Geoseq maps a reference sequence against the sequencing data. It is web-based, and holds pre-computed data from public libraries. The analysis reduces the input sequence to tiles and measures the coverage of each tile in a sequence library through the use of suffix arrays. The user can upload custom target sequences or use gene/miRNA names for the search and get back results as plots and spreadsheet files. Geoseq organizes the public sequencing data using a controlled vocabulary, allowing identification of relevant libraries by organism, tissue and type of experiment. Analysis of small sets of sequences against deep-sequencing datasets, as well as identification of public datasets of interest, is simplified by Geoseq. We applied Geoseq to, a) identify differential isoform expression in mRNA-seq datasets, b) identify miRNAs (microRNAs) in libraries, and identify mature and star sequences in miRNAS and c) to identify potentially mis-annotated miRNAs. The ease of using Geoseq for these analyses suggests its utility and uniqueness as an analysis tool.

Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing.

PubMed

Sahoo, Malaya K; Holubar, Marisa; Huang, ChunHong; Mohamed-Hadley, Alisha; Liu, Yuanyuan; Waggoner, Jesse J; Troy, Stephanie B; Garcia-Garcia, Lourdes; Ferreyra-Reyes, Leticia; Maldonado, Yvonne; Pinsky, Benjamin A

2017-07-01

Oral poliovirus vaccine can mutate to regain neurovirulence. To date, evaluation of these mutations has been performed primarily on culture-enriched isolates by using conventional Sanger sequencing. We therefore developed a culture-independent, deep-sequencing method targeting the 5' untranslated region (UTR) and P1 genomic region to characterize vaccine-related poliovirus variants. Error analysis of the deep-sequencing method demonstrated reliable detection of poliovirus mutations at levels of <1%, depending on read depth. Sequencing of viral nucleic acids from the stool of vaccinated, asymptomatic children and their close contacts collected during a prospective cohort study in Veracruz, Mexico, revealed no vaccine-derived polioviruses. This was expected given that the longest duration between sequenced sample collection and the end of the most recent national immunization week was 66 days. However, we identified many low-level variants (<5%) distributed across the 5' UTR and P1 genomic region in all three Sabin serotypes, as well as vaccine-related viruses with multiple canonical mutations associated with phenotypic reversion present at high levels (>90%). These results suggest that monitoring emerging vaccine-related poliovirus variants by deep sequencing may aid in the poliovirus endgame and efforts to ensure global polio eradication. Copyright © 2017 Sahoo et al.

Rational Protein Engineering Guided by Deep Mutational Scanning

PubMed Central

Shin, HyeonSeok; Cho, Byung-Kwan

2015-01-01

Sequence–function relationship in a protein is commonly determined by the three-dimensional protein structure followed by various biochemical experiments. However, with the explosive increase in the number of genome sequences, facilitated by recent advances in sequencing technology, the gap between protein sequences available and three-dimensional structures is rapidly widening. A recently developed method termed deep mutational scanning explores the functional phenotype of thousands of mutants via massive sequencing. Coupled with a highly efficient screening system, this approach assesses the phenotypic changes made by the substitution of each amino acid sequence that constitutes a protein. Such an informational resource provides the functional role of each amino acid sequence, thereby providing sufficient rationale for selecting target residues for protein engineering. Here, we discuss the current applications of deep mutational scanning and consider experimental design. PMID:26404267

Deep Sequencing in Infectious Diseases: Immune and Pathogen Repertoires for the Improvement of Patient Outcomes.

PubMed

Burkholder, William F; Newell, Evan W; Poidinger, Michael; Chen, Swaine; Fink, Katja

2017-01-01

The inaugural workshop "Deep Sequencing in Infectious Diseases: Immune and Pathogen Repertoires for the Improvement of Patient Outcomes" was held in Singapore on 13-14 October 2016. The aim of the workshop was to discuss the latest trends in using high-throughput sequencing, bioinformatics, and allied technologies to analyze immune and pathogen repertoires and their interplay within the host, bringing together key international players in the field and Singapore-based researchers and clinician-scientists. The focus was in particular on the application of these technologies for the improvement of patient diagnosis, prognosis and treatment, and for other broad public health outcomes. The presentations by scientists and clinicians showed the potential of deep sequencing technology to capture the coevolution of adaptive immunity and pathogens. For clinical applications, some key challenges remain, such as the long turnaround time and relatively high cost of deep sequencing for pathogen identification and characterization and the lack of international standardization in immune repertoire analysis.

Deep Sequencing in Infectious Diseases: Immune and Pathogen Repertoires for the Improvement of Patient Outcomes

PubMed Central

Burkholder, William F.; Newell, Evan W.; Poidinger, Michael; Chen, Swaine; Fink, Katja

2017-01-01

The inaugural workshop “Deep Sequencing in Infectious Diseases: Immune and Pathogen Repertoires for the Improvement of Patient Outcomes” was held in Singapore on 13–14 October 2016. The aim of the workshop was to discuss the latest trends in using high-throughput sequencing, bioinformatics, and allied technologies to analyze immune and pathogen repertoires and their interplay within the host, bringing together key international players in the field and Singapore-based researchers and clinician-scientists. The focus was in particular on the application of these technologies for the improvement of patient diagnosis, prognosis and treatment, and for other broad public health outcomes. The presentations by scientists and clinicians showed the potential of deep sequencing technology to capture the coevolution of adaptive immunity and pathogens. For clinical applications, some key challenges remain, such as the long turnaround time and relatively high cost of deep sequencing for pathogen identification and characterization and the lack of international standardization in immune repertoire analysis. PMID:28620372

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

SEPT9 Mutations and a Conserved 17q25 Sequence in Sporadic and Hereditary Brachial Plexus Neuropathy

PubMed Central

Klein, Christopher J.; Wu, Yanhong; Cunningham, Julie M.; Windebank, Anthony J.; Dyck, P. James B.; Friedenberg, Scott M.; Klein, Diane M.; Dyck, Peter J.

2009-01-01

Background The clinical characteristics of sporadic brachial plexus neuropathy (S-BPN) and hereditary brachial plexus neuropathy (H-BPN) are similar. At times of attack inflammation in brachial plexus nerves has been identified in both conditions. SEPT-9 mutations (Arg88Trp, Ser93Phe, 5UTR-131G to C) occur in some families with H-BPN. These mutations were not found in American H-BPN kindreds with a conserved 500 Kb sequence of DNA at 17q25 (the location of SEPT-9) where a founder mutation has been suggested. Objective To study 17q25 and SEPT-9 in S-BPN (56 patients) and H-BPN (13 kindreds). Methods Allele analysis at 17q25, SEPT-9 DNA sequencing and mRNA analysis from lymphoblast cultures. Results A conserved 17q25 sequence was found in 5 of 13 H-BPN kindreds and one S-BPN patient. This conserved sequence was not found in the family with a SEPT-9 mutation (Arg88Trp) or controls (182). SEPT-9 mRNA expression did not differ between forms of H-BPN and controls. No known mutations of SEPT-9 were found in S-BPN. Conclusions/Relevance Rare S-BPN patients have the same conserved 17q25 sequence found in many American H-BPN kindreds. BPN patients with this conserved sequence do not appear to have SEPT-9 mutations or alterations of its mRNA expression levels in lymphoblast cultures. BPN patients with this conserved sequence may have the most common genetic cause in the Americas by a founder effect mutation. PMID:19204161

Development of a microcapillary column for detecting targeted messenger RNA molecules.

PubMed

Ohnishi, Michihiro

2006-03-24

A capillary column in a rapid-flow system has been developed for detecting targeted messenger RNA (mRNA) molecules. The column has a structure made of two beds-one bed of porous microbeads and one bed of microbeads with a polythymidine base sequence. The targeted eukaryotic mRNA molecules are detected by two-step hybridization (sandwich hybridization) composed of polyadenosine selection of mRNA molecules and formation of a probe-target (targeted mRNA) hybrid. The sandwich hybridization, which is accomplished within 1 h, was tested using synthetic polydeoxynucleotides. Ten picomoles of the targeted polydeoxynucleotide were detected.

Molecular cloning and sequence analysis of two carbonic anhydrase in the swimming crab Portunus trituberculatus and its expression in response to salinity and pH stress.

PubMed

Pan, Luqing; Hu, Dongxu; Liu, Maoqi; Hu, Yanyan; Liu, Shengnan

2016-01-15

Carbonic anhydrase (CA) is involved in ion transport, acid-base balance and pH regulation by catalyzing the interconversion of CO2 and HCO3(-). In this study, full-length cDNA sequences of two CA isoforms were identified from Portunus trituberculatus. One was Portunus trituberculatus cytoplasmic carbonic anydrase (PtCAc) and the other one was Portunus trituberculatus glycosyl-phosphatidylinositol-linked carbonic anhydrase (PtCAg). The sequence of PtCAc was formed by an ORF of 816 bp, encoding a protein of 30.18 kDa. The PtCAg was constituted by an ORF of 927 bp, encoding a protein of 34.09 kDa. The deduced amino acid sequences of the two CA isoforms were compared to other crustacean' CA sequences. Both of them reflected high conservation of the residues and domains essential to the function of the two enzymes. The tissue expression analysis of PtCAc and PtCAg were detected in gill, muscle, hepatopancreas, hemocytes and gonad. PtCAc and PtCAg gene expressions were studied under salinity and pH challenge. The results showed that when salinity decreased (30 to 20 ppt), the mRNA expression of PtCAc increased significantly at 24 and 48 h, and the highest value appeared at 24h. The mRNA expression of PtCAg had the same situation with PtCAc. However, when salinity increased (30 to 35 ppt), only the mRNA expression of PtCAc increased significantly at 48 h. When pH changed, only the mRNA expression of PtCAc increased significantly at 12h, which was under low pH situation. The mRNA expression of PtCAg increased significantly at 12-48 h, and there was no significant difference of the expression between the pH challenged group and the control group in other experimental time. The results provided the base of understanding CA' function and the underlying mechanism in response to environmental changes in crustaceans. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Waltman, Peter H.; Guo, Jian; Reistetter, Emily Nahas

Micromonas is a unicellular green alga that belongs to the prasinophytes, a sister lineage to land plants. This picoeukaryotic (<2 μm diameter) alga is widespread in the marine environment but still not understood at the cellular level. Here, we examine the mRNA and protein level changes that take place over the course of the day-night cycle using mid-exponential nutrient replete cultures of Micromonas pusilla CCMP1545 grown and analyzed in biological triplicate. During the experiment, samples were collected at key transition points during the diel for evaluation using high-throughput LC-MS proteomics. We also sequenced matched mRNA samples from the same timemore » points, using pair-ended directional Illumina RNA-Seq to investigate the dynamics and relationship between the mRNA and protein expression programs of M. pusilla. Similar to a prior study of the marine cyanobacterium Prochlorococcus, we found significant divergence in the mRNA and proteomics expression dynamics in response to the light:dark cycle. Additionally, expressional responses of genes and the proteins they encoded could also be variable within the same metabolic pathway, such as the oxygenic photosynthesis pathway. A regression framework was used to predict protein levels using both mRNA expression and gene-specific sequence-based features. Several features in the genome sequence were found to influence protein abundance including the codon usage and the length of the 3’ UTR. Collectively, our studies provide insights into the regulation of the proteome over a diel as relationships between the transcriptional and translational programs in the widespread marine green alga Micromonas.« less

mRNA localization to the mitochondrial surface allows the efficient translocation inside the organelle of a nuclear recoded ATP6 protein

PubMed Central

Kaltimbacher, Valérie; Bonnet, Crystel; Lecoeuvre, Gaëlle; Forster, Valérie; Sahel, José-Alain; Corral-Debrinski, Marisol

2006-01-01

As previously established in yeast, two sequences within mRNAs are responsible for their specific localization to the mitochondrial surface—the region coding for the mitochondrial targeting sequence and the 3′UTR. This phenomenon is conserved in human cells. Therefore, we decided to use mRNA localization as a tool to address to mitochondria, a protein that is not normally imported. For this purpose, we associated a nuclear recoded ATP6 gene with the mitochondrial targeting sequence and the 3′UTR of the nuclear SOD2 gene, which mRNA exclusively localizes to the mitochondrial surface in HeLa cells. The ATP6 gene is naturally located into the organelle and encodes a highly hydrophobic protein of the respiratory chain complex V. In this study, we demonstrated that hybrid ATP6 mRNAs, as the endogenous SOD2 mRNA, localize to the mitochondrial surface in human cells. Remarkably, fusion proteins localize to mitochondria in vivo. Indeed, ATP6 precursors synthesized in the cytoplasm were imported into mitochondria in a highly efficient way, especially when both the MTS and the 3′UTR of the SOD2 gene were associated with the re-engineered ATP6 gene. Hence, these data indicate that mRNA targeting to the mitochondrial surface represents an attractive strategy for allowing the mitochondrial import of proteins originally encoded by the mitochondrial genome without any amino acid change in the protein that could interfere with its biologic activity. PMID:16751614

cDNA cloning of carrot extracellular beta-fructosidase and its expression in response to wounding and bacterial infection.

PubMed

Sturm, A; Chrispeels, M J

1990-11-01

We isolated a full-length cDNA for apoplastic (extracellular or cell wall-bound) beta-fructosidase (invertase), determined its nucleotide sequence, and used it as a probe to measure changes in mRNA as a result of wounding of carrot storage roots and infection of carrot plants with the bacterial pathogen Erwinia carotovora. The derived amino acid sequence of extracellular beta-fructosidase shows that it is a basic protein (pl 9.9) with a signal sequence for entry into the endoplasmic reticulum and a propeptide at the N terminus that is not present in the mature protein. Amino acid sequence comparison with yeast and bacterial invertases shows that the overall homology is only about 28%, but that there are short conserved motifs, one of which is at the active site. Maturing carrot storage roots contain barely detectable levels of mRNA for extracellular beta-fructosidase and these levels rise slowly but dramatically after wounding with maximal expression after 12 hours. Infection of roots and leaves of carrot plants with E. carotovora results in a very fast increase in the mRNA levels with maximal expression after 1 hour. These results indicate that apoplastic beta-fructosidase is probably a new and hitherto unrecognized pathogenesis-related protein [Van Loon, L.C. (1985). Plant Mol. Biol. 4, 111-116]. Suspension-cultured carrot cells contain high levels of mRNA for extracellular beta-fructosidase and these levels remain the same whether the cells are grown on sucrose, glucose, or fructose.

cDNA cloning of carrot extracellular beta-fructosidase and its expression in response to wounding and bacterial infection.

PubMed Central

Sturm, A; Chrispeels, M J

1990-01-01

We isolated a full-length cDNA for apoplastic (extracellular or cell wall-bound) beta-fructosidase (invertase), determined its nucleotide sequence, and used it as a probe to measure changes in mRNA as a result of wounding of carrot storage roots and infection of carrot plants with the bacterial pathogen Erwinia carotovora. The derived amino acid sequence of extracellular beta-fructosidase shows that it is a basic protein (pl 9.9) with a signal sequence for entry into the endoplasmic reticulum and a propeptide at the N terminus that is not present in the mature protein. Amino acid sequence comparison with yeast and bacterial invertases shows that the overall homology is only about 28%, but that there are short conserved motifs, one of which is at the active site. Maturing carrot storage roots contain barely detectable levels of mRNA for extracellular beta-fructosidase and these levels rise slowly but dramatically after wounding with maximal expression after 12 hours. Infection of roots and leaves of carrot plants with E. carotovora results in a very fast increase in the mRNA levels with maximal expression after 1 hour. These results indicate that apoplastic beta-fructosidase is probably a new and hitherto unrecognized pathogenesis-related protein [Van Loon, L.C. (1985). Plant Mol. Biol. 4, 111-116]. Suspension-cultured carrot cells contain high levels of mRNA for extracellular beta-fructosidase and these levels remain the same whether the cells are grown on sucrose, glucose, or fructose. PMID:2152110

Tau mRNA 3'UTR-to-CDS ratio is increased in Alzheimer disease.

PubMed

García-Escudero, Vega; Gargini, Ricardo; Martín-Maestro, Patricia; García, Esther; García-Escudero, Ramón; Avila, Jesús

2017-08-10

Neurons frequently show an imbalance in expression of the 3' untranslated region (3'UTR) relative to the coding DNA sequence (CDS) region of mature messenger RNAs (mRNA). The ratio varies among different cells or parts of the brain. The Map2 protein levels per cell depend on the 3'UTR-to-CDS ratio rather than the total mRNA amount, which suggests powerful regulation of protein expression by 3'UTR sequences. Here we found that MAPT (the microtubule-associated protein tau gene) 3'UTR levels are particularly high with respect to other genes; indeed, the 3'UTR-to-CDS ratio of MAPT is balanced in healthy brain in mouse and human. The tau protein accumulates in Alzheimer diseased brain. We nonetheless observed that the levels of RNA encoding MAPT/tau were diminished in these patients' brains. To explain this apparently contradictory result, we studied MAPT mRNA stoichiometry in coding and non-coding regions, and found that the 3'UTR-to-CDS ratio was higher in the hippocampus of Alzheimer disease patients, with higher tau protein but lower total mRNA levels. Our data indicate that changes in the 3'UTR-to-CDS ratio have a regulatory role in the disease. Future research should thus consider not only mRNA levels, but also the ratios between coding and non-coding regions. Copyright © 2017 Elsevier B.V. All rights reserved.

Human placental lactogen mRNA and its structural genes during pregnancy: quantitation with a complementary DNA.

PubMed Central

McWilliams, D; Callahan, R C; Boime, I

1977-01-01

A complementary DNA (cDNA) strand was transcribed from human placental lactogen (hPL) mRNA. Based on alkaline sucrose gradient centrifugation, the size of the cDNA was about 8 S, which would represent at least 80% of the hPL mRNA. Previously we showed that four to five times more hPL was synthesized in cell-free extracts derived from term as compared to first trimester placentas. Hybridization of the cDNA with RNA derived from placental tissue revealed that there was about four times more hPL mRNA sequences in total RNA from term placenta than in a comparable quantity of total first trimester RNA. Only background hybridization was observed when the cDNA was incubated with RNA prepared from human kidney. To test if this differential accumulation of hPL mRNA was the result of an amplification of hPL genes, we hybridized the labeled cDNA with cellular DNA from first trimester and term placentas and with DNA isolated from human brain. In all cases, the amount of hPL sequences was approximately two copies per haploid genome. Thus, the enhanced synthesis of hPL mRNA appears to result from a transcriptional activation rather than an amplification of the hPL gene. The increase likely reflects placental differentiation in which the proportion of syncytial trophoblast increases at term. Images PMID:66681

Highly abundant and stage-specific mRNAs in the obligate pathogen Bremia lactucae.

PubMed

Judelson, H S; Michelmore, R W

1990-01-01

Germinating spores of the obligate pathogen Bremia lactucae (lettuce downy mildew) contain several unusually abundant species of mRNA. Thirty-nine cDNA clones corresponding to prevalent transcripts were isolated from a library synthesized using poly(A)+ RNA from germinating spores; these clones represented only five distinct classes. Each corresponding mRNA accounted for from 0.4 to 9 percent by mass of poly(A)+ RNA from germinating spores and together represented greater than 20 percent of the mRNA. The expression of the corresponding genes, and a gene encoding Hsp70, was analyzed in spores during germination and during growth in planta. The Hsp70 mRNA and mRNA from one abundant cDNA clone (ham34) were expressed constitutively. Two clones (ham9 and ham12) hybridized only to mRNA from spores and germinating spores. Two clones (ham37 and ham27) showed hybridization specific to germinating spores. Quantification of the number of genes homologous to each cDNA clone indicated that four clones corresponded to one or two copies per haploid genome, and one hybridized to an approximately 11-member family of genes. A sequence of the gene corresponding to ham34 was obtained to investigate its function and to identify sequences conferring high levels of gene expression for use in constructing vectors for the transformation of B. lactucae.

DNA Replication Profiling Using Deep Sequencing.

PubMed

Saayman, Xanita; Ramos-Pérez, Cristina; Brown, Grant W

2018-01-01

Profiling of DNA replication during progression through S phase allows a quantitative snap-shot of replication origin usage and DNA replication fork progression. We present a method for using deep sequencing data to profile DNA replication in S. cerevisiae.

Relationship between mRNA secondary structure and sequence variability in Chloroplast genes: possible life history implications.

PubMed

Krishnan, Neeraja M; Seligmann, Hervé; Rao, Basuthkar J

2008-01-28

Synonymous sites are freer to vary because of redundancy in genetic code. Messenger RNA secondary structure restricts this freedom, as revealed by previous findings in mitochondrial genes that mutations at third codon position nucleotides in helices are more selected against than those in loops. This motivated us to explore the constraints imposed by mRNA secondary structure on evolutionary variability at all codon positions in general, in chloroplast systems. We found that the evolutionary variability and intrinsic secondary structure stability of these sequences share an inverse relationship. Simulations of most likely single nucleotide evolution in Psilotum nudum and Nephroselmis olivacea mRNAs, indicate that helix-forming propensities of mutated mRNAs are greater than those of the natural mRNAs for short sequences and vice-versa for long sequences. Moreover, helix-forming propensity estimated by the percentage of total mRNA in helices increases gradually with mRNA length, saturating beyond 1000 nucleotides. Protection levels of functionally important sites vary across plants and proteins: r-strategists minimize mutation costs in large genes; K-strategists do the opposite. Mrna length presumably predisposes shorter mRNAs to evolve under different constraints than longer mRNAs. The positive correlation between secondary structure protection and functional importance of sites suggests that some sites might be conserved due to packing-protection constraints at the nucleic acid level in addition to protein level constraints. Consequently, nucleic acid secondary structure a priori biases mutations. The converse (exposure of conserved sites) apparently occurs in a smaller number of cases, indicating a different evolutionary adaptive strategy in these plants. The differences between the protection levels of functionally important sites for r- and K-strategists reflect their respective molecular adaptive strategies. These converge with increasing domestication levels of K-strategists, perhaps because domestication increases reproductive output.

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.

Selection of mRNA 5'-untranslated region sequence with high translation efficiency through ribosome display

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

Mie, Masayasu; Shimizu, Shun; Takahashi, Fumio

2008-08-15

The 5'-untranslated region (5'-UTR) of mRNAs functions as a translation enhancer, promoting translation efficiency. Many in vitro translation systems exhibit a reduced efficiency in protein translation due to decreased translation initiation. The use of a 5'-UTR sequence with high translation efficiency greatly enhances protein production in these systems. In this study, we have developed an in vitro selection system that favors 5'-UTRs with high translation efficiency using a ribosome display technique. A 5'-UTR random library, comprised of 5'-UTRs tagged with a His-tag and Renilla luciferase (R-luc) fusion, were in vitro translated in rabbit reticulocytes. By limiting the translation period, onlymore » mRNAs with high translation efficiency were translated. During translation, mRNA, ribosome and translated R-luc with His-tag formed ternary complexes. They were collected with translated His-tag using Ni-particles. Extracted mRNA from ternary complex was amplified using RT-PCR and sequenced. Finally, 5'-UTR with high translation efficiency was obtained from random 5'-UTR library.« less

DeepARG: a deep learning approach for predicting antibiotic resistance genes from metagenomic data.

PubMed

Arango-Argoty, Gustavo; Garner, Emily; Pruden, Amy; Heath, Lenwood S; Vikesland, Peter; Zhang, Liqing

2018-02-01

Growing concerns about increasing rates of antibiotic resistance call for expanded and comprehensive global monitoring. Advancing methods for monitoring of environmental media (e.g., wastewater, agricultural waste, food, and water) is especially needed for identifying potential resources of novel antibiotic resistance genes (ARGs), hot spots for gene exchange, and as pathways for the spread of ARGs and human exposure. Next-generation sequencing now enables direct access and profiling of the total metagenomic DNA pool, where ARGs are typically identified or predicted based on the "best hits" of sequence searches against existing databases. Unfortunately, this approach produces a high rate of false negatives. To address such limitations, we propose here a deep learning approach, taking into account a dissimilarity matrix created using all known categories of ARGs. Two deep learning models, DeepARG-SS and DeepARG-LS, were constructed for short read sequences and full gene length sequences, respectively. Evaluation of the deep learning models over 30 antibiotic resistance categories demonstrates that the DeepARG models can predict ARGs with both high precision (> 0.97) and recall (> 0.90). The models displayed an advantage over the typical best hit approach, yielding consistently lower false negative rates and thus higher overall recall (> 0.9). As more data become available for under-represented ARG categories, the DeepARG models' performance can be expected to be further enhanced due to the nature of the underlying neural networks. Our newly developed ARG database, DeepARG-DB, encompasses ARGs predicted with a high degree of confidence and extensive manual inspection, greatly expanding current ARG repositories. The deep learning models developed here offer more accurate antimicrobial resistance annotation relative to current bioinformatics practice. DeepARG does not require strict cutoffs, which enables identification of a much broader diversity of ARGs. The DeepARG models and database are available as a command line version and as a Web service at http://bench.cs.vt.edu/deeparg .

Cloning of Russian sturgeon (Acipenser gueldenstaedtii) growth hormone and insulin-like growth factor I and their expression in male and female fish during the first period of growth.

PubMed

Yom Din, S; Hurvitz, A; Goldberg, D; Jackson, K; Levavi-Sivan, B; Degani, G

2008-03-01

In this study, the GH and IGF-I of the Russian sturgeon (rs), Acipenser gueldenstaedtii, were cloned and sequenced, and their mRNA gene expression determined. In addition, to improve our understanding of the GH function, the expression of this hormone was assessed in young males and females. Moreover, IGF-I expression was quantified in young males and compared to that in older ones. The nucleotide sequence of the rsGH cDNA was 980 bp long and had an open reading frame of 642 bp, beginning with the first ATG codon at position 39 and ending with the stop codon at position 683. A putative polyadenylation signal, AATAAA, was recognized 42 bp upstream of the poly (A) tail. The position of the signal- peptide cleavage site was predicted to be at position 111, yielding a signal peptide of 24 amino-acids (aa) and a mature peptide of 190 aa. When the rsGH aa sequence was compared with other species, the highest degree of identity was found to be with mammalians (66-70% identity), followed by anguilliformes and amphibia (61%) and other fish (39-47%). The level of rsGH mRNA was discovered to be similar in pituitaries of females and males of 5 age groups (1, 2, 3, 4, and 5- yr-old). In females and males, the levels did not change dramatically during the first 5 yr of growth. The partial nucleotide sequence of the rsIGF-I was 445 bp long and had an open reading frame of 396 bp, beginning with the ATG codon at position 50. The position of the signal-peptide cleavage site was predicted to be at position 187, yielding a signal peptide of 44 aa. The highest level of IGF-I mRNA expression was recorded in the kidney of adult sturgeons. The IGF-I mRNA expression levels in the intestine, pituitary gland, and liver were not significantly different. Low levels of expression were found in the brain, heart, and muscle. In most tissues, there was no significant difference between mRNA levels of one and 5-yr-old fish. In conclusion, based on the GH-sequence analysis, A. gueldenstaedtii is genetically distant from other teleosts. The expression of the GH mRNA was similar in males and females, and its level remained constant during the first 5 yr of growth. While the IGF-I mRNA expression differed amongst various tissues, the level in each tissue was similar in 1 and 5-yr-old fish.

Understanding the complex evolution of rapidly mutating viruses with deep sequencing: Beyond the analysis of viral diversity.

PubMed

Leung, Preston; Eltahla, Auda A; Lloyd, Andrew R; Bull, Rowena A; Luciani, Fabio

2017-07-15

With the advent of affordable deep sequencing technologies, detection of low frequency variants within genetically diverse viral populations can now be achieved with unprecedented depth and efficiency. The high-resolution data provided by next generation sequencing technologies is currently recognised as the gold standard in estimation of viral diversity. In the analysis of rapidly mutating viruses, longitudinal deep sequencing datasets from viral genomes during individual infection episodes, as well as at the epidemiological level during outbreaks, now allow for more sophisticated analyses such as statistical estimates of the impact of complex mutation patterns on the evolution of the viral populations both within and between hosts. These analyses are revealing more accurate descriptions of the evolutionary dynamics that underpin the rapid adaptation of these viruses to the host response, and to drug therapies. This review assesses recent developments in methods and provide informative research examples using deep sequencing data generated from rapidly mutating viruses infecting humans, particularly hepatitis C virus (HCV), human immunodeficiency virus (HIV), Ebola virus and influenza virus, to understand the evolution of viral genomes and to explore the relationship between viral mutations and the host adaptive immune response. Finally, we discuss limitations in current technologies, and future directions that take advantage of publically available large deep sequencing datasets. Copyright © 2016 Elsevier B.V. All rights reserved.

DEEP MOTIF DASHBOARD: VISUALIZING AND UNDERSTANDING GENOMIC SEQUENCES USING DEEP NEURAL NETWORKS.

PubMed

Lanchantin, Jack; Singh, Ritambhara; Wang, Beilun; Qi, Yanjun

2017-01-01

Deep neural network (DNN) models have recently obtained state-of-the-art prediction accuracy for the transcription factor binding (TFBS) site classification task. However, it remains unclear how these approaches identify meaningful DNA sequence signals and give insights as to why TFs bind to certain locations. In this paper, we propose a toolkit called the Deep Motif Dashboard (DeMo Dashboard) which provides a suite of visualization strategies to extract motifs, or sequence patterns from deep neural network models for TFBS classification. We demonstrate how to visualize and understand three important DNN models: convolutional, recurrent, and convolutional-recurrent networks. Our first visualization method is finding a test sequence's saliency map which uses first-order derivatives to describe the importance of each nucleotide in making the final prediction. Second, considering recurrent models make predictions in a temporal manner (from one end of a TFBS sequence to the other), we introduce temporal output scores, indicating the prediction score of a model over time for a sequential input. Lastly, a class-specific visualization strategy finds the optimal input sequence for a given TFBS positive class via stochastic gradient optimization. Our experimental results indicate that a convolutional-recurrent architecture performs the best among the three architectures. The visualization techniques indicate that CNN-RNN makes predictions by modeling both motifs as well as dependencies among them.

smRNAome profiling to identify conserved and novel microRNAs in Stevia rebaudiana Bertoni

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) constitute a family of small RNA (sRNA) population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. Extensive studies on miRNAs have been performed in different plants such as Arabidopsis thaliana, Oryza sativa etc. and volume of the miRNA database, mirBASE, has been increasing on day to day basis. Stevia rebaudiana Bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed sweetening property with no calorific value. Several studies have been carried out for understanding molecular mechanism involved in biosynthesis of these glycosides, however, information about miRNAs has been lacking in S. rebaudiana. Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs irrespective of availability of genome sequence data. Results To identify miRNAs in S. rebaudiana, sRNA library was constructed and sequenced using Illumina genome analyzer II. A total of 30,472,534 reads representing 2,509,190 distinct sequences were obtained from sRNA library. Based on sequence similarity, we identified 100 miRNAs belonging to 34 highly conserved families. Also, we identified 12 novel miRNAs whose precursors were potentially generated from stevia EST and nucleotide sequences. All novel sequences have not been earlier described in other plant species. Putative target genes were predicted for most conserved and novel miRNAs. The predicted targets are mainly mRNA encoding enzymes regulating essential plant metabolic and signaling pathways. Conclusions This study led to the identification of 34 highly conserved miRNA families and 12 novel potential miRNAs indicating that specific miRNAs exist in stevia species. Our results provided information on stevia miRNAs and their targets building a foundation for future studies to understand their roles in key stevia traits. PMID:23116282

smRNAome profiling to identify conserved and novel microRNAs in Stevia rebaudiana Bertoni.

PubMed

Mandhan, Vibha; Kaur, Jagdeep; Singh, Kashmir

2012-11-01

MicroRNAs (miRNAs) constitute a family of small RNA (sRNA) population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. Extensive studies on miRNAs have been performed in different plants such as Arabidopsis thaliana, Oryza sativa etc. and volume of the miRNA database, mirBASE, has been increasing on day to day basis. Stevia rebaudiana Bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed sweetening property with no calorific value. Several studies have been carried out for understanding molecular mechanism involved in biosynthesis of these glycosides, however, information about miRNAs has been lacking in S. rebaudiana. Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs irrespective of availability of genome sequence data. To identify miRNAs in S. rebaudiana, sRNA library was constructed and sequenced using Illumina genome analyzer II. A total of 30,472,534 reads representing 2,509,190 distinct sequences were obtained from sRNA library. Based on sequence similarity, we identified 100 miRNAs belonging to 34 highly conserved families. Also, we identified 12 novel miRNAs whose precursors were potentially generated from stevia EST and nucleotide sequences. All novel sequences have not been earlier described in other plant species. Putative target genes were predicted for most conserved and novel miRNAs. The predicted targets are mainly mRNA encoding enzymes regulating essential plant metabolic and signaling pathways. This study led to the identification of 34 highly conserved miRNA families and 12 novel potential miRNAs indicating that specific miRNAs exist in stevia species. Our results provided information on stevia miRNAs and their targets building a foundation for future studies to understand their roles in key stevia traits.

Improved detection of CXCR4-using HIV by V3 genotyping: application of population-based and "deep" sequencing to plasma RNA and proviral DNA.

PubMed

Swenson, Luke C; Moores, Andrew; Low, Andrew J; Thielen, Alexander; Dong, Winnie; Woods, Conan; Jensen, Mark A; Wynhoven, Brian; Chan, Dennison; Glascock, Christopher; Harrigan, P Richard

2010-08-01

Tropism testing should rule out CXCR4-using HIV before treatment with CCR5 antagonists. Currently, the recombinant phenotypic Trofile assay (Monogram) is most widely utilized; however, genotypic tests may represent alternative methods. Independent triplicate amplifications of the HIV gp120 V3 region were made from either plasma HIV RNA or proviral DNA. These underwent standard, population-based sequencing with an ABI3730 (RNA n = 63; DNA n = 40), or "deep" sequencing with a Roche/454 Genome Sequencer-FLX (RNA n = 12; DNA n = 12). Position-specific scoring matrices (PSSMX4/R5) (-6.96 cutoff) and geno2pheno[coreceptor] (5% false-positive rate) inferred tropism from V3 sequence. These methods were then independently validated with a separate, blinded dataset (n = 278) of screening samples from the maraviroc MOTIVATE trials. Standard sequencing of HIV RNA with PSSM yielded 69% sensitivity and 91% specificity, relative to Trofile. The validation dataset gave 75% sensitivity and 83% specificity. Proviral DNA plus PSSM gave 77% sensitivity and 71% specificity. "Deep" sequencing of HIV RNA detected >2% inferred-CXCR4-using virus in 8/8 samples called non-R5 by Trofile, and <2% in 4/4 samples called R5. Triplicate analyses of V3 standard sequence data detect greater proportions of CXCR4-using samples than previously achieved. Sequencing proviral DNA and "deep" V3 sequencing may also be useful tools for assessing tropism.

Reduced TCOF1 mRNA level in a rhesus macaque with Treacher Collins-like syndrome: further evidence for haploinsufficiency of treacle as the cause of disease.

PubMed

Shows, Kathryn H; Ward, Christy; Summers, Laura; Li, Lin; Ziegler, Gregory R; Hendrickx, Andrew G; Shiang, Rita

2006-02-01

Mutations in the human gene TCOF1 cause a mandibulofacial dysostosis known as Treacher Collins syndrome (TCS). An infant rhesus macaque (Macaca mulatta) that displayed the TCS phenotype was identified at the California National Primate Research Center. The TCOF1 coding region was cloned from a normal rhesus macaque and sequenced. The rhesus macaque homolog of TCOF1 is 91.6% identical in cDNA sequence and 93.8% identical in translated protein sequence compared to human TCOF1. Sequencing of TCOF1 in the TCS-affected rhesus macaque showed no mutations within the coding region or splice sites; however, real-time quantitative PCR showed an 87% reduction of spleen TCOF1 mRNA level in the TCS affected macaque when compared with normal macaque spleen.

Cloning, sequencing and expression in MEL cells of a cDNA encoding the mouse ribosomal protein S5.

PubMed

Vanegas, N; Castañeda, V; Santamaría, D; Hernández, P; Schvartzman, J B; Krimer, D B

1997-06-05

We describe the isolation and characterization of a cDNA encoding the mouse S5 ribosomal protein. It was isolated from a MEL (murine erythroleukemia) cell cDNA library by differential hybridization as a down regulated sequence during HMBA-induced differentiation. Northern series analysis showed that S5 mRNA expression is reduced 5-fold throughout the differentiation process. The mouse S5 mRNA is 760 bp long and encodes for a 204 amino acid protein with 94% homology with the human and rat S5.

Global impact of RNA splicing on transcriptome remodeling in the heart.

PubMed

Gao, Chen; Wang, Yibin

2012-08-01

In the eukaryotic transcriptome, both the numbers of genes and different RNA species produced by each gene contribute to the overall complexity. These RNA species are generated by the utilization of different transcriptional initiation or termination sites, or more commonly, from different messenger RNA (mRNA) splicing events. Among the 30,000+ genes in human genome, it is estimated that more than 95% of them can generate more than one gene product via alternative RNA splicing. The protein products generated from different RNA splicing variants can have different intracellular localization, activity, or tissue-distribution. Therefore, alternative RNA splicing is an important molecular process that contributes to the overall complexity of the genome and the functional specificity and diversity among different cell types. In this review, we will discuss current efforts to unravel the full complexity of the cardiac transcriptome using a deep-sequencing approach, and highlight the potential of this technology to uncover the global impact of RNA splicing on the transcriptome during development and diseases of the heart.

The effect of Bacopa monnieri on gene expression levels in SH-SY5Y human neuroblastoma cells.

PubMed

Leung, How-Wing; Foo, Gabriel; Banumurthy, Gokulakrishna; Chai, Xiaoran; Ghosh, Sujoy; Mitra-Ganguli, Tora; VanDongen, Antonius M J

2017-01-01

Bacopa monnieri is a plant used as a nootropic in Ayurveda, a 5000-year-old system of traditional Indian medicine. Although both animal and clinical studies supported its role as a memory enhancer, the molecular and cellular mechanism underlying Bacopa's nootropic action are not understood. In this study, we used deep sequencing (RNA-Seq) to identify the transcriptome changes upon Bacopa treatment on SH-SY5Y human neuroblastoma cells. We identified several genes whose expression levels were regulated by Bacopa. Biostatistical analysis of the RNA-Seq data identified biological pathways and molecular functions that were regulated by Bacopa, including regulation of mRNA translation and transmembrane transport, responses to oxidative stress and protein misfolding. Pathway analysis using the Ingenuity platform suggested that Bacopa may protect against brain damage and improve brain development. These newly identified molecular and cellular determinants may contribute to the nootropic action of Bacopa and open up a new direction of investigation into its mechanism of action.

The effect of Bacopa monnieri on gene expression levels in SH-SY5Y human neuroblastoma cells

PubMed Central

Foo, Gabriel; Banumurthy, Gokulakrishna; Chai, Xiaoran; Ghosh, Sujoy

2017-01-01

Bacopa monnieri is a plant used as a nootropic in Ayurveda, a 5000-year-old system of traditional Indian medicine. Although both animal and clinical studies supported its role as a memory enhancer, the molecular and cellular mechanism underlying Bacopa’s nootropic action are not understood. In this study, we used deep sequencing (RNA-Seq) to identify the transcriptome changes upon Bacopa treatment on SH-SY5Y human neuroblastoma cells. We identified several genes whose expression levels were regulated by Bacopa. Biostatistical analysis of the RNA-Seq data identified biological pathways and molecular functions that were regulated by Bacopa, including regulation of mRNA translation and transmembrane transport, responses to oxidative stress and protein misfolding. Pathway analysis using the Ingenuity platform suggested that Bacopa may protect against brain damage and improve brain development. These newly identified molecular and cellular determinants may contribute to the nootropic action of Bacopa and open up a new direction of investigation into its mechanism of action. PMID:28832626

Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster

PubMed Central

Dunn, Joshua G; Foo, Catherine K; Belletier, Nicolette G; Gavis, Elizabeth R; Weissman, Jonathan S

2013-01-01

Ribosomes can read through stop codons in a regulated manner, elongating rather than terminating the nascent peptide. Stop codon readthrough is essential to diverse viruses, and phylogenetically predicted to occur in a few hundred genes in Drosophila melanogaster, but the importance of regulated readthrough in eukaryotes remains largely unexplored. Here, we present a ribosome profiling assay (deep sequencing of ribosome-protected mRNA fragments) for Drosophila melanogaster, and provide the first genome-wide experimental analysis of readthrough. Readthrough is far more pervasive than expected: the vast majority of readthrough events evolved within D. melanogaster and were not predicted phylogenetically. The resulting C-terminal protein extensions show evidence of selection, contain functional subcellular localization signals, and their readthrough is regulated, arguing for their importance. We further demonstrate that readthrough occurs in yeast and humans. Readthrough thus provides general mechanisms both to regulate gene expression and function, and to add plasticity to the proteome during evolution. DOI: http://dx.doi.org/10.7554/eLife.01179.001 PMID:24302569

Staufen-mediated mRNA decay

PubMed Central

Park, Eonyoung; Maquat, Lynne E.

2013-01-01

Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base-pairing of 3'UTR sequences or by intermolecular base-pairing of 3'UTR sequences with a long noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Since both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1, SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. PMID:23681777

Stimulation of S14 mRNA and lipogenesis in brown fat by hypothyroidism, cold exposure, and cafeteria feeding: evidence supporting a general role for S14 in lipogenesis and lipogenesis in the maintenance of thermogenesis

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

Freake, H.C.; Oppenheimer, J.H.

1987-05-01

In liver, thyroid hormone rapidly induces S14 mRNA, which encodes a small acidic protein. This sequence is abundantly expressed only in lipogenic tissues and is thought to have some function in fat metabolism. In the euthyroid rat, we measured 20-fold higher levels of S14 mRNA in interscapular brown adipose tissue than liver. Furthermore, whereas in liver or epididymal fat, hypothyroidism resulted in an 80% fall in S14 mRNA, in brown fat the level of this sequence increased a further 3-fold. In all three tissues, the expression of S14 mRNA correlated well with lipogenesis, as assessed by /sup 3/H/sub 2/O incorporation.more » Physiological activation of brown fat by chronic cold exposure or cafeteria feeding increased the concentration of S14 mRNA in this tissue and again this was accompanied by a greater rate of fatty acid synthesis. Overall, in liver and white and brown adipose tissue, S14 mRNA and lipogenesis were well correlated and strongly suggest a function of the S14 protein related to fat synthesis. These studies suggest that the S14 protein and lipogenesis may be important for thyroid hormone-induced and brown adipose tissue thermogenesis and that stimulation of these functions in hypothyroid brown fat is a consequence of decreased thyroid hormone-induced thermogenesis elsewhere.« less

Ferritin gene transcription is regulated by iron in soybean cell cultures.

PubMed Central

Lescure, A M; Proudhon, D; Pesey, H; Ragland, M; Theil, E C; Briat, J F

1991-01-01

Iron-regulated ferritin synthesis in animals is dominated by translational control of stored mRNA; iron-induced transcription of ferritin genes, when it occurs, changes the subunit composition of ferritin mRNA and protein and is coupled to translational control. Ferritins in plants and animals have evolved from a common progenitor, based on the similarity of protein sequence; however, sequence divergence occurs in the C termini; structure prediction suggests that plant ferritin has the E-helix, which, in horse ferritin, forms a large channel at the tetrameric interface. In contemporary plants, a transit peptide is encoded by ferritin mRNA to target the protein to plastids. Iron-regulated synthesis of ferritin in plants and animals appears to be very different since the 50- to 60-fold increases of ferritin protein, previously observed to be induced by iron in cultured soybean cells, is accompanied by an equivalent accumulation of hybridizable ferritin mRNA and by increased transcription of ferritin genes. Ferritin mRNA from iron-induced cells and the constitutive ferritin mRNA from soybean hypocotyls are identical. The iron-induced protein is translocated normally to plastids. Differences in animal ferritin structure coincide with the various iron storage functions (reserve for iron proteins and detoxification). In contrast, the constancy of structure of soybean ferritin, iron-induced and constitutive, coupled with the potential for vacuolar storage of excess iron in plants suggest that rapid synthesis of ferritin from a stored ferritin mRNA may not be needed in plants for detoxification of iron. Images PMID:1896472

High-throughput sequencing identification and characterization of potentially adhesion-related small RNAs in Streptococcus mutans.

PubMed

Zhu, Wenhui; Liu, Shanshan; Liu, Jia; Zhou, Yan; Lin, Huancai

2018-05-01

Adherence capacity is one of the principal virulence factors of Streptococcus mutans, and adhesion virulence factors are controlled by small RNAs (sRNAs) at the post-transcriptional level in various bacteria. Here, we aimed to identify and decipher putative adhesion-related sRNAs in clinical strains of S. mutans. RNA deep-sequencing was performed to identify potential sRNAs under different adhesion conditions. The expression of sRNAs was analysed by quantitative real-time PCR (qRT-PCR), and bioinformatic methods were used to predict the functional characteristics of sRNAs. A total of 736 differentially expressed candidate sRNAs were predicted, and these included 352 sRNAs located on the antisense to mRNA (AM) and 384 sRNAs in intergenic regions (IGRs). The top 7 differentially expressed sRNAs were successfully validated by qRT-PCR in UA159, and 2 of these were further confirmed in 100 clinical isolates. Moreover, the sequences of two sRNAs were conserved in other Streptococcus species, indicating a conserved role in such closely related species. A good correlation between the expression of sRNAs and the adhesion of 100 clinical strains was observed, which, combined with GO and KEGG, provides a perspective for the comprehension of sRNA function annotation. This study revealed a multitude of novel putative adhesion-related sRNAs in S. mutans and contributed to a better understanding of information concerning the transcriptional regulation of adhesion in S. mutans.

Comparison of ribosomal RNA removal methods for transcriptome sequencing workflows in teleost fish

USDA-ARS?s Scientific Manuscript database

RNA sequencing (RNA-Seq) is becoming the standard for transcriptome analysis. Removal of contaminating ribosomal RNA (rRNA) is a priority in the preparation of libraries suitable for sequencing. rRNAs are commonly removed from total RNA via either mRNA selection or rRNA depletion. These methods have...

Enhanced sensitivity for detection of low-level germline mosaic RB1 mutations in sporadic retinoblastoma cases using deep semiconductor sequencing.

PubMed

Chen, Zhao; Moran, Kimberly; Richards-Yutz, Jennifer; Toorens, Erik; Gerhart, Daniel; Ganguly, Tapan; Shields, Carol L; Ganguly, Arupa

2014-03-01

Sporadic retinoblastoma (RB) is caused by de novo mutations in the RB1 gene. Often, these mutations are present as mosaic mutations that cannot be detected by Sanger sequencing. Next-generation deep sequencing allows unambiguous detection of the mosaic mutations in lymphocyte DNA. Deep sequencing of the RB1 gene on lymphocyte DNA from 20 bilateral and 70 unilateral RB cases was performed, where Sanger sequencing excluded the presence of mutations. The individual exons of the RB1 gene from each sample were amplified, pooled, ligated to barcoded adapters, and sequenced using semiconductor sequencing on an Ion Torrent Personal Genome Machine. Six low-level mosaic mutations were identified in bilateral RB and four in unilateral RB cases. The incidence of low-level mosaic mutation was estimated to be 30% and 6%, respectively, in sporadic bilateral and unilateral RB cases, previously classified as mutation negative. The frequency of point mutations detectable in lymphocyte DNA increased from 96% to 97% for bilateral RB and from 13% to 18% for unilateral RB. The use of deep sequencing technology increased the sensitivity of the detection of low-level germline mosaic mutations in the RB1 gene. This finding has significant implications for improved clinical diagnosis, genetic counseling, surveillance, and management of RB. © 2013 WILEY PERIODICALS, INC.

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.

Draft Genome Sequence of Deep-Sea Alteromonas sp. Strain V450 Isolated from the Marine Sponge Leiodermatium sp.

PubMed Central

Barrett, Nolan H.; McCarthy, Peter J.

2017-01-01

ABSTRACT The proteobacterium Alteromonas sp. strain V450 was isolated from the Atlantic deep-sea sponge Leiodermatium sp. Here, we report the draft genome sequence of this strain, with a genome size of approx. 4.39 Mb and a G+C content of 44.01%. The results will aid deep-sea microbial ecology, evolution, and sponge-microbe association studies. PMID:28153886

Translation of the mRNA of the maize transcriptional activator Opaque-2 is inhibited by upstream open reading frames present in the leader sequence.

PubMed Central

Lohmer, S; Maddaloni, M; Motto, M; Salamini, F; Thompson, R D

1993-01-01

The protein encoded by the Opaque-2 (O2) gene is a transcription factor, translated from an mRNA that possesses an unusually long 5' leader sequence containing three upstream open reading frames (uORFs). The efficiency of translation of O2 mRNA has been tested in vivo by a transient assay in which the level of activation of the b32 promoter, a natural target of O2 protein, is measured. We show that uORF-less O2 alleles possess a higher transactivation value than the wild-type allele and that the reduction in transactivation due to the uORFs is a cis-dominant effect. The data presented indicate that both uORF1 and uORF2 are involved in the reducing effect and suggest that both are likely to be translated. PMID:8439744

Signal sequence-independent targeting of MID2 mRNA to the endoplasmic reticulum by the yeast RNA-binding protein Khd1p.

PubMed

Syed, Muhammad Ibrahim; Moorthy, Balaji T; Jenner, Andreas; Fetka, Ingrid; Jansen, Ralf-Peter

2018-05-17

Localization of mRNAs depends on specific RNA-binding proteins (RBPs) and critically contributes not only to cell polarization but also to basal cell function. The yeast RBP Khd1p binds to several hundred mRNAs, the majority of which encodes secreted or membrane proteins. We demonstrate that a subfraction of Khd1p associates with artificial liposomes and endoplasmic reticulum (ER), and that Khd1p endomembrane association is partially dependent on its binding to RNA. ER targeting of at least two mRNAs, MID2 and SLG1/WSC1, requires KHD1 but is independent of their translation. Together, our results suggest interdependence of Khd1p and mRNA for their targeting to the ER and presents additional evidence for signal sequence-independent, RBP-mediated mRNA targeting. © 2018 Federation of European Biochemical Societies.

Identification and expression analysis of cDNA encoding insulin-like growth factor 2 in horses

PubMed Central

KIKUCHI, Kohta; SASAKI, Keisuke; AKIZAWA, Hiroki; TSUKAHARA, Hayato; BAI, Hanako; TAKAHASHI, Masashi; NAMBO, Yasuo; HATA, Hiroshi; KAWAHARA, Manabu

2017-01-01

Insulin-like growth factor 2 (IGF2) is responsible for a broad range of physiological processes during fetal development and adulthood, but genomic analyses of IGF2 containing the 5ʹ- and 3ʹ-untranslated regions (UTRs) in equines have been limited. In this study, we characterized the IGF2 mRNA containing the UTRs, and determined its expression pattern in the fetal tissues of horses. The complete equine IGF2 mRNA sequence harboring another exon approximately 2.8 kb upstream from the canonical transcription start site was identified as a new transcript variant. As this upstream exon did not contain the start codon, the amino acid sequence was identical to the canonical variant. Analysis of the deduced amino acid sequence revealed that the protein possessed two major domains, IlGF and IGF2_C, and analysis of IGF2 sequence polymorphism in fetal tissues of Hokkaido native horse and Thoroughbreds revealed a single nucleotide polymorphism (T to C transition) at position 398 in Thoroughbreds, which caused an amino acid substitution at position 133 in the IGF2 sequence. Furthermore, the expression pattern of the IGF2 mRNA in the fetal tissues of horses was determined for the first time, and was found to be consistent with those of other species. Taken together, these results suggested that the transcriptional and translational products of the IGF2 gene have conserved functions in the fetal development of mammals, including horses. PMID:29151450

The ribosome as a missing link in prebiotic evolution II: Ribosomes encode ribosomal proteins that bind to common regions of their own mRNAs and rRNAs.

PubMed

Root-Bernstein, Robert; Root-Bernstein, Meredith

2016-05-21

We have proposed that the ribosome may represent a missing link between prebiotic chemistries and the first cells. One of the predictions that follows from this hypothesis, which we test here, is that ribosomal RNA (rRNA) must have encoded the proteins necessary for ribosomal function. In other words, the rRNA also functioned pre-biotically as mRNA. Since these ribosome-binding proteins (rb-proteins) must bind to the rRNA, but the rRNA also functioned as mRNA, it follows that rb-proteins should bind to their own mRNA as well. This hypothesis can be contrasted to a "null" hypothesis in which rb-proteins evolved independently of the rRNA sequences and therefore there should be no necessary similarity between the rRNA to which rb-proteins bind and the mRNA that encodes the rb-protein. Five types of evidence reported here support the plausibility of the hypothesis that the mRNA encoding rb-proteins evolved from rRNA: (1) the ubiquity of rb-protein binding to their own mRNAs and autogenous control of their own translation; (2) the higher-than-expected incidence of Arginine-rich modules associated with RNA binding that occurs in rRNA-encoded proteins; (3) the fact that rRNA-binding regions of rb-proteins are homologous to their mRNA binding regions; (4) the higher than expected incidence of rb-protein sequences encoded in rRNA that are of a high degree of homology to their mRNA as compared with a random selection of other proteins; and (5) rRNA in modern prokaryotes and eukaryotes encodes functional proteins. None of these results can be explained by the null hypothesis that assumes independent evolution of rRNA and the mRNAs encoding ribosomal proteins. Also noteworthy is that very few proteins bind their own mRNAs that are not associated with ribosome function. Further tests of the hypothesis are suggested: (1) experimental testing of whether rRNA-encoded proteins bind to rRNA at their coding sites; (2) whether tRNA synthetases, which are also known to bind to their own mRNAs, are encoded by the tRNA sequences themselves; (3) and the prediction that archaeal and prokaryotic (DNA-based) genomes were built around rRNA "genes" so that rRNA-related sequences will be found to make up an unexpectedly high proportion of these genomes. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Molecular cloning and functional analysis of MRLC2 in Tianfu, Boer, and Chengdu Ma goats.

PubMed

Xu, H G; Xu, G Y; Wan, L; Ma, J

2013-03-15

To determine the molecular basis of heterosis in goats, fluorescence quantitative polymerase chain reaction (PCR) was performed to investigate myosin-regulatory light chain 2 (MRLC2) gene expression in the longissimus dorsi muscle tissues of the Tianfu goat and its parents, the Boer and Chengdu Ma goats. The goat MRLC2 gene was differentially expressed in the crossbreed, and the purebred mRNA were isolated and identified using fluorescence quantitative reverse transcription-PCR (RT-PCR). The complete coding sequence of MRLC2 was obtained using the cDNA method, and the full-length coding sequence consisted of 513 bp encoding 172 amino acids. The EF-hand superfamily domain of the MRLC2 protein is well conserved in caprine and other animals. The deduced amino acid sequence of MRLC2 shared significant identity with MRLC2 from other mammals. Phylogenetic tree analysis revealed that the MRLC2 protein was closely related to MRLC2 in other mammals. Several predicted miRNA target sites were found in the coding sequence of caprine MRLC2 mRNA. Analysis by RT-PCR showed that MRLC2 mRNA was present in the heart, stomach, liver, spleen, lung, small intestine, kidney, leg muscle, abdominal muscle, and longissimus dorsi muscles. In particular, the high expression of MRLC2 mRNA was detected in the longissimus dorsi, leg muscle, abdominal muscle, stomach, and heart, but low levels of expression were also observed in the liver, spleen, lung, small intestine, and kidney. The expression of the MRLC2 gene was upregulated in the longissimus dorsi muscle of Boer and Tianfu goats, and it was moderately upregulated in Chengdu Ma goats.

The nif Gene Operon of the Methanogenic Archaeon Methanococcus maripaludis

PubMed Central

Kessler, Peter S.; Blank, Carrine; Leigh, John A.

1998-01-01

Nitrogen fixation occurs in two domains, Archaea and Bacteria. We have characterized a nif (nitrogen fixation) gene cluster in the methanogenic archaeon Methanococcus maripaludis. Sequence analysis revealed eight genes, six with sequence similarity to known nif genes and two with sequence similarity to glnB. The gene order, nifH, ORF105 (similar to glnB), ORF121 (similar to glnB), nifD, nifK, nifE, nifN, and nifX, was the same as that found in part in other diazotrophic methanogens and except for the presence of the glnB-like genes, also resembled the order found in many members of the Bacteria. Using transposon insertion mutagenesis, we determined that an 8-kb region required for nitrogen fixation corresponded to the nif gene cluster. Northern analysis revealed the presence of either a single 7.6-kb nif mRNA transcript or 10 smaller mRNA species containing portions of the large transcript. Polar effects of transposon insertions demonstrated that all of these mRNAs arose from a single promoter region, where transcription initiated 80 bp 5′ to nifH. Distinctive features of the nif gene cluster include the presence of the six primary nif genes in a single operon, the placement of the two glnB-like genes within the cluster, the apparent physical separation of the cluster from any other nif genes that might be in the genome, the fragmentation pattern of the mRNA, and the regulation of expression by a repression mechanism described previously. Our study and others with methanogenic archaea reporting multiple mRNAs arising from gene clusters with only a single putative promoter sequence suggest that mRNA processing following transcription may be a common occurrence in methanogens. PMID:9515920

Sequence and expression analyses of porcine ISG15 and ISG43 genes.

PubMed

Huang, Jiangnan; Zhao, Shuhong; Zhu, Mengjin; Wu, Zhenfang; Yu, Mei

2009-08-01

The coding sequences of porcine interferon-stimulated gene 15 (ISG15) and the interferon-stimulated gene (ISG43) were cloned from swine spleen mRNA. The amino acid sequences deduced from porcine ISG15 and ISG43 genes coding sequence shared 24-75% and 29-83% similarity with ISG15s and ISG43s from other vertebrates, respectively. Structural analyses revealed that porcine ISG15 comprises two ubiquitin homologues motifs (UBQ) domain and a conserved C-terminal LRLRGG conjugating motif. Porcine ISG43 contains an ubiquitin-processing proteases-like domain. Phylogenetic analyses showed that porcine ISG15 and ISG43 were mostly related to rat ISG15 and cattle ISG43, respectively. Using quantitative real-time PCR assay, significant increased expression levels of porcine ISG15 and ISG43 genes were detected in porcine kidney endothelial cells (PK15) cells treated with poly I:C. We also observed the enhanced mRNA expression of three members of dsRNA pattern-recognition receptors (PRR), TLR3, DDX58 and IFIH1, which have been reported to act as critical receptors in inducing the mRNA expression of ISG15 and ISG43 genes. However, we did not detect any induced mRNA expression of IFNalpha and IFNbeta, suggesting that transcriptional activations of ISG15 and ISG43 were mediated through IFN-independent signaling pathway in the poly I:C treated PK15 cells. Association analyses in a Landrace pig population revealed that ISG15 c.347T>C (BstUI) polymorphism and the ISG43 c.953T>G (BccI) polymorphism were significantly associated with hematological parameters and immune-related traits.

Characterization of Bunyamwera virus S RNA that is transcribed and replicated by the L protein expressed from recombinant vaccinia virus.

PubMed

Jin, H; Elliott, R M

1993-03-01

Analysis of the 5' termini of Bunyamwera virus S segment mRNAs by cloning and sequence analysis revealed the presence of nonviral, heterogeneous sequences 12 to 17 bases long. This is similar to reports for other members of the family Bunyaviridae and is taken to indicate that mRNA transcription is primed by a "cap-snatching" mechanism. The 3' end of the Bunyamwera virus S mRNA was mapped, by using an RNase protection assay, to 100 to 110 nucleotides upstream of the 3' end of the template. Previously we reported expression of the Bunyamwera virus L (polymerase) protein by recombinant vaccinia virus and demonstrated that the recombinant L protein was functional in terms of RNA synthesis activity in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, J. Virol. 65: 4182-4189, 1991). In the present study we further analyze the RNAs made by using this system and show that positive-sense RNAs contain 5' nonviral sequences. Hence the initiation of mRNA transcription by the recombinant L protein resembles that seen during authentic bunyavirus infection and suggests that the L protein has the endonuclease activity which generates the primers. Some of these positive-sense transcripts terminated at the mRNA termination site, but the majority read through to the end of the template. No primer sequences were found at the 5' terminal of negative-sense RNAs. The recombinant L protein was able to replicate negative-sense RNA supplied by transfected virion-derived nucleocapsids, and both positive- and negative-sense RNAs were synthesized. These results indicate that the recombinant L protein has both transcriptase and replicase activities.

Characterization of Bunyamwera virus S RNA that is transcribed and replicated by the L protein expressed from recombinant vaccinia virus.

PubMed Central

Jin, H; Elliott, R M

1993-01-01

Analysis of the 5' termini of Bunyamwera virus S segment mRNAs by cloning and sequence analysis revealed the presence of nonviral, heterogeneous sequences 12 to 17 bases long. This is similar to reports for other members of the family Bunyaviridae and is taken to indicate that mRNA transcription is primed by a "cap-snatching" mechanism. The 3' end of the Bunyamwera virus S mRNA was mapped, by using an RNase protection assay, to 100 to 110 nucleotides upstream of the 3' end of the template. Previously we reported expression of the Bunyamwera virus L (polymerase) protein by recombinant vaccinia virus and demonstrated that the recombinant L protein was functional in terms of RNA synthesis activity in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, J. Virol. 65: 4182-4189, 1991). In the present study we further analyze the RNAs made by using this system and show that positive-sense RNAs contain 5' nonviral sequences. Hence the initiation of mRNA transcription by the recombinant L protein resembles that seen during authentic bunyavirus infection and suggests that the L protein has the endonuclease activity which generates the primers. Some of these positive-sense transcripts terminated at the mRNA termination site, but the majority read through to the end of the template. No primer sequences were found at the 5' terminal of negative-sense RNAs. The recombinant L protein was able to replicate negative-sense RNA supplied by transfected virion-derived nucleocapsids, and both positive- and negative-sense RNAs were synthesized. These results indicate that the recombinant L protein has both transcriptase and replicase activities. Images PMID:8437222

Combinatory RNA-Sequencing Analyses Reveal a Dual Mode of Gene Regulation by ADAR1 in Gastric Cancer.

PubMed

Cho, Charles J; Jung, Jaeeun; Jiang, Lushang; Lee, Eun Ji; Kim, Dae-Soo; Kim, Byung Sik; Kim, Hee Sung; Jung, Hwoon-Yong; Song, Ho-June; Hwang, Sung Wook; Park, Yangsoon; Jung, Min Kyo; Pack, Chan Gi; Myung, Seung-Jae; Chang, Suhwan

2018-04-25

Adenosine deaminase acting on RNA 1 (ADAR1) is known to mediate deamination of adenosine-to-inosine through binding to double-stranded RNA, the phenomenon known as RNA editing. Currently, the function of ADAR1 in gastric cancer is unclear. This study was aimed at investigating RNA editing-dependent and editing-independent functions of ADAR1 in gastric cancer, especially focusing on its influence on editing of 3' untranslated regions (UTRs) and subsequent changes in expression of messenger RNAs (mRNAs) as well as microRNAs (miRNAs). RNA-sequencing and small RNA-sequencing were performed on AGS and MKN-45 cells with a stable ADAR1 knockdown. Changed frequencies of editing and mRNA and miRNA expression were then identified by bioinformatic analyses. Targets of RNA editing were further validated in patients' samples. In the Alu region of both gastric cell lines, editing was most commonly of the A-to-I type in 3'-UTR or intron. mRNA and protein levels of PHACTR4 increased in ADAR1 knockdown cells, because of the loss of seed sequences in 3'-UTR of PHACTR4 mRNA that are required for miRNA-196a-3p binding. Immunohistochemical analyses of tumor and paired normal samples from 16 gastric cancer patients showed that ADAR1 expression was higher in tumors than in normal tissues and inversely correlated with PHACTR4 staining. On the other hand, decreased miRNA-148a-3p expression in ADAR1 knockdown cells led to increased mRNA and protein expression of NFYA, demonstrating ADAR1's editing-independent function. ADAR1 regulates post-transcriptional gene expression in gastric cancer through both RNA editing-dependent and editing-independent mechanisms.

Decoy Oligonucleotide Rescues IGF1R Expression from MicroRNA-223 Suppression

PubMed Central

Wang, Rong; He, Bao Mei; Qi, Bing; Xu, Chang Jun; Wu, Xing Zhong

2013-01-01

A mature miRNA generally suppresses hundreds of mRNA targets. To evaluate the selective effect of synthetic oligonucleotide decoys on hsa-miR-223 activity, reporters containing 3’ untranslated regions (UTR) of IGF1R, FOXO1, POLR3G, FOXO3, CDC27, FBXW7 and PAXIP1 mRNAs were constructed for the luciferase assay. The oligonucleotide decoys were designed and synthesized according to mature miR-223 sequence and its target mRNA sequence. Quantitative RT-PCR & western analysis were used to measure miR-223-targeted mRNA expression, Interestingly, apart from the antisense oligonucleotide, decoy nucleotides which were complementary to the 5’, central or 3’ region of mature miR-223 suppressed miR-223 targeting the 3’UTR of IGF1R, FOXO1, FOXO3, CDC27, POLR3G, and FBXW7 mRNAs and rescued the expression of these genes to varying degrees from miR-223 suppression at both mRNA and protein levels. All decoys had no effect on PAXIP1 which was not targeted by miR-223. The decoy 1 that was based on the sequence of IGF1R 3’UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4. Decoy 1 also rescued the expression of FOXO3 and POLR3G of which their 3’UTRs have similar binding sites for miR-223 with IGF1R 3’UTR. However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression. These data support that the sequence-specific decoy oligonucleotides might represent exogenous competing RNA which selectively inhibits microRNA targeting. PMID:24324762

Decoy oligonucleotide rescues IGF1R expression from MicroRNA-223 suppression.

PubMed

Wu, Li Hui; Cai, Qian Qian; Dong, Yi Wei; Wang, Rong; He, Bao Mei; Qi, Bing; Xu, Chang Jun; Wu, Xing Zhong

2013-01-01

A mature miRNA generally suppresses hundreds of mRNA targets. To evaluate the selective effect of synthetic oligonucleotide decoys on hsa-miR-223 activity, reporters containing 3' untranslated regions (UTR) of IGF1R, FOXO1, POLR3G, FOXO3, CDC27, FBXW7 and PAXIP1 mRNAs were constructed for the luciferase assay. The oligonucleotide decoys were designed and synthesized according to mature miR-223 sequence and its target mRNA sequence. Quantitative RT-PCR & western analysis were used to measure miR-223-targeted mRNA expression, Interestingly, apart from the antisense oligonucleotide, decoy nucleotides which were complementary to the 5', central or 3' region of mature miR-223 suppressed miR-223 targeting the 3'UTR of IGF1R, FOXO1, FOXO3, CDC27, POLR3G, and FBXW7 mRNAs and rescued the expression of these genes to varying degrees from miR-223 suppression at both mRNA and protein levels. All decoys had no effect on PAXIP1 which was not targeted by miR-223. The decoy 1 that was based on the sequence of IGF1R 3'UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4. Decoy 1 also rescued the expression of FOXO3 and POLR3G of which their 3'UTRs have similar binding sites for miR-223 with IGF1R 3'UTR. However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression. These data support that the sequence-specific decoy oligonucleotides might represent exogenous competing RNA which selectively inhibits microRNA targeting.

Identification of Prostate Cancer-Specific microDNAs

DTIC Science & Technology

2016-02-01

circular DNA by rolling circle amplification (RCA) and then amplified DNA fragments were subject to deep sequencing. Deep sequencing of the...demonstrate the existence of microDNAs in prostate cancer. We adopted multiple displacement amplification (MDA) with random 2 primers for enriched...prostate cancer cells through multiple displacement amplification and next generation sequencing. R e la ti v e c e ll g ro w th ( % ) 0 20

Sequence-specific bias correction for RNA-seq data using recurrent neural networks.

PubMed

Zhang, Yao-Zhong; Yamaguchi, Rui; Imoto, Seiya; Miyano, Satoru

2017-01-25

The recent success of deep learning techniques in machine learning and artificial intelligence has stimulated a great deal of interest among bioinformaticians, who now wish to bring the power of deep learning to bare on a host of bioinformatical problems. Deep learning is ideally suited for biological problems that require automatic or hierarchical feature representation for biological data when prior knowledge is limited. In this work, we address the sequence-specific bias correction problem for RNA-seq data redusing Recurrent Neural Networks (RNNs) to model nucleotide sequences without pre-determining sequence structures. The sequence-specific bias of a read is then calculated based on the sequence probabilities estimated by RNNs, and used in the estimation of gene abundance. We explore the application of two popular RNN recurrent units for this task and demonstrate that RNN-based approaches provide a flexible way to model nucleotide sequences without knowledge of predetermined sequence structures. Our experiments show that training a RNN-based nucleotide sequence model is efficient and RNN-based bias correction methods compare well with the-state-of-the-art sequence-specific bias correction method on the commonly used MAQC-III data set. RNNs provides an alternative and flexible way to calculate sequence-specific bias without explicitly pre-determining sequence structures.

NF-κB controls four genes encoding core enzymes of tricarboxylic acid cycle.

PubMed

Zhou, Fei; Xu, Xinhui; Wu, Jian; Wang, Danyang; Wang, Jinke

2017-07-20

NF-κB may promote tumor progression by altering cell metabolism. Hence, finding its target genes that are involved in cell metabolism is helpful for understanding its role in tumor growth. Here we discovered four metabolism-related target genes of this transcription factor. By analyzing a chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) data that characterizing the global binding sites (BSs) of NF-κB RelA in the TNFα-stimulated HeLa cells, we found that four genes that encode core enzymes of the tricarboxylic acid (TCA) cycle, including IDH1, IDH3A, ACO2, and SUCLA2, were multiply bound by this transcription factor. The subsequent bioinformatic analysis revealed that the NF-κB BSs contained many canonical κB sequences and the NF-κB-like DNA-binding motifs. Detection of ChIPed DNA with polymerase chain reaction (ChIP-PCR) also indicated that the NF-κB BSs were bound by NF-κB in both TNFα-treated HeLa and HepG2 cells. The reporter construct showed that the NF-κB BSs could activate the luciferase expression in cells in a NF-κB-specific manner. The quantitative PCR and Western blot detections demonstrated that NF-κB could regulate the expressions of IDH1, IDH3A, and ACO2 genes at both mRNA and protein levels and that of SUCLA2 gene at mRNA level in the TNFα-treated HeLa and HepG2 cells. Based on these investigations we identified the four genes as new target genes of NF-κB. The finding provides new insights into the role of NF-κB in cellular energetic metabolism, which may be beneficial for understanding the metabolic physiology of tumor growth. Copyright © 2017. Published by Elsevier B.V.

An Efficient Strategy of Screening for Pathogens in Wild-Caught Ticks and Mosquitoes by Reusing Small RNA Deep Sequencing Data

PubMed Central

An, Xiaoping; Fan, Hang; Ma, Maijuan; Anderson, Benjamin D.; Jiang, Jiafu; Liu, Wei; Cao, Wuchun; Tong, Yigang

2014-01-01

This paper explored our hypothesis that sRNA (18∼30 bp) deep sequencing technique can be used as an efficient strategy to identify microorganisms other than viruses, such as prokaryotic and eukaryotic pathogens. In the study, the clean reads derived from the sRNA deep sequencing data of wild-caught ticks and mosquitoes were compared against the NCBI nucleotide collection (non-redundant nt database) using Blastn. The blast results were then analyzed with in-house Python scripts. An empirical formula was proposed to identify the putative pathogens. Results showed that not only viruses but also prokaryotic and eukaryotic species of interest can be screened out and were subsequently confirmed with experiments. Specially, a novel Rickettsia spp. was indicated to exist in Haemaphysalis longicornis ticks collected in Beijing. Our study demonstrated the reuse of sRNA deep sequencing data would have the potential to trace the origin of pathogens or discover novel agents of emerging/re-emerging infectious diseases. PMID:24618575

Draft Genome Sequence of Deep-Sea Alteromonas sp. Strain V450 Isolated from the Marine Sponge Leiodermatium sp.

PubMed

Wang, Guojun; Barrett, Nolan H; McCarthy, Peter J

2017-02-02

The proteobacterium Alteromonas sp. strain V450 was isolated from the Atlantic deep-sea sponge Leiodermatium sp. Here, we report the draft genome sequence of this strain, with a genome size of approx. 4.39 Mb and a G+C content of 44.01%. The results will aid deep-sea microbial ecology, evolution, and sponge-microbe association studies. Copyright © 2017 Wang et al.

Genome-wide identification and functional prediction of nitrogen-responsive intergenic and intronic long non-coding RNAs in maize (Zea mays L.).

PubMed

Lv, Yuanda; Liang, Zhikai; Ge, Min; Qi, Weicong; Zhang, Tifu; Lin, Feng; Peng, Zhaohua; Zhao, Han

2016-05-11

Nitrogen (N) is an essential and often limiting nutrient to plant growth and development. Previous studies have shown that the mRNA expressions of numerous genes are regulated by nitrogen supplies; however, little is known about the expressed non-coding elements, for example long non-coding RNAs (lncRNAs) that control the response of maize (Zea mays L.) to nitrogen. LncRNAs are a class of non-coding RNAs larger than 200 bp, which have emerged as key regulators in gene expression. In this study, we surveyed the intergenic/intronic lncRNAs in maize B73 leaves at the V7 stage under conditions of N-deficiency and N-sufficiency using ribosomal RNA depletion and ultra-deep total RNA sequencing approaches. By integration with mRNA expression profiles and physiological evaluations, 7245 lncRNAs and 637 nitrogen-responsive lncRNAs were identified that exhibited unique expression patterns. Co-expression network analysis showed that the nitrogen-responsive lncRNAs were enriched mainly in one of the three co-expressed modules. The genes in the enriched module are mainly involved in NADH dehydrogenase activity, oxidative phosphorylation and the nitrogen compounds metabolic process. We identified a large number of lncRNAs in maize and illustrated their potential regulatory roles in response to N stress. The results lay the foundation for further in-depth understanding of the molecular mechanisms of lncRNAs' role in response to nitrogen stresses.

miRBase: integrating microRNA annotation and deep-sequencing data.

PubMed

Kozomara, Ana; Griffiths-Jones, Sam

2011-01-01

miRBase is the primary online repository for all microRNA sequences and annotation. The current release (miRBase 16) contains over 15,000 microRNA gene loci in over 140 species, and over 17,000 distinct mature microRNA sequences. Deep-sequencing technologies have delivered a sharp rise in the rate of novel microRNA discovery. We have mapped reads from short RNA deep-sequencing experiments to microRNAs in miRBase and developed web interfaces to view these mappings. The user can view all read data associated with a given microRNA annotation, filter reads by experiment and count, and search for microRNAs by tissue- and stage-specific expression. These data can be used as a proxy for relative expression levels of microRNA sequences, provide detailed evidence for microRNA annotations and alternative isoforms of mature microRNAs, and allow us to revisit previous annotations. miRBase is available online at: http://www.mirbase.org/.

Transcriptome sequences resolve deep relationships of the grape family.

PubMed

Wen, Jun; Xiong, Zhiqiang; Nie, Ze-Long; Mao, Likai; Zhu, Yabing; Kan, Xian-Zhao; Ickert-Bond, Stefanie M; Gerrath, Jean; Zimmer, Elizabeth A; Fang, Xiao-Dong

2013-01-01

Previous phylogenetic studies of the grape family (Vitaceae) yielded poorly resolved deep relationships, thus impeding our understanding of the evolution of the family. Next-generation sequencing now offers access to protein coding sequences very easily, quickly and cost-effectively. To improve upon earlier work, we extracted 417 orthologous single-copy nuclear genes from the transcriptomes of 15 species of the Vitaceae, covering its phylogenetic diversity. The resulting transcriptome phylogeny provides robust support for the deep relationships, showing the phylogenetic utility of transcriptome data for plants over a time scale at least since the mid-Cretaceous. The pros and cons of transcriptome data for phylogenetic inference in plants are also evaluated.

Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region

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

Pelletier, J.; Kaplan, G.; Racaniello, V.R.

1988-03-01

Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, the authors determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. They found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element mapsmore » between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. They also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation.« less

Draft Genome Sequence of Pseudomonas oceani DSM 100277T, a Deep-Sea Bacterium

PubMed Central

2018-01-01

ABSTRACT Pseudomonas oceani DSM 100277T was isolated from deep seawater in the Okinawa Trough at 1390 m. P. oceani belongs to the Pseudomonas pertucinogena group. Here, we report the draft genome sequence of P. oceani, which has an estimated size of 4.1 Mb and exhibits 3,790 coding sequences, with a G+C content of 59.94 mol%. PMID:29650573

A Bioinformatic Pipeline for Monitoring of the Mutational Stability of Viral Drug Targets with Deep-Sequencing Technology.

PubMed

Kravatsky, Yuri; Chechetkin, Vladimir; Fedoseeva, Daria; Gorbacheva, Maria; Kravatskaya, Galina; Kretova, Olga; Tchurikov, Nickolai

2017-11-23

The efficient development of antiviral drugs, including efficient antiviral small interfering RNAs (siRNAs), requires continuous monitoring of the strict correspondence between a drug and the related highly variable viral DNA/RNA target(s). Deep sequencing is able to provide an assessment of both the general target conservation and the frequency of particular mutations in the different target sites. The aim of this study was to develop a reliable bioinformatic pipeline for the analysis of millions of short, deep sequencing reads corresponding to selected highly variable viral sequences that are drug target(s). The suggested bioinformatic pipeline combines the available programs and the ad hoc scripts based on an original algorithm of the search for the conserved targets in the deep sequencing data. We also present the statistical criteria for the threshold of reliable mutation detection and for the assessment of variations between corresponding data sets. These criteria are robust against the possible sequencing errors in the reads. As an example, the bioinformatic pipeline is applied to the study of the conservation of RNA interference (RNAi) targets in human immunodeficiency virus 1 (HIV-1) subtype A. The developed pipeline is freely available to download at the website http://virmut.eimb.ru/. Brief comments and comparisons between VirMut and other pipelines are also presented.

Metatranscriptomics of Soil Eukaryotic Communities.

PubMed

Yadav, Rajiv K; Bragalini, Claudia; Fraissinet-Tachet, Laurence; Marmeisse, Roland; Luis, Patricia

2016-01-01

Functions expressed by eukaryotic organisms in soil can be specifically studied by analyzing the pool of eukaryotic-specific polyadenylated mRNA directly extracted from environmental samples. In this chapter, we describe two alternative protocols for the extraction of high-quality RNA from soil samples. Total soil RNA or mRNA can be converted to cDNA for direct high-throughput sequencing. Polyadenylated mRNA-derived full-length cDNAs can also be cloned in expression plasmid vectors to constitute soil cDNA libraries, which can be subsequently screened for functional gene categories. Alternatively, the diversity of specific gene families can also be explored following cDNA sequence capture using exploratory oligonucleotide probes.

Identification of sequence motifs in oligonucleotides whose presence is correlated with antisense activity

PubMed Central

Matveeva, O. V.; Tsodikov, A. D.; Giddings, M.; Freier, S. M.; Wyatt, J. R.; Spiridonov, A. N.; Shabalina, S. A.; Gesteland, R. F.; Atkins, J. F.

2000-01-01

Design of antisense oligonucleotides targeting any mRNA can be much more efficient when several activity-enhancing motifs are included and activity-decreasing motifs are avoided. This conclusion was made after statistical analysis of data collected from >1000 experiments with phosphorothioate-modified oligonucleotides. Highly significant positive correlation between the presence of motifs CCAC, TCCC, ACTC, GCCA and CTCT in the oligonucleotide and its antisense efficiency was demonstrated. In addition, negative correlation was revealed for the motifs GGGG, ACTG, AAA and TAA. It was found that the likelihood of activity of an oligonucleotide against a desired mRNA target is sequence motif content dependent. PMID:10908347

Vascular endothelial cells express isoforms of protein kinase A inhibitor.

PubMed

Lum, Hazel; Hao, Zengping; Gayle, Dave; Kumar, Priyadarsini; Patterson, Carolyn E; Uhler, Michael D

2002-01-01

The expression and function of the endogenous inhibitor of cAMP-dependent protein kinase (PKI) in endothelial cells are unknown. In this study, overexpression of rabbit muscle PKI gene into endothelial cells inhibited the cAMP-mediated increase and exacerbated thrombin-induced decrease in endothelial barrier function. We investigated PKI expression in human pulmonary artery (HPAECs), foreskin microvessel (HMECs), and brain microvessel endothelial cells (HBMECs). RT-PCR using specific primers for human PKI alpha, human PKI gamma, and mouse PKI beta sequences detected PKI alpha and PKI gamma mRNA in all three cell types. Sequencing and BLAST analysis indicated that forward and reverse DNA strands for PKI alpha and PKI gamma were of >96% identity with database sequences. RNase protection assays showed protection of the 542 nucleotides in HBMEC and HPAEC PKI alpha mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKI gamma mRNA. Western blot analysis indicated that PKI gamma protein was detected in all three cell types, whereas PKI alpha was found in HBMECs. In summary, endothelial cells from three different vascular beds express PKI alpha and PKI gamma, which may be physiologically important in endothelial barrier function.

Base substitutions at scissile bond sites are sufficient to alter RNA-binding and cleavage activity of RNase III.

PubMed

Kim, Kyungsub; Sim, Se-Hoon; Jeon, Che Ok; Lee, Younghoon; Lee, Kangseok

2011-02-01

RNase III, a double-stranded RNA-specific endoribonuclease, degrades bdm mRNA via cleavage at specific sites. To better understand the mechanism of cleavage site selection by RNase III, we performed a genetic screen for sequences containing mutations at the bdm RNA cleavage sites that resulted in altered mRNA stability using a transcriptional bdm'-'cat fusion construct. While most of the isolated mutants showed the increased bdm'-'cat mRNA stability that resulted from the inability of RNase III to cleave the mutated sequences, one mutant sequence (wt-L) displayed in vivo RNA stability similar to that of the wild-type sequence. In vivo and in vitro analyses of the wt-L RNA substrate showed that it was cut only once on the RNA strand to the 5'-terminus by RNase III, while the binding constant of RNase III to this mutant substrate was moderately increased. A base substitution at the uncleaved RNase III cleavage site in wt-L mutant RNA found in another mutant lowered the RNA-binding affinity by 11-fold and abolished the hydrolysis of scissile bonds by RNase III. Our results show that base substitutions at sites forming the scissile bonds are sufficient to alter RNA cleavage as well as the binding activity of RNase III. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus.

PubMed

Belak, Zachery R; Ovsenek, Nicholas; Eskiw, Christopher H

2018-05-23

Yin-Yang 1 (YY1) is a highly conserved transcription factor possessing RNA-binding activity. A putative YY1 homologue was previously identified in the developmental model organism Strongylocentrotus purpuratus (the purple sea urchin) by genomic sequencing. We identified a high degree of sequence similarity with YY1 homologues of vertebrate origin which shared 100% protein sequence identity over the DNA- and RNA-binding zinc-finger region with high similarity in the N-terminal transcriptional activation domain. SpYY1 demonstrated identical DNA- and RNA-binding characteristics between Xenopus laevis and S. purpuratus indicating that it maintains similar functional and biochemical properties across widely divergent deuterostome species. SpYY1 binds to the consensus YY1 DNA element, and also to U-rich RNA sequences. Although we detected SpYY1 RNA-binding activity in ova lysates and observed cytoplasmic localization, SpYY1 was not associated with maternal mRNA in ova. SpYY1 expressed in Xenopus oocytes was excluded from the nucleus and associated with maternally expressed cytoplasmic mRNA molecules. These data demonstrate the existence of an YY1 homologue in S. purpuratus with similar structural and biochemical features to those of the well-studied vertebrate YY1; however, the data reveal major differences in the biological role of YY1 in the regulation of maternally expressed mRNA in the two species.

Deep Motif Dashboard: Visualizing and Understanding Genomic Sequences Using Deep Neural Networks

PubMed Central

Lanchantin, Jack; Singh, Ritambhara; Wang, Beilun; Qi, Yanjun

2018-01-01

Deep neural network (DNN) models have recently obtained state-of-the-art prediction accuracy for the transcription factor binding (TFBS) site classification task. However, it remains unclear how these approaches identify meaningful DNA sequence signals and give insights as to why TFs bind to certain locations. In this paper, we propose a toolkit called the Deep Motif Dashboard (DeMo Dashboard) which provides a suite of visualization strategies to extract motifs, or sequence patterns from deep neural network models for TFBS classification. We demonstrate how to visualize and understand three important DNN models: convolutional, recurrent, and convolutional-recurrent networks. Our first visualization method is finding a test sequence’s saliency map which uses first-order derivatives to describe the importance of each nucleotide in making the final prediction. Second, considering recurrent models make predictions in a temporal manner (from one end of a TFBS sequence to the other), we introduce temporal output scores, indicating the prediction score of a model over time for a sequential input. Lastly, a class-specific visualization strategy finds the optimal input sequence for a given TFBS positive class via stochastic gradient optimization. Our experimental results indicate that a convolutional-recurrent architecture performs the best among the three architectures. The visualization techniques indicate that CNN-RNN makes predictions by modeling both motifs as well as dependencies among them. PMID:27896980

Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

PubMed Central

Kilimann, M W; DeGennaro, L J

1985-01-01

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. PMID:3933975

Heparanase mRNA expression and point mutation in hepatocellular carcinoma

PubMed Central

Chen, Xiao-Peng; Liu, Yin-Bib; Rui, Jing; Peng, Shu-You; Peng, Cheng-Hong; Zhou, Zi-Yan; Shi, Liang-Hui; Shen, Hong-Wei; Xu, Bin

2004-01-01

AIM: To explore the expression of heparanase mRNA and point mutation in hepatocellular carcinoma (HCC). METHODS: Reverse transcription polymerase chain reaction was used to measure the expression of heparanase mRNA in the primary tumor tissues and surrounding liver tissues of 33 HCC patients. T-A cloning and sequencing were used to detect whether there was any mutation in the amplified PCR products. RESULTS: The expression of heparanase mRNA was positive in 16 primary tumor tissues of HCC, and the positive rate was 48.5%, which was significantly higher than that in the surrounding liver parenchyma (P < 0.01). The positive rate for heparanase gene in high-tendency to metastatic recurrence group (71.4%, 10/14) was obviously higher than that in low-tendency to metastatic recurrence group (31.6%, 6/19) (P = 0.023). The positive rate for heparanase gene in patients with metastatic recurrence during postoperative follow-up (78.6%, 11/14) was also significantly higher than that in those without metastatic recurrence (21.4%, 3/14) (P = 0.003). Sequence analysis of the HPA PCR products was made in 7 patients, and 2-point mutations were found in 4 patients, one of which was sense mutation, neither base insertion nor deletion was detected. The mutation rate was 57.1% (4/7). CONCLUSION: The expression rate of heparanase mRNA increases in HCC, and HPA mRNA may be one of the reliable markers for the metastatic activity gained by the liver tumor cells and could be used clinically in predicting metastatic recurrence of HCC. Point mutation may be one of the causes for enhanced heparanase mRNA expression. PMID:15334672

Aftershock occurrence rate decay for individual sequences and catalogs

NASA Astrophysics Data System (ADS)

Nyffenegger, Paul A.

One of the earliest observations of the Earth's seismicity is that the rate of aftershock occurrence decays with time according to a power law commonly known as modified Omori-law (MOL) decay. However, the physical reasons for aftershock occurrence and the empirical decay in rate remain unclear despite numerous models that yield similar rate decay behavior. Key problems in relating the observed empirical relationship to the physical conditions of the mainshock and fault are the lack of studies including small magnitude mainshocks and the lack of uniformity between studies. We use simulated aftershock sequences to investigate the factors which influence the maximum likelihood (ML) estimate of the Omori-law p value, the parameter describing aftershock occurrence rate decay, for both individual aftershock sequences and "stacked" or superposed sequences. Generally the ML estimate of p is accurate, but since the ML estimated uncertainty is unaffected by whether the sequence resembles an MOL model, a goodness-of-fit test such as the Anderson-Darling statistic is necessary. While stacking aftershock sequences permits the study of entire catalogs and sequences with small aftershock populations, stacking introduces artifacts. The p value for stacked sequences is approximately equal to the mean of the individual sequence p values. We apply single-link cluster analysis to identify all aftershock sequences from eleven regional seismicity catalogs. We observe two new mathematically predictable empirical relationships for the distribution of aftershock sequence populations. The average properties of aftershock sequences are not correlated with tectonic environment, but aftershock populations and p values do show a depth dependence. The p values show great variability with time, and large values or changes in p sometimes precedes major earthquakes. Studies of teleseismic earthquake catalogs over the last twenty years have led seismologists to question seismicity models and aftershock sequence decay for deep sequences. For seven exceptional deep sequences, we conclude that MOL decay adequately describes these sequences, and little difference exists compared to shallow sequences. However, they do include larger aftershock populations compared to most deep sequences. These results imply that p values for deep sequences are larger than those for intermediate depth sequences.

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

Enhanced UGT1A1 Gene and Protein Expression in Endometriotic Lesions.

PubMed

Piccinato, Carla A; Neme, Rosa M; Torres, Natália; da Silva Victor, Elivane; Brudniewski, Heloísa F; Rosa E Silva, Júlio C; Ferriani, Rui A

2018-01-01

The cellular function in endometriosis lesions depends on a highly estrogenic milieu. Lately, it is becoming evident that, besides the circulating levels of estrogens, the balance of synthesis versus inactivation (metabolism) of estrogens by intralesion steroid-metabolizing enzymes also determines the local net estrogen availability. In order to extend the knowledge of the role of estrogen-metabolizing enzymes in endometriosis, we investigated the gene and protein expression of a key uridine diphospho-glucuronosyltransferase (UGT) for estrogen glucuronidation, UGT1A1, in eutopic endometrial samples obtained from nonaffected and endometriosis-affected women and also from endometriotic lesions. Although UGT1A1 messenger RNA (mRNA) expression was detected at similar frequencies in endometriotic lesions and in eutopic endometrial samples, the levels of mRNA expression were greater in deep-infiltrating endometriotic lesions and in non-deep-infiltrating lesions when compared with either control endometrium or eutopic endometrium from women with endometriosis. Overall, we observed that protein expression of UGT1A1 was significantly more frequent in samples from endometriotic lesions in comparison with endometria. In addition, expression of UGT1A1 protein was greater in deep-infiltrating than in non-deep-infiltrating endometriotic lesions. We suggest that the finding of increased expression of UGT1A1 in lesions versus endometria might be related to impairment of regulatory mechanisms, in response to a highly estrogenic milieu, and that this enzyme may be a new target for therapy.

Protein Secondary Structure Prediction Using Deep Convolutional Neural Fields.

PubMed

Wang, Sheng; Peng, Jian; Ma, Jianzhu; Xu, Jinbo

2016-01-11

Protein secondary structure (SS) prediction is important for studying protein structure and function. When only the sequence (profile) information is used as input feature, currently the best predictors can obtain ~80% Q3 accuracy, which has not been improved in the past decade. Here we present DeepCNF (Deep Convolutional Neural Fields) for protein SS prediction. DeepCNF is a Deep Learning extension of Conditional Neural Fields (CNF), which is an integration of Conditional Random Fields (CRF) and shallow neural networks. DeepCNF can model not only complex sequence-structure relationship by a deep hierarchical architecture, but also interdependency between adjacent SS labels, so it is much more powerful than CNF. Experimental results show that DeepCNF can obtain ~84% Q3 accuracy, ~85% SOV score, and ~72% Q8 accuracy, respectively, on the CASP and CAMEO test proteins, greatly outperforming currently popular predictors. As a general framework, DeepCNF can be used to predict other protein structure properties such as contact number, disorder regions, and solvent accessibility.

Protein Secondary Structure Prediction Using Deep Convolutional Neural Fields

NASA Astrophysics Data System (ADS)

Wang, Sheng; Peng, Jian; Ma, Jianzhu; Xu, Jinbo

2016-01-01

Protein secondary structure (SS) prediction is important for studying protein structure and function. When only the sequence (profile) information is used as input feature, currently the best predictors can obtain ~80% Q3 accuracy, which has not been improved in the past decade. Here we present DeepCNF (Deep Convolutional Neural Fields) for protein SS prediction. DeepCNF is a Deep Learning extension of Conditional Neural Fields (CNF), which is an integration of Conditional Random Fields (CRF) and shallow neural networks. DeepCNF can model not only complex sequence-structure relationship by a deep hierarchical architecture, but also interdependency between adjacent SS labels, so it is much more powerful than CNF. Experimental results show that DeepCNF can obtain ~84% Q3 accuracy, ~85% SOV score, and ~72% Q8 accuracy, respectively, on the CASP and CAMEO test proteins, greatly outperforming currently popular predictors. As a general framework, DeepCNF can be used to predict other protein structure properties such as contact number, disorder regions, and solvent accessibility.

Deep learning methods for protein torsion angle prediction.

PubMed

Li, Haiou; Hou, Jie; Adhikari, Badri; Lyu, Qiang; Cheng, Jianlin

2017-09-18

Deep learning is one of the most powerful machine learning methods that has achieved the state-of-the-art performance in many domains. Since deep learning was introduced to the field of bioinformatics in 2012, it has achieved success in a number of areas such as protein residue-residue contact prediction, secondary structure prediction, and fold recognition. In this work, we developed deep learning methods to improve the prediction of torsion (dihedral) angles of proteins. We design four different deep learning architectures to predict protein torsion angles. The architectures including deep neural network (DNN) and deep restricted Boltzmann machine (DRBN), deep recurrent neural network (DRNN) and deep recurrent restricted Boltzmann machine (DReRBM) since the protein torsion angle prediction is a sequence related problem. In addition to existing protein features, two new features (predicted residue contact number and the error distribution of torsion angles extracted from sequence fragments) are used as input to each of the four deep learning architectures to predict phi and psi angles of protein backbone. The mean absolute error (MAE) of phi and psi angles predicted by DRNN, DReRBM, DRBM and DNN is about 20-21° and 29-30° on an independent dataset. The MAE of phi angle is comparable to the existing methods, but the MAE of psi angle is 29°, 2° lower than the existing methods. On the latest CASP12 targets, our methods also achieved the performance better than or comparable to a state-of-the art method. Our experiment demonstrates that deep learning is a valuable method for predicting protein torsion angles. The deep recurrent network architecture performs slightly better than deep feed-forward architecture, and the predicted residue contact number and the error distribution of torsion angles extracted from sequence fragments are useful features for improving prediction accuracy.

Composite transcriptome assembly of RNA-seq data in a sheep model for delayed bone healing.

PubMed

Jäger, Marten; Ott, Claus-Eric; Grünhagen, Johannes; Hecht, Jochen; Schell, Hanna; Mundlos, Stefan; Duda, Georg N; Robinson, Peter N; Lienau, Jasmin

2011-03-24

The sheep is an important model organism for many types of medically relevant research, but molecular genetic experiments in the sheep have been limited by the lack of knowledge about ovine gene sequences. Prior to our study, mRNA sequences for only 1,556 partial or complete ovine genes were publicly available. Therefore, we developed a composite de novo transcriptome assembly method for next-generation sequence data to combine known ovine mRNA and EST sequences, mRNA sequences from mouse and cow, and sequences assembled de novo from short read RNA-Seq data into a composite reference transcriptome, and identified transcripts from over 12 thousand previously undescribed ovine genes. Gene expression analysis based on these data revealed substantially different expression profiles in standard versus delayed bone healing in an ovine tibial osteotomy model. Hundreds of transcripts were differentially expressed between standard and delayed healing and between the time points of the standard and delayed healing groups. We used the sheep sequences to design quantitative RT-PCR assays with which we validated the differential expression of 26 genes that had been identified by RNA-seq analysis. A number of clusters of characteristic expression profiles could be identified, some of which showed striking differences between the standard and delayed healing groups. Gene Ontology (GO) analysis showed that the differentially expressed genes were enriched in terms including extracellular matrix, cartilage development, contractile fiber, and chemokine activity. Our results provide a first atlas of gene expression profiles and differentially expressed genes in standard and delayed bone healing in a large-animal model and provide a number of clues as to the shifts in gene expression that underlie delayed bone healing. In the course of our study, we identified transcripts of 13,987 ovine genes, including 12,431 genes for which no sequence information was previously available. This information will provide a basis for future molecular research involving the sheep as a model organism.

Composite transcriptome assembly of RNA-seq data in a sheep model for delayed bone healing

PubMed Central

2011-01-01

Background The sheep is an important model organism for many types of medically relevant research, but molecular genetic experiments in the sheep have been limited by the lack of knowledge about ovine gene sequences. Results Prior to our study, mRNA sequences for only 1,556 partial or complete ovine genes were publicly available. Therefore, we developed a composite de novo transcriptome assembly method for next-generation sequence data to combine known ovine mRNA and EST sequences, mRNA sequences from mouse and cow, and sequences assembled de novo from short read RNA-Seq data into a composite reference transcriptome, and identified transcripts from over 12 thousand previously undescribed ovine genes. Gene expression analysis based on these data revealed substantially different expression profiles in standard versus delayed bone healing in an ovine tibial osteotomy model. Hundreds of transcripts were differentially expressed between standard and delayed healing and between the time points of the standard and delayed healing groups. We used the sheep sequences to design quantitative RT-PCR assays with which we validated the differential expression of 26 genes that had been identified by RNA-seq analysis. A number of clusters of characteristic expression profiles could be identified, some of which showed striking differences between the standard and delayed healing groups. Gene Ontology (GO) analysis showed that the differentially expressed genes were enriched in terms including extracellular matrix, cartilage development, contractile fiber, and chemokine activity. Conclusions Our results provide a first atlas of gene expression profiles and differentially expressed genes in standard and delayed bone healing in a large-animal model and provide a number of clues as to the shifts in gene expression that underlie delayed bone healing. In the course of our study, we identified transcripts of 13,987 ovine genes, including 12,431 genes for which no sequence information was previously available. This information will provide a basis for future molecular research involving the sheep as a model organism. PMID:21435219

High precision in protein contact prediction using fully convolutional neural networks and minimal sequence features.

PubMed

Jones, David T; Kandathil, Shaun M

2018-04-26

In addition to substitution frequency data from protein sequence alignments, many state-of-the-art methods for contact prediction rely on additional sources of information, or features, of protein sequences in order to predict residue-residue contacts, such as solvent accessibility, predicted secondary structure, and scores from other contact prediction methods. It is unclear how much of this information is needed to achieve state-of-the-art results. Here, we show that using deep neural network models, simple alignment statistics contain sufficient information to achieve state-of-the-art precision. Our prediction method, DeepCov, uses fully convolutional neural networks operating on amino-acid pair frequency or covariance data derived directly from sequence alignments, without using global statistical methods such as sparse inverse covariance or pseudolikelihood estimation. Comparisons against CCMpred and MetaPSICOV2 show that using pairwise covariance data calculated from raw alignments as input allows us to match or exceed the performance of both of these methods. Almost all of the achieved precision is obtained when considering relatively local windows (around 15 residues) around any member of a given residue pairing; larger window sizes have comparable performance. Assessment on a set of shallow sequence alignments (fewer than 160 effective sequences) indicates that the new method is substantially more precise than CCMpred and MetaPSICOV2 in this regime, suggesting that improved precision is attainable on smaller sequence families. Overall, the performance of DeepCov is competitive with the state of the art, and our results demonstrate that global models, which employ features from all parts of the input alignment when predicting individual contacts, are not strictly needed in order to attain precise contact predictions. DeepCov is freely available at https://github.com/psipred/DeepCov. d.t.jones@ucl.ac.uk.

A HuD-ZBP1 ribonucleoprotein complex localizes GAP-43 mRNA into axons through its 3' untranslated region AU-rich regulatory element.

PubMed

Yoo, Soonmoon; Kim, Hak H; Kim, Paul; Donnelly, Christopher J; Kalinski, Ashley L; Vuppalanchi, Deepika; Park, Michael; Lee, Seung J; Merianda, Tanuja T; Perrone-Bizzozero, Nora I; Twiss, Jeffery L

2013-09-01

Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β-actin and GAP-43 mRNAs. β-actin 3'UTR has a defined element for interaction with ZBP1, but GAP-43 mRNA shows no homology to this RNA sequence. Here, we show that an AU-rich regulatory element (ARE) in GAP-43's 3'UTR is necessary and sufficient for its axonal localization. Axonal GAP-43 mRNA levels increase after in vivo injury, and GAP-43 mRNA shows an increased half-life in regenerating axons. GAP-43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 co-immunoprecipitate in an RNA-dependent fashion. Reporter mRNA with the GAP-43 ARE competes with endogenous β-actin mRNA for axonal localization and decreases axon length and branching similar to the β-actin 3'UTR competing with endogenous GAP-43 mRNA. Conversely, over-expressing GAP-43 coding sequence with its 3'UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP-43's 3'UTR. We have recently found that over-expression of GAP-43 using an axonally targeted construct with the 3'UTRs of GAP-43 promoted elongating growth of axons, while restricting the mRNA to the cell body with the 3'UTR of γ-actin had minimal effect on axon length. In this study, we show that the ARE in GAP-43's 3'UTR is responsible for localization of GAP-43 mRNA into axons and is sufficient for GAP-43 protein's role in elongating axonal growth. © 2013 International Society for Neurochemistry.

A combination of LongSAGE with Solexa sequencing is well suited to explore the depth and the complexity of transcriptome

PubMed Central

Hanriot, Lucie; Keime, Céline; Gay, Nadine; Faure, Claudine; Dossat, Carole; Wincker, Patrick; Scoté-Blachon, Céline; Peyron, Christelle; Gandrillon, Olivier

2008-01-01

Background "Open" transcriptome analysis methods allow to study gene expression without a priori knowledge of the transcript sequences. As of now, SAGE (Serial Analysis of Gene Expression), LongSAGE and MPSS (Massively Parallel Signature Sequencing) are the mostly used methods for "open" transcriptome analysis. Both LongSAGE and MPSS rely on the isolation of 21 pb tag sequences from each transcript. In contrast to LongSAGE, the high throughput sequencing method used in MPSS enables the rapid sequencing of very large libraries containing several millions of tags, allowing deep transcriptome analysis. However, a bias in the complexity of the transcriptome representation obtained by MPSS was recently uncovered. Results In order to make a deep analysis of mouse hypothalamus transcriptome avoiding the limitation introduced by MPSS, we combined LongSAGE with the Solexa sequencing technology and obtained a library of more than 11 millions of tags. We then compared it to a LongSAGE library of mouse hypothalamus sequenced with the Sanger method. Conclusion We found that Solexa sequencing technology combined with LongSAGE is perfectly suited for deep transcriptome analysis. In contrast to MPSS, it gives a complex representation of transcriptome as reliable as a LongSAGE library sequenced by the Sanger method. PMID:18796152

Virtual Northern analysis of the human genome.

PubMed

Hurowitz, Evan H; Drori, Iddo; Stodden, Victoria C; Donoho, David L; Brown, Patrick O

2007-05-23

We applied the Virtual Northern technique to human brain mRNA to systematically measure human mRNA transcript lengths on a genome-wide scale. We used separation by gel electrophoresis followed by hybridization to cDNA microarrays to measure 8,774 mRNA transcript lengths representing at least 6,238 genes at high (>90%) confidence. By comparing these transcript lengths to the Refseq and H-Invitational full-length cDNA databases, we found that nearly half of our measurements appeared to represent novel transcript variants. Comparison of length measurements determined by hybridization to different cDNAs derived from the same gene identified clones that potentially correspond to alternative transcript variants. We observed a close linear relationship between ORF and mRNA lengths in human mRNAs, identical in form to the relationship we had previously identified in yeast. Some functional classes of protein are encoded by mRNAs whose untranslated regions (UTRs) tend to be longer or shorter than average; these functional classes were similar in both human and yeast. Human transcript diversity is extensive and largely unannotated. Our length dataset can be used as a new criterion for judging the completeness of cDNAs and annotating mRNA sequences. Similar relationships between the lengths of the UTRs in human and yeast mRNAs and the functions of the proteins they encode suggest that UTR sequences serve an important regulatory role among eukaryotes.

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

Feyereisen-Koener, J.M.

Double-stranded cDNA was prepared from infectious hematopoietic necrosis virus mRNA and cloned into the plasmid vector pUC8. A coprotein (G-protein) of infectious hematopoietic necrosis virus was selected by hybridization to a /sup 32/P-labeled probe. The restriction map and nucleotide sequence of the mRNA encoding the glycoprotein of infectious hematopoietic necrosis virus was determined using this full-length cDNA clone.

The Beads of Translation: Using Beads to Translate mRNA into a Polypeptide Bracelet

ERIC Educational Resources Information Center

Dunlap, Dacey; Patrick, Patricia

2012-01-01

During this activity, by making beaded bracelets that represent the steps of translation, students simulate the creation of an amino acid chain. They are given an mRNA sequence that they translate into a corresponding polypeptide chain (beads). This activity focuses on the events and sites of translation. The activity provides students with a…

Staufen-mediated mRNA decay.

PubMed

Park, Eonyoung; Maquat, Lynne E

2013-01-01

Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'-UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base pairing of 3'-UTR sequences or by intermolecular base pairing of 3'-UTR sequences with a long-noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Because both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1; SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. Copyright © 2013 John Wiley & Sons, Ltd.

RNA metabolism in the regulation of protein synthesis in plants. Progress report, 1975-1979

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

Key, J L

1979-01-01

The major objectives of the research for the contract period covered by this report were (1) to gain an insight into the sequence organization of the DNA of soybean, emphasizing the arrangement of single copy or unique sequences and repetitive sequences of DNA throughout the genome, (2) to characterize soybean RNAs relative to nucleotide sequence complexity and kinetics of synthesis and turnover of poly A/sup +/ mRNA, and (3) to study ribosomal proteins directed to an analysis of possible changes in proteins which relate to the activation of 80S ribosomes and thus mRNA utilization and protein synthesis in response tomore » environmental stimuli. Even with greatly reduced funding compared to that requested, objectives 1 and 2 were substantially accomplished. Because of reduced funding and the 20-month no cost extension, relatively little progress was made on objective 3. Accordingly objectives 1 and 2 will be summarized in some detail; a brief account of progress is presented on objective 3.« less

RBSDesigner: software for designing synthetic ribosome binding sites that yields a desired level of protein expression.

PubMed

Na, Dokyun; Lee, Doheon

2010-10-15

RBSDesigner predicts the translation efficiency of existing mRNA sequences and designs synthetic ribosome binding sites (RBSs) for a given coding sequence (CDS) to yield a desired level of protein expression. The program implements the mathematical model for translation initiation described in Na et al. (Mathematical modeling of translation initiation for the estimation of its efficiency to computationally design mRNA sequences with a desired expression level in prokaryotes. BMC Syst. Biol., 4, 71). The program additionally incorporates the effect on translation efficiency of the spacer length between a Shine-Dalgarno (SD) sequence and an AUG codon, which is crucial for the incorporation of fMet-tRNA into the ribosome. RBSDesigner provides a graphical user interface (GUI) for the convenient design of synthetic RBSs. RBSDesigner is written in Python and Microsoft Visual Basic 6.0 and is publicly available as precompiled stand-alone software on the web (http://rbs.kaist.ac.kr). dhlee@kaist.ac.kr

RNA Editing in Plant Mitochondria

NASA Astrophysics Data System (ADS)

Hiesel, Rudolf; Wissinger, Bernd; Schuster, Wolfgang; Brennicke, Axel

1989-12-01

Comparative sequence analysis of genomic and complementary DNA clones from several mitochondrial genes in the higher plant Oenothera revealed nucleotide sequence divergences between the genomic and the messenger RNA-derived sequences. These sequence alterations could be most easily explained by specific post-transcriptional nucleotide modifications. Most of the nucleotide exchanges in coding regions lead to altered codons in the mRNA that specify amino acids better conserved in evolution than those encoded by the genomic DNA. Several instances show that the genomic arginine codon CGG is edited in the mRNA to the tryptophan codon TGG in amino acid positions that are highly conserved as tryptophan in the homologous proteins of other species. This editing suggests that the standard genetic code is used in plant mitochondria and resolves the frequent coincidence of CGG codons and tryptophan in different plant species. The apparently frequent and non-species-specific equivalency of CGG and TGG codons in particular suggests that RNA editing is a common feature of all higher plant mitochondria.

A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing.

PubMed

Margaglione, M; Santacroce, R; Colaizzo, D; Seripa, D; Vecchione, G; Lupone, M R; De Lucia, D; Fortina, P; Grandone, E; Perricone, C; Di Minno, G

2000-10-01

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the gamma-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated gamma-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

Light-modulated abundance of an mRNA encoding a calmodulin-regulated, chromatin-associated NTPase in pea

NASA Technical Reports Server (NTRS)

Hsieh, H. L.; Tong, C. G.; Thomas, C.; Roux, S. J.

1996-01-01

A CDNA encoding a 47 kDa nucleoside triphosphatase (NTPase) that is associated with the chromatin of pea nuclei has been cloned and sequenced. The translated sequence of the cDNA includes several domains predicted by known biochemical properties of the enzyme, including five motifs characteristic of the ATP-binding domain of many proteins, several potential casein kinase II phosphorylation sites, a helix-turn-helix region characteristic of DNA-binding proteins, and a potential calmodulin-binding domain. The deduced primary structure also includes an N-terminal sequence that is a predicted signal peptide and an internal sequence that could serve as a bipartite-type nuclear localization signal. Both in situ immunocytochemistry of pea plumules and immunoblots of purified cell fractions indicate that most of the immunodetectable NTPase is within the nucleus, a compartment proteins typically reach through nuclear pores rather than through the endoplasmic reticulum pathway. The translated sequence has some similarity to that of human lamin C, but not high enough to account for the earlier observation that IgG against human lamin C binds to the NTPase in immunoblots. Northern blot analysis shows that the NTPase MRNA is strongly expressed in etiolated plumules, but only poorly or not at all in the leaf and stem tissues of light-grown plants. Accumulation of NTPase mRNA in etiolated seedlings is stimulated by brief treatments with both red and far-red light, as is characteristic of very low-fluence phytochrome responses. Southern blotting with pea genomic DNA indicates the NTPase is likely to be encoded by a single gene.

How the Sequence of a Gene Specifies Structural Symmetry in Proteins

PubMed Central

Shen, Xiaojuan; Huang, Tongcheng; Wang, Guanyu; Li, Guanglin

2015-01-01

Internal symmetry is commonly observed in the majority of fundamental protein folds. Meanwhile, sufficient evidence suggests that nascent polypeptide chains of proteins have the potential to start the co-translational folding process and this process allows mRNA to contain additional information on protein structure. In this paper, we study the relationship between gene sequences and protein structures from the viewpoint of symmetry to explore how gene sequences code for structural symmetry in proteins. We found that, for a set of two-fold symmetric proteins from left-handed beta-helix fold, intragenic symmetry always exists in their corresponding gene sequences. Meanwhile, codon usage bias and local mRNA structure might be involved in modulating translation speed for the formation of structural symmetry: a major decrease of local codon usage bias in the middle of the codon sequence can be identified as a common feature; and major or consecutive decreases in local mRNA folding energy near the boundaries of the symmetric substructures can also be observed. The results suggest that gene duplication and fusion may be an evolutionarily conserved process for this protein fold. In addition, the usage of rare codons and the formation of higher order of secondary structure near the boundaries of symmetric substructures might have coevolved as conserved mechanisms to slow down translation elongation and to facilitate effective folding of symmetric substructures. These findings provide valuable insights into our understanding of the mechanisms of translation and its evolution, as well as the design of proteins via symmetric modules. PMID:26641668

Cloning and sequence analysis of a cDNA clone coding for the mouse GM2 activator protein.

PubMed Central

Bellachioma, G; Stirling, J L; Orlacchio, A; Beccari, T

1993-01-01

A cDNA (1.1 kb) containing the complete coding sequence for the mouse GM2 activator protein was isolated from a mouse macrophage library using a cDNA for the human protein as a probe. There was a single ATG located 12 bp from the 5' end of the cDNA clone followed by an open reading frame of 579 bp. Northern blot analysis of mouse macrophage RNA showed that there was a single band with a mobility corresponding to a size of 2.3 kb. We deduce from this that the mouse mRNA, in common with the mRNA for the human GM2 activator protein, has a long 3' untranslated sequence of approx. 1.7 kb. Alignment of the mouse and human deduced amino acid sequences showed 68% identity overall and 75% identity for the sequence on the C-terminal side of the first 31 residues, which in the human GM2 activator protein contains the signal peptide. Hydropathicity plots showed great similarity between the mouse and human sequences even in regions of low sequence similarity. There is a single N-glycosylation site in the mouse GM2 activator protein sequence (Asn151-Phe-Thr) which differs in its location from the single site reported in the human GM2 activator protein sequence (Asn63-Val-Thr). Images Figure 1 PMID:7689829

Deep Impact Sequence Planning Using Multi-Mission Adaptable Planning Tools With Integrated Spacecraft Models

NASA Technical Reports Server (NTRS)

Wissler, Steven S.; Maldague, Pierre; Rocca, Jennifer; Seybold, Calina

2006-01-01

The Deep Impact mission was ambitious and challenging. JPL's well proven, easily adaptable multi-mission sequence planning tools combined with integrated spacecraft subsystem models enabled a small operations team to develop, validate, and execute extremely complex sequence-based activities within very short development times. This paper focuses on the core planning tool used in the mission, APGEN. It shows how the multi-mission design and adaptability of APGEN made it possible to model spacecraft subsystems as well as ground assets throughout the lifecycle of the Deep Impact project, starting with models of initial, high-level mission objectives, and culminating in detailed predictions of spacecraft behavior during mission-critical activities.

Transcriptome Sequences Resolve Deep Relationships of the Grape Family

PubMed Central

Wen, Jun; Xiong, Zhiqiang; Nie, Ze-Long; Mao, Likai; Zhu, Yabing; Kan, Xian-Zhao; Ickert-Bond, Stefanie M.; Gerrath, Jean; Zimmer, Elizabeth A.; Fang, Xiao-Dong

2013-01-01

Previous phylogenetic studies of the grape family (Vitaceae) yielded poorly resolved deep relationships, thus impeding our understanding of the evolution of the family. Next-generation sequencing now offers access to protein coding sequences very easily, quickly and cost-effectively. To improve upon earlier work, we extracted 417 orthologous single-copy nuclear genes from the transcriptomes of 15 species of the Vitaceae, covering its phylogenetic diversity. The resulting transcriptome phylogeny provides robust support for the deep relationships, showing the phylogenetic utility of transcriptome data for plants over a time scale at least since the mid-Cretaceous. The pros and cons of transcriptome data for phylogenetic inference in plants are also evaluated. PMID:24069307

Deep Learning and Its Applications in Biomedicine.

PubMed

Cao, Chensi; Liu, Feng; Tan, Hai; Song, Deshou; Shu, Wenjie; Li, Weizhong; Zhou, Yiming; Bo, Xiaochen; Xie, Zhi

2018-02-01

Advances in biological and medical technologies have been providing us explosive volumes of biological and physiological data, such as medical images, electroencephalography, genomic and protein sequences. Learning from these data facilitates the understanding of human health and disease. Developed from artificial neural networks, deep learning-based algorithms show great promise in extracting features and learning patterns from complex data. The aim of this paper is to provide an overview of deep learning techniques and some of the state-of-the-art applications in the biomedical field. We first introduce the development of artificial neural network and deep learning. We then describe two main components of deep learning, i.e., deep learning architectures and model optimization. Subsequently, some examples are demonstrated for deep learning applications, including medical image classification, genomic sequence analysis, as well as protein structure classification and prediction. Finally, we offer our perspectives for the future directions in the field of deep learning. Copyright © 2018. Production and hosting by Elsevier B.V.

Impact of embryo number and maternal undernutrition around the time of conception on insulin signaling and gluconeogenic factors and microRNAs in the liver of fetal sheep.

PubMed

Lie, Shervi; Morrison, Janna L; Williams-Wyss, Olivia; Suter, Catherine M; Humphreys, David T; Ozanne, Susan E; Zhang, Song; MacLaughlin, Severence M; Kleemann, David O; Walker, Simon K; Roberts, Claire T; McMillen, I Caroline

2014-05-01

This study aimed to determine whether exposure of the oocyte and/or embryo to maternal undernutrition results in the later programming of insulin action in the liver and factors regulating gluconeogenesis. To do this, we collect livers from singleton and twin fetal sheep that were exposed to periconceptional (PCUN; -60 to 7 days) or preimplantation (PIUN; 0-7 days) undernutrition at 136-138 days of gestation (term = 150 days). The mRNA and protein abundance of insulin signaling and gluconeogenic factors were then quantified using qRT-PCR and Western blotting, respectively, and global microRNA expression was quantified using deep sequencing methodology. We found that hepatic PEPCK-C mRNA (P < 0.01) and protein abundance and the protein abundance of IRS-1 (P < 0.01), p110β (P < 0.05), PTEN (P < 0.05), CREB (P < 0.01), and pCREB (Ser(133); P < 0.05) were decreased in the PCUN and PIUN singletons. In contrast, hepatic protein abundance of IRS-1 (P < 0.01), p85 (P < 0.01), p110β (P < 0.001), PTEN (P < 0.01), Akt2 (P < 0.01), p-Akt (Ser(473); P < 0.01), and p-FOXO-1 (Thr24) (P < 0.01) was increased in twins. There was a decrease in PEPCK-C mRNA (P < 0.01) but, paradoxically, an increase in PEPCK-C protein (P < 0.001) in twins. Both PCUN and PIUN altered the hepatic expression of 23 specific microRNAs. We propose that the differential impact of maternal undernutrition in the presence of one or two embryos on mRNAs and proteins involved in the insulin signaling and gluconeogenesis is explained by changes in the expression of a suite of specific candidate microRNAs.

Impact of embryo number and maternal undernutrition around the time of conception on insulin signaling and gluconeogenic factors and microRNAs in the liver of fetal sheep

PubMed Central

Lie, Shervi; Morrison, Janna L.; Williams-Wyss, Olivia; Suter, Catherine M.; Humphreys, David T.; Ozanne, Susan E.; Zhang, Song; MacLaughlin, Severence M.; Kleemann, David O.; Walker, Simon K.; Roberts, Claire T.

2014-01-01

This study aimed to determine whether exposure of the oocyte and/or embryo to maternal undernutrition results in the later programming of insulin action in the liver and factors regulating gluconeogenesis. To do this, we collect livers from singleton and twin fetal sheep that were exposed to periconceptional (PCUN; −60 to 7 days) or preimplantation (PIUN; 0–7 days) undernutrition at 136–138 days of gestation (term = 150 days). The mRNA and protein abundance of insulin signaling and gluconeogenic factors were then quantified using qRT-PCR and Western blotting, respectively, and global microRNA expression was quantified using deep sequencing methodology. We found that hepatic PEPCK-C mRNA (P < 0.01) and protein abundance and the protein abundance of IRS-1 (P < 0.01), p110β (P < 0.05), PTEN (P < 0.05), CREB (P < 0.01), and pCREB (Ser133; P < 0.05) were decreased in the PCUN and PIUN singletons. In contrast, hepatic protein abundance of IRS-1 (P < 0.01), p85 (P < 0.01), p110β (P < 0.001), PTEN (P < 0.01), Akt2 (P < 0.01), p-Akt (Ser473; P < 0.01), and p-FOXO-1 (Thr24) (P < 0.01) was increased in twins. There was a decrease in PEPCK-C mRNA (P < 0.01) but, paradoxically, an increase in PEPCK-C protein (P < 0.001) in twins. Both PCUN and PIUN altered the hepatic expression of 23 specific microRNAs. We propose that the differential impact of maternal undernutrition in the presence of one or two embryos on mRNAs and proteins involved in the insulin signaling and gluconeogenesis is explained by changes in the expression of a suite of specific candidate microRNAs. PMID:24496309

Evaluation of ribosomal RNA removal protocols for Salmonella RNA-Seq projects

USDA-ARS?s Scientific Manuscript database

Next generation sequencing is a powerful technology and its application to sequencing entire RNA populations of food-borne pathogens will provide valuable insights. A problem unique to prokaryotic RNA-Seq is the massive abundance of ribosomal RNA. Unlike eukaryotic messenger RNA (mRNA), bacterial ...

cDNA sequences and organization of IgM heavy chain genes in two holostean fish.

PubMed

Wilson, M R; van Ravenstein, E; Miller, N W; Clem, L W; Middleton, D L; Warr, G W

1995-01-01

Immunoglobulin M heavy chain (mu) sequences of two holostean fish, the bowfin, Amia calva, and the longnose gar, Lepisosteus osseus, were amplified from spleen mRNA by RACE-PCR, cloned, and sequenced. Each mu chain showed the conserved four constant domain structure typical of a secreted mu chain. Southern blot analyses with specific heavy chain variable (VH) and constant (CH) region probes suggest that both fish possess an IgH locus that resembles that of the teleosts, amphibians, and mammals in its organization. The overall sequence similarity of gar and bowfin mu chains was 60% and 48% at the nucleotide and amino acid levels, respectively, while similarity to the mu chains of teleosts and elasmobranchs was lower. The bowfin mu chain possesses a distinctive proline-rich sequence at the C mu 1/C mu 2 boundary; a shorter proline-rich sequence is present at this position in the gar mu chain. Both gar and bowfin show, in their C mu 4 sequences, motifs that could serve as cryptic splice donor sites for the production of mRNA encoding the membrane-bound form of the mu chains, and the bowfin also shows a potential cryptic splice donor site in the C mu 3 exon.

Final report: FASEB Summer Research Conference on ''Post-transcriptional control of gene expression: Effectors of mRNA decay'' [agenda and attendees list

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

Maquat, Lynne

2002-12-01

The goal of this meeting was to provide an interactive forum for scientists working on prokaryotic and eukaryotic mRNA decay. A special seminar presented by a leader in the field of mRNA decay in S. cerevisiae focused on what is known and what needs to be determined, not only for yeast but for other organisms. The large attendance (110 participants) reflects the awareness that mRNA decay is a key player in gene regulation in a way that is affected by the many steps that precede mRNA formation. Sessions were held on the following topics: mRNA transport and mRNP; multicomponent eukaryoticmore » nucleases; nonsense-mediated mRNA decay and nonsense-associated altered splicing; Cis-acting sequences/Trans-acting factors of mRNA decay; translational accuracy; multicomponent bacterial nucleases; interplay between mRNA polyadenylation, translation and decay in prokaryotes and prokaryotic organelles; and RNA interference and other RNA mediators of gene expression. In addition to the talks and two poster sessions, there were three round tables: (1) Does translation occur in the nucleus? (2) Differences and similarities in the mechanisms of mRNA decay in different eukaryotes, and (3) RNA surveillance in bacteria?« less

SEQassembly: A Practical Tools Program for Coding Sequences Splicing

NASA Astrophysics Data System (ADS)

Lee, Hongbin; Yang, Hang; Fu, Lei; Qin, Long; Li, Huili; He, Feng; Wang, Bo; Wu, Xiaoming

CDS (Coding Sequences) is a portion of mRNA sequences, which are composed by a number of exon sequence segments. The construction of CDS sequence is important for profound genetic analysis such as genotyping. A program in MATLAB environment is presented, which can process batch of samples sequences into code segments under the guide of reference exon models, and splice these code segments of same sample source into CDS according to the exon order in queue file. This program is useful in transcriptional polymorphism detection and gene function study.

Emergent HIV-1 Drug Resistance Mutations Were Not Present at Low-Frequency at Baseline in Non-Nucleoside Reverse Transcriptase Inhibitor-Treated Subjects in the STaR Study

PubMed Central

Porter, Danielle P.; Daeumer, Martin; Thielen, Alexander; Chang, Silvia; Martin, Ross; Cohen, Cal; Miller, Michael D.; White, Kirsten L.

2015-01-01

At Week 96 of the Single-Tablet Regimen (STaR) study, more treatment-naïve subjects that received rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF) developed resistance mutations compared to those treated with efavirenz (EFV)/FTC/TDF by population sequencing. Furthermore, more RPV/FTC/TDF-treated subjects with baseline HIV-1 RNA >100,000 copies/mL developed resistance compared to subjects with baseline HIV-1 RNA ≤100,000 copies/mL. Here, deep sequencing was utilized to assess the presence of pre-existing low-frequency variants in subjects with and without resistance development in the STaR study. Deep sequencing (Illumina MiSeq) was performed on baseline and virologic failure samples for all subjects analyzed for resistance by population sequencing during the clinical study (n = 33), as well as baseline samples from control subjects with virologic response (n = 118). Primary NRTI or NNRTI drug resistance mutations present at low frequency (≥2% to 20%) were detected in 6.6% of baseline samples by deep sequencing, all of which occurred in control subjects. Deep sequencing results were generally consistent with population sequencing but detected additional primary NNRTI and NRTI resistance mutations at virologic failure in seven samples. HIV-1 drug resistance mutations emerging while on RPV/FTC/TDF or EFV/FTC/TDF treatment were not present at low frequency at baseline in the STaR study. PMID:26690199

Emergent HIV-1 Drug Resistance Mutations Were Not Present at Low-Frequency at Baseline in Non-Nucleoside Reverse Transcriptase Inhibitor-Treated Subjects in the STaR Study.

PubMed

Porter, Danielle P; Daeumer, Martin; Thielen, Alexander; Chang, Silvia; Martin, Ross; Cohen, Cal; Miller, Michael D; White, Kirsten L

2015-12-07

At Week 96 of the Single-Tablet Regimen (STaR) study, more treatment-naïve subjects that received rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF) developed resistance mutations compared to those treated with efavirenz (EFV)/FTC/TDF by population sequencing. Furthermore, more RPV/FTC/TDF-treated subjects with baseline HIV-1 RNA >100,000 copies/mL developed resistance compared to subjects with baseline HIV-1 RNA ≤100,000 copies/mL. Here, deep sequencing was utilized to assess the presence of pre-existing low-frequency variants in subjects with and without resistance development in the STaR study. Deep sequencing (Illumina MiSeq) was performed on baseline and virologic failure samples for all subjects analyzed for resistance by population sequencing during the clinical study (n = 33), as well as baseline samples from control subjects with virologic response (n = 118). Primary NRTI or NNRTI drug resistance mutations present at low frequency (≥2% to 20%) were detected in 6.6% of baseline samples by deep sequencing, all of which occurred in control subjects. Deep sequencing results were generally consistent with population sequencing but detected additional primary NNRTI and NRTI resistance mutations at virologic failure in seven samples. HIV-1 drug resistance mutations emerging while on RPV/FTC/TDF or EFV/FTC/TDF treatment were not present at low frequency at baseline in the STaR study.

VirusDetect: An automated pipeline for efficient virus discovery using deep sequencing of small RNAs

USDA-ARS?s Scientific Manuscript database

Accurate detection of viruses in plants and animals is critical for agriculture production and human health. Deep sequencing and assembly of virus-derived siRNAs has proven to be a highly efficient approach for virus discovery. However, to date no computational tools specifically designed for both k...

Natural Variation in Brachypodium disctachyon: Deep Sequencing of Highly Diverse Natural Accessions (2013 DOE JGI Genomics of Energy and Environment 8th Annual User Meeting)

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

Gordon, Sean

2013-03-01

Sean Gordon of the USDA on Natural variation in Brachypodium disctachyon: Deep Sequencing of Highly Diverse Natural Accessions at the 8th Annual Genomics of Energy Environment Meeting on March 27, 2013 in Walnut Creek, CA.

Microbial Diversity in Deep-sea Methane Seep Sediments Presented by SSU rRNA Gene Tag Sequencing

PubMed Central

Nunoura, Takuro; Takaki, Yoshihiro; Kazama, Hiromi; Hirai, Miho; Ashi, Juichiro; Imachi, Hiroyuki; Takai, Ken

2012-01-01

Microbial community structures in methane seep sediments in the Nankai Trough were analyzed by tag-sequencing analysis for the small subunit (SSU) rRNA gene using a newly developed primer set. The dominant members of Archaea were Deep-sea Hydrothermal Vent Euryarchaeotic Group 6 (DHVEG 6), Marine Group I (MGI) and Deep Sea Archaeal Group (DSAG), and those in Bacteria were Alpha-, Gamma-, Delta- and Epsilonproteobacteria, Chloroflexi, Bacteroidetes, Planctomycetes and Acidobacteria. Diversity and richness were examined by 8,709 and 7,690 tag-sequences from sediments at 5 and 25 cm below the seafloor (cmbsf), respectively. The estimated diversity and richness in the methane seep sediment are as high as those in soil and deep-sea hydrothermal environments, although the tag-sequences obtained in this study were not sufficient to show whole microbial diversity in this analysis. We also compared the diversity and richness of each taxon/division between the sediments from the two depths, and found that the diversity and richness of some taxa/divisions varied significantly along with the depth. PMID:22510646

Deep Recurrent Neural Networks for Human Activity Recognition

PubMed Central

Murad, Abdulmajid

2017-01-01

Adopting deep learning methods for human activity recognition has been effective in extracting discriminative features from raw input sequences acquired from body-worn sensors. Although human movements are encoded in a sequence of successive samples in time, typical machine learning methods perform recognition tasks without exploiting the temporal correlations between input data samples. Convolutional neural networks (CNNs) address this issue by using convolutions across a one-dimensional temporal sequence to capture dependencies among input data. However, the size of convolutional kernels restricts the captured range of dependencies between data samples. As a result, typical models are unadaptable to a wide range of activity-recognition configurations and require fixed-length input windows. In this paper, we propose the use of deep recurrent neural networks (DRNNs) for building recognition models that are capable of capturing long-range dependencies in variable-length input sequences. We present unidirectional, bidirectional, and cascaded architectures based on long short-term memory (LSTM) DRNNs and evaluate their effectiveness on miscellaneous benchmark datasets. Experimental results show that our proposed models outperform methods employing conventional machine learning, such as support vector machine (SVM) and k-nearest neighbors (KNN). Additionally, the proposed models yield better performance than other deep learning techniques, such as deep believe networks (DBNs) and CNNs. PMID:29113103

Deep Recurrent Neural Networks for Human Activity Recognition.

PubMed

Murad, Abdulmajid; Pyun, Jae-Young

2017-11-06

Adopting deep learning methods for human activity recognition has been effective in extracting discriminative features from raw input sequences acquired from body-worn sensors. Although human movements are encoded in a sequence of successive samples in time, typical machine learning methods perform recognition tasks without exploiting the temporal correlations between input data samples. Convolutional neural networks (CNNs) address this issue by using convolutions across a one-dimensional temporal sequence to capture dependencies among input data. However, the size of convolutional kernels restricts the captured range of dependencies between data samples. As a result, typical models are unadaptable to a wide range of activity-recognition configurations and require fixed-length input windows. In this paper, we propose the use of deep recurrent neural networks (DRNNs) for building recognition models that are capable of capturing long-range dependencies in variable-length input sequences. We present unidirectional, bidirectional, and cascaded architectures based on long short-term memory (LSTM) DRNNs and evaluate their effectiveness on miscellaneous benchmark datasets. Experimental results show that our proposed models outperform methods employing conventional machine learning, such as support vector machine (SVM) and k-nearest neighbors (KNN). Additionally, the proposed models yield better performance than other deep learning techniques, such as deep believe networks (DBNs) and CNNs.

Draft Genome Sequence of Pseudomonas oceani DSM 100277T, a Deep-Sea Bacterium.

PubMed

García-Valdés, Elena; Gomila, Margarita; Mulet, Magdalena; Lalucat, Jorge

2018-04-12

Pseudomonas oceani DSM 100277 T was isolated from deep seawater in the Okinawa Trough at 1390 m. P. oceani belongs to the Pseudomonas pertucinogena group. Here, we report the draft genome sequence of P. oceani , which has an estimated size of 4.1 Mb and exhibits 3,790 coding sequences, with a G+C content of 59.94 mol%. Copyright © 2018 García-Valdés et al.

Deep Ion Torrent sequencing identifies soil fungal community shifts after frequent prescribed fires in a southeastern US forest ecosystem.

PubMed

Brown, Shawn P; Callaham, Mac A; Oliver, Alena K; Jumpponen, Ari

2013-12-01

Prescribed burning is a common management tool to control fuel loads, ground vegetation, and facilitate desirable game species. We evaluated soil fungal community responses to long-term prescribed fire treatments in a loblolly pine forest on the Piedmont of Georgia and utilized deep Internal Transcribed Spacer Region 1 (ITS1) amplicon sequencing afforded by the recent Ion Torrent Personal Genome Machine (PGM). These deep sequence data (19,000 + reads per sample after subsampling) indicate that frequent fires (3-year fire interval) shift soil fungus communities, whereas infrequent fires (6-year fire interval) permit system resetting to a state similar to that without prescribed fire. Furthermore, in nonmetric multidimensional scaling analyses, primarily ectomycorrhizal taxa were correlated with axes associated with long fire intervals, whereas soil saprobes tended to be correlated with the frequent fire recurrence. We conclude that (1) multiplexed Ion Torrent PGM analyses allow deep cost effective sequencing of fungal communities but may suffer from short read lengths and inconsistent sequence quality adjacent to the sequencing adaptor; (2) frequent prescribed fires elicit a shift in soil fungal communities; and (3) such shifts do not occur when fire intervals are longer. Our results emphasize the general responsiveness of these forests to management, and the importance of fire return intervals in meeting management objectives. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

RNA-Seq analysis to capture the transcriptome landscape of a single cell

PubMed Central

Tang, Fuchou; Barbacioru, Catalin; Nordman, Ellen; Xu, Nanlan; Bashkirov, Vladimir I; Lao, Kaiqin; Surani, M. Azim

2013-01-01

We describe here a protocol for digital transcriptome analysis in a single mouse blastomere using a deep sequencing approach. An individual blastomere was first isolated and put into lysate buffer by mouth pipette. Reverse transcription was then performed directly on the whole cell lysate. After this, the free primers were removed by Exonuclease I and a poly(A) tail was added to the 3′ end of the first-strand cDNA by Terminal Deoxynucleotidyl Transferase. Then the single cell cDNAs were amplified by 20 plus 9 cycles of PCR. Then 100-200 ng of these amplified cDNAs were used to construct a sequencing library. The sequencing library can be used for deep sequencing using the SOLiD system. Compared with the cDNA microarray technique, our assay can capture up to 75% more genes expressed in early embryos. The protocol can generate deep sequencing libraries within 6 days for 16 single cell samples. PMID:20203668

Deep sequencing reveals double mutations in cis of MPL exon 10 in myeloproliferative neoplasms.

PubMed

Pietra, Daniela; Brisci, Angela; Rumi, Elisa; Boggi, Sabrina; Elena, Chiara; Pietrelli, Alessandro; Bordoni, Roberta; Ferrari, Maurizio; Passamonti, Francesco; De Bellis, Gianluca; Cremonesi, Laura; Cazzola, Mario

2011-04-01

Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome.

Simplified Identification of mRNA or DNA in Whole Cells

NASA Technical Reports Server (NTRS)

Almeida, Eduardo; Kadambi, Geeta

2007-01-01

A recently invented method of detecting a selected messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA) sequence offers two important advantages over prior such methods: it is simpler and can be implemented by means of compact equipment. The simplification and miniaturization achieved by this invention are such that this method is suitable for use outside laboratories, in field settings in which space and power supplies may be limited. The present method is based partly on hybridization of nucleic acid, which is a powerful technique for detection of specific complementary nucleic acid sequences and is increasingly being used for detection of changes in gene expression in microarrays containing thousands of gene probes.

Triazole-linked DNA as a primer surrogate in the synthesis of first-strand cDNA.

PubMed

Fujino, Tomoko; Yasumoto, Ken-ichi; Yamazaki, Naomi; Hasome, Ai; Sogawa, Kazuhiro; Isobe, Hiroyuki

2011-11-04

A phosphate-eliminated nonnatural oligonucleotide serves as a primer surrogate in reverse transcription reaction of mRNA. Despite of the nonnatural triazole linkages in the surrogate, the reverse transcriptase effectively elongated cDNA sequences on the 3'-downstream of the primer by transcription of the complementary sequence of mRNA. A structure-activity comparison with the reference natural oligonucleotides shows the superior priming activity of the surrogate containing triazole-linkages. The nonnatural linkages also protect the transcribed cDNA from digestion reactions with 5'-exonuclease and enable us to remove noise transcripts of unknown origins. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular identification of catalases from Nicotiana plumbaginifolia (L.).

PubMed

Willekens, H; Villarroel, R; Van Montagu, M; Inzé, D; Van Camp, W

1994-09-19

We have isolated three different catalase cDNAs from Nicotiana plumbaginifolia (cat1, cat2, and cat3) and a partial sequence of a fourth catalase gene (cat4) that shows no discernible expression based on Northern analysis. The catalase sequences were used to determine the similarity with other plant catalases and to study the transcriptional response to paraquat, 3-aminotriazole, and salicylic acid. 3-Aminotriazole induces mRNA levels of cat1, cat2 and cat3, indicating that a reduction in catalase activity positively affects catalase mRNA abundance. Salicylic acid that binds catalase in vitro, had no effect on catalase transcript levels at physiological concentrations. Paraquat resulted in the induction of cat1.

The Choice of Alternative 5' Splice Sites in Influenza Virus M1 mRNA is Regulated by the Viral Polymerase Complex

NASA Astrophysics Data System (ADS)

Shih, Shin-Ru; Nemeroff, Martin E.; Krug, Robert M.

1995-07-01

The influenza virus M1 mRNA has two alternative 5' splice sites: a distal 5' splice site producing mRNA_3 that has the coding potential for 9 amino acids and a proximal 5' splice site producing M2 mRNA encoding the essential M2 ion-channel protein. Only mRNA_3 was made in uninfected cells transfected with DNA expressing M1 mRNA. Similarly, using nuclear extracts from uninfected cells, in vitro splicing of M1 mRNA yielded only mRNA_3. Only when the mRNA_3 5' splice site was inactivated by mutation was M2 mRNA made in uninfected cells and in uninfected cell extracts. In influenza virus-infected cells, M2 mRNA was made, but only after a delay, suggesting that newly synthesized viral gene product(s) were needed to activate the M2 5' splice site. We present strong evidence that these gene products are the complex of the three polymerase proteins, the same complex that functions in the transcription and replication of the viral genome. Gel shift experiments showed that the viral polymerase complex bound to the 5' end of the viral M1 mRNA in a sequence-specific and cap-dependent manner. During in vitro splicing catalyzed by uninfected cell extracts, the binding of the viral polymerase complex blocked the mRNA_3 5' splice site, resulting in the switch to the M2 mRNA 5' splice site and the production of M2 mRNA.

The primary structure of L37--a rat ribosomal protein with a zinc finger-like motif.

PubMed

Chan, Y L; Paz, V; Olvera, J; Wool, I G

1993-04-30

The amino acid sequence of the rat 60S ribosomal subunit protein L37 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L37 has 96 amino acids, the NH2-terminal methionine is removed after translation of the mRNA, and has a molecular weight of 10,939. Ribosomal protein L37 has a single zinc finger-like motif of the C2-C2 type. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 13 or 14 copies of the L37 gene. The mRNA for the protein is about 500 nucleotides in length. Rat L37 is related to Saccharomyces cerevisiae ribosomal protein YL35 and to Caenorhabditis elegans L37. We have identified in the data base a DNA sequence that encodes the chicken homolog of rat L37.

X-linked lymphocyte regulated gene 5c-like (Xlr5c-like) Is a Novel Target of Progesterone Action in Granulosa Cells of Periovulatory Rat Ovaries

PubMed Central

Mishra, Birendra; Park, Ji Yeon; Wilson, Kalin; Jo, Misung

2015-01-01

Progesterone (P4), acting through its nuclear receptor (PGR), plays an essential role in ovulation by mediating the expression of genes involved in ovulation and/or luteal formation. To identify ovulatory specific PGR-regulated genes, a preliminary microarray analysis was performed using rat granulosa cells treated with hCG ± RU486 (PGR antagonist). The transcript most highly down-regulated by RU486 was an EST (Expressed Sequence Tag) sequence (gb: BI289578.1) that matches with predicted sequence for Xlr5c-like mRNA. Since nothing is known about Xlr5c-like, we first characterized the expression pattern of Xlr5c-like mRNA in the rat ovary. The level of mRNA for Xlr5c-like is transiently up-regulated in granulosa cells of periovulatory follicles after hCG stimulation in PMSG-primed rat ovaries. The transient induction of Xlr5c-like mRNA was mimicked by hCG treatment in cultured granulosa cells from preovulatory ovaries. We further demonstrated that the LH-activated PKA, MEK, PI3K, and p38 signaling is involved in the increase in Xlr5c-like mRNA. The increase in Xlr5c-like mRNA was abolished by RU486. The inhibitory effect of RU486 was reversed by MPA (synthetic progestin), but not by dexamethasone (synthetic glucocorticoid). Furthermore, mutation of SP1/SP3 and PGR response element sites in the promoter region of Xlr5c-like decreased Xlr5c-like reporter activity. RU486 also inhibited Xlr5c-like reporter activity. ChIP assay verified the binding of PGR and SP3 to the Xlr5c-like promoter in periovulatory granulosa cells. Functionally, siRNA-mediated Xlr5c-like knockdown in granulosa cell cultures resulted in reduced levels of mRNA for Snap25, Cxcr4, and Adamts1. Recombinant Xlr5c-like protein expressed using an adenoviral approach was localized predominantly to the nucleus and to a lesser extent to the cytoplasm of rat granulosa cells. In conclusion, this is the first report showing the spatiotemporally regulated expression of Xlr5c-like mRNA by hCG in rat periovulatory ovaries. P4/PGR mediates the LH-induced increase in Xlr5c-like mRNA. In turn, Xlr5c-like is involved in regulating the expression of specific ovulatory genes such as Snap25, Cxcr4, and Adamts1, possibly acting in the nucleus of periovulatory granulosa cells. PMID:26004213

Sounds of silence: synonymous nucleotides as a key to biological regulation and complexity

PubMed Central

Shabalina, Svetlana A.; Spiridonov, Nikolay A.; Kashina, Anna

2013-01-01

Messenger RNA is a key component of an intricate regulatory network of its own. It accommodates numerous nucleotide signals that overlap protein coding sequences and are responsible for multiple levels of regulation and generation of biological complexity. A wealth of structural and regulatory information, which mRNA carries in addition to the encoded amino acid sequence, raises the question of how these signals and overlapping codes are delineated along non-synonymous and synonymous positions in protein coding regions, especially in eukaryotes. Silent or synonymous codon positions, which do not determine amino acid sequences of the encoded proteins, define mRNA secondary structure and stability and affect the rate of translation, folding and post-translational modifications of nascent polypeptides. The RNA level selection is acting on synonymous sites in both prokaryotes and eukaryotes and is more common than previously thought. Selection pressure on the coding gene regions follows three-nucleotide periodic pattern of nucleotide base-pairing in mRNA, which is imposed by the genetic code. Synonymous positions of the coding regions have a higher level of hybridization potential relative to non-synonymous positions, and are multifunctional in their regulatory and structural roles. Recent experimental evidence and analysis of mRNA structure and interspecies conservation suggest that there is an evolutionary tradeoff between selective pressure acting at the RNA and protein levels. Here we provide a comprehensive overview of the studies that define the role of silent positions in regulating RNA structure and processing that exert downstream effects on proteins and their functions. PMID:23293005

Single step production of Cas9 mRNA for zygote injection.

PubMed

Redel, Bethany K; Beaton, Benjamin P; Spate, Lee D; Benne, Joshua A; Murphy, Stephanie L; O'Gorman, Chad W; Spate, Anna M; Prather, Randall S; Wells, Kevin D

2018-03-01

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

Expression of very low density lipoprotein receptor mRNA in circulating human monocytes: its up-regulation by hypoxia.

PubMed

Nakazato, K; Ishibashi, T; Nagata, K; Seino, Y; Wada, Y; Sakamoto, T; Matsuoka, R; Teramoto, T; Sekimata, M; Homma, Y; Maruyama, Y

2001-04-01

Although very low density lipoprotein (VLDL) receptor expression by macrophages has been shown in the vascular wall, it is not clear whether or not circulating monocytes express the VLDL receptor. We investigated the expression of VLDL receptor mRNA in human peripheral blood monocytes and monocyte-derived macrophages by reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing after subcloning of PCR product. VLDL receptor mRNA was detected both in peripheral blood monocytes and monocyte-derived macrophages. Expression of VLDL receptor mRNA was upregulated by hypoxia in monocytes, whereas treatment with oxidized LDL, interleukin-1beta or monocyte chemoattractant protein-1 did not affect the levels of VLDL receptor mRNA in monocytes and macrophages. The present study shows a novel response of VLDL receptor mRNA to hypoxia, suggesting a role for VLDL receptor in the metabolism of lipoproteins in the vascular wall and the development of atherosclerosis.

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 ...

Whole-gene CFTR sequencing combined with digital RT-PCR improves genetic diagnosis of cystic fibrosis.

PubMed

Straniero, Letizia; Soldà, Giulia; Costantino, Lucy; Seia, Manuela; Melotti, Paola; Colombo, Carla; Asselta, Rosanna; Duga, Stefano

2016-12-01

Despite extensive screening, 1-5% of cystic fibrosis (CF) patients lack a definite molecular diagnosis. Next-generation sequencing (NGS) is making affordable genetic testing based on the identification of variants in extended genomic regions. In this frame, we analyzed 23 CF patients and one carrier by whole-gene CFTR resequencing: 4 were previously characterized and served as controls; 17 were cases lacking a complete diagnosis after a full conventional CFTR screening; 3 were consecutive subjects referring to our centers, not previously submitted to any screening. We also included in the custom NGS design the coding portions of the SCNN1A, SCNN1B and SCNN1G genes, encoding the subunits of the sodium channel ENaC, which were found to be mutated in CF-like patients. Besides 2 novel SCNN1B missense mutations, we identified 22 previously-known CFTR mutations, including 2 large deletions (whose breakpoints were precisely mapped), and novel deep-intronic variants, whose role on splicing was excluded by ex-vivo analyses. Finally, for 2 patients, compound heterozygotes for a CFTR mutation and the intron-9c.1210-34TG [11-12] T 5 allele-known to be associated with decreased CFTR mRNA levels-the molecular diagnosis was implemented by measuring the residual level of wild-type transcript by digital reverse transcription polymerase chain reaction performed on RNA extracted from nasal brushing.

Characterization of the cDNA coding for rat brain cysteine sulfinate decarboxylase: brain and liver enzymes are identical proteins encoded by two distinct mRNAs.

PubMed

Tappaz, M; Bitoun, M; Reymond, I; Sergeant, A

1999-09-01

Cysteine sulfinate decarboxylase (CSD) is considered as the rate-limiting enzyme in the biosynthesis of taurine, a possible osmoregulator in brain. Through cloning and sequencing of RT-PCR and RACE-PCR products of rat brain mRNAs, a 2,396-bp cDNA sequence was obtained encoding a protein of 493 amino acids (calculated molecular mass, 55.2 kDa). The corresponding fusion protein showed a substrate specificity similar to that of the endogenous enzyme. The sequence of the encoded protein is identical to that encoded by liver CSD cDNA. Among other characterized amino acid decarboxylases, CSD shows the highest homology (54%) with either isoform of glutamic acid decarboxylase (GAD65 and GAD67). A single mRNA band, approximately 2.5 kb, was detected by northern blot in RNA extracts of brain, liver, and kidney. However, brain and liver CSD cDNA sequences differed in the 5' untranslated region. This indicates two forms of CSD mRNA. Analysis of PCR-amplified products of genomic DNA suggests that the brain form results from the use of a 3' alternative internal splicing site within an exon specifically found in liver CSD mRNA. Through selective RT-PCR the brain form was detected in brain only, whereas the liver form was found in liver and kidney. These results indicate a tissue-specific regulation of CSD genomic expression.

Expression of arginine kinase enzymatic activity and mRNA in gills of the euryhaline crabs Carcinus maenas and Callinectes sapidus.

PubMed

Kotlyar, S; Weihrauch, D; Paulsen, R S; Towle, D W

2000-08-01

Phosphagen kinases catalyze the reversible dephosphorylation of guanidino phosphagens such as phosphocreatine and phosphoarginine, contributing to the restoration of adenosine triphosphate concentrations in cells experiencing high and variable demands on their reserves of high-energy phosphates. The major invertebrate phosphagen kinase, arginine kinase, is expressed in the gills of two species of euryhaline crabs, the blue crab Callinectes sapidus and the shore crab Carcinus maenas, in which energy-requiring functions include monovalent ion transport, acid-base balance, nitrogen excretion and gas exchange. The enzymatic activity of arginine kinase approximately doubles in the ion-transporting gills of C. sapidus, a strong osmoregulator, when the crabs are transferred from high to low salinity, but does not change in C. maenas, a more modest osmoregulator. Amplification and sequencing of arginine kinase cDNA from both species, accomplished by reverse transcription of gill mRNA and the polymerase chain reaction, revealed an open reading frame coding for a 357-amino-acid protein. The predicted amino acid sequences showed a minimum of 75 % identity with arginine kinase sequences of other arthropods. Ten of the 11 amino acid residues believed to participate in arginine binding are completely conserved among the arthropod sequences analyzed. An estimation of arginine kinase mRNA abundance indicated that acclimation salinity has no effect on arginine kinase gene transcription. Thus, the observed enhancement of enzyme activity in C. sapidus probably results from altered translation rates or direct activation of pre-existing enzyme protein.

Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum)

NASA Technical Reports Server (NTRS)

Shibata, K.; Abe, S.; Davies, E.

2001-01-01

Partial amino acid sequences of a 49 kDa apyrase (ATP diphosphohydrolase, EC 3.6.1.5) from the cytoskeletal fraction of etiolated pea stems were used to derive oligonucleotide DNA primers to generate a cDNA fragment of pea apyrase mRNA by RT-PCR and these primers were used to screen a pea stem cDNA library. Two almost identical cDNAs differing in just 6 nucleotides within the coding regions were found, and these cDNA sequences were used to clone genomic fragments by PCR. Two nearly identical gene fragments containing 8 exons and 7 introns were obtained. One of them (H-type) encoded the mRNA sequence described by Hsieh et al. (1996) (DDBJ/EMBL/GenBank Z32743), while the other (S-type) differed by the same 6 nucleotides as the mRNAs, suggesting that these genes may be alleles. The six nucleotide differences between these two alleles were found solely in the first exon, and these mutation sites had two types of consensus sequences. These mRNAs were found with varying lengths of 3' untranslated regions (3'-UTR). There are some similarities between the 3'-UTR of these mRNAs and those of actin and actin binding proteins in plants. The putative roles of the 3'-UTR and alternative polyadenylation sites are discussed in relation to their possible role in targeting the mRNAs to different subcellular compartments.

RaptorX-Property: a web server for protein structure property prediction.

PubMed

Wang, Sheng; Li, Wei; Liu, Shiwang; Xu, Jinbo

2016-07-08

RaptorX Property (http://raptorx2.uchicago.edu/StructurePropertyPred/predict/) is a web server predicting structure property of a protein sequence without using any templates. It outperforms other servers, especially for proteins without close homologs in PDB or with very sparse sequence profile (i.e. carries little evolutionary information). This server employs a powerful in-house deep learning model DeepCNF (Deep Convolutional Neural Fields) to predict secondary structure (SS), solvent accessibility (ACC) and disorder regions (DISO). DeepCNF not only models complex sequence-structure relationship by a deep hierarchical architecture, but also interdependency between adjacent property labels. Our experimental results show that, tested on CASP10, CASP11 and the other benchmarks, this server can obtain ∼84% Q3 accuracy for 3-state SS, ∼72% Q8 accuracy for 8-state SS, ∼66% Q3 accuracy for 3-state solvent accessibility, and ∼0.89 area under the ROC curve (AUC) for disorder prediction. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Deep sequencing analysis of viral infection and evolution allows rapid and detailed characterization of viral mutant spectrum.

PubMed

Isakov, Ofer; Bordería, Antonio V; Golan, David; Hamenahem, Amir; Celniker, Gershon; Yoffe, Liron; Blanc, Hervé; Vignuzzi, Marco; Shomron, Noam

2015-07-01

The study of RNA virus populations is a challenging task. Each population of RNA virus is composed of a collection of different, yet related genomes often referred to as mutant spectra or quasispecies. Virologists using deep sequencing technologies face major obstacles when studying virus population dynamics, both experimentally and in natural settings due to the relatively high error rates of these technologies and the lack of high performance pipelines. In order to overcome these hurdles we developed a computational pipeline, termed ViVan (Viral Variance Analysis). ViVan is a complete pipeline facilitating the identification, characterization and comparison of sequence variance in deep sequenced virus populations. Applying ViVan on deep sequenced data obtained from samples that were previously characterized by more classical approaches, we uncovered novel and potentially crucial aspects of virus populations. With our experimental work, we illustrate how ViVan can be used for studies ranging from the more practical, detection of resistant mutations and effects of antiviral treatments, to the more theoretical temporal characterization of the population in evolutionary studies. Freely available on the web at http://www.vivanbioinfo.org : nshomron@post.tau.ac.il Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

The 5'-untranslated region of p23 mRNA from the Ehrlich ascites tumor is involved in translation control of the growth related protein p23.

PubMed

Böhm, H; Gross, B; Gaestel, M; Bommer, U A; Ryffel, G; Bielka, H

1991-01-01

The growth-related protein p23 of the Ehrlich ascites tumor (EAT) is preferentially expressed in the exponentially growing tumor; its synthesis is translationally controlled. p23 mRNA is efficiently translated in the wheat germ cell-free lysate. In contrast, p23 mRNA present in poly(A)+RNA isolated from EAT is not translated in cell-free systems of EAT and reticulocytes. Moreover, translation of a p23 transcript is inhibited in the presence of total poly(A)+RNA. This inhibition is abolished by the removal of the 5'-UTR of the p23 transcript. Solution hybridization/RNase protection experiments point to the presence of a nucleotide sequence complementary to the 5'-UTR of p23 mRNA which might be involved in p23 mRNA inhibition.

a Simple Symmetric Algorithm Using a Likeness with Introns Behavior in RNA Sequences

NASA Astrophysics Data System (ADS)

Regoli, Massimo

2009-02-01

The RNA-Crypto System (shortly RCS) is a symmetric key algorithm to cipher data. The idea for this new algorithm starts from the observation of nature. In particular from the observation of RNA behavior and some of its properties. The RNA sequences has some sections called Introns. Introns, derived from the term "intragenic regions", are non-coding sections of precursor mRNA (pre-mRNA) or other RNAs, that are removed (spliced out of the RNA) before the mature RNA is formed. Once the introns have been spliced out of a pre-mRNA, the resulting mRNA sequence is ready to be translated into a protein. The corresponding parts of a gene are known as introns as well. The nature and the role of Introns in the pre-mRNA is not clear and it is under ponderous researches by Biologists but, in our case, we will use the presence of Introns in the RNA-Crypto System output as a strong method to add chaotic non coding information and an unnecessary behaviour in the access to the secret key to code the messages. In the RNA-Crypto System algoritnm the introns are sections of the ciphered message with non-coding information as well as in the precursor mRNA.

cDNA nucleotide sequence coding for stearoyl-CoA desaturase and its expression in the zebrafish (Danio rerio) embryo.

PubMed

Hsieh, S L; Liu, R W; Wu, C H; Cheng, W T; Kuo, Ching-Ming

2003-12-01

A cDNA sequence of stearoyl-CoA desaturase (SCD) was determined from zebrafish (Danio rerio) and compared to the corresponding genes in several teleosts. Zebrafish SCD cDNA has a size of 1,061 bp, encodes a polypeptide of 325 amino acids, and shares 88, 85, 84, and 83% similarities with tilapia (Oreochromis mossambicus), grass carp (Ctenopharyngodon idella), common carp (Cyprinus carpio), and milkfish (Chanos chanos), respectively. This 1,061 bp sequence specifies a protein that, in common with other fatty acid desaturases, contains three histidine boxes, believed to be involved in catalysis. These observations suggested that SCD genes are highly conserved. In addition, an oligonucleotide probe complementary to zebrafish SCD mRNA was hybridized to mRNA of approximately 396 bases with Northern blot analysis. The Northern blot and RT-PCR analyses showed that the SCD mRNA was expressed predominantly in the liver, intestine, gill, and muscle, while a lower level was found in the brain. Furthermore, we utilized whole-mount in situ hybridization and real-time quantitative RT-PCR to identify expression of the zebrafish SCD gene at five different stages of development. This revealed that very high levels of transcripts were found in zebrafish at all stages during embryogenesis and early development. Copyright 2003 Wiley-Liss, Inc.

Identification of ribonucleotide reductase mutation causing temperature-sensitivity of herpes simplex virus isolates from whitlow by deep sequencing.

PubMed

Daikoku, Tohru; Oyama, Yukari; Yajima, Misako; Sekizuka, Tsuyoshi; Kuroda, Makoto; Shimada, Yuka; Takehara, Kazuhiko; Miwa, Naoko; Okuda, Tomoko; Sata, Tetsutaro; Shiraki, Kimiyasu

2015-06-01

Herpes simplex virus 2 caused a genital ulcer, and a secondary herpetic whitlow appeared during acyclovir therapy. The secondary and recurrent whitlow isolates were acyclovir-resistant and temperature-sensitive in contrast to a genital isolate. We identified the ribonucleotide reductase mutation responsible for temperature-sensitivity by deep-sequencing analysis.

Metatranscriptomic analysis of diverse microbial communities reveals core metabolic pathways and microbiome-specific functionality.

PubMed

Jiang, Yue; Xiong, Xuejian; Danska, Jayne; Parkinson, John

2016-01-12

Metatranscriptomics is emerging as a powerful technology for the functional characterization of complex microbial communities (microbiomes). Use of unbiased RNA-sequencing can reveal both the taxonomic composition and active biochemical functions of a complex microbial community. However, the lack of established reference genomes, computational tools and pipelines make analysis and interpretation of these datasets challenging. Systematic studies that compare data across microbiomes are needed to demonstrate the ability of such pipelines to deliver biologically meaningful insights on microbiome function. Here, we apply a standardized analytical pipeline to perform a comparative analysis of metatranscriptomic data from diverse microbial communities derived from mouse large intestine, cow rumen, kimchi culture, deep-sea thermal vent and permafrost. Sequence similarity searches allowed annotation of 19 to 76% of putative messenger RNA (mRNA) reads, with the highest frequency in the kimchi dataset due to its relatively low complexity and availability of closely related reference genomes. Metatranscriptomic datasets exhibited distinct taxonomic and functional signatures. From a metabolic perspective, we identified a common core of enzymes involved in amino acid, energy and nucleotide metabolism and also identified microbiome-specific pathways such as phosphonate metabolism (deep sea) and glycan degradation pathways (cow rumen). Integrating taxonomic and functional annotations within a novel visualization framework revealed the contribution of different taxa to metabolic pathways, allowing the identification of taxa that contribute unique functions. The application of a single, standard pipeline confirms that the rich taxonomic and functional diversity observed across microbiomes is not simply an artefact of different analysis pipelines but instead reflects distinct environmental influences. At the same time, our findings show how microbiome complexity and availability of reference genomes can impact comprehensive annotation of metatranscriptomes. Consequently, beyond the application of standardized pipelines, additional caution must be taken when interpreting their output and performing downstream, microbiome-specific, analyses. The pipeline used in these analyses along with a tutorial has been made freely available for download from our project website: http://www.compsysbio.org/microbiome .

Chiron: translating nanopore raw signal directly into nucleotide sequence using deep learning.

PubMed

Teng, Haotian; Cao, Minh Duc; Hall, Michael B; Duarte, Tania; Wang, Sheng; Coin, Lachlan J M

2018-05-01

Sequencing by translocating DNA fragments through an array of nanopores is a rapidly maturing technology that offers faster and cheaper sequencing than other approaches. However, accurately deciphering the DNA sequence from the noisy and complex electrical signal is challenging. Here, we report Chiron, the first deep learning model to achieve end-to-end basecalling and directly translate the raw signal to DNA sequence without the error-prone segmentation step. Trained with only a small set of 4,000 reads, we show that our model provides state-of-the-art basecalling accuracy, even on previously unseen species. Chiron achieves basecalling speeds of more than 2,000 bases per second using desktop computer graphics processing units.

Molecular Cloning and Ethylene Induction of mRNA Encoding a Phytoalexin Elicitor-Releasing Factor, beta-1,3-Endoglucanase, in Soybean.

PubMed

Takeuchi, Y; Yoshikawa, M; Takeba, G; Tanaka, K; Shibata, D; Horino, O

1990-06-01

Soybean (Glycine max) beta-1,3-endoglucanase (EC 3.2. 1.39) is involved in one of the earliest plant-pathogen interactions that may lead to active disease resistance by releasing elicitor-active carbohydrates from the cell walls of fungal pathogens. Ethylene induced beta-1,3-endoglucanase activity to 2- to 3-fold higher levels in cotyledons of soybean seedlings. A specific polyclonal antiserum raised against purified soybean beta-1,3-endoglucanase was used to immunoprecipitate in vitro translation products, demonstrating that ethylene induction increased translatable beta-1,3-endoglucanase mRNA. Several cDNA clones for the endoglucanase gene were obtained by antibody screening of a lambda-gt11 expression library prepared from soybean cotyledons. Hybrid-select translation experiments indicated that the cloned cDNA encoded a 36-kilodalton precursor protein product that was specifically immunoprecipitated with beta-1,3-endoglucanase antiserum. Escherichia coli cells expressing the cloned cDNA also synthesized an immunologically positive protein. Nucleotide sequence of three independent clones revealed a single uninterrupted open reading frame of 1041 nucleotides, corresponding to a polypeptide of 347 residue long. The primary amino acid sequence of beta-1,3-endoglucanase as deduced from the nucleotide sequence was confirmed by direct amino acid sequencing of trypsin digests of the glucanase. The soybean beta-1,3-endoglucanase exhibited 53% amino acid homology to a beta-1,3-glucanase cloned from cultured tobacco cells and 48% homology to a beta-(1,3-1,4)-glucanase from barley. Utilizing the largest cloned cDNA (pEG488) as a hybridization probe, it was found that the increase in translatable beta-1,3-endoglucanase mRNA seen upon ethylene treatment of soybean seedlings was due to 50- to 100-fold increase in steady state mRNA levels, indicating that ethylene regulates gene expression of this enzyme important in disease resistance at the level of gene transcription.

Profiling of drought-responsive microRNA and mRNA in tomato using high-throughput sequencing.

PubMed

Liu, Minmin; Yu, Huiyang; Zhao, Gangjun; Huang, Qiufeng; Lu, Yongen; Ouyang, Bo

2017-06-26

Abiotic stresses cause severe loss of crop production. Among them, drought is one of the most frequent environmental stresses, which limits crop growth, development and productivity. Plant drought tolerance is fine-tuned by a complex gene regulatory network. Understanding the molecular regulation of this polygenic trait is crucial for the eventual success to improve plant yield and quality. Recent studies have demonstrated that microRNAs play critical roles in plant drought tolerance. However, little is known about the microRNA in drought response of the model plant tomato. Here, we described the profiling of drought-responsive microRNA and mRNA in tomato using high-throughput next-generation sequencing. Drought stress was applied on the seedlings of M82, a drought-sensitive cultivated tomato genotype, and IL9-1, a drought-tolerant introgression line derived from the stress-resistant wild species Solanum pennellii LA0716 and M82. Under drought, IL9-1 performed superior than M82 regarding survival rate, H 2 O 2 elimination and leaf turgor maintenance. A total of four small RNA and eight mRNA libraries were constructed and sequenced using Illumina sequencing technology. 105 conserved and 179 novel microRNAs were identified, among them, 54 and 98 were differentially expressed upon drought stress, respectively. The majority of the differentially-expressed conserved microRNAs was up-regulated in IL9-1 whereas down-regulated in M82. Under drought stress, 2714 and 1161 genes were found to be differentially expressed in M82 and IL9-1, respectively, and many of their homologues are involved in plant stress, such as genes encoding transcription factor and protein kinase. Various pathways involved in abiotic stress were revealed by Gene Ontology and pathway analysis. The mRNA sequencing results indicated that most of the target genes were regulated by their corresponding microRNAs, which suggested that microRNAs may play essential roles in the drought tolerance of tomato. In this study, numerous microRNAs and mRNAs involved in the drought response of tomato were identified using high-throughput sequencing, which will provide new insights into the complex regulatory network of plant adaption to drought stress. This work will also help to exploit new players functioning in plant drought-stress tolerance.

The RNA helicase RHAU (DHX36) suppresses expression of the transcription factor PITX1.

PubMed

Booy, Evan P; Howard, Ryan; Marushchak, Oksana; Ariyo, Emmanuel O; Meier, Markus; Novakowski, Stefanie K; Deo, Soumya R; Dzananovic, Edis; Stetefeld, Jörg; McKenna, Sean A

2014-03-01

RNA Helicase associated with AU-rich element (RHAU) (DHX36) is a DEAH (Aspartic acid, Glumatic Acid, Alanine, Histidine)-box RNA helicase that can bind and unwind G4-quadruplexes in DNA and RNA. To detect novel RNA targets of RHAU, we performed an RNA co-immunoprecipitation screen and identified the PITX1 messenger RNA (mRNA) as specifically and highly enriched. PITX1 is a homeobox transcription factor with roles in both development and cancer. Primary sequence analysis identified three probable quadruplexes within the 3'-untranslated region of the PITX1 mRNA. Each of these sequences, when isolated, forms stable quadruplex structures that interact with RHAU. We provide evidence that these quadruplexes exist in the endogenous mRNA; however, we discovered that RHAU is tethered to the mRNA via an alternative non-quadruplex-forming region. RHAU knockdown by small interfering RNA results in significant increases in PITX1 protein levels with only marginal changes in mRNA, suggesting a role for RHAU in translational regulation. Involvement of components of the microRNA machinery is supported by similar and non-additive increases in PITX1 protein expression on Dicer and combined RHAU/Dicer knockdown. We also demonstrate a requirement of argonaute-2, a key RNA-induced silencing complex component, to mediate RHAU-dependent changes in PITX1 protein levels. These results demonstrate a novel role for RHAU in microRNA-mediated translational regulation at a quadruplex-containing 3'-untranslated region.

Virtual Northern Analysis of the Human Genome

PubMed Central

Hurowitz, Evan H.; Drori, Iddo; Stodden, Victoria C.; Donoho, David L.; Brown, Patrick O.

2007-01-01

Background We applied the Virtual Northern technique to human brain mRNA to systematically measure human mRNA transcript lengths on a genome-wide scale. Methodology/Principal Findings We used separation by gel electrophoresis followed by hybridization to cDNA microarrays to measure 8,774 mRNA transcript lengths representing at least 6,238 genes at high (>90%) confidence. By comparing these transcript lengths to the Refseq and H-Invitational full-length cDNA databases, we found that nearly half of our measurements appeared to represent novel transcript variants. Comparison of length measurements determined by hybridization to different cDNAs derived from the same gene identified clones that potentially correspond to alternative transcript variants. We observed a close linear relationship between ORF and mRNA lengths in human mRNAs, identical in form to the relationship we had previously identified in yeast. Some functional classes of protein are encoded by mRNAs whose untranslated regions (UTRs) tend to be longer or shorter than average; these functional classes were similar in both human and yeast. Conclusions/Significance Human transcript diversity is extensive and largely unannotated. Our length dataset can be used as a new criterion for judging the completeness of cDNAs and annotating mRNA sequences. Similar relationships between the lengths of the UTRs in human and yeast mRNAs and the functions of the proteins they encode suggest that UTR sequences serve an important regulatory role among eukaryotes. PMID:17520019

Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data.

PubMed

Krøigård, Anne Bruun; Thomassen, Mads; Lænkholm, Anne-Vibeke; Kruse, Torben A; Larsen, Martin Jakob

2016-01-01

Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths.

Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data

PubMed Central

Krøigård, Anne Bruun; Thomassen, Mads; Lænkholm, Anne-Vibeke; Kruse, Torben A.; Larsen, Martin Jakob

2016-01-01

Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths. PMID:27002637

mRNA trans-splicing in gene therapy for genetic diseases.

PubMed

Berger, Adeline; Maire, Séverine; Gaillard, Marie-Claude; Sahel, José-Alain; Hantraye, Philippe; Bemelmans, Alexis-Pierre

2016-07-01

Spliceosome-mediated RNA trans-splicing, or SMaRT, is a promising strategy to design innovative gene therapy solutions for currently intractable genetic diseases. SMaRT relies on the correction of mutations at the post-transcriptional level by modifying the mRNA sequence. To achieve this, an exogenous RNA is introduced into the target cell, usually by means of gene transfer, to induce a splice event in trans between the exogenous RNA and the target endogenous pre-mRNA. This produces a chimeric mRNA composed partly of exons of the latter, and partly of exons of the former, encoding a sequence free of mutations. The principal challenge of SMaRT technology is to achieve a reaction as complete as possible, i.e., resulting in 100% repairing of the endogenous mRNA target. The proof of concept of SMaRT feasibility has already been established in several models of genetic diseases caused by recessive mutations. In such cases, in fact, the repair of only a portion of the mutant mRNA pool may be sufficient to obtain a significant therapeutic effect. However in the case of dominant mutations, the target cell must be freed from the majority of mutant mRNA copies, requiring a highly efficient trans-splicing reaction. This likely explains why only a few examples of SMaRT approaches targeting dominant mutations are reported in the literature. In this review, we explain in details the mechanism of trans-splicing, review the different strategies that are under evaluation to lead to efficient trans-splicing, and discuss the advantages and limitations of SMaRT. WIREs RNA 2016, 7:487-498. doi: 10.1002/wrna.1347 For further resources related to this article, please visit the WIREs website. © 2016 The Authors. WIREs RNA published by Wiley Periodicals, Inc.

KSRP Modulation of GAP-43 mRNA Stability Restricts Axonal Outgrowth in Embryonic Hippocampal Neurons

PubMed Central

Bird, Clark W.; Gardiner, Amy S.; Bolognani, Federico; Tanner, Daniel C.; Chen, Ching-Yi; Lin, Wei-Jye; Yoo, Soonmoon; Twiss, Jeffery L.; Perrone- Bizzozero, Nora

2013-01-01

The KH-type splicing regulatory protein (KSRP) promotes the decay of AU-rich element (ARE)-containing mRNAs. Although KSRP is expressed in the nervous system, very little is known about its role in neurons. In this study, we examined whether KSRP regulates the stability of the ARE-containing GAP-43 mRNA. We found that KSRP destabilizes this mRNA by binding to its ARE, a process that requires the presence of its fourth KH domain (KH4). Furthermore, KSRP competed with the stabilizing factor HuD for binding to these sequences. We also examined the functional consequences of KSRP overexpression and knockdown on the differentiation of primary hippocampal neurons in culture. Overexpression of full length KSRP or KSRP without its nuclear localization signal hindered axonal outgrowth in these cultures, while overexpression of a mutant protein without the KH4 domain that has less affinity for binding to GAP-43′s ARE had no effect. In contrast, depletion of KSRP led to a rise in GAP-43 mRNA levels and a dramatic increase in axonal length, both in KSRP shRNA transfected cells and neurons cultured from Ksrp+/− and Ksrp −/−embryos. Finally we found that overexpression of GAP-43 rescued the axonal outgrowth deficits seen with KSRP overexpression, but only when cells were transfected with GAP-43 constructs containing 3′ UTR sequences targeting the transport of this mRNA to axons. Together, our results suggest that KSRP is an important regulator of mRNA stability and axonal length that works in direct opposition to HuD to regulate the levels of GAP-43 and other ARE-containing neuronal mRNAs. PMID:24244461

The evolution and functional characterization of lined seahorse (Hippocampus erectus) CCKs involved in fasting and thermal stress response.

PubMed

Zhang, Huixian; Qin, Geng; Sun, Jinhui; Zhang, Bo; Lin, Qiang

2018-01-01

The peptide cholecystokinin (CCK) plays an important role in the regulation of vertebrate appetite and feeding behaviour. In the present study, the full-length cDNA and genomic DNA sequences of two CCK precursors were cloned and analysed in the Syngnathidae fish, the lined seahorse (Hippocampus erectus). Both CCK1 and CCK2 in the seahorse consist of four exons. The sequence of the octapeptide of seahorse CCK1 (DYMGWMDF) was the same as that of the chicken and human, while the octapeptide of seahorse CCK2 (DYEGWMDF) was unique among vertebrates. According to the phylogenetic analysis, two types of CCKs were produced by teleost-specific genome duplication (TGD). Both CCK1 and CCK2 were highly expressed in the brain, while detectable amounts of CCK1 mRNA in the brood pouch and CCK2 mRNA in the intestine were also found. Both CCK1 and CCK2 mRNA levels significantly increased during the transition from endogenous to exogenous nutrition. Additionally, fasting induced a significant increase in the CCK1 mRNA expression in the brain of juvenile seahorses but had no effect on CCK2 transcript levels. In addition, the CCK1 and CCK2 mRNA levels in the seahorse brain significantly increased after a high-temperature treatment. Thus, the mRNA expression of CCK had obvious tissue specificities and this preliminary study opens new avenues for further functional studies on the endocrine regulations of CCK in the transition from endogenous to exogenous nutrition, food intake regulation and metabolism in the seahorse. Copyright © 2017 Elsevier Inc. All rights reserved.

Pre-Mrna Introns as a Model for Cryptographic Algorithm:. Theory and Experiments

NASA Astrophysics Data System (ADS)

Regoli, Massimo

2010-01-01

The RNA-Crypto System (shortly RCS) is a symmetric key algorithm to cipher data. The idea for this new algorithm starts from the observation of nature. In particular from the observation of RNA behavior and some of its properties. In particular the RNA sequences have some sections called Introns. Introns, derived from the term "intragenic regions", are non-coding sections of precursor mRNA (pre-mRNA) or other RNAs, that are removed (spliced out of the RNA) before the mature RNA is formed. Once the introns have been spliced out of a pre-mRNA, the resulting mRNA sequence is ready to be translated into a protein. The corresponding parts of a gene are known as introns as well. The nature and the role of Introns in the pre-mRNA is not clear and it is under ponderous researches by Biologists but, in our case, we will use the presence of Introns in the RNA-Crypto System output as a strong method to add chaotic non coding information and an unnecessary behaviour in the access to the secret key to code the messages. In the RNA-Crypto System algorithm the introns are sections of the ciphered message with non-coding information as well as in the precursor mRNA.

Variations in endothelin receptor B subtype 2 (EDNRB2) coding sequences and mRNA expression levels in 4 Muscovy duck plumage colour phenotypes.

PubMed

Wu, N; Qin, H; Wang, M; Bian, Y; Dong, B; Sun, G; Zhao, W; Chang, G; Xu, Q; Chen, G

2017-04-01

1. Endothelin receptor B subtype 2 (EDNRB2) is a paralog of EDNRB, which encodes a 7-transmembrane G-protein coupled receptor. Previous studies reported that EDNRB was essential for melanoblast migration in mammals and ducks. 2. Muscovy ducks have different plumage colour phenotypes. Variations in EDNRB2 coding sequences (CDSs) and mRNA expression levels were investigated in 4 different Muscovy duck plumage colour phenotypes, including black, black mutant, silver and white head. 3. The EDNRB2 gene from Muscovy duck was cloned; it had a length of 6435 bp and encoded 437 amino acids. The coding region was screened and potential single nucleotide polymorphisms were identified. Eight mutations were obtained, including one missense variant (c.64C > T) and 7 synonymous substitutions. The substitutions were associated with plumage colour phenotypes. 4. The EDNRB2 mRNA expression levels were compared between feather pulp from black birds and black mutant birds. The results indicated that EDNRB2 transcripts in feather pulp were significantly higher in black feathers than in white feathers. 5. The results determined the variation of EDNRB2 CDS and mRNA expression in Muscovy ducks of various plumage colours.

Hypoxia-responsive miRNAs target argonaute 1 to promote angiogenesis

PubMed Central

Chen, Zhen; Lai, Tsung-Ching; Jan, Yi-Hua; Lin, Feng-Mao; Wang, Wei-Chi; Xiao, Han; Wang, Yun-Ting; Sun, Wei; Cui, Xiaopei; Li, Ying-Shiuan; Fang, Tzan; Zhao, Hongwei; Padmanabhan, Chellappan; Sun, Ruobai; Wang, Danny Ling; Jin, Hailing; Chau, Gar-Yang; Huang, Hsien-Da; Hsiao, Michael; Shyy, John Y-J.

2013-01-01

Despite a general repression of translation under hypoxia, cells selectively upregulate a set of hypoxia-inducible genes. Results from deep sequencing revealed that Let-7 and miR-103/107 are hypoxia-responsive microRNAs (HRMs) that are strongly induced in vascular endothelial cells. In silico bioinformatics and in vitro validation showed that these HRMs are induced by HIF1α and target argonaute 1 (AGO1), which anchors the microRNA-induced silencing complex (miRISC). HRM targeting of AGO1 resulted in the translational desuppression of VEGF mRNA. Inhibition of HRM or overexpression of AGO1 without the 3′ untranslated region decreased hypoxia-induced angiogenesis. Conversely, AGO1 knockdown increased angiogenesis under normoxia in vivo. In addition, data from tumor xenografts and human cancer specimens indicate that AGO1-mediated translational desuppression of VEGF may be associated with tumor angiogenesis and poor prognosis. These findings provide evidence for an angiogenic pathway involving HRMs that target AGO1 and suggest that this pathway may be a suitable target for anti- or proangiogenesis strategies. PMID:23426184

Mutations Affecting Expression of the rosy Locus in Drosophila melanogaster

PubMed Central

Lee, Chong Sung; Curtis, Daniel; McCarron, Margaret; Love, Carol; Gray, Mark; Bender, Welcome; Chovnick, Arthur

1987-01-01

The rosy locus in Drosophila melanogaster codes for the enzyme xanthine dehydrogenase (XDH). Previous studies defined a "control element" near the 5' end of the gene, where variant sites affected the amount of rosy mRNA and protein produced. We have determined the DNA sequence of this region from both genomic and cDNA clones, and from the ry+10 underproducer strain. This variant strain had many sequence differences, so that the site of the regulatory change could not be fixed. A mutagenesis was also undertaken to isolate new regulatory mutations. We induced 376 new mutations with 1-ethyl-1-nitrosourea (ENU) and screened them to isolate those that reduced the amount of XDH protein produced, but did not change the properties of the enzyme. Genetic mapping was used to find mutations located near the 5' end of the gene. DNA from each of seven mutants was cloned and sequenced through the 5' region. Mutant base changes were identified in all seven; they appear to affect splicing and translation of the rosy mRNA. In a related study (T. P. Keith et al. 1987), the genomic and cDNA sequences are extended through the 3' end of the gene; the combined sequences define the processing pattern of the rosy transcript and predict the amino acid sequence of XDH. PMID:3036645

RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection.

PubMed

Takahashi, Hirokazu; Ohkawachi, Masahiko; Horio, Kyohei; Kobori, Toshiro; Aki, Tsunehiro; Matsumura, Yukihiko; Nakashimada, Yutaka; Okamura, Yoshiko

2018-05-17

RNA-primed rolling circle amplification (RPRCA) is a useful laboratory method for RNA detection; however, the detection of RNA is limited by the lack of information on 3'-terminal sequences. We uncovered that conventional RPRCA using pre-circularized probes could potentially detect the internal sequence of target RNA molecules in combination with RNase H. However, the specificity for mRNA detection was low, presumably due to non-specific hybridization of non-target RNA with the circular probe. To overcome this technical problem, we developed a method for detecting a sequence of interest in target RNA molecules via RNase H-assisted RPRCA using padlocked probes. When padlock probes are hybridized to the target RNA molecule, they are converted to the circular form by SplintR ligase. Subsequently, RNase H creates nick sites only in the hybridized RNA sequence, and single-stranded DNA is finally synthesized from the nick site by phi29 DNA polymerase. This method could specifically detect at least 10 fmol of the target RNA molecule without reverse transcription. Moreover, this method detected GFP mRNA present in 10 ng of total RNA isolated from Escherichia coli without background DNA amplification. Therefore, this method can potentially detect almost all types of RNA molecules without reverse transcription and reveal full-length sequence information.

Modeling positional effects of regulatory sequences with spline transformations increases prediction accuracy of deep neural networks

PubMed Central

Avsec, Žiga; Cheng, Jun; Gagneur, Julien

2018-01-01

Abstract Motivation Regulatory sequences are not solely defined by their nucleic acid sequence but also by their relative distances to genomic landmarks such as transcription start site, exon boundaries or polyadenylation site. Deep learning has become the approach of choice for modeling regulatory sequences because of its strength to learn complex sequence features. However, modeling relative distances to genomic landmarks in deep neural networks has not been addressed. Results Here we developed spline transformation, a neural network module based on splines to flexibly and robustly model distances. Modeling distances to various genomic landmarks with spline transformations significantly increased state-of-the-art prediction accuracy of in vivo RNA-binding protein binding sites for 120 out of 123 proteins. We also developed a deep neural network for human splice branchpoint based on spline transformations that outperformed the current best, already distance-based, machine learning model. Compared to piecewise linear transformation, as obtained by composition of rectified linear units, spline transformation yields higher prediction accuracy as well as faster and more robust training. As spline transformation can be applied to further quantities beyond distances, such as methylation or conservation, we foresee it as a versatile component in the genomics deep learning toolbox. Availability and implementation Spline transformation is implemented as a Keras layer in the CONCISE python package: https://github.com/gagneurlab/concise. Analysis code is available at https://github.com/gagneurlab/Manuscript_Avsec_Bioinformatics_2017. Contact avsec@in.tum.de or gagneur@in.tum.de Supplementary information Supplementary data are available at Bioinformatics online. PMID:29155928

MUFOLD-SS: New deep inception-inside-inception networks for protein secondary structure prediction.

PubMed

Fang, Chao; Shang, Yi; Xu, Dong

2018-05-01

Protein secondary structure prediction can provide important information for protein 3D structure prediction and protein functions. Deep learning offers a new opportunity to significantly improve prediction accuracy. In this article, a new deep neural network architecture, named the Deep inception-inside-inception (Deep3I) network, is proposed for protein secondary structure prediction and implemented as a software tool MUFOLD-SS. The input to MUFOLD-SS is a carefully designed feature matrix corresponding to the primary amino acid sequence of a protein, which consists of a rich set of information derived from individual amino acid, as well as the context of the protein sequence. Specifically, the feature matrix is a composition of physio-chemical properties of amino acids, PSI-BLAST profile, and HHBlits profile. MUFOLD-SS is composed of a sequence of nested inception modules and maps the input matrix to either eight states or three states of secondary structures. The architecture of MUFOLD-SS enables effective processing of local and global interactions between amino acids in making accurate prediction. In extensive experiments on multiple datasets, MUFOLD-SS outperformed the best existing methods and other deep neural networks significantly. MUFold-SS can be downloaded from http://dslsrv8.cs.missouri.edu/~cf797/MUFoldSS/download.html. © 2018 Wiley Periodicals, Inc.

Development of polymorphic genic-SSR markers by cDNA library sequencing in boxwood, Buxus spp. (Buxaceae)

USDA-ARS?s Scientific Manuscript database

Genic microsatellites or simple sequence repeat (genic-SSR) markers were developed in boxwood (Buxus taxa) for genetic diversity analysis, identification of taxa, and to facilitate breeding. cDNA libraries were developed from mRNA extracted from leaves of Buxus sempervirens ‘Vardar Valley’ and seque...

Relationship between expression of muscle-specific uncoupling protein 2 messenger RNA and genetic selection toward growth in channel catfish.

PubMed

Kobayashi, Y; Peterson, B C; Waldbieser, G C

2015-04-01

This study tested the hypothesis that increased growth in channel catfish is associated with expression of the genes that code for uncoupling proteins (UCP) 2 and 3, members of the mitochondrial channel proteins involved in nutrient sensing and metabolism. The specific objective was to contrast the levels of UCP2 messenger RNA (mRNA) in fast vs slow growing catfish as well as in fed vs fasted catfish. Two distinct UCP2 transcripts were identified and named UCP2a and UCP2b, respectively. Nucleotide and amino acid sequence of catfish UCP2s were highly similar to UCP2 and other UCPs from other fish and mammals (>75%). Expression of UCP2a mRNA was detectable at very low levels in various metabolically active tissues, whereas the expression of UCP2b mRNA was readily detectable in the muscle and heart. In a 21-wk feeding study, fish that grew faster had a greater percent body fat at the end of the study (P < 0.01). Expression of UCP2b mRNA tended to be lower (P < 0.10) in fast growing fish in the middle of the study although levels were similar at the beginning and the end of the study. In the fed vs fasted study, expression of UCP2b mRNA in muscle was increased (P < 0.05) in fish assigned to 30 d of fasting. Our results suggest that, based on the nucleotide and amino acid sequence similarities and tissue mRNA distribution, catfish UCP2b may be the analog to UCP3. Moreover, our results suggest selection toward growth and associated fat accumulation appears to be independent of muscle UCP2b mRNA expression and UCP2b-mediated mechanisms. Copyright © 2015 Elsevier Inc. All rights reserved.

Control of aflatoxin production of Aspergillus flavus and Aspergillus parasiticus using RNA silencing technology by targeting aflD (nor-1) gene.

PubMed

Abdel-Hadi, Ahmed M; Caley, Daniel P; Carter, David R F; Magan, Naresh

2011-06-01

Aspergillus flavus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B(1 )(AFB(1)), and aflatoxin G(1) (AFG(1)) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB(1) production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB(1 )production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB(1) production by A. flavus EGP9 and AFG(1 )production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG(1) production by A. parasiticus SSWT 2999. Changes in AFB(1) production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between these structural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technology.

Inhibition of Escherichia coli viability by external guide sequences complementary to two essential genes

PubMed Central

McKinney, Jeffrey; Guerrier-Takada, Cecilia; Wesolowski, Donna; Altman, Sidney

2001-01-01

Narrow spectrum antimicrobial activity has been designed to reduce the expression of two essential genes, one coding for the protein subunit of RNase P (C5 protein) and one for gyrase (gyrase A). In both cases, external guide sequences (EGS) have been designed to complex with either mRNA. Using the EGS technology, the level of microbial viability is reduced to less than 10% of the wild-type strain. The EGSs are additive when used together and depend on the number of nucleotides paired when attacking gyrase A mRNA. In the case of gyrase A, three nucleotides unpaired out of a 15-mer EGS still favor complete inhibition by the EGS but five unpaired nucleotides do not. PMID:11381134

MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression

PubMed Central

Grace, Christy R.; Ferreira, Antonio M.; Waddell, M. Brett; Ridout, Granger; Naeve, Deanna; Leuze, Michael; LoCascio, Philip F.; Panetta, John C.; Wilkinson, Mark R.; Pui, Ching-Hon; Naeve, Clayton W.; Uberbacher, Edward C.; Bonten, Erik J.; Evans, William E.

2016-01-01

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10−16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription. PMID:26844769

Fungal diversity in deep-sea sediments of a hydrothermal vent system in the Southwest Indian Ridge

NASA Astrophysics Data System (ADS)

Xu, Wei; Gong, Lin-feng; Pang, Ka-Lai; Luo, Zhu-Hua

2018-01-01

Deep-sea hydrothermal sediment is known to support remarkably diverse microbial consortia. In deep sea environments, fungal communities remain less studied despite their known taxonomic and functional diversity. High-throughput sequencing methods have augmented our capacity to assess eukaryotic diversity and their functions in microbial ecology. Here we provide the first description of the fungal community diversity found in deep sea sediments collected at the Southwest Indian Ridge (SWIR) using culture-dependent and high-throughput sequencing approaches. A total of 138 fungal isolates were cultured from seven different sediment samples using various nutrient media, and these isolates were identified to 14 fungal taxa, including 11 Ascomycota taxa (7 genera) and 3 Basidiomycota taxa (2 genera) based on internal transcribed spacers (ITS1, ITS2 and 5.8S) of rDNA. Using illumina HiSeq sequencing, a total of 757,467 fungal ITS2 tags were recovered from the samples and clustered into 723 operational taxonomic units (OTUs) belonging to 79 taxa (Ascomycota and Basidiomycota contributed to 99% of all samples) based on 97% sequence similarity. Results from both approaches suggest that there is a high fungal diversity in the deep-sea sediments collected in the SWIR and fungal communities were shown to be slightly different by location, although all were collected from adjacent sites at the SWIR. This study provides baseline data of the fungal diversity and biogeography, and a glimpse to the microbial ecology associated with the deep-sea sediments of the hydrothermal vent system of the Southwest Indian Ridge.

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

Inaugural Genomics Automation Congress and the coming deluge of sequencing data.

PubMed

Creighton, Chad J

2010-10-01

Presentations at Select Biosciences's first 'Genomics Automation Congress' (Boston, MA, USA) in 2010 focused on next-generation sequencing and the platforms and methodology around them. The meeting provided an overview of sequencing technologies, both new and emerging. Speakers shared their recent work on applying sequencing to profile cells for various levels of biomolecular complexity, including DNA sequences, DNA copy, DNA methylation, mRNA and microRNA. With sequencing time and costs continuing to drop dramatically, a virtual explosion of very large sequencing datasets is at hand, which will probably present challenges and opportunities for high-level data analysis and interpretation, as well as for information technology infrastructure.

High-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

PubMed Central

2010-01-01

Background Bathymodiolus azoricus is a deep-sea hydrothermal vent mussel found in association with large faunal communities living in chemosynthetic environments at the bottom of the sea floor near the Azores Islands. Investigation of the exceptional physiological reactions that vent mussels have adopted in their habitat, including responses to environmental microbes, remains a difficult challenge for deep-sea biologists. In an attempt to reveal genes potentially involved in the deep-sea mussel innate immunity we carried out a high-throughput sequence analysis of freshly collected B. azoricus transcriptome using gills tissues as the primary source of immune transcripts given its strategic role in filtering the surrounding waterborne potentially infectious microorganisms. Additionally, a substantial EST data set was produced and from which a comprehensive collection of genes coding for putative proteins was organized in a dedicated database, "DeepSeaVent" the first deep-sea vent animal transcriptome database based on the 454 pyrosequencing technology. Results A normalized cDNA library from gills tissue was sequenced in a full 454 GS-FLX run, producing 778,996 sequencing reads. Assembly of the high quality reads resulted in 75,407 contigs of which 3,071 were singletons. A total of 39,425 transcripts were conceptually translated into amino-sequences of which 22,023 matched known proteins in the NCBI non-redundant protein database, 15,839 revealed conserved protein domains through InterPro functional classification and 9,584 were assigned with Gene Ontology terms. Queries conducted within the database enabled the identification of genes putatively involved in immune and inflammatory reactions which had not been previously evidenced in the vent mussel. Their physical counterpart was confirmed by semi-quantitative quantitative Reverse-Transcription-Polymerase Chain Reactions (RT-PCR) and their RNA transcription level by quantitative PCR (qPCR) experiments. Conclusions We have established the first tissue transcriptional analysis of a deep-sea hydrothermal vent animal and generated a searchable catalog of genes that provides a direct method of identifying and retrieving vast numbers of novel coding sequences which can be applied in gene expression profiling experiments from a non-conventional model organism. This provides the most comprehensive sequence resource for identifying novel genes currently available for a deep-sea vent organism, in particular, genes putatively involved in immune and inflammatory reactions in vent mussels. The characterization of the B. azoricus transcriptome will facilitate research into biological processes underlying physiological adaptations to hydrothermal vent environments and will provide a basis for expanding our understanding of genes putatively involved in adaptations processes during post-capture long term acclimatization experiments, at "sea-level" conditions, using B. azoricus as a model organism. PMID:20937131

DeepGO: predicting protein functions from sequence and interactions using a deep ontology-aware classifier.

PubMed

Kulmanov, Maxat; Khan, Mohammed Asif; Hoehndorf, Robert; Wren, Jonathan

2018-02-15

A large number of protein sequences are becoming available through the application of novel high-throughput sequencing technologies. Experimental functional characterization of these proteins is time-consuming and expensive, and is often only done rigorously for few selected model organisms. Computational function prediction approaches have been suggested to fill this gap. The functions of proteins are classified using the Gene Ontology (GO), which contains over 40 000 classes. Additionally, proteins have multiple functions, making function prediction a large-scale, multi-class, multi-label problem. We have developed a novel method to predict protein function from sequence. We use deep learning to learn features from protein sequences as well as a cross-species protein-protein interaction network. Our approach specifically outputs information in the structure of the GO and utilizes the dependencies between GO classes as background information to construct a deep learning model. We evaluate our method using the standards established by the Computational Assessment of Function Annotation (CAFA) and demonstrate a significant improvement over baseline methods such as BLAST, in particular for predicting cellular locations. Web server: http://deepgo.bio2vec.net, Source code: https://github.com/bio-ontology-research-group/deepgo. robert.hoehndorf@kaust.edu.sa. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Sequence of the cDNA of a human dihydrodiol dehydrogenase isoform (AKR1C2) and tissue distribution of its mRNA.

PubMed Central

Shiraishi, H; Ishikura, S; Matsuura, K; Deyashiki, Y; Ninomiya, M; Sakai, S; Hara, A

1998-01-01

Human liver contains three isoforms (DD1, DD2 and DD4) of dihydrodiol dehydrogenase with 20alpha- or 3alpha-hydroxysteroid dehydrogenase activity; the dehydrogenases belong to the aldo-oxo reductase (AKR) superfamily. cDNA species encoding DD1 and DD4 have been identified. However, four cDNA species with more than 99% sequence identity have been cloned and are compatible with a partial amino acid sequence of DD2. In this study we have isolated a cDNA clone encoding DD2, which was confirmed by comparison of the properties of the recombinant and hepatic enzymes. This cDNA showed differences of one, two, four and five nucleotides from the previously reported four cDNA species for a dehydrogenase of human colon carcinoma HT29 cells, human prostatic 3alpha-hydroxysteroid dehydrogenase, a human liver 3alpha-hydroxysteroid dehydrogenase-like protein and chlordecone reductase-like protein respectively. Expression of mRNA species for the five similar cDNA species in 20 liver samples and 10 other different tissue samples was examined by reverse transcriptase-mediated PCR with specific primers followed by diagnostic restriction with endonucleases. All the tissues expressed only one mRNA species corresponding to the newly identified cDNA for DD2: mRNA transcripts corresponding to the other cDNA species were not detected. We suggest that the new cDNA is derived from the principal gene for DD2, which has been named AKR1C2 by a new nomenclature for the AKR superfamily. It is possible that some of the other cDNA species previously reported are rare allelic variants of this gene. PMID:9716498

Structural Requirement in Clostridium perfringens Collagenase mRNA 5′ Leader Sequence for Translational Induction through Small RNA-mRNA Base Pairing

PubMed Central

Nomura, Nobuhiko; Nakamura, Kouji

2013-01-01

The Gram-positive anaerobic bacterium Clostridium perfringens is pathogenic to humans and animals, and the production of its toxins is strictly regulated during the exponential phase. We recently found that the 5′ leader sequence of the colA transcript encoding collagenase, which is a major toxin of this organism, is processed and stabilized in the presence of the small RNA VR-RNA. The primary colA 5′-untranslated region (5′UTR) forms a long stem-loop structure containing an internal bulge and masks its own ribosomal binding site. Here we found that VR-RNA directly regulates colA expression through base pairing with colA mRNA in vivo. However, when the internal bulge structure was closed by point mutations in colA mRNA, translation ceased despite the presence of VR-RNA. In addition, a mutation disrupting the colA stem-loop structure induced mRNA processing and ColA-FLAG translational activation in the absence of VR-RNA, indicating that the stem-loop and internal bulge structure of the colA 5′ leader sequence is important for regulation by VR-RNA. On the other hand, processing was required for maximal ColA expression but was not essential for VR-RNA-dependent colA regulation. Finally, colA processing and translational activation were induced at a high temperature without VR-RNA. These results suggest that inhibition of the colA 5′ leader structure through base pairing is the primary role of VR-RNA in colA regulation and that the colA 5′ leader structure is a possible thermosensor. PMID:23585542

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

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.

The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

PubMed Central

Williams, N P; Mueller, P P; Hinnebusch, A G

1988-01-01

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function. Images PMID:3065626

Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin

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

Monaco, L.; Murtagh, J.J.; Newman, K.B.

1990-03-01

ADP-ribosylation factors (ARFs) are {approx}20-kDa proteins that act as GTP-dependent allosteric activators of cholera toxin. With deoxyinosine-containing degenerate oligonucleotide primers corresponding to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids, with intervening sequences. Amplification products that differed in size because of differences in intron sizes were separated by agarose gel electrophoresis. One amplified DNA contained no introns and had a sequence different from those of known AFRs. Based on this sequence, selective oligonucleotide probes were prepared and usedmore » to isolate clone {Psi}ARF 4, a putative ARF pseudogene, from a human genomic library in {lambda} phage EMBL3. Reverse transcription-PCR was then used to clone from human poly(A){sup +} RNA the cDNA corresponding to the expressed homolog of {Psi}ARF 4, referred to as human ARF 4. It appears that {Psi}ARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome. Human ARF 4 differs from previously identified mammalian ARFs 1, 2, and 3. Hybridization of ARF 4-specific oligonucleotide probes with human, bovine, and rat RNA revealed a single 1.8-kilobase mRNA, which was clearly distinguished from the 1.9-kilobase mRNA for ARF 1 in these tissues. The PCR provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance.« less

Conserved small mRNA with an unique, extended Shine-Dalgarno sequence

PubMed Central

Hahn, Julia; Migur, Anzhela; von Boeselager, Raphael Freiherr; Kubatova, Nina; Kubareva, Elena; Schwalbe, Harald

2017-01-01

ABSTRACT Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3′-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level. PMID:27834614

Overproduction of S-adenosylmethionine decarboxylase in ethylglyoxal-bis(guanylhydrazone)-resistant mouse FM3A cells.

PubMed

Suzuki, T; Sadakata, Y; Kashiwagi, K; Hoshino, K; Kakinuma, Y; Shirahata, A; Igarashi, K

1993-07-15

A variant cell line, termed SAM-1, which overproduced S-adenosylmethionine decarboxylase (AdoMetDC), was isolated by treatment of mouse FM3A cells with N-methyl-N'-nitro-N-nitrosoguanidine and subsequent incubation with ethylglyoxal bis(guanylhydrazone), an inhibitor of the enzyme. The cells were resistant to ethylglyoxal bis(guanylhydrazone), and showed AdoMetDC activity approximately five-times higher than control cells. The rate of AdoMetDC synthesis and the amount of AdoMetDC existing in SAM-1 cells were about five-times those in control cells. The amount of AdoMetDC mRNA existing in SAM-1 cells was five-times more than that in control cells. The amount of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine, an irreversible inhibitor of AdoMetDC, necessary to inhibit cell growth was also five-times more in SAM-1 cells than in control cells. However, the following were the same in both SAM-1 and control cells; the amount of genomic DNA for AdoMetDC, the size and nucleotide sequence of 5' untranslated region of AdoMetDC mRNA, the deduced amino acid sequence (334 residues) from the nucleotide sequence of AdoMetDC cDNA and the degradation rate (t1/2 = about 4 h) of AdoMetDC. In addition, AdoMetDC mRNA in control cells was slightly more stable than that in SAM-1 cells. The results indicate that the overproduction of AdoMetDC in SAM-1 cells was caused by the increase of AdoMetDC mRNA. The variant cell line is convenient for studying the regulation of AdoMetDC and the physiological function of polyamines.

Widespread signatures of local mRNA folding structure selection in four Dengue virus serotypes

PubMed Central

2015-01-01

Background It is known that mRNA folding can affect and regulate various gene expression steps both in living organisms and in viruses. Previous studies have recognized functional RNA structures in the genome of the Dengue virus. However, these studies usually focused either on the viral untranslated regions or on very specific and limited regions at the beginning of the coding sequences, in a limited number of strains, and without considering evolutionary selection. Results Here we performed the first large scale comprehensive genomics analysis of selection for local mRNA folding strength in the Dengue virus coding sequences, based on a total of 1,670 genomes and 4 serotypes. Our analysis identified clusters of positions along the coding regions that may undergo a conserved evolutionary selection for strong or weak local folding maintained across different viral variants. Specifically, 53-66 clusters for strong folding and 49-73 clusters for weak folding (depending on serotype) aggregated of positions with a significant conservation of folding energy signals (related to partially overlapping local genomic regions) were recognized. In addition, up to 7% of these positions were found to be conserved in more than 90% of the viral genomes. Although some of the identified positions undergo frequent synonymous / non-synonymous substitutions, the selection for folding strength therein is preserved, and thus cannot be trivially explained based on sequence conservation alone. Conclusions The fact that many of the positions with significant folding related signals are conserved among different Dengue variants suggests that a better understanding of the mRNA structures in the corresponding regions may promote the development of prospective anti- Dengue vaccination strategies. The comparative genomics approach described here can be employed in the future for detecting functional regions in other pathogens with very high mutations rates. PMID:26449467

The complete sequence and structural analysis of human apolipoprotein B-100: relationship between apoB-100 and apoB-48 forms.

PubMed Central

Cladaras, C; Hadzopoulou-Cladaras, M; Nolte, R T; Atkinson, D; Zannis, V I

1986-01-01

We have isolated and sequenced overlapping cDNA clones covering the entire sequence of human apolipoprotein B-100 (apoB-100). DNA sequence analysis and determination of the mRNA transcription initiation site by S1 nuclease mapping showed that the apoB mRNA consists of 14,112 nucleotides including the 5' and 3' untranslated regions which are 128 and 301 nucleotides respectively. The DNA-derived protein sequence shows that apoB-100 is 513,000 daltons and contains 4560 amino acids including a 24-amino-acid-long signal peptide. The mol. wt of apoB-100 implies that there is one apoB molecule per LDL particle. Computer analysis of the predicted secondary structure of the protein showed that some of the potential alpha helical and beta sheet structures are amphipathic, whereas others have non-amphipathic neutral to apolar character. These latter regions may contribute to the formation of the lipid-binding domains of apoB-100. The protein contains 25 cysteines and 20 potential N-glycosylation sites. The majority of cysteines are distributed in the amino terminal portion of the protein. Four of the potential glycosylation sites are in predicted beta turn structures and may represent true glycosylation positions. ApoB lacks the tandem repeats which are characteristic of other apolipoproteins. The mean hydrophobicity the mean value of H1 and helical hydrophobic moment the mean value of microH profiles of apoB showed the presence of several potential helical regions with strong polar character and high hydrophobic moment. The region with the highest hydrophobic moment, between amino acid residues 3352 and 3369, contains five closely spaced, positively charged residues, and has sequence homology to the LDL receptor binding site of apoE. This region is flanked by three neighbouring regions with positively charged amino acids and high hydrophobic moment that are located between residues 3174 and 3681. One or more of these closely spaced apoB sequences may be involved in the formation of the LDL receptor-binding domain of apoB-100. Blotting analysis of intestinal RNA and hybridization of the blots with carboxy apoB cDNA probes produced a single 15-kb hybridization band whereas hybridization with amino terminal probes produced two hybridization bands of 15 and 8 kb. Our data indicate that both forms of apoB mRNA contain common sequences which extend from the amino terminal of apoB-100 to the vicinity of nucleotide residue 6300. These two messages may have resulted from differential splicing of the same primary apoB mRNA transcript. Images Fig. 4. Fig. 6. PMID:3030729

Modeling RNA interference in mammalian cells

PubMed Central

2011-01-01

Background RNA interference (RNAi) is a regulatory cellular process that controls post-transcriptional gene silencing. During RNAi double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous mRNA via the generation of smaller dsRNA oligomers of length between 21-23nt (siRNAs). siRNAs are then loaded onto the RNA-Induced Silencing multiprotein Complex (RISC), which uses the siRNA antisense strand to specifically recognize mRNA species which exhibit a complementary sequence. Once the siRNA loaded-RISC binds the target mRNA, the mRNA is cleaved and degraded, and the siRNA loaded-RISC can degrade additional mRNA molecules. Despite the widespread use of siRNAs for gene silencing, and the importance of dosage for its efficiency and to avoid off target effects, none of the numerous mathematical models proposed in literature was validated to quantitatively capture the effects of RNAi on the target mRNA degradation for different concentrations of siRNAs. Here, we address this pressing open problem performing in vitro experiments of RNAi in mammalian cells and testing and comparing different mathematical models fitting experimental data to in-silico generated data. We performed in vitro experiments in human and hamster cell lines constitutively expressing respectively EGFP protein or tTA protein, measuring both mRNA levels, by quantitative Real-Time PCR, and protein levels, by FACS analysis, for a large range of concentrations of siRNA oligomers. Results We tested and validated four different mathematical models of RNA interference by quantitatively fitting models' parameters to best capture the in vitro experimental data. We show that a simple Hill kinetic model is the most efficient way to model RNA interference. Our experimental and modeling findings clearly show that the RNAi-mediated degradation of mRNA is subject to saturation effects. Conclusions Our model has a simple mathematical form, amenable to analytical investigations and a small set of parameters with an intuitive physical meaning, that makes it a unique and reliable mathematical tool. The findings here presented will be a useful instrument for better understanding RNAi biology and as modelling tool in Systems and Synthetic Biology. PMID:21272352

Sensitive Deep-Sequencing-Based HIV-1 Genotyping Assay To Simultaneously Determine Susceptibility to Protease, Reverse Transcriptase, Integrase, and Maturation Inhibitors, as Well as HIV-1 Coreceptor Tropism

PubMed Central

Gibson, Richard M.; Meyer, Ashley M.; Winner, Dane; Archer, John; Feyertag, Felix; Ruiz-Mateos, Ezequiel; Leal, Manuel; Robertson, David L.; Schmotzer, Christine L.

2014-01-01

With 29 individual antiretroviral drugs available from six classes that are approved for the treatment of HIV-1 infection, a combination of different phenotypic and genotypic tests is currently needed to monitor HIV-infected individuals. In this study, we developed a novel HIV-1 genotypic assay based on deep sequencing (DeepGen HIV) to simultaneously assess HIV-1 susceptibilities to all drugs targeting the three viral enzymes and to predict HIV-1 coreceptor tropism. Patient-derived gag-p2/NCp7/p1/p6/pol-PR/RT/IN- and env-C2V3 PCR products were sequenced using the Ion Torrent Personal Genome Machine. Reads spanning the 3′ end of the Gag, protease (PR), reverse transcriptase (RT), integrase (IN), and V3 regions were extracted, truncated, translated, and assembled for genotype and HIV-1 coreceptor tropism determination. DeepGen HIV consistently detected both minority drug-resistant viruses and non-R5 HIV-1 variants from clinical specimens with viral loads of ≥1,000 copies/ml and from B and non-B subtypes. Additional mutations associated with resistance to PR, RT, and IN inhibitors, previously undetected by standard (Sanger) population sequencing, were reliably identified at frequencies as low as 1%. DeepGen HIV results correlated with phenotypic (original Trofile, 92%; enhanced-sensitivity Trofile assay [ESTA], 80%; TROCAI, 81%; and VeriTrop, 80%) and genotypic (population sequencing/Geno2Pheno with a 10% false-positive rate [FPR], 84%) HIV-1 tropism test results. DeepGen HIV (83%) and Trofile (85%) showed similar concordances with the clinical response following an 8-day course of maraviroc monotherapy (MCT). In summary, this novel all-inclusive HIV-1 genotypic and coreceptor tropism assay, based on deep sequencing of the PR, RT, IN, and V3 regions, permits simultaneous multiplex detection of low-level drug-resistant and/or non-R5 viruses in up to 96 clinical samples. This comprehensive test, the first of its class, will be instrumental in the development of new antiretroviral drugs and, more importantly, will aid in the treatment and management of HIV-infected individuals. PMID:24468782

Dendrites, deep learning, and sequences in the hippocampus.

PubMed

Bhalla, Upinder S

2017-10-12

The hippocampus places us both in time and space. It does so over remarkably large spans: milliseconds to years, and centimeters to kilometers. This works for sensory representations, for memory, and for behavioral context. How does it fit in such wide ranges of time and space scales, and keep order among the many dimensions of stimulus context? A key organizing principle for a wide sweep of scales and stimulus dimensions is that of order in time, or sequences. Sequences of neuronal activity are ubiquitous in sensory processing, in motor control, in planning actions, and in memory. Against this strong evidence for the phenomenon, there are currently more models than definite experiments about how the brain generates ordered activity. The flip side of sequence generation is discrimination. Discrimination of sequences has been extensively studied at the behavioral, systems, and modeling level, but again physiological mechanisms are fewer. It is against this backdrop that I discuss two recent developments in neural sequence computation, that at face value share little beyond the label "neural." These are dendritic sequence discrimination, and deep learning. One derives from channel physiology and molecular signaling, the other from applied neural network theory - apparently extreme ends of the spectrum of neural circuit detail. I suggest that each of these topics has deep lessons about the possible mechanisms, scales, and capabilities of hippocampal sequence computation. © 2017 Wiley Periodicals, Inc.

Antisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation.

PubMed

Nuzzo, Francesca; Radu, Claudia; Baralle, Marco; Spiezia, Luca; Hackeng, Tilman M; Simioni, Paolo; Castoldi, Elisabetta

2013-11-28

Antisense molecules are emerging as a powerful tool to correct splicing defects. Recently, we identified a homozygous deep-intronic mutation (F5 c.1296+268A>G) activating a cryptic donor splice site in a patient with severe coagulation factor V (FV) deficiency and life-threatening bleeding episodes. Here, we assessed the ability of 2 mutation-specific antisense molecules (a morpholino oligonucleotide [MO] and an engineered U7 small nuclear RNA [snRNA]) to correct this splicing defect. COS-1 and HepG2 cells transfected with a F5 minigene construct containing the patient's mutation expressed aberrant messenger RNA (mRNA) in excess of normal mRNA. Treatment with mutation-specific antisense MO (1-5 µM) or a construct expressing antisense U7snRNA (0.25-2 µg) dose-dependently increased the relative amount of correctly spliced mRNA by 1 to 2 orders of magnitude, whereas control MO and U7snRNA were ineffective. Patient-derived megakaryocytes obtained by differentiation of circulating progenitor cells did not express FV, but became positive for FV at immunofluorescence staining after administration of antisense MO or U7snRNA. However, treatment adversely affected cell viability, mainly because of the transfection reagents used to deliver the antisense molecules. Our data provide in vitro and ex vivo proof of principle for the efficacy of RNA therapy in severe FV deficiency, but additional cytotoxicity studies are warranted.

A robust and cost-effective approach to sequence and analyze complete genomes of small RNA viruses

USDA-ARS?s Scientific Manuscript database

Background: Next-generation sequencing (NGS) allows ultra-deep sequencing of nucleic acids. The use of sequence-independent amplification of viral nucleic acids without utilization of target-specific primers provides advantages over traditional sequencing methods and allows detection of unsuspected ...

De novo transcriptome assembly and positive selection analysis of an individual deep-sea fish.

PubMed

Lan, Yi; Sun, Jin; Xu, Ting; Chen, Chong; Tian, Renmao; Qiu, Jian-Wen; Qian, Pei-Yuan

2018-05-24

High hydrostatic pressure and low temperatures make the deep sea a harsh environment for life forms. Actin organization and microtubules assembly, which are essential for intracellular transport and cell motility, can be disrupted by high hydrostatic pressure. High hydrostatic pressure can also damage DNA. Nucleic acids exposed to low temperatures can form secondary structures that hinder genetic information processing. To study how deep-sea creatures adapt to such a hostile environment, one of the most straightforward ways is to sequence and compare their genes with those of their shallow-water relatives. We captured an individual of the fish species Aldrovandia affinis, which is a typical deep-sea inhabitant, from the Okinawa Trough at a depth of 1550 m using a remotely operated vehicle (ROV). We sequenced its transcriptome and analyzed its molecular adaptation. We obtained 27,633 protein coding sequences using an Illumina platform and compared them with those of several shallow-water fish species. Analysis of 4918 single-copy orthologs identified 138 positively selected genes in A. affinis, including genes involved in microtubule regulation. Particularly, functional domains related to cold shock as well as DNA repair are exposed to positive selection pressure in both deep-sea fish and hadal amphipod. Overall, we have identified a set of positively selected genes related to cytoskeleton structures, DNA repair and genetic information processing, which shed light on molecular adaptation to the deep sea. These results suggest that amino acid substitutions of these positively selected genes may contribute crucially to the adaptation of deep-sea animals. Additionally, we provide a high-quality transcriptome of a deep-sea fish for future deep-sea studies.

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.

Detection of large numbers of novel sequences in the metatranscriptomes of complex marine microbial communities.

PubMed

Gilbert, Jack A; Field, Dawn; Huang, Ying; Edwards, Rob; Li, Weizhong; Gilna, Paul; Joint, Ian

2008-08-22

Sequencing the expressed genetic information of an ecosystem (metatranscriptome) can provide information about the response of organisms to varying environmental conditions. Until recently, metatranscriptomics has been limited to microarray technology and random cloning methodologies. The application of high-throughput sequencing technology is now enabling access to both known and previously unknown transcripts in natural communities. We present a study of a complex marine metatranscriptome obtained from random whole-community mRNA using the GS-FLX Pyrosequencing technology. Eight samples, four DNA and four mRNA, were processed from two time points in a controlled coastal ocean mesocosm study (Bergen, Norway) involving an induced phytoplankton bloom producing a total of 323,161,989 base pairs. Our study confirms the finding of the first published metatranscriptomic studies of marine and soil environments that metatranscriptomics targets highly expressed sequences which are frequently novel. Our alternative methodology increases the range of experimental options available for conducting such studies and is characterized by an exceptional enrichment of mRNA (99.92%) versus ribosomal RNA. Analysis of corresponding metagenomes confirms much higher levels of assembly in the metatranscriptomic samples and a far higher yield of large gene families with >100 members, approximately 91% of which were novel. This study provides further evidence that metatranscriptomic studies of natural microbial communities are not only feasible, but when paired with metagenomic data sets, offer an unprecedented opportunity to explore both structure and function of microbial communities--if we can overcome the challenges of elucidating the functions of so many never-seen-before gene families.

Revealing the unexplored fungal communities in deep groundwater of crystalline bedrock fracture zones in Olkiluoto, Finland.

PubMed

Sohlberg, Elina; Bomberg, Malin; Miettinen, Hanna; Nyyssönen, Mari; Salavirta, Heikki; Vikman, Minna; Itävaara, Merja

2015-01-01

The diversity and functional role of fungi, one of the ecologically most important groups of eukaryotic microorganisms, remains largely unknown in deep biosphere environments. In this study we investigated fungal communities in packer-isolated bedrock fractures in Olkiluoto, Finland at depths ranging from 296 to 798 m below surface level. DNA- and cDNA-based high-throughput amplicon sequencing analysis of the fungal internal transcribed spacer (ITS) gene markers was used to examine the total fungal diversity and to identify the active members in deep fracture zones at different depths. Results showed that fungi were present in fracture zones at all depths and fungal diversity was higher than expected. Most of the observed fungal sequences belonged to the phylum Ascomycota. Phyla Basidiomycota and Chytridiomycota were only represented as a minor part of the fungal community. Dominating fungal classes in the deep bedrock aquifers were Sordariomycetes, Eurotiomycetes, and Dothideomycetes from the Ascomycota phylum and classes Microbotryomycetes and Tremellomycetes from the Basidiomycota phylum, which are the most frequently detected fungal taxa reported also from deep sea environments. In addition some fungal sequences represented potentially novel fungal species. Active fungi were detected in most of the fracture zones, which proves that fungi are able to maintain cellular activity in these oligotrophic conditions. Possible roles of fungi and their origin in deep bedrock groundwater can only be speculated in the light of current knowledge but some species may be specifically adapted to deep subsurface environment and may play important roles in the utilization and recycling of nutrients and thus sustaining the deep subsurface microbial community.

Inference of RNA decay rate from transcriptional profiling highlights the regulatory programs of Alzheimer's disease.

PubMed

Alkallas, Rached; Fish, Lisa; Goodarzi, Hani; Najafabadi, Hamed S

2017-10-13

The abundance of mRNA is mainly determined by the rates of RNA transcription and decay. Here, we present a method for unbiased estimation of differential mRNA decay rate from RNA-sequencing data by modeling the kinetics of mRNA metabolism. We show that in all primary human tissues tested, and particularly in the central nervous system, many pathways are regulated at the mRNA stability level. We present a parsimonious regulatory model consisting of two RNA-binding proteins and four microRNAs that modulate the mRNA stability landscape of the brain, which suggests a new link between RBFOX proteins and Alzheimer's disease. We show that downregulation of RBFOX1 leads to destabilization of mRNAs encoding for synaptic transmission proteins, which may contribute to the loss of synaptic function in Alzheimer's disease. RBFOX1 downregulation is more likely to occur in older and female individuals, consistent with the association of Alzheimer's disease with age and gender."mRNA abundance is determined by the rates of transcription and decay. Here, the authors propose a method for estimating the rate of differential mRNA decay from RNA-seq data and model mRNA stability in the brain, suggesting a link between mRNA stability and Alzheimer's disease."

DsrA regulatory RNA represses both hns and rbsD mRNAs through distinct mechanisms in Escherichia coli.

PubMed

Lalaouna, David; Morissette, Audrey; Carrier, Marie-Claude; Massé, Eric

2015-10-01

The 87 nucleotide long DsrA sRNA has been mostly studied for its translational activation of the transcriptional regulator RpoS. However, it also represses hns mRNA, which encodes H-NS, a major regulator that affects expression of nearly 5% of Escherichia coli genes. A speculative model previously suggested that DsrA would block hns mRNA translation by binding simultaneously to start and stop codon regions of hns mRNA (coaxial model). Here, we show that DsrA efficiently blocked translation of hns mRNA by base-pairing immediately downstream of the start codon. In addition, DsrA induced hns mRNA degradation by actively recruiting the RNA degradosome complex. Data presented here led to a model of DsrA action on hns mRNA, which supports a canonical mechanism of sRNA-induced mRNA degradation by binding to the translation initiation region. Furthermore, using MS2-affinity purification coupled with RNA sequencing technology (MAPS), we also demonstrated that DsrA targets rbsD mRNA, involved in ribose utilization. Surprisingly, DsrA base pairs far downstream of rbsD start codon and induces rapid degradation of the transcript. Thus, our study enables us to draw an extended DsrA targetome. © 2015 John Wiley & Sons Ltd.

Stabilization of Clostridium perfringens collagenase mRNA by VR-RNA-dependent cleavage in 5' leader sequence.

PubMed

Obana, Nozomu; Shirahama, Yu; Abe, Kimihiro; Nakamura, Kouji

2010-09-01

The small RNA (sRNA), VR-RNA that is directly regulated by the VirR/VirS two-component system, regulates many genes including toxin genes such as collagenase (colA) and phospholipase C (plc) in Clostridium perfringens. Although the VR-RNA 3' region is sufficient to regulate the colA and plc genes, the molecular mechanism of toxin gene regulation by VR-RNA remains unclear. Here, we found that colA mRNA is cleaved at position -79 and -78 from the A of the first codon (ATG) in the presence of VR-RNA. The processed transcripts were stable compared with longer intact transcripts. On the other hand, colA mRNA was labile in a VR-RNA-deficient strain, and processed transcripts were undetectable. The stability and processing of colA mRNA were restored by transformation of the 3' region of VR-RNA-expression vector. The 3' region of VR-RNA and colA mRNA had significant complementation and interacted in vitro. These results show that VR-RNA base pairs with colA mRNA and induces cleavage in the 5' untranslated region (UTR) of colA mRNA, which leads to the stabilization of colA mRNA and the activation of colA expression. © 2010 Blackwell Publishing Ltd.

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

Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection.

PubMed

Showalter, A M; Bell, J N; Cramer, C L; Bailey, J A; Varner, J E; Lamb, C J

1985-10-01

Hydroxyproline-rich glycoproteins (HRGPs) are important structural components of plant cell walls and also accumulate in response to infection as an apparent defense mechanism. Accumulation of HRGP mRNA in biologically stressed bean (Phaseolus vulgaris L.) cells was monitored by blot hybridization with (32)P-labeled tomato genomic HRGP sequences. Elicitor treatment of suspension-cultured cells caused a marked increase in hybridizable HRGP mRNA. The response was less rapid but more prolonged than that observed for mRNAs encoding enzymes of phytoalexin biosynthesis. HRGP mRNA also accumulated during race:cultivar-specific interactions between bean hypocotyls and the partially biotrophic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. In an incompatible interaction (host resistant) there was an early increase in HRGP mRNA correlated with expression of hypersensitive resistance; whereas, in a compatible interaction (host susceptible), marked accumulation of HRGP mRNA occurred as a delayed response at the onset of lesion formation. In both interactions, mRNA accumulation was observed in uninfected cells distant from the site of fungal inoculation, indicating intercellular transmission of an elicitation signal.

Recruitment of Staufen2 Enhances Dendritic Localization of an Intron-Containing CaMKIIα mRNA.

PubMed

Ortiz, Raúl; Georgieva, Maya V; Gutiérrez, Sara; Pedraza, Neus; Fernández-Moya, Sandra M; Gallego, Carme

2017-07-05

Regulation of mRNA localization is a conserved cellular process observed in many types of cells and organisms. Asymmetrical mRNA distribution plays a particularly important role in the nervous system, where local translation of localized mRNA represents a key mechanism in synaptic plasticity. CaMKIIα is a very abundant mRNA detected in neurites, consistent with its crucial role at glutamatergic synapses. Here, we report the presence of CaMKIIα mRNA isoforms that contain intron i16 in dendrites, RNA granules, and synaptoneurosomes from primary neurons and brain. This subpopulation of unspliced mRNA preferentially localizes to distal dendrites in a synaptic-activity-dependent manner. Staufen2, a well-established marker of RNA transport in dendrites, interacts with intron i16 sequences and enhances its distal dendritic localization, pointing to the existence of intron-mediated mechanisms in the molecular pathways that modulate dendritic transport and localization of synaptic mRNAs. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Ultra-deep mutant spectrum profiling: improving sequencing accuracy using overlapping read pairs.

PubMed

Chen-Harris, Haiyin; Borucki, Monica K; Torres, Clinton; Slezak, Tom R; Allen, Jonathan E

2013-02-12

High throughput sequencing is beginning to make a transformative impact in the area of viral evolution. Deep sequencing has the potential to reveal the mutant spectrum within a viral sample at high resolution, thus enabling the close examination of viral mutational dynamics both within- and between-hosts. The challenge however, is to accurately model the errors in the sequencing data and differentiate real viral mutations, particularly those that exist at low frequencies, from sequencing errors. We demonstrate that overlapping read pairs (ORP) -- generated by combining short fragment sequencing libraries and longer sequencing reads -- significantly reduce sequencing error rates and improve rare variant detection accuracy. Using this sequencing protocol and an error model optimized for variant detection, we are able to capture a large number of genetic mutations present within a viral population at ultra-low frequency levels (<0.05%). Our rare variant detection strategies have important implications beyond viral evolution and can be applied to any basic and clinical research area that requires the identification of rare mutations.

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.

A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization

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

Qiu, Chen; McCann, Kathleen L.; Wine, Robert N.

Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins bind sequence specifically to mRNA targets using a single-stranded RNA-binding domain comprising eight Pumilio (PUM) repeats. PUM repeats have now been identified in proteins that function in pre-rRNA processing, including human Puf-A and yeast Puf6. This is a role not previously ascribed to PUF proteins. In this paper we present crystal structures of human Puf-A that reveal a class of nucleic acid-binding proteins with 11 PUM repeats arranged in an “L”-like shape. In contrast to classical PUF proteins, Puf-A forms sequence-independent interactions with DNA or RNA, mediated by conservedmore » basic residues. We demonstrate that equivalent basic residues in yeast Puf6 are important for RNA binding, pre-rRNA processing, and mRNA localization. Finally, PUM repeats can be assembled into alternative folds that bind to structured nucleic acids in addition to forming canonical eight-repeat crescent-shaped RNA-binding domains found in classical PUF proteins.« less

A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization

DOE PAGES

Qiu, Chen; McCann, Kathleen L.; Wine, Robert N.; ...

2014-12-15

Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins bind sequence specifically to mRNA targets using a single-stranded RNA-binding domain comprising eight Pumilio (PUM) repeats. PUM repeats have now been identified in proteins that function in pre-rRNA processing, including human Puf-A and yeast Puf6. This is a role not previously ascribed to PUF proteins. In this paper we present crystal structures of human Puf-A that reveal a class of nucleic acid-binding proteins with 11 PUM repeats arranged in an “L”-like shape. In contrast to classical PUF proteins, Puf-A forms sequence-independent interactions with DNA or RNA, mediated by conservedmore » basic residues. We demonstrate that equivalent basic residues in yeast Puf6 are important for RNA binding, pre-rRNA processing, and mRNA localization. Finally, PUM repeats can be assembled into alternative folds that bind to structured nucleic acids in addition to forming canonical eight-repeat crescent-shaped RNA-binding domains found in classical PUF proteins.« less

Linear RNA amplification for the production of microarray hybridization probes.

PubMed

Klebes, Ansgar; Kornberg, Thomas B

2008-01-01

To understand Drosophila development and other genetically controlled processes, it is often desirable to identify differences in gene expression levels. An experimental approach to investigate these processes is to catalog the transcriptome by hybridization of mRNA to DNA microbar-rays. In these experiments mRNA-derived hybridization probes are produced and hybridized to an array of DNA spots on a solid support. The labeled cDNAs of the complex hybridization probe will bind to their complementary sequences and provide quantification of the relative concentration of the corresponding transcript in the starting material. However, such approaches are often limited by the scarcity of the experimental sample because standard methods of probe preparation require microgram quantities of mRNA template. Linear RNA amplification can alleviate such limitations to support the generation of microarray hybridization probes from a few 100 pg of mRNA. These smaller quantities can be isolated from a few 100 cells. Here, we present a linear amplification protocol designed to preserve both the relative abundance of transcripts as well as their sequence complexity.

[Effects of bushen yinao tablet on physiology and cerebral gene expression in senescence-accelerated mice].

PubMed

Zhang, Chong; Wang, Jin-gang; Yang, Ting

2006-06-01

To study the effects of Bushen Yin' ao Tablet (BSYNT) on physiology and cerebral gene expression in senescence-accelerated mice (SAM). The change of cerebral tissues mRNA expression in SAM was analyzed and compared by messenger ribonucleic acids reverse transcription differential display polymerase chain reaction (mRNA DDRT-PCR) between the medicated group and the control group. BSYNT could increase the level of hemoglobin (Hb) and amount of erythrocyte (RBC) of blood deficiency mice, improve the spatial learning and memory function and the escape response by conditional stimulus. In this study, 14 differential display bands had been discerned, and three of them had been sequenced. The sequence of the three fragments was similar to fatty acid binding protein 7, ubiquinol-cytochrome C reductase complex (7. 2 kD) and 60S ribosomal protein L21 respectively. And the homogeneity was 97% , 100% , and 99% , respectively. BSYNT has effect on the physiological changing of mice, and its effect on cerebral tissues mRNA expression maybe play an important role in anti-aging on the molecular level.

Calcium-Dependent Protein Kinase Genes in Corn Roots

NASA Technical Reports Server (NTRS)

Takezawa, D.; Patil, S.; Bhatia, A.; Poovaiah, B. W.

1996-01-01

Two cDNAs encoding Ca-2(+) - Dependent Protein Kinases (CDPKs), Corn Root Protein Kinase 1 and 2 (CRPK 1, CRPK 2) were isolated from the root tip library of corn (Zea mays L., cv. Merit) and their nucleotide sequences were determined. Deduced amino acid sequences of both the clones have features characteristic of plant CDPKS, including all 11 conserved serine/threonine kinase subdomains, a junction domain and a calmodulin-like domain with four Ca-2(+), -binding sites. Northern analysis revealed that CRPKI mRNA is preferentially expressed in roots, especially in the root tip; whereas, the expression of CRPK2 mRNA was very low in all the tissues tested. In situ hybridization experiments revealed that CRPKI mRNA is highly expressed in the root apex, as compared to other parts of the root. Partially purified CDPK from the root tip phosphorylates syntide-2, a common peptide substrate for plant CDPKs, and the phosphorylation was stimulated 7-fold by the addition of Ca-2(+). Our results show that two CDPK isoforms are expressed in corn roots and they may be involved in the Ca-2(+)-dependent signal transduction process.

Demonstration of mRNA editing and localization of guide RNA genes in kinetoplast-mitochondria of the plant trypanosomatid Phytomonas serpens.

PubMed

Maslov, D A; Hollar, L; Haghighat, P; Nawathean, P

1998-06-01

Maxicircle molecules of kDNA in several isolates of Phytomonas were detected by hybridization with the 12S rRNA gene probe from Leishmania tarentolae. The estimated size of maxicircles is isolate-specific and varies from 27 to 36 kb. Fully edited and polyadenylated mRNA for kinetoplast-encoded ribosomal protein S12 (RPS12) was found in the steady-state kinetoplast RNA isolated from Phytomonas serpens strain 1G. Two minicircles (1.45 kb) from this strain were also sequenced. Each minicircle contains two 120 bp conserved regions positioned 180 degrees apart, a region enriched with G and T bases and a variable region. One minicircle encodes a gRNA for the first block of editing of RPSl2 mRNA, and the other encodes a gRNA with unknown function. A gRNA gene for the second block of RPSl2 was found on a minicircle sequenced previously. On each minicircle, a gRNA gene is located in the variable region in a similar position and orientation with respect to the conserved regions.

Low levels of PRB3 mRNA are associated with dopamine-agonist resistance and tumor recurrence in prolactinomas.

PubMed

Wang, Fei; Gao, Hua; Li, Chuzhong; Bai, Jiwei; Lu, Runchun; Cao, Lei; Wu, Yongtu; Hong, Lichuan; Wu, Yonggang; Lan, Xiaolei; Zhang, Yazhuo

2014-01-01

Prolactinomas, or prolactin-secreting adenomas, constitute the most common type of hyperfunctioning pituitary adenoma. Dopamine agonists are used as first-line medication for prolactinomas, but the tumors are resistant to the therapy in 5-18 % of patients. To explore potential mechanisms of resistance to bromocriptine (a dopamine agonist), we analyzed six responsive prolactinomas and six resistant prolactinomas by whole-exome sequencing. We identified ten genes with sequence variants that were differentially found in the two groups of tumors. The expression of these genes was then quantified by real-time reverse-transcription PCR (RT-qPCR) in the 12 prolactinomas and in six normal pituitary glands. The mRNA levels of one of the genes, PRB3, were about fourfold lower in resistant prolactinomas than in the responsive tumors (p = 0.02). Furthermore, low PRB3 expression was also associated with tumor recurrence. Our results suggest that low levels of PRB3 mRNA may have a role in dopamine-agonist resistance and tumor recurrence of prolactinomas.

Molecular mechanism for the operation of nitrogen control in cyanobacteria.

PubMed Central

Luque, I; Flores, E; Herrero, A

1994-01-01

In cyanobacteria, ammonium exerts a negative regulation of the expression of proteins involved in the assimilation of nitrogen sources alternative to ammonium. In Synechococcus, mRNA levels of genes encoding proteins for nitrate and ammonium assimilation were observed to be negatively regulated by ammonium, and ammonium-regulated transcription start points were identified for those genes. The NtcA protein is a positive regulator of genes subjected to nitrogen control by ammonium. Mutants lacking NtcA exhibited only basal mRNA levels of the regulated genes, even in the absence of ammonium, indicating that NtcA exerts its regulatory action by positively influencing mRNA levels of the nitrogen-regulated genes. NtcA was observed to bind directly to the promoters of nitrogen-regulated genes, and the palindromic DNA sequence GTAN8TAC was identified as a sequence signature for NtcA-target sites. The structure of the nitrogen-, NtcA-regulated promoters of Synechococcus was determined to be constituted by a -10, Pribnow-like box in the form TAN3T, and an NtcA-binding site that substituted for the canonical -35 box. Images PMID:8026471

Deep RNNs for video denoising

NASA Astrophysics Data System (ADS)

Chen, Xinyuan; Song, Li; Yang, Xiaokang

2016-09-01

Video denoising can be described as the problem of mapping from a specific length of noisy frames to clean one. We propose a deep architecture based on Recurrent Neural Network (RNN) for video denoising. The model learns a patch-based end-to-end mapping between the clean and noisy video sequences. It takes the corrupted video sequences as the input and outputs the clean one. Our deep network, which we refer to as deep Recurrent Neural Networks (deep RNNs or DRNNs), stacks RNN layers where each layer receives the hidden state of the previous layer as input. Experiment shows (i) the recurrent architecture through temporal domain extracts motion information and does favor to video denoising, and (ii) deep architecture have large enough capacity for expressing mapping relation between corrupted videos as input and clean videos as output, furthermore, (iii) the model has generality to learned different mappings from videos corrupted by different types of noise (e.g., Poisson-Gaussian noise). By training on large video databases, we are able to compete with some existing video denoising methods.

Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

PubMed Central

Toro-Ascuy, Daniela; Rojas-Araya, Bárbara; Valiente-Echeverría, Fernando; Soto-Rifo, Ricardo

2016-01-01

The human immunodeficiency virus type-1 (HIV-1) unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs) containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1), Staufen double-stranded RNA binding protein 1/2 (STAU1/2), or components of miRNA-induced silencing complex (miRISC) and processing bodies (PBs). More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A), allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries. PMID:27886048

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.

A sense oligonucleotide to inducible nitric oxide synthase mRNA increases the survival rate of rats in septic shock.

PubMed

Okuyama, Tetsuya; Nakatake, Richi; Kaibori, Masaki; Okumura, Tadayoshi; Kon, Masanori; Nishizawa, Mikio

2018-01-30

Natural antisense transcripts (asRNAs) that do not encode proteins are transcribed from rat, mouse, and human genes, encoding inducible nitric oxide synthase (iNOS), which catalyzes the production of the inflammatory mediator nitric oxide (NO). In septic shock, NO is excessively produced in hepatocytes and macrophages. The iNOS asRNA interacts with and stabilizes iNOS mRNA. We found that single-stranded 'sense' oligonucleotides corresponding to the iNOS mRNA sequence reduced iNOS mRNA levels by interfering with the mRNA-asRNA interactions in rat hepatocytes. The iNOS sense oligonucleotides that were substituted with phosphorothioate bonds and locked nucleic acids efficiently decreased the levels of iNOS mRNA and iNOS protein. In this study, the gene expression patterns in the livers of two endotoxemia model rats with acute liver failure were compared. Next, we optimized the sequence and modification of the iNOS sense oligonucleotides in interleukin 1β-treated rat hepatocytes. When a sense oligonucleotide was simultaneously administered with d-galactosamine and bacterial lipopolysaccharide (LPS) to rats, their survival rate significantly increased compared to the rats administered d-galactosamine and LPS alone. In the livers of the sense oligonucleotide-administered rats, apoptosis in the hepatocytes markedly decreased. These results suggest that natural antisense transcript-targeted regulation technology using iNOS sense oligonucleotides may be used to treat human inflammatory diseases, such as sepsis and septic shock. Copyright © 2017 Elsevier Inc. All rights reserved.

Progranulin mutation causes frontotemporal dementia in the Swedish Karolinska family.

PubMed

Chiang, Huei-Hsin; Rosvall, Lina; Brohede, Jesper; Axelman, Karin; Björk, Behnosh F; Nennesmo, Inger; Robins, Tiina; Graff, Caroline

2008-11-01

Frontotemporal dementia (FTD) is a neurodegenerative disease characterized by cognitive impairment, language dysfunction, and/or changes in personality. Recently it has been shown that progranulin (GRN) mutations can cause FTD as well as other neurodegenerative phenotypes. DNA from 30 family members, of whom seven were diagnosed with FTD, in the Karolinska family was available for GRN sequencing. Fibroblast cell mRNA from one affected family member and six control individuals was available for relative quantitative real-time polymerase chain reaction to investigate the effect of the mutation. Furthermore, the cDNA of an affected individual was sequenced. Clinical and neuropathologic findings of a previously undescribed family branch are presented. A frameshift mutation in GRN (g.102delC) was detected in all affected family members and absent in four unaffected family members older than 70 years. Real-time polymerase chain reaction data showed an approximately 50% reduction of GRN fibroblast mRNA in an affected individual. The mutated mRNA transcripts were undetectable by cDNA sequencing. Segregation and RNA analyses showed that the g.102delC mutation, previously reported, causes FTD in the Karolinska family. Our findings add further support to the significance of GRN in FTD etiology and the presence of modifying genes, which emphasize the need for further studies into the mechanisms of clinical heterogeneity. However, the results already call for attention to the complexity of predictive genetic testing of GRN mutations.

Detection and Analysis of Circular RNAs by RT-PCR.

PubMed

Panda, Amaresh C; Gorospe, Myriam

2018-03-20

Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al ., 2017b; Panda et al ., 2017c). However, the validation of true circRNAs identified by RNA sequencing requires other molecular biology techniques including reverse transcription (RT) followed by conventional or quantitative (q) polymerase chain reaction (PCR), and Northern blot analysis (Jeck and Sharpless, 2014). RT-qPCR analysis of circular RNAs using divergent primers has been widely used for the detection, validation, and sometimes quantification of circRNAs (Abdelmohsen et al ., 2015 and 2017; Panda et al ., 2017b). As detailed here, divergent primers designed to span the circRNA backsplice junction sequence can specifically amplify the circRNAs and not the counterpart linear RNA. In sum, RT-PCR analysis using divergent primers allows direct detection and quantification of circRNAs.

deepTools2: a next generation web server for deep-sequencing data analysis.

PubMed

Ramírez, Fidel; Ryan, Devon P; Grüning, Björn; Bhardwaj, Vivek; Kilpert, Fabian; Richter, Andreas S; Heyne, Steffen; Dündar, Friederike; Manke, Thomas

2016-07-08

We present an update to our Galaxy-based web server for processing and visualizing deeply sequenced data. Its core tool set, deepTools, allows users to perform complete bioinformatic workflows ranging from quality controls and normalizations of aligned reads to integrative analyses, including clustering and visualization approaches. Since we first described our deepTools Galaxy server in 2014, we have implemented new solutions for many requests from the community and our users. Here, we introduce significant enhancements and new tools to further improve data visualization and interpretation. deepTools continue to be open to all users and freely available as a web service at deeptools.ie-freiburg.mpg.de The new deepTools2 suite can be easily deployed within any Galaxy framework via the toolshed repository, and we also provide source code for command line usage under Linux and Mac OS X. A public and documented API for access to deepTools functionality is also available. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Deep ART Neural Model for Biologically Inspired Episodic Memory and Its Application to Task Performance of Robots.

PubMed

Park, Gyeong-Moon; Yoo, Yong-Ho; Kim, Deok-Hwa; Kim, Jong-Hwan; Gyeong-Moon Park; Yong-Ho Yoo; Deok-Hwa Kim; Jong-Hwan Kim; Yoo, Yong-Ho; Park, Gyeong-Moon; Kim, Jong-Hwan; Kim, Deok-Hwa

2018-06-01

Robots are expected to perform smart services and to undertake various troublesome or difficult tasks in the place of humans. Since these human-scale tasks consist of a temporal sequence of events, robots need episodic memory to store and retrieve the sequences to perform the tasks autonomously in similar situations. As episodic memory, in this paper we propose a novel Deep adaptive resonance theory (ART) neural model and apply it to the task performance of the humanoid robot, Mybot, developed in the Robot Intelligence Technology Laboratory at KAIST. Deep ART has a deep structure to learn events, episodes, and even more like daily episodes. Moreover, it can retrieve the correct episode from partial input cues robustly. To demonstrate the effectiveness and applicability of the proposed Deep ART, experiments are conducted with the humanoid robot, Mybot, for performing the three tasks of arranging toys, making cereal, and disposing of garbage.

Temporal and spatial regulation of mRNA export: Single particle RNA-imaging provides new tools and insights

PubMed Central

Heinrich, Stephanie; Derrer, Carina Patrizia; Lari, Azra; Weis, Karsten; Montpetit, Ben

2017-01-01

The transport of messenger RNAs (mRNAs) from the nucleus to cytoplasm is an essential step in the gene expression program of all eukaryotes. Recent technological advances in the areas of RNA-labeling, microscopy, and sequencing are leading to novel insights about mRNA biogenesis and export. This includes quantitative single molecule imaging (SMI) of RNA molecules in live cells, which is providing knowledge of the spatial and temporal dynamics of the export process. As this information becomes available, it leads to new questions, the reinterpretation of previous findings, and revised models of mRNA export. In this review, we will briefly highlight some of these recent findings and discuss how live cell SMI approaches may be used to further our current understanding of mRNA export and gene expression. PMID:28052353

Construction of a Lotus japonicus late nodulin expressed sequence tag library and identification of novel nodule-specific genes.

PubMed Central

Szczyglowski, K; Hamburger, D; Kapranov, P; de Bruijn, F J

1997-01-01

A range of novel expressed sequence tags (ESTs) associated with late developmental events during nodule organogenesis in the legume Lotus japonicus were identified using mRNA differential display; 110 differentially displayed polymerase chain reaction products were cloned and analyzed. Of 88 unique cDNAs obtained, 22 shared significant homology to DNA/protein sequences in the respective databases. This group comprises, among others, a nodule-specific homolog of protein phosphatase 2C, a peptide transporter protein, and a nodule-specific form of cytochrome P450. RNA gel-blot analysis of 16 differentially displayed ESTs confirmed their nodule-specific expression pattern. The kinetics of mRNA accumulation of the majority of the ESTs analyzed were found to resemble the expression pattern observed for the L. japonicus leghemoglobin gene. These results indicate that the newly isolated molecular markers correspond to genes induced during late developmental stages of L. japonicus nodule organogenesis and provide important, novel tools for the study of nodulation. PMID:9276951

Investigation of mRNA quadruplex formation in Escherichia coli.

PubMed

Wieland, Markus; Hartig, Jörg S

2009-01-01

The protocol presented here allows for the investigation of the formation of unusual nucleic acid structures in the 5'-untranslated region (UTR) of bacteria by correlating gene expression levels to the in vitro stability of the respective structure. In particular, we describe the introduction of G-quadruplex forming sequences close to the ribosome-binding site (RBS) on the mRNA of a reporter gene and the subsequent read-out of the expression levels. Insertion of a stable secondary structure results in the cloaking of RBS and eventually reduced gene expression levels. The structures and stability of the introduced sequences are further characterized by circular dichroism (CD) spectroscopy and thermal melting experiments. The extent of inhibition is then correlated to the stability of the respective quadruplex structure, allowing judgement of whether factors other than thermodynamic stability affect the formation of a given quadruplex sequence in vivo. Measuring gene expression levels takes 2 d including cloning; CD experiments take 5 hours per experiment.

Optimization of whole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysis.

PubMed

Wu, Jieying; Gao, Weimin; Zhang, Weiwen; Meldrum, Deirdre R

2011-01-01

Limitation in sample quality and quantity is one of the big obstacles for applying metatranscriptomic technologies to explore gene expression and functionality of microbial communities in natural environments. In this study, several amplification methods were evaluated for whole-transcriptome amplification of deep-sea microbial samples, which are of low cell density and high impurity. The best amplification method was identified and incorporated into a complete protocol to isolate and amplify deep-sea microbial samples. In the protocol, total RNA was first isolated by a modified method combining Trizol (Invitrogen, CA) and RNeasy (QIAGEN, CA) method, amplified with a WT-Ovation™ Pico RNA Amplification System (NuGEN, CA), and then converted to double-strand DNA from single-strand cDNA with a WT-Ovation™ Exon Module (NuGEN, CA). The products from the whole-transcriptome amplification of deep-sea microbial samples were assessed first through random clone library sequencing. The BLAST search results showed that marine-based sequences are dominant in the libraries, consistent with the ecological source of the samples. The products were then used for next-generation Roche GS FLX Titanium sequencing to obtain metatranscriptome data. Preliminary analysis of the metatranscriptomic data showed good sequencing quality. Although the protocol was designed and demonstrated to be effective for deep-sea microbial samples, it should be applicable to similar samples from other extreme environments in exploring community structure and functionality of microbial communities. Copyright © 2010 Elsevier B.V. All rights reserved.

Quantitative imaging of single mRNA splice variants in living cells

NASA Astrophysics Data System (ADS)

Lee, Kyuwan; Cui, Yi; Lee, Luke P.; Irudayaraj, Joseph

2014-06-01

Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.

Technical variations in low-input RNA-seq methodologies.

PubMed

Bhargava, Vipul; Head, Steven R; Ordoukhanian, Phillip; Mercola, Mark; Subramaniam, Shankar

2014-01-14

Recent advances in RNA-seq methodologies from limiting amounts of mRNA have facilitated the characterization of rare cell-types in various biological systems. So far, however, technical variations in these methods have not been adequately characterized, vis-à-vis sensitivity, starting with reduced levels of mRNA. Here, we generated sequencing libraries from limiting amounts of mRNA using three amplification-based methods, viz. Smart-seq, DP-seq and CEL-seq, and demonstrated significant technical variations in these libraries. Reduction in mRNA levels led to inefficient amplification of the majority of low to moderately expressed transcripts. Furthermore, noise in primer hybridization and/or enzyme incorporation was magnified during the amplification step resulting in significant distortions in fold changes of the transcripts. Consequently, the majority of the differentially expressed transcripts identified were either high-expressed and/or exhibited high fold changes. High technical variations ultimately masked subtle biological differences mandating the development of improved amplification-based strategies for quantitative transcriptomics from limiting amounts of mRNA.

The complete mitochondrial genome of the deep-sea sponge Poecillastra laminaris (Astrophorida, Vulcanellidae).

PubMed

Zeng, Cong; Thomas, Leighton J; Kelly, Michelle; Gardner, Jonathan P A

2016-05-01

The complete mitochondrial genome of a New Zealand specimen of the deep-sea sponge Poecillastra laminaris (Sollas, 1886) (Astrophorida, Vulcanellidae), from the Colville Ridge, New Zealand, was sequenced using the 454 Life Science pyrosequencing system. To identify homologous mitochondrial sequences, the 454 reads were mapped to the complete mitochondrial genome sequence of Geodia neptuni (GeneBank No. NC_006990). The P. laminaris genome is 18,413 bp in length and includes 14 protein-coding genes, 24 transfer RNA genes and 2 ribosomal RNA genes. Gene order resembled that of other demosponges. The base composition of the genome is A (29.1%), T (35.2%), C (14.0%) and G (21.7%). This is the second published mitogenome for a sponge of the order Astrophorida and will be useful in future phylogenetic analysis of deep-sea sponges.

MusiteDeep: a deep-learning framework for general and kinase-specific phosphorylation site prediction.

PubMed

Wang, Duolin; Zeng, Shuai; Xu, Chunhui; Qiu, Wangren; Liang, Yanchun; Joshi, Trupti; Xu, Dong

2017-12-15

Computational methods for phosphorylation site prediction play important roles in protein function studies and experimental design. Most existing methods are based on feature extraction, which may result in incomplete or biased features. Deep learning as the cutting-edge machine learning method has the ability to automatically discover complex representations of phosphorylation patterns from the raw sequences, and hence it provides a powerful tool for improvement of phosphorylation site prediction. We present MusiteDeep, the first deep-learning framework for predicting general and kinase-specific phosphorylation sites. MusiteDeep takes raw sequence data as input and uses convolutional neural networks with a novel two-dimensional attention mechanism. It achieves over a 50% relative improvement in the area under the precision-recall curve in general phosphorylation site prediction and obtains competitive results in kinase-specific prediction compared to other well-known tools on the benchmark data. MusiteDeep is provided as an open-source tool available at https://github.com/duolinwang/MusiteDeep. xudong@missouri.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Fungal diversity in deep-sea sediments associated with asphalt seeps at the Sao Paulo Plateau

NASA Astrophysics Data System (ADS)

Nagano, Yuriko; Miura, Toshiko; Nishi, Shinro; Lima, Andre O.; Nakayama, Cristina; Pellizari, Vivian H.; Fujikura, Katsunori

2017-12-01

We investigated the fungal diversity in a total of 20 deep-sea sediment samples (of which 14 samples were associated with natural asphalt seeps and 6 samples were not associated) collected from two different sites at the Sao Paulo Plateau off Brazil by Ion Torrent PGM targeting ITS region of ribosomal RNA. Our results suggest that diverse fungi (113 operational taxonomic units (OTUs) based on clustering at 97% sequence similarity assigned into 9 classes and 31 genus) are present in deep-sea sediment samples collected at the Sao Paulo Plateau, dominated by Ascomycota (74.3%), followed by Basidiomycota (11.5%), unidentified fungi (7.1%), and sequences with no affiliation to any organisms in the public database (7.1%). However, it was revealed that only three species, namely Penicillium sp., Cadophora malorum and Rhodosporidium diobovatum, were dominant, with the majority of OTUs remaining a minor community. Unexpectedly, there was no significant difference in major fungal community structure between the asphalt seep and non-asphalt seep sites, despite the presence of mass hydrocarbon deposits and the high amount of macro organisms surrounding the asphalt seeps. However, there were some differences in the minor fungal communities, with possible asphalt degrading fungi present specifically in the asphalt seep sites. In contrast, some differences were found between the two different sampling sites. Classification of OTUs revealed that only 47 (41.6%) fungal OTUs exhibited >97% sequence similarity, in comparison with pre-existing ITS sequences in public databases, indicating that a majority of deep-sea inhabiting fungal taxa still remain undescribed. Although our knowledge on fungi and their role in deep-sea environments is still limited and scarce, this study increases our understanding of fungal diversity and community structure in deep-sea environments.

CrossQuery: a web tool for easy associative querying of transcriptome data.

PubMed

Wagner, Toni U; Fischer, Andreas; Thoma, Eva C; Schartl, Manfred

2011-01-01

Enormous amounts of data are being generated by modern methods such as transcriptome or exome sequencing and microarray profiling. Primary analyses such as quality control, normalization, statistics and mapping are highly complex and need to be performed by specialists. Thereafter, results are handed back to biomedical researchers, who are then confronted with complicated data lists. For rather simple tasks like data filtering, sorting and cross-association there is a need for new tools which can be used by non-specialists. Here, we describe CrossQuery, a web tool that enables straight forward, simple syntax queries to be executed on transcriptome sequencing and microarray datasets. We provide deep-sequencing data sets of stem cell lines derived from the model fish Medaka and microarray data of human endothelial cells. In the example datasets provided, mRNA expression levels, gene, transcript and sample identification numbers, GO-terms and gene descriptions can be freely correlated, filtered and sorted. Queries can be saved for later reuse and results can be exported to standard formats that allow copy-and-paste to all widespread data visualization tools such as Microsoft Excel. CrossQuery enables researchers to quickly and freely work with transcriptome and microarray data sets requiring only minimal computer skills. Furthermore, CrossQuery allows growing association of multiple datasets as long as at least one common point of correlated information, such as transcript identification numbers or GO-terms, is shared between samples. For advanced users, the object-oriented plug-in and event-driven code design of both server-side and client-side scripts allow easy addition of new features, data sources and data types.

Transcriptome profiling analysis of Mactra veneriformis by deep sequencing after exposure to 2,2',4,4'-tetrabromodiphenyl ether

NASA Astrophysics Data System (ADS)

Shi, Pengju; Dong, Shihang; Zhang, Huanjun; Wang, Peiliang; Niu, Zhuang; Fang, Yan

2018-03-01

Polybrominated diphenyl ethers (PBDEs) are ubiquitous global pollutants, which are known to have immune, development, reproduction, and endocrine toxicity in aquatic organisms, including bivalves. 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is the predominant PBDE congener detected in environmental samples and the tissues of organisms. However, the mechanism of its toxicity remains unclear. In this study, high-throughput sequencing was performed using the clam Mactra veneriformis, a good model for toxicological research, to clarify the transcriptomic response to BDE-47 and the mechanism responsible for the toxicity of BDE-47. The clams were exposed to 5 μg/L BDE-47 for 3 days and the digestive glands were sampled for high-throughput sequencing analysis. We obtained 127 648, 154 225, and 124 985 unigenes by de novo assembly of the control group reads (CG), BDE-47 group reads (BDEG), and control and BDE-47 reads (CG & BDEG), respectively. We annotated 32 176 unigenes from the CG & BDEG reads using the NR database. We categorized 24 401 unigenes into 25 functional COG clusters and 21 749 unigenes were assigned to 259 KEGG pathways. Moreover, 17 625 differentially expressed genes (DEGs) were detected, with 10 028 upregulated DEGs and 7 597 downregulated DEGs. Functional enrichment analysis showed that the DEGs were involved with detoxification, antioxidant defense, immune response, apoptosis, and other functions. The mRNA expression levels of 26 DEGs were verified by quantitative real-time PCR, which demonstrated the high agreement between the two methods. These results provide a good basis for future research using the M. veneriformis model into the mechanism of PBDEs toxicity and molecular biomarkers for BDE-47 pollution. The regulation and interaction of the DEGs would be studied in the future for clarifying the mechanism of PBDEs toxicity.

Vertical Segregation and Phylogenetic Characterization of Ammonia-Oxidizing Bacteria and Archaea in the Sediment of a Freshwater Aquaculture Pond

PubMed Central

Lu, Shimin; Liu, Xingguo; Ma, Zhuojun; Liu, Qigen; Wu, Zongfan; Zeng, Xianlei; Shi, Xu; Gu, Zhaojun

2016-01-01

Pond aquaculture is the major freshwater aquaculture method in China. Ammonia-oxidizing communities inhabiting pond sediments play an important role in controlling culture water quality. However, the distribution and activities of ammonia-oxidizing microbial communities along sediment profiles are poorly understood in this specific environment. Vertical variations in the abundance, transcription, potential ammonia oxidizing rate, and community composition of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in sediment samples (0–50 cm depth) collected from a freshwater aquaculture pond were investigated. The concentrations of the AOA amoA gene were higher than those of the AOB by an order of magnitude, which suggested that AOA, as opposed to AOB, were the numerically predominant ammonia-oxidizing organisms in the surface sediment. This could be attributed to the fact that AOA are more resistant to low levels of dissolved oxygen. However, the concentrations of the AOB amoA mRNA were higher than those of the AOA by 2.5- to 39.9-fold in surface sediments (0–10 cm depth), which suggests that the oxidation of ammonia was mainly performed by AOB in the surface sediments, and by AOA in the deeper sediments, where only AOA could be detected. Clone libraries of AOA and AOB amoA sequences indicated that the diversity of AOA and AOB decreased with increasing depth. The AOB community consisted of two groups: the Nitrosospira and Nitrosomonas clusters, and Nitrosomonas were predominant in the freshwater pond sediment. All AOA amoA gene sequences in the 0–2 cm deep sediment were grouped into the Nitrososphaera cluster, while other AOA sequences in deeper sediments (10–15 and 20–25 cm depths) were grouped into the Nitrosopumilus cluster. PMID:26834709

Deep Sequencing Reveals Uncharted Isoform Heterogeneity of the Protein-Coding Transcriptome in Cerebral Ischemia.

PubMed

Bhattarai, Sunil; Aly, Ahmed; Garcia, Kristy; Ruiz, Diandra; Pontarelli, Fabrizio; Dharap, Ashutosh

2018-06-03

Gene expression in cerebral ischemia has been a subject of intense investigations for several years. Studies utilizing probe-based high-throughput methodologies such as microarrays have contributed significantly to our existing knowledge but lacked the capacity to dissect the transcriptome in detail. Genome-wide RNA-sequencing (RNA-seq) enables comprehensive examinations of transcriptomes for attributes such as strandedness, alternative splicing, alternative transcription start/stop sites, and sequence composition, thus providing a very detailed account of gene expression. Leveraging this capability, we conducted an in-depth, genome-wide evaluation of the protein-coding transcriptome of the adult mouse cortex after transient focal ischemia at 6, 12, or 24 h of reperfusion using RNA-seq. We identified a total of 1007 transcripts at 6 h, 1878 transcripts at 12 h, and 1618 transcripts at 24 h of reperfusion that were significantly altered as compared to sham controls. With isoform-level resolution, we identified 23 splice variants arising from 23 genes that were novel mRNA isoforms. For a subset of genes, we detected reperfusion time-point-dependent splice isoform switching, indicating an expression and/or functional switch for these genes. Finally, for 286 genes across all three reperfusion time-points, we discovered multiple, distinct, simultaneously expressed and differentially altered isoforms per gene that were generated via alternative transcription start/stop sites. Of these, 165 isoforms derived from 109 genes were novel mRNAs. Together, our data unravel the protein-coding transcriptome of the cerebral cortex at an unprecedented depth to provide several new insights into the flexibility and complexity of stroke-related gene transcription and transcript organization.

Gene and genon concept: coding versus regulation

PubMed Central

2007-01-01

We analyse here the definition of the gene in order to distinguish, on the basis of modern insight in molecular biology, what the gene is coding for, namely a specific polypeptide, and how its expression is realized and controlled. Before the coding role of the DNA was discovered, a gene was identified with a specific phenotypic trait, from Mendel through Morgan up to Benzer. Subsequently, however, molecular biologists ventured to define a gene at the level of the DNA sequence in terms of coding. As is becoming ever more evident, the relations between information stored at DNA level and functional products are very intricate, and the regulatory aspects are as important and essential as the information coding for products. This approach led, thus, to a conceptual hybrid that confused coding, regulation and functional aspects. In this essay, we develop a definition of the gene that once again starts from the functional aspect. A cellular function can be represented by a polypeptide or an RNA. In the case of the polypeptide, its biochemical identity is determined by the mRNA prior to translation, and that is where we locate the gene. The steps from specific, but possibly separated sequence fragments at DNA level to that final mRNA then can be analysed in terms of regulation. For that purpose, we coin the new term “genon”. In that manner, we can clearly separate product and regulative information while keeping the fundamental relation between coding and function without the need to introduce a conceptual hybrid. In mRNA, the program regulating the expression of a gene is superimposed onto and added to the coding sequence in cis - we call it the genon. The complementary external control of a given mRNA by trans-acting factors is incorporated in its transgenon. A consequence of this definition is that, in eukaryotes, the gene is, in most cases, not yet present at DNA level. Rather, it is assembled by RNA processing, including differential splicing, from various pieces, as steered by the genon. It emerges finally as an uninterrupted nucleic acid sequence at mRNA level just prior to translation, in faithful correspondence with the amino acid sequence to be produced as a polypeptide. After translation, the genon has fulfilled its role and expires. The distinction between the protein coding information as materialised in the final polypeptide and the processing information represented by the genon allows us to set up a new information theoretic scheme. The standard sequence information determined by the genetic code expresses the relation between coding sequence and product. Backward analysis asks from which coding region in the DNA a given polypeptide originates. The (more interesting) forward analysis asks in how many polypeptides of how many different types a given DNA segment is expressed. This concerns the control of the expression process for which we have introduced the genon concept. Thus, the information theoretic analysis can capture the complementary aspects of coding and regulation, of gene and genon. PMID:18087760

Gene expression profiling of flax (Linum usitatissimum L.) under edaphic stress.

PubMed

Dmitriev, Alexey A; Kudryavtseva, Anna V; Krasnov, George S; Koroban, Nadezhda V; Speranskaya, Anna S; Krinitsina, Anastasia A; Belenikin, Maxim S; Snezhkina, Anastasiya V; Sadritdinova, Asiya F; Kishlyan, Natalya V; Rozhmina, Tatiana A; Yurkevich, Olga Yu; Muravenko, Olga V; Bolsheva, Nadezhda L; Melnikova, Nataliya V

2016-11-16

Cultivated flax (Linum usitatissimum L.) is widely used for production of textile, food, chemical and pharmaceutical products. However, various stresses decrease flax production. Search for genes, which are involved in stress response, is necessary for breeding of adaptive cultivars. Imbalanced concentration of nutrient elements in soil decrease flax yields and also results in heritable changes in some flax lines. The appearance of Linum Insertion Sequence 1 (LIS-1) is the most studied modification. However, LIS-1 function is still unclear. High-throughput sequencing of transcriptome of flax plants grown under normal (N), phosphate deficient (P), and nutrient excess (NPK) conditions was carried out using Illumina platform. The assembly of transcriptome was performed, and a total of 34924, 33797, and 33698 unique transcripts for N, P, and NPK sequencing libraries were identified, respectively. We have not revealed any LIS-1 derived mRNA in our sequencing data. The analysis of high-throughput sequencing data allowed us to identify genes with potentially differential expression under imbalanced nutrition. For further investigation with qPCR, 15 genes were chosen and their expression levels were evaluated in the extended sampling of 31 flax plants. Significant expression alterations were revealed for genes encoding WRKY and JAZ protein families under P and NPK conditions. Moreover, the alterations of WRKY family genes differed depending on LIS-1 presence in flax plant genome. Besides, we revealed slight and LIS-1 independent mRNA level changes of KRP2 and ING1 genes, which are adjacent to LIS-1, under nutrition stress. Differentially expressed genes were identified in flax plants, which were grown under phosphate deficiency and excess nutrition, on the basis of high-throughput sequencing and qPCR data. We showed that WRKY and JAS gene families participate in flax response to imbalanced nutrient content in soil. Besides, we have not identified any mRNA, which could be derived from LIS-1, in our transcriptome sequencing data. Expression of LIS-1 flanking genes, ING1 and KRP2, was suggested not to be nutrient stress-induced. Obtained results provide new insights into edaphic stress response in flax and the role of LIS-1 in these process.

LookSeq: a browser-based viewer for deep sequencing data.

PubMed

Manske, Heinrich Magnus; Kwiatkowski, Dominic P

2009-11-01

Sequencing a genome to great depth can be highly informative about heterogeneity within an individual or a population. Here we address the problem of how to visualize the multiple layers of information contained in deep sequencing data. We propose an interactive AJAX-based web viewer for browsing large data sets of aligned sequence reads. By enabling seamless browsing and fast zooming, the LookSeq program assists the user to assimilate information at different levels of resolution, from an overview of a genomic region to fine details such as heterogeneity within the sample. A specific problem, particularly if the sample is heterogeneous, is how to depict information about structural variation. LookSeq provides a simple graphical representation of paired sequence reads that is more revealing about potential insertions and deletions than are conventional methods.

An improved strategy and a useful housekeeping gene for RNA analysis from formalin-fixed, paraffin-embedded tissues by PCR.

PubMed

Finke, J; Fritzen, R; Ternes, P; Lange, W; Dölken, G

1993-03-01

Specific amplification of nucleic acid sequences by PCR has been extensively used for the detection of gene rearrangements and gene expression. Although successful amplification of DNA sequences has been carried out with DNA prepared from formalin-fixed, paraffin-embedded (FFPE) tissues, there are only a few reports regarding RNA analysis in this kind of material. We describe a procedure for RNA extraction from different types of FFPE tissues, involving digestion with proteinase K followed by guanidinium-thiocyanate acid phenol extraction and DNase I digestion. These RNA preparations are suitable for PCR analysis of mRNA and even of intronless genes. Furthermore, the universally expressed porphobilinogen deaminase mRNA proved to be useful as a positive control because of the lack of pseudogenes.

A manganese-dependent ribozyme in the 3'-untranslated region of Xenopus Vg1 mRNA.

PubMed

Kolev, Nikolay G; Hartland, Emilia I; Huber, Paul W

2008-10-01

The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage. We show here that this ribozyme occurs naturally in the 3'-UTR of Vg1 and beta-actin mRNAs. In accord with earlier studies with model RNAs, cleavage occurs only in the presence of manganese or cadmium ions and proceeds optimally near 30 degrees C and physiological pH. The time course of cleavage in Vg1 mRNA best fits a two-step process in which both steps are first-order. In Vg1 mRNA, the ribozyme is positioned adjacent to a polyadenylation signal, but has no influence on translation of the mRNA in Xenopus oocytes. Putative GAAA ribozyme structures are also near polyadenylation sites in yeast and rat actin mRNAs. Analysis of sequences in the PolyA Cleavage Site and 3'-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts. Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3'-end processing of eukaryotic mRNAs.

Reversion of multidrug resistance in the P-glycoprotein-positive human pancreatic cell line (EPP85-181RDB) by introduction of a hammerhead ribozyme.

PubMed Central

Holm, P. S.; Scanlon, K. J.; Dietel, M.

1994-01-01

A major problem in cytostatic treatment of many tumours is the development of multidrug resistance (MDR4). This is most often accompanied by the overexpression of a membrane transport protein, P-glycoprotein, and its encoding mRNA. In order to reverse the resistant phenotype in cell cultures, we constructed a specific hammerhead ribozyme possessing catalytic activity that cleaves the 3'-end of the GUC sequence in codon 880 of the mdr1 mRNA. We demonstrated that the constructed ribozyme is able to cleave a reduced substrate mdr1 mRNA at the GUC position under physiological conditions in a cell-free system. A DNA sequence encoding the ribozyme gene was then incorporated into a mammalian expression vector (pH beta APr-1 neo) and transfected into the human pancreatic carcinoma cell line EPP85-181RDB, which is resistant to daunorubicin and expresses the MDR phenotype. The expressed ribozyme decreased the level of mdr1 mRNA expression, inhibited the formation of P-glycoprotein and reduced the cell's resistance to daunorubicin dramatically; this means that the resistant cells were 1,600-fold more resistant than the parental cell line (EPP85-181P), whereas those cell clones that showed ribozyme expression were only 5.3-fold more resistant than the parental cell line. Images Figure 1 Figure 3 Figure 2 PMID:7914421

Effect of long real space flight on the whole genome mRNA expression properties in medaka Oryzias latipes

NASA Astrophysics Data System (ADS)

Kozlova, Olga; Gusev, Oleg; Levinskikh, Margarita; Sychev, Vladimir; Poddubko, Svetlana

The current study is addressed to the complex analysis of whole genome mRNA expression profile and properties of splicing variants formation in different organs of medaka fish exposed to prolonged space flight in the frame of joint Russia-Japan research program “Aquarium-AQH”. The fish were kept in the AQH joint-aquariums system in October-December 2013, followed by fixation in RNA-preserving buffers and freezing during the space flight. The samples we returned to the Earth frozen in March 2013 and mRNAs from four fish were sequenced in organ-specific manner using HiSeq Illumina sequencing platform. The ground group fish treated in the same way was used as a control. The comparison between the groups revealed space group-specific specific mRNA expression pattern. More than 50 genes (including several types of myosins) were down-regulated in the space group. Moreover, we found an evidence for formation of space group-specific splicing variants of mRNA. Taking together, the data suggest that in spite of aquatic environment, space flight-associated factors have a strong effect on the activity of fish genome. This work was supported in part by subsidy of the Russian Government to support the Program of competitive growth of Kazan Federal University among world class academic centres and universities.

A retinoic acid-inducible mRNA from F9 teratocarcinoma cells encodes a novel protease inhibitor homologue.

PubMed

Wang, S Y; Gudas, L J

1990-09-15

We have previously isolated several cDNA clones specific for mRNA species that increase in abundance during the retinoic acid-associated differentiation of F9 teratocarcinoma stem cells. One of these mRNAs, J6, encodes a approximately 40 kDa protein as assayed by hybrid selection and in vitro translation (Wang, S.-Y., LaRosa, G., and Gudas, L. J. (1985) Dev. Biol. 107, 75-86). The time course of J6 mRNA expression is similar to those of both laminin B1 and collagen IV (alpha 1) messages following retinoic acid addition. To address the functional role of this protein, we have isolated a full-length cDNA clone complementary to this approximately 40-kDa protein mRNA. Sequence analysis reveals an open reading frame of 406 amino acids (Mr 45,652). The carboxyl-terminal portion of this predicted protein contains a region that is homologous to the reactive sites found among members of the serpin (serine protease inhibitor) family. The predicted reactive site (P1-P1') of this J6 protein is Arg-Ser, which is the same as that of antithrombin III. Like ovalbumin and human monocyte-derived plasminogen activator inhibitor (mPAI-2), which are members of the serpin gene family, the J6 protein appears to have no typical amino-terminal signal sequence.

Identification of a cytochrome P450 gene in the earthworm Eisenia fetida and its mRNA expression under enrofloxacin stress.

PubMed

Li, Yinsheng; Zhao, Chun; Lu, Xiaoxu; Ai, Xiaojie; Qiu, Jiangping

2018-04-15

Cytochrome P450 (CYP450) enzymes are a family of hemoproteins primarily responsible for detoxification functions. Earthworms have been used as a bioindicator of soil pollution in numerous studies, but no CYP450 gene has so far been cloned. RT-PCR and RACE-PCR were employed to construct and sequence the CYP450 gene DNA from the extracted mRNA in the earthworm Eisenia fetida. The cloned gene (EW1) has an open reading frame of 477bp. The 3'-terminal region contained both the consensus and the signature sequences characteristic of CYP450. It was closely related to the CYP450 gene from the flatworm genus Opisthorchis felineus with 87% homology. The predicted structure of the putative protein was 97% homologous to human CYP450 family 27. This gene has been deposited in GenBank (accession no. KM881474). Earthworms (E. fetida) were then exposed to 1, 10, 100, and 500mgkg -1 enrofloxacin in soils to explore the mRNA expression by real time qPCR. The effect of enrofloxacin on mRNA expression levels of EW1 exhibited a marked hormesis pattern across the enrofloxacin dose range tested. This is believed to be the first reported CYP450 gene in earthworms, with reference value for molecular studies on detoxification processes in earthworms. Copyright © 2017 Elsevier Inc. All rights reserved.

Translational resistivity/conductivity of coding sequences during exponential growth of Escherichia coli.

PubMed

Takai, Kazuyuki

2017-01-21

Codon adaptation index (CAI) has been widely used for prediction of expression of recombinant genes in Escherichia coli and other organisms. However, CAI has no mechanistic basis that rationalizes its application to estimation of translational efficiency. Here, I propose a model based on which we could consider how codon usage is related to the level of expression during exponential growth of bacteria. In this model, translation of a gene is considered as an analog of electric current, and an analog of electric resistance corresponding to each gene is considered. "Translational resistance" is dependent on the steady-state concentration and the sequence of the mRNA species, and "translational resistivity" is dependent only on the mRNA sequence. The latter is the sum of two parts: one is the resistivity for the elongation reaction (coding sequence resistivity), and the other comes from all of the other steps of the decoding reaction. This electric circuit model clearly shows that some conditions should be met for codon composition of a coding sequence to correlate well with its expression level. On the other hand, I calculated relative frequency of each of the 61 sense codon triplets translated during exponential growth of E. coli from a proteomic dataset covering over 2600 proteins. A tentative method for estimating relative coding sequence resistivity based on the data is presented. Copyright © 2016. Published by Elsevier Ltd.

Development and utilization of novel intron length polymorphic markers in foxtail millet (Setaria italica (L.) P. Beauv.).

PubMed

Gupta, Sarika; Kumari, Kajal; Das, Jyotirmoy; Lata, Charu; Puranik, Swati; Prasad, Manoj

2011-07-01

Introns are noncoding sequences in a gene that are transcribed to precursor mRNA but spliced out during mRNA maturation and are abundant in eukaryotic genomes. The availability of codominant molecular markers and saturated genetic linkage maps have been limited in foxtail millet (Setaria italica (L.) P. Beauv.). Here, we describe the development of 98 novel intron length polymorphic (ILP) markers in foxtail millet using sequence information of the model plant rice. A total of 575 nonredundant expressed sequence tag (EST) sequences were obtained, of which 327 and 248 unique sequences were from dehydration- and salinity-stressed suppression subtractive hybridization libraries, respectively. The BLAST analysis of 98 EST sequences suggests a nearly defined function for about 64% of them, and they were grouped into 11 different functional categories. All 98 ILP primer pairs showed a high level of cross-species amplification in two millets and two nonmillets species ranging from 90% to 100%, with a mean of ∼97%. The mean observed heterozygosity and Nei's average gene diversity 0.016 and 0.171, respectively, established the efficiency of the ILP markers for distinguishing the foxtail millet accessions. Based on 26 ILP markers, a reasonable dendrogram of 45 foxtail millet accessions was constructed, demonstrating the utility of ILP markers in germplasm characterizations and genomic relationships in millets and nonmillets species.

MicroRNAs form triplexes with double stranded DNA at sequence-specific binding sites; a eukaryotic mechanism via which microRNAs could directly alter gene expression

DOE PAGES

Paugh, Steven W.; Coss, David R.; Bao, Ju; ...

2016-02-04

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA). Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence that microRNAs form triple-helical structures with duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show thatmore » several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 x 10 -16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. As a result, this work has thus revealed a new mechanism by which microRNAs can interact with gene promoter regions to modify gene transcription.« less

Molecular characterization of long direct repeat (LDR) sequences expressing a stable mRNA encoding for a 35-amino-acid cell-killing peptide and a cis-encoded small antisense RNA in Escherichia coli.

PubMed

Kawano, Mitsuoki; Oshima, Taku; Kasai, Hiroaki; Mori, Hirotada

2002-07-01

Genome sequence analyses of Escherichia coli K-12 revealed four copies of long repetitive elements. These sequences are designated as long direct repeat (LDR) sequences. Three of the repeats (LDR-A, -B, -C), each approximately 500 bp in length, are located as tandem repeats at 27.4 min on the genetic map. Another copy (LDR-D), 450 bp in length and nearly identical to LDR-A, -B and -C, is located at 79.7 min, a position that is directly opposite the position of LDR-A, -B and -C. In this study, we demonstrate that LDR-D encodes a 35-amino-acid peptide, LdrD, the overexpression of which causes rapid cell killing and nucleoid condensation of the host cell. Northern blot and primer extension analysis showed constitutive transcription of a stable mRNA (approximately 370 nucleotides) encoding LdrD and an unstable cis-encoded antisense RNA (approximately 60 nucleotides), which functions as a trans-acting regulator of ldrD translation. We propose that LDR encodes a toxin-antitoxin module. LDR-homologous sequences are not pre-sent on any known plasmids but are conserved in Salmonella and other enterobacterial species.

MicroRNAs form triplexes with double stranded DNA at sequence-specific binding sites; a eukaryotic mechanism via which microRNAs could directly alter gene expression

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

Paugh, Steven W.; Coss, David R.; Bao, Ju

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA). Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence that microRNAs form triple-helical structures with duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show thatmore » several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 x 10 -16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. As a result, this work has thus revealed a new mechanism by which microRNAs can interact with gene promoter regions to modify gene transcription.« less

Structural basis of UGUA recognition by the Nudix protein CFIm25 and implications for a regulatory role in mRNA 3′ processing

PubMed Central

Yang, Qin; Gilmartin, Gregory M.; Doublié, Sylvie

2010-01-01

Human Cleavage Factor Im (CFIm) is an essential component of the pre-mRNA 3′ processing complex that functions in the regulation of poly(A) site selection through the recognition of UGUA sequences upstream of the poly(A) site. Although the highly conserved 25 kDa subunit (CFIm25) of the CFIm complex possesses a characteristic α/β/α Nudix fold, CFIm25 has no detectable hydrolase activity. Here we report the crystal structures of the human CFIm25 homodimer in complex with UGUAAA and UUGUAU RNA sequences. CFIm25 is the first Nudix protein to be reported to bind RNA in a sequence-specific manner. The UGUA sequence contributes to binding specificity through an intramolecular G:A Watson–Crick/sugar-edge base interaction, an unusual pairing previously found to be involved in the binding specificity of the SAM-III riboswitch. The structures, together with mutational data, suggest a novel mechanism for the simultaneous sequence-specific recognition of two UGUA elements within the pre-mRNA. Furthermore, the mutually exclusive binding of RNA and the signaling molecule Ap4A (diadenosine tetraphosphate) by CFIm25 suggests a potential role for small molecules in the regulation of mRNA 3′ processing. PMID:20479262

Structural basis of UGUA recognition by the Nudix protein CFI(m)25 and implications for a regulatory role in mRNA 3' processing.

PubMed

Yang, Qin; Gilmartin, Gregory M; Doublié, Sylvie

2010-06-01

Human Cleavage Factor Im (CFI(m)) is an essential component of the pre-mRNA 3' processing complex that functions in the regulation of poly(A) site selection through the recognition of UGUA sequences upstream of the poly(A) site. Although the highly conserved 25 kDa subunit (CFI(m)25) of the CFI(m) complex possesses a characteristic alpha/beta/alpha Nudix fold, CFI(m)25 has no detectable hydrolase activity. Here we report the crystal structures of the human CFI(m)25 homodimer in complex with UGUAAA and UUGUAU RNA sequences. CFI(m)25 is the first Nudix protein to be reported to bind RNA in a sequence-specific manner. The UGUA sequence contributes to binding specificity through an intramolecular G:A Watson-Crick/sugar-edge base interaction, an unusual pairing previously found to be involved in the binding specificity of the SAM-III riboswitch. The structures, together with mutational data, suggest a novel mechanism for the simultaneous sequence-specific recognition of two UGUA elements within the pre-mRNA. Furthermore, the mutually exclusive binding of RNA and the signaling molecule Ap(4)A (diadenosine tetraphosphate) by CFI(m)25 suggests a potential role for small molecules in the regulation of mRNA 3' processing.

Unveiling the Biodiversity of Deep-Sea Nematodes through Metabarcoding: Are We Ready to Bypass the Classical Taxonomy?

PubMed Central

2015-01-01

Nematodes inhabiting benthic deep-sea ecosystems account for >90% of the total metazoan abundances and they have been hypothesised to be hyper-diverse, but their biodiversity is still largely unknown. Metabarcoding could facilitate the census of biodiversity, especially for those tiny metazoans for which morphological identification is difficult. We compared, for the first time, different DNA extraction procedures based on the use of two commercial kits and a previously published laboratory protocol and tested their suitability for sequencing analyses of 18S rDNA of marine nematodes. We also investigated the reliability of Roche 454 sequencing analyses for assessing the biodiversity of deep-sea nematode assemblages previously morphologically identified. Finally, intra-genomic variation in 18S rRNA gene repeats was investigated by Illumina MiSeq in different deep-sea nematode morphospecies to assess the influence of polymorphisms on nematode biodiversity estimates. Our results indicate that the two commercial kits should be preferred for the molecular analysis of biodiversity of deep-sea nematodes since they consistently provide amplifiable DNA suitable for sequencing. We report that the morphological identification of deep-sea nematodes matches the results obtained by metabarcoding analysis only at the order-family level and that a large portion of Operational Clustered Taxonomic Units (OCTUs) was not assigned. We also show that independently from the cut-off criteria and bioinformatic pipelines used, the number of OCTUs largely exceeds the number of individuals and that 18S rRNA gene of different morpho-species of nematodes displayed intra-genomic polymorphisms. Our results indicate that metabarcoding is an important tool to explore the diversity of deep-sea nematodes, but still fails in identifying most of the species due to limited number of sequences deposited in the public databases, and in providing quantitative data on the species encountered. These aspects should be carefully taken into account before using metabarcoding in quantitative ecological research and monitoring programmes of marine biodiversity. PMID:26701112

Unveiling the Biodiversity of Deep-Sea Nematodes through Metabarcoding: Are We Ready to Bypass the Classical Taxonomy?

PubMed

Dell'Anno, Antonio; Carugati, Laura; Corinaldesi, Cinzia; Riccioni, Giulia; Danovaro, Roberto

2015-01-01

Nematodes inhabiting benthic deep-sea ecosystems account for >90% of the total metazoan abundances and they have been hypothesised to be hyper-diverse, but their biodiversity is still largely unknown. Metabarcoding could facilitate the census of biodiversity, especially for those tiny metazoans for which morphological identification is difficult. We compared, for the first time, different DNA extraction procedures based on the use of two commercial kits and a previously published laboratory protocol and tested their suitability for sequencing analyses of 18S rDNA of marine nematodes. We also investigated the reliability of Roche 454 sequencing analyses for assessing the biodiversity of deep-sea nematode assemblages previously morphologically identified. Finally, intra-genomic variation in 18S rRNA gene repeats was investigated by Illumina MiSeq in different deep-sea nematode morphospecies to assess the influence of polymorphisms on nematode biodiversity estimates. Our results indicate that the two commercial kits should be preferred for the molecular analysis of biodiversity of deep-sea nematodes since they consistently provide amplifiable DNA suitable for sequencing. We report that the morphological identification of deep-sea nematodes matches the results obtained by metabarcoding analysis only at the order-family level and that a large portion of Operational Clustered Taxonomic Units (OCTUs) was not assigned. We also show that independently from the cut-off criteria and bioinformatic pipelines used, the number of OCTUs largely exceeds the number of individuals and that 18S rRNA gene of different morpho-species of nematodes displayed intra-genomic polymorphisms. Our results indicate that metabarcoding is an important tool to explore the diversity of deep-sea nematodes, but still fails in identifying most of the species due to limited number of sequences deposited in the public databases, and in providing quantitative data on the species encountered. These aspects should be carefully taken into account before using metabarcoding in quantitative ecological research and monitoring programmes of marine biodiversity.

Kinetics of hairpin ribozyme cleavage in yeast.

PubMed Central

Donahue, C P; Fedor, M J

1997-01-01

Hairpin ribozymes catalyze a self-cleavage reaction that provides a simple model for quantitative analyses of intracellular mechanisms of RNA catalysis. Decay rates of chimeric mRNAs containing self-cleaving ribozymes give a direct measure of intracellular cleavage kinetics in yeast. Intracellular ribozyme-mediated cleavage occurs at similar rates and shows similar inhibition by ribozyme mutations as ribozyme-mediated reactions in vitro, but only when ribozymes are located in a favorable mRNA sequence context. The impact of cleavage on mRNA abundance is shown to depend directly on intrinsic mRNA stability. Surprisingly, cleavage products are no more labile than uncleaved mRNAs despite the loss of terminal cap structures or poly (A). PMID:9292496

Discovery and characterization of miRNA genes in atlantic salmon (Salmo salar) by use of a deep sequencing approach

PubMed Central

2013-01-01

Background MicroRNAs (miRNAs) are an abundant class of endogenous small RNA molecules that downregulate gene expression at the posttranscriptional level. They play important roles in multiple biological processes by regulating genes that control developmental timing, growth, stem cell division and apoptosis by binding to the mRNA of target genes. Despite the position Atlantic salmon (Salmo salar) has as an economically important domesticated animal, there has been little research on miRNAs in this species. Knowledge about miRNAs and their target genes may be used to control health and to improve performance of economically important traits. However, before their biological function can be unravelled they must be identified and annotated. The aims of this study were to identify and characterize miRNA genes in Atlantic salmon by deep sequencing analysis of small RNA libraries from nine different tissues. Results A total of 180 distinct mature miRNAs belonging to 106 families of evolutionary conserved miRNAs, and 13 distinct novel mature miRNAs were discovered and characterized. The mature miRNAs corresponded to 521 putative precursor sequences located at unique genome locations. About 40% of these precursors were part of gene clusters, and the majority of the Salmo salar gene clusters discovered were conserved across species. Comparison of expression levels in samples from different tissues applying DESeq indicated that there were tissue specific expression differences in three conserved and one novel miRNA. Ssa-miR 736 was detected in heart tissue only, while two other clustered miRNAs (ssa-miR 212 and132) seems to be at a higher expression level in brain tissue. These observations correlate well with their expected functions as regulators of signal pathways in cardiac and neuronal cells, respectively. Ssa-miR 8163 is one of the novel miRNAs discovered and its function remains unknown. However, differential expression analysis using DESeq suggests that this miRNA is enriched in liver tissue and the precursor was mapped to intron 7 of the transferrin gene. Conclusions The identification and annotation of evolutionary conserved and novel Salmo salar miRNAs as well as the characterization of miRNA gene clusters provide biological knowledge that will greatly facilitate further functional studies on miRNAs in this species. PMID:23865519

Molecular Phylogenetic Analysis of Archaeal Intron-Containing Genes Coding for rRNA Obtained from a Deep-Subsurface Geothermal Water Pool

PubMed Central

Takai, Ken; Horikoshi, Koki

1999-01-01

Molecular phylogenetic analysis of a naturally occurring microbial community in a deep-subsurface geothermal environment indicated that the phylogenetic diversity of the microbial population in the environment was extremely limited and that only hyperthermophilic archaeal members closely related to Pyrobaculum were present. All archaeal ribosomal DNA sequences contained intron-like sequences, some of which had open reading frames with repeated homing-endonuclease motifs. The sequence similarity analysis and the phylogenetic analysis of these homing endonucleases suggested the possible phylogenetic relationship among archaeal rRNA-encoded homing endonucleases. PMID:10584021

Genomic variation in macrophage-cultured European porcine reproductive and respiratory syndrome virus Olot/91 revealed using ultra-deep next generation sequencing.

PubMed

Lu, Zen H; Brown, Alexander; Wilson, Alison D; Calvert, Jay G; Balasch, Monica; Fuentes-Utrilla, Pablo; Loecherbach, Julia; Turner, Frances; Talbot, Richard; Archibald, Alan L; Ait-Ali, Tahar

2014-03-04

Porcine Reproductive and Respiratory Syndrome (PRRS) is a disease of major economic impact worldwide. The etiologic agent of this disease is the PRRS virus (PRRSV). Increasing evidence suggest that microevolution within a coexisting quasispecies population can give rise to high sequence heterogeneity in PRRSV. We developed a pipeline based on the ultra-deep next generation sequencing approach to first construct the complete genome of a European PRRSV, strain Olot/9, cultured on macrophages and then capture the rare variants representative of the mixed quasispecies population. Olot/91 differs from the reference Lelystad strain by about 5% and a total of 88 variants, with frequencies as low as 1%, were detected in the mixed population. These variants included 16 non-synonymous variants concentrated in the genes encoding structural and nonstructural proteins; including Glycoprotein 2a and 5. Using an ultra-deep sequencing methodology, the complete genome of Olot/91 was constructed without any prior knowledge of the sequence. Rare variants that constitute minor fractions of the heterogeneous PRRSV population could successfully be detected to allow further exploration of microevolutionary events.

PolyA_DB 3 catalogs cleavage and polyadenylation sites identified by deep sequencing in multiple genomes

PubMed Central

Wang, Ruijia; Nambiar, Ram; Zheng, Dinghai

2018-01-01

Abstract PolyA_DB is a database cataloging cleavage and polyadenylation sites (PASs) in several genomes. Previous versions were based mainly on expressed sequence tags (ESTs), which had a limited amount and could lead to inaccurate PAS identification due to the presence of internal A-rich sequences in transcripts. Here, we present an updated version of the database based solely on deep sequencing data. First, PASs are mapped by the 3′ region extraction and deep sequencing (3′READS) method, ensuring unequivocal PAS identification. Second, a large volume of data based on diverse biological samples increases PAS coverage by 3.5-fold over the EST-based version and provides PAS usage information. Third, strand-specific RNA-seq data are used to extend annotated 3′ ends of genes to obtain more thorough annotations of alternative polyadenylation (APA) sites. Fourth, conservation information of PAS across mammals sheds light on significance of APA sites. The database (URL: http://www.polya-db.org/v3) currently holds PASs in human, mouse, rat and chicken, and has links to the UCSC genome browser for further visualization and for integration with other genomic data. PMID:29069441

Deep whole-genome sequencing of 90 Han Chinese genomes.

PubMed

Lan, Tianming; Lin, Haoxiang; Zhu, Wenjuan; Laurent, Tellier Christian Asker Melchior; Yang, Mengcheng; Liu, Xin; Wang, Jun; Wang, Jian; Yang, Huanming; Xu, Xun; Guo, Xiaosen

2017-09-01

Next-generation sequencing provides a high-resolution insight into human genetic information. However, the focus of previous studies has primarily been on low-coverage data due to the high cost of sequencing. Although the 1000 Genomes Project and the Haplotype Reference Consortium have both provided powerful reference panels for imputation, low-frequency and novel variants remain difficult to discover and call with accuracy on the basis of low-coverage data. Deep sequencing provides an optimal solution for the problem of these low-frequency and novel variants. Although whole-exome sequencing is also a viable choice for exome regions, it cannot account for noncoding regions, sometimes resulting in the absence of important, causal variants. For Han Chinese populations, the majority of variants have been discovered based upon low-coverage data from the 1000 Genomes Project. However, high-coverage, whole-genome sequencing data are limited for any population, and a large amount of low-frequency, population-specific variants remain uncharacterized. We have performed whole-genome sequencing at a high depth (∼×80) of 90 unrelated individuals of Chinese ancestry, collected from the 1000 Genomes Project samples, including 45 Northern Han Chinese and 45 Southern Han Chinese samples. Eighty-three of these 90 have been sequenced by the 1000 Genomes Project. We have identified 12 568 804 single nucleotide polymorphisms, 2 074 210 short InDels, and 26 142 structural variations from these 90 samples. Compared to the Han Chinese data from the 1000 Genomes Project, we have found 7 000 629 novel variants with low frequency (defined as minor allele frequency < 5%), including 5 813 503 single nucleotide polymorphisms, 1 169 199 InDels, and 17 927 structural variants. Using deep sequencing data, we have built a greatly expanded spectrum of genetic variation for the Han Chinese genome. Compared to the 1000 Genomes Project, these Han Chinese deep sequencing data enhance the characterization of a large number of low-frequency, novel variants. This will be a valuable resource for promoting Chinese genetics research and medical development. Additionally, it will provide a valuable supplement to the 1000 Genomes Project, as well as to other human genome projects. © The Authors 2017. Published by Oxford University Press.

MicroRNA and Transcription Factor: Key Players in Plant Regulatory Network.

PubMed

Samad, Abdul F A; Sajad, Muhammad; Nazaruddin, Nazaruddin; Fauzi, Izzat A; Murad, Abdul M A; Zainal, Zamri; Ismail, Ismanizan

2017-01-01

Recent achievements in plant microRNA (miRNA), a large class of small and non-coding RNAs, are very exciting. A wide array of techniques involving forward genetic, molecular cloning, bioinformatic analysis, and the latest technology, deep sequencing have greatly advanced miRNA discovery. A tiny miRNA sequence has the ability to target single/multiple mRNA targets. Most of the miRNA targets are transcription factors (TFs) which have paramount importance in regulating the plant growth and development. Various families of TFs, which have regulated a range of regulatory networks, may assist plants to grow under normal and stress environmental conditions. This present review focuses on the regulatory relationships between miRNAs and different families of TFs like; NF-Y, MYB, AP2, TCP, WRKY, NAC, GRF, and SPL. For instance NF-Y play important role during drought tolerance and flower development, MYB are involved in signal transduction and biosynthesis of secondary metabolites, AP2 regulate the floral development and nodule formation, TCP direct leaf development and growth hormones signaling. WRKY have known roles in multiple stress tolerances, NAC regulate lateral root formation, GRF are involved in root growth, flower, and seed development, and SPL regulate plant transition from juvenile to adult. We also studied the relation between miRNAs and TFs by consolidating the research findings from different plant species which will help plant scientists in understanding the mechanism of action and interaction between these regulators in the plant growth and development under normal and stress environmental conditions.

Optimization of conditions to sequence long cDNAs from viruses

USDA-ARS?s Scientific Manuscript database

Fourth generation sequencing with the Minion nanopore sequencer provides opportunity to obtain deep coverage and long read for single molecules. This will benefit studies on RNA viruses. In the past, Sanger, Illumina, and Ion Torrent sequencing have been utilized to study RNA viruses. Both technique...

SNP discovery through de novo deep sequencing using the next generation of DNA sequencers

USDA-ARS?s Scientific Manuscript database

The production of high volumes of DNA sequence data using new technologies has permitted more efficient identification of single nucleotide polymorphisms in vertebrate genomes. This chapter presented practical methodology for production and analysis of DNA sequence data for SNP discovery....

Engineered proteins with PUF scaffold to manipulate RNA metabolism

PubMed Central

Wang, Yang; Wang, Zefeng; Tanaka Hall, Traci M.

2013-01-01

Pumilio/fem-3 mRNA binding factor (FBF) proteins are characterized by a sequence-specific RNA-binding domain. This unique single-stranded RNA recognition module, whose sequence specificity can be reprogrammed, has been fused with functional modules to engineer protein factors with various functions. Here we summarize the advancement in developing RNA regulatory tools and opportunities for the future. PMID:23731364

Identification and transcription profiling of NDUFS8 in Aedes taeniorhynchus (Diptera:Culididae): developmental regulation and environmental response

USDA-ARS?s Scientific Manuscript database

The cDNA of a NADH dehydrogenase -ubiquinone Fe-S protein 8 subunit (NDUFS8) gene from Aedes (Ochlerotatus) taeniorhynchus Wiedemann has been cloned and sequenced. The full-length mRNA sequence (824 bp) of AetNDUFS8 encodes an open reading region of 651 bp (i.e., 217 amino acids). To detect whether ...

Late-onset spastic paraplegia: Aberrant SPG11 transcripts generated by a novel splice site donor mutation.

PubMed

Kawarai, Toshitaka; Miyamoto, Ryosuke; Mori, Atsuko; Oki, Ryosuke; Tsukamoto-Miyashiro, Ai; Matsui, Naoko; Miyazaki, Yoshimichi; Orlacchio, Antonio; Izumi, Yuishin; Nishida, Yoshihiko; Kaji, Ryuji

2015-12-15

We identified a novel homozygous mutation in the splice site donor (SSD) of intron 30 (c.5866+1G>A) in consanguineous Japanese SPG11 siblings showing late-onset spastic paraplegia using the whole-exome sequencing. Phenotypic variability was observed, including age-at-onset, dysarthria and pes cavus. Coding DNA sequencing revealed that the mutation affected the recognition of the constitutive SSD of intron 30, splicing upstream onto a nearby cryptic SSD in exon 30. The use of constitutive splice sites of intron 29 was confirmed by sequencing. The mutant transcripts are mostly subject to degradation by the nonsense-mediated mRNA decay system. SPG11 transcripts, escaping from the nonsense-mediated mRNA decay pathway, would generate a truncated protein (p.Tyr1900Phefs5X) containing the first 1899 amino acids and followed by 4 aberrant amino acids. This study showed a successful clinical application of whole-exome sequencing in spastic paraplegia and demonstrated a further evidence of allelic heterogeneity in SPG11. The confirmation of aberrant transcript by splice site mutation is a prerequisite for a more precise molecular diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Genetics of bacteria that utilize one carbon compounds: Final report, March 1, 1982-February 29, 1988

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

Hanson, R.S.

Broad host range plasmid vectors useful for cloning genes from bacteria that grow on methane and methanol were constructed. We have cloned and mapped nineteen genes required for the growth of Methylobacterium organophilum strain XX on methanol. Nineteen genes were found in seven linkage groups on the M. organophilum genome and were separated by 40 kb or more. Eleven genes were required for the synthesis of methanol dehydrogenase (MDH) and were located in three unlinked gene clusters. The MDH structural gene was localized on a 2.5 kb DNA fragment. The gene was sequenced and contains a 175 bp untranslated leadermore » sequence, a signal sequence and the structural gene. MDH messenger RNA (mRNA) has a half life of approximately 20 min. and is present at approximately 2% of the cellular mRNA. The structural gene for the ..gamma.. subunit of methane monoxygenases has been cloned from Methylosporovibrio. Methane monooxygenase subunits have been purified by Prof. J. Lipscomb's laboratory and are being sequenced to construct DNA probes to identify cloned subunit genes. New facultative methylotrophic bacteria were isolated and characterized. Several amino acid auxotrophs have been isolated. 11 refs.« less

Deep Sequencing-Based Analysis of the Cymbidium ensifolium Floral Transcriptome

PubMed Central

Li, Xiaobai; Luo, Jie; Yan, Tianlian; Xiang, Lin; Jin, Feng; Qin, Dehui; Sun, Chongbo; Xie, Ming

2013-01-01

Cymbidium ensifolium is a Chinese Cymbidium with an elegant shape, beautiful appearance, and a fragrant aroma. C. ensifolium has a long history of cultivation in China and it has excellent commercial value as a potted plant and cut flower. The development of C. ensifolium genomic resources has been delayed because of its large genome size. Taking advantage of technical and cost improvement of RNA-Seq, we extracted total mRNA from flower buds and mature flowers and obtained a total of 9.52 Gb of filtered nucleotides comprising 98,819,349 filtered reads. The filtered reads were assembled into 101,423 isotigs, representing 51,696 genes. Of the 101,423 isotigs, 41,873 were putative homologs of annotated sequences in the public databases, of which 158 were associated with floral development and 119 were associated with flowering. The isotigs were categorized according to their putative functions. In total, 10,212 of the isotigs were assigned into 25 eukaryotic orthologous groups (KOGs), 41,690 into 58 gene ontology (GO) terms, and 9,830 into 126 Arabidopsis Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and 9,539 isotigs into 123 rice pathways. Comparison of the isotigs with those of the two related orchid species P. equestris and C. sinense showed that 17,906 isotigs are unique to C. ensifolium. In addition, a total of 7,936 SSRs and 16,676 putative SNPs were identified. To our knowledge, this transcriptome database is the first major genomic resource for C. ensifolium and the most comprehensive transcriptomic resource for genus Cymbidium. These sequences provide valuable information for understanding the molecular mechanisms of floral development and flowering. Sequences predicted to be unique to C. ensifolium would provide more insights into C. ensifolium gene diversity. The numerous SNPs and SSRs identified in the present study will contribute to marker development for C. ensifolium. PMID:24392013

MRI markers of small vessel disease in lobar and deep hemispheric intracerebral hemorrhage.

PubMed

Smith, Eric E; Nandigam, Kaveer R N; Chen, Yu-Wei; Jeng, Jed; Salat, David; Halpin, Amy; Frosch, Matthew; Wendell, Lauren; Fazen, Louis; Rosand, Jonathan; Viswanathan, Anand; Greenberg, Steven M

2010-09-01

MRI evidence of small vessel disease is common in intracerebral hemorrhage (ICH). We hypothesized that ICH caused by cerebral amyloid angiopathy (CAA) or hypertensive vasculopathy would have different distributions of MRI T2 white matter hyperintensity (WMH) and microbleeds. Data were analyzed from 133 consecutive patients with primary supratentorial ICH and adequate MRI sequences. CAA was diagnosed using the Boston criteria. WMH segmentation was performed using a validated semiautomated method. WMH and microbleeds were compared according to site of symptomatic hematoma origin (lobar versus deep) or by pattern of hemorrhages, including both hematomas and microbleeds, on MRI gradient recalled echo sequence (grouped as lobar only-probable CAA, lobar only-possible CAA, deep hemispheric only, or mixed lobar and deep hemorrhages). Patients with lobar and deep hemispheric hematoma had similar median normalized WMH volumes (19.5 cm versus 19.9 cm(3), P=0.74) and prevalence of >or=1 microbleed (54% versus 52%, P=0.99). The supratentorial WMH distribution was similar according to hemorrhage location category; however, the prevalence of brain stem T2 hyperintensity was lower in lobar hematoma versus deep hematoma (54% versus 70%, P=0.004). Mixed ICH was common (23%). Patients with mixed ICH had large normalized WMH volumes and a posterior distribution of cortical hemorrhages similar to that seen in CAA. WMH distribution is largely similar between CAA-related and non-CAA-related ICH. Mixed lobar and deep hemorrhages are seen on MRI gradient recalled echo sequence in up to one fourth of patients; in these patients, both hypertension and CAA may be contributing to the burden of WMH.

Preparation of anti-mouse caspase-12 mRNA hammerhead ribozyme and identification of its activity in vitro

PubMed Central

Jiang, Shan; Xie, Qing; Zhang, Wei; Zhou, Xia-Qiu; Jin, You-Xin

2005-01-01

AIM: To prepare and identify specific anti-mouse caspase-12 hammerhead ribozymes in vitro, in order to select a more effective ribozyme against mouse caspase-12 as a potential tool to rescue cells from endoplasmic reticulum stress induced apoptosis. METHODS: Two hammerhead ribozymes directed separately against 138 and 218 site of nucleotide of mouse caspase-12 mRNA were designed by computer software, and their DNA sequences were synthesized. The synthesized ribozymes were cloned into an eukaryotic expression vector-neorpBSKU6 and embedded in U6 SnRNA context for further study. Mouse caspase-12 gene segment was cloned into PGEM-T vector under the control of T7 RNA polymerase promoter (containing gene sequence from positions nt 41 to nt 894) as target. In vitro transcription both the ribozymes and target utilize T7 promoter. The target was labeled with [α-32P]UTP, while ribozymes were not labeled. After gel purification the RNAs were dissolved in RNase free water. Ribozyme and target were incubated for 90 min at 37°C in reaction buffer (40 mmol/L Tris-HCL, pH 7.5, 10 mmol/L Mg2+). Molar ratio of ribozyme vs target was 30:1. Samples were analyzed on 6% PAGE (containing 8 mol/L urea). RESULTS: Both caspase-12 and ribozyme gene sequences were successfully cloned into expression vector confirmed by sequencing. Ribozymes and caspase-12 mRNA were obtained by in vitro transcription. Cleavage experiment showed that in a physiological similar condition (37°C, pH 7.5), Rz138 and Rz218 both cleaved targets at predicted sites, for Rz138 the cleavage efficiency was about 100%, for Rz218 the value was 36.66%. CONCLUSION: Rz138 prepared in vitro can site specific cleave mouse caspase-12 mRNA with an excellent efficiency. It shows a potential to suppress the expression of caspase-12 in vivo, thus provided a new way to protect cells from ER stress induced apoptosis. PMID:15996037

G to A substitution in 5{prime} donor splice site of introns 18 and 48 of COL1A1 gene of type I collagen results in different splicing alternatives in osteogenesis imperfecta type I cell strains

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

Willing, M.; Deschenes, S.

We have identified a G to A substitution in the 5{prime} donor splice site of intron 18 of one COL1A1 allele in two unrelated families with osteogenesis imperfecta (OI) type I. A third OI type I family has a G to A substitution at the identical position in intron 48 of one COL1A1 allele. Both mutations abolish normal splicing and lead to reduced steady-state levels of mRNA from the mutant COL1A1 allele. The intron 18 mutation leads to both exon 18 skipping in the mRNA and to utilization of a single alternative splice site near the 3{prime} end of exonmore » 18. The latter results in deletion of the last 8 nucleotides of exon 18 from the mRNA, a shift in the translational reading-frame, and the creation of a premature termination codon in exon 19. Of the potential alternative 5{prime} splice sites in exon 18 and intron 18, the one utilized has a surrounding nucleotide sequence which most closely resembles that of the natural splice site. Although a G to A mutation was detected at the identical position in intron 48 of one COL1A1 allele in another OI type I family, nine complex alternative splicing patterns were identified by sequence analysis of cDNA clones derived from fibroblast mRNA from this cell strain. All result in partial or complete skipping of exon 48, with in-frame deletions of portions of exons 47 and/or 49. The different patterns of RNA splicing were not explained by their sequence homology with naturally occuring 5{prime} splice sites, but rather by recombination between highly homologous exon sequences, suggesting that we may not have identified the major splicing alternative(s) in this cell strain. Both G to A mutations result in decreased production of type I collagen, the common biochemical correlate of OI type I.« less

Identification of Smoking-Associated Differentially Methylated Regions Using Reduced Representation Bisulfite Sequencing and Cell type-Specific Enhancer Activation and Gene Expression.

PubMed

Wan, Ma; Bennett, Brian D; Pittman, Gary S; Campbell, Michelle R; Reynolds, Lindsay M; Porter, Devin K; Crowl, Christopher L; Wang, Xuting; Su, Dan; Englert, Neal A; Thompson, Isabel J; Liu, Yongmei; Bell, Douglas A

2018-04-27

Cigarette smoke is a causal factor in cancers and cardiovascular disease. Smoking-associated differentially methylated regions (SM-DMRs) have been observed in disease studies, but the causal link between altered DNA methylation and transcriptional change is obscure. Our objectives were to finely resolve SM-DMRs and to interrogate the mechanistic link between SM-DMRs and altered transcription of enhancer noncoding RNA (eRNA) and mRNA in human circulating monocytes. We integrated SM-DMRs identified by reduced representation bisulfite sequencing (RRBS) of circulating CD14+ monocyte DNA collected from two independent human studies [ n =38 from Clinical Research Unit (CRU) and n =55 from the Multi-Ethnic Study of Atherosclerosis (MESA), about half of whom were active smokers] with gene expression for protein-coding genes and noncoding RNAs measured by RT-PCR or RNA sequencing. Candidate SM-DMRs were compared with RRBS of purified CD4+ T cells, CD8+ T cells, CD15+ granulocytes, CD19+ B cells, and CD56+ NK cells ( n =19 females, CRU). DMRs were validated using pyrosequencing or bisulfite amplicon sequencing in up to 85 CRU volunteers, who also provided saliva DNA. RRBS identified monocyte SM-DMRs frequently located in putative gene regulatory regions. The most significant monocyte DMR occurred at a poised enhancer in the aryl-hydrocarbon receptor repressor gene ( AHRR ) and it was also detected in both granulocytes and saliva DNA. To our knowledge, we identify for the first time that SM-DMRs in or near AHRR , C5orf55-EXOC-AS , and SASH1 were associated with increased noncoding eRNA as well as mRNA in monocytes. Functionally, the AHRR SM-DMR appeared to up-regulate AHRR mRNA through activating the AHRR enhancer, as suggested by increased eRNA in the monocytes, but not granulocytes, from smokers compared with nonsmokers. Our findings suggest that AHRR SM-DMR up-regulates AHRR mRNA in a monocyte-specific manner by activating the AHRR enhancer. Cell type-specific activation of enhancers at SM-DMRs may represent a mechanism driving smoking-related disease. https://doi.org/10.1289/EHP2395.

hnRNP L binds to CA repeats in the 3'UTR of bcl-2 mRNA

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

Lee, Dong-Hyoung; Lim, Mi-Hyun; Youn, Dong-Ye

We previously reported that the CA-repeat sequence in the 3'-untranslated region (3'UTR) of bcl-2 mRNA is involved in the decay of bcl-2 mRNA. However, the trans-acting factor for the CA element in bcl-2 mRNA remains unidentified. The heterogeneous nuclear ribonucleoprotein L (hnRNP L), an intron splicing factor, has been reported to bind to CA repeats and CA clusters in the 3'UTR of several genes. We reported herein that the CA repeats of bcl-2 mRNA have the potential to form a distinct ribonuclear protein complex in cytoplasmic extracts of MCF-7 cells, as evidenced by RNA electrophoretic mobility shift assays (REMSA). Amore » super-shift assay using the hnRNP L antibody completely shifted the complex. Immunoprecipitation with the hnRNP L antibody and MCF-7 cells followed by RT-PCR revealed that hnRNP L interacts with endogenous bcl-2 mRNA in vivo. Furthermore, the suppression of hnRNP L in MCF-7 cells by the transfection of siRNA for hnRNP L resulted in a delay in the degradation of RNA transcripts including CA repeats of bcl-2 mRNA in vitro, suggesting that the interaction between hnRNPL and CA repeats of bcl-2 mRNA participates in destabilizing bcl-2 mRNA.« less

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

Deep Sequencing Analysis of Apple Infecting Viruses in Korea

PubMed Central

Cho, In-Sook; Igori, Davaajargal; Lim, Seungmo; Choi, Gug-Seoun; Hammond, John; Lim, Hyoun-Sub; Moon, Jae Sun

2016-01-01

Deep sequencing has generated 52 contigs derived from five viruses; Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple green crinkle associated virus (AGCaV), and Apricot latent virus (ApLV) were identified from eight apple samples showing small leaves and/or growth retardation. Nucleotide (nt) sequence identity of the assembled contigs was from 68% to 99% compared to the reference sequences of the five respective viral genomes. Sequences of ASPV and ASGV were the most abundantly represented by the 52 contigs assembled. The presence of the five viruses in the samples was confirmed by RT-PCR using specific primers based on the sequences of each assembled contig. All five viruses were detected in three of the samples, whereas all samples had mixed infections with at least two viruses. The most frequently detected virus was ASPV, followed by ASGV, ApLV, ACLSV, and AGCaV which were withal found in mixed infections in the tested samples. AGCaV was identified in assembled contigs ID 1012480 and 93549, which showed 82% and 78% nt sequence identity with ORF1 of AGCaV isolate Aurora-1. ApLV was identified in three assembled contigs, ID 65587, 1802365, and 116777, which showed 77%, 78%, and 76% nt sequence identity respectively with ORF1 of ApLV isolate LA2. Deep sequencing assay was shown to be a valuable and powerful tool for detection and identification of known and unknown virome in infected apple trees, here identifying ApLV and AGCaV in commercial orchards in Korea for the first time. PMID:27721694

Deep sequencing approaches for the analysis of prokaryotic transcriptional boundaries and dynamics.

PubMed

James, Katherine; Cockell, Simon J; Zenkin, Nikolay

2017-05-01

The identification of the protein-coding regions of a genome is straightforward due to the universality of start and stop codons. However, the boundaries of the transcribed regions, conditional operon structures, non-coding RNAs and the dynamics of transcription, such as pausing of elongation, are non-trivial to identify, even in the comparatively simple genomes of prokaryotes. Traditional methods for the study of these areas, such as tiling arrays, are noisy, labour-intensive and lack the resolution required for densely-packed bacterial genomes. Recently, deep sequencing has become increasingly popular for the study of the transcriptome due to its lower costs, higher accuracy and single nucleotide resolution. These methods have revolutionised our understanding of prokaryotic transcriptional dynamics. Here, we review the deep sequencing and data analysis techniques that are available for the study of transcription in prokaryotes, and discuss the bioinformatic considerations of these analyses. Copyright © 2017 Elsevier Inc. All rights reserved.

Insertion sequences enrichment in extreme Red sea brine pool vent.

PubMed

Elbehery, Ali H A; Aziz, Ramy K; Siam, Rania

2017-03-01

Mobile genetic elements are major agents of genome diversification and evolution. Limited studies addressed their characteristics, including abundance, and role in extreme habitats. One of the rare natural habitats exposed to multiple-extreme conditions, including high temperature, salinity and concentration of heavy metals, are the Red Sea brine pools. We assessed the abundance and distribution of different mobile genetic elements in four Red Sea brine pools including the world's largest known multiple-extreme deep-sea environment, the Red Sea Atlantis II Deep. We report a gradient in the abundance of mobile genetic elements, dramatically increasing in the harshest environment of the pool. Additionally, we identified a strong association between the abundance of insertion sequences and extreme conditions, being highest in the harshest and deepest layer of the Red Sea Atlantis II Deep. Our comparative analyses of mobile genetic elements in secluded, extreme and relatively non-extreme environments, suggest that insertion sequences predominantly contribute to polyextremophiles genome plasticity.

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

Delta ribozyme has the ability to cleave in transan mRNA.

PubMed Central

Roy, G; Ananvoranich, S; Perreault, J P

1999-01-01

We report here the first demonstration of the cleavage of an mRNA in trans by delta ribozyme derived from the antigenomic version of the human hepatitis delta virus (HDV). We characterized potential delta ribozyme cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using oligonucleotide binding shift assays and ribonuclease H hydrolysis. Ribozymes were synthesized based on the structural data and then tested for their ability to cleave the mRNA. Of the nine ribozymes examined, three specifically cleaved a derivative HDV mRNA. All three active ribozymes gave consistent indications that they cleaved single-stranded regions. Kinetic characterization of the ability of ribozymes to cleave both the full-length mRNA and either wild-type or mutant small model substrate suggests: (i) delta ribozyme has turnovers, that is to say, several mRNA molecules can be successively cleaved by one ribozyme molecule; and (ii) the substrate specificity of delta ribozyme cleavage is not restricted to C/UGN6. Specifically, substrates with a higher guanosine residue content upstream of the cleavage site (i.e. positions -4 to -2) were always cleaved more efficiently than wild-type substrate. This work shows that delta ribozyme constitutes a potential catalytic RNA for further gene-inactivation therapy. PMID:9927724

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

Spermidine/spermine N1-acetyltransferase (SSAT) activity in human small-cell lung carcinoma cells following transfection with a genomic SSAT construct.

PubMed

Murray-Stewart, Tracy; Applegren, Nancy B; Devereux, Wendy; Hacker, Amy; Smith, Renee; Wang, Yanlin; Casero, Robert A

2003-07-15

Spermidine/spermine N (1)-acetyltransferase (SSAT) activity is typically highly inducible in non-small-cell lung carcinomas in response to treatment with anti-tumour polyamine analogues, and this induction is associated with subsequent cell death. In contrast, cells of the small-cell lung carcinoma (SCLC) phenotype generally do not respond to these compounds with an increase in SSAT activity, and usually are only moderately affected with respect to growth. The goal of the present study was to produce an SSAT-overexpressing SCLC cell line to further investigate the role of SSAT in response to these anti-tumour analogues. To accomplish this, NCI-H82 SCLC cells were stably transfected with plasmids containing either the SSAT genomic sequence or the corresponding cDNA sequence. Individual clones were selected based on their ability to show induced SSAT activity in response to exposure to a polyamine analogue, and an increase in the steady-state SSAT mRNA level. Cells transfected with the genomic sequence exhibited a significant increase in basal SSAT mRNA expression, as well as enhanced SSAT activity, intracellular polyamine pool depletion and growth inhibition following treatment with the analogue N (1), N (11)-bis(ethyl)norspermine. Cells containing the transfected cDNA also exhibited an increase in the basal SSAT mRNA level, but remained phenotypically similar to vector control cells with respect to their response to analogue exposure. These studies indicate that both the genomic SSAT sequence and polyamine analogue exposure play a role in the transcriptional and post-transcriptional regulation and subsequent induction of SSAT activity in these cells. Furthermore, this is the first production of a cell line capable of SSAT protein induction from a generally unresponsive parent line.

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.

High glucose augments angiotensinogen in human renal proximal tubular cells through hepatocyte nuclear factor-5

PubMed Central

Wang, Juan; Shibayama, Yuki; Kobori, Hiroyuki; Liu, Ya; Kobara, Hideki; Masaki, Tsutomu; Wang, Zhiyu

2017-01-01

High glucose has been demonstrated to induce angiotensinogen (AGT) synthesis in the renal proximal tubular cells (RPTCs) of rats, which may further activate the intrarenal renin-angiotensin system (RAS) and contribute to diabetic nephropathy. This study aimed to investigate the effects of high glucose on AGT in the RPTCs of human origin and identify the glucose-responsive transcriptional factor(s) that bind(s) to the DNA sequences of AGT promoter in human RPTCs. Human kidney (HK)-2 cells were treated with normal glucose (5.5 mM) and high glucose (15.0 mM), respectively. Levels of AGT mRNA and AGT secretion of HK-2 cells were measured by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Consecutive 5’-end deletion mutant constructs and different site-directed mutagenesis products of human AGT promoter sequences were respectively transfected into HK-2 cells, followed by AGT promoter activity measurement through dual luciferase assay. High glucose significantly augmented the levels of AGT mRNA and AGT secretion of HK-2 cells, compared with normal glucose treatment. High glucose also significantly augmented AGT promoter activity in HK-2 cells transfected with the constructs of human AGT promoter sequences, compared with normal glucose treatment. Hepatocyte nuclear factor (HNF)-5 was found to be one of the glucose-responsive transcriptional factors of AGT in human RPTCs, since the mutation of its binding sites within AGT promoter sequences abolished the above effects of high glucose on AGT promoter activity as well as levels of AGT mRNA and its secretion. The present study has demonstrated, for the first time, that high glucose augments AGT in human RPTCs through HNF-5, which provides a potential therapeutic target for diabetic nephropathy. PMID:29053707

Comparison of Human and Guinea Pig Acetylcholinesterase Sequences and Rates of Oxime-Assisted Reactivation

DTIC Science & Technology

2010-01-01

of appropriate animal model systems. For OP poisoning, the guinea pig (Cavia porcellus) is a commonly used animal model because guinea pigs more...endogenous bioscavenger in vivo. Although guinea pigs historically have been used to test OP poisoning therapies, it has been found recently that guinea pig AChE...transcribed mRNA encoding guinea pig AChE, amplified the resulting cDNA, and sequenced this product. The nucleotide and deduced amino acid sequences of

Evaluation of Cytokine Synthesis in Human Whole Blood by Enzyme Linked Immunoassay (ELISA), Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), and Flow Cytometry

DTIC Science & Technology

2007-05-08

deoxynucleotide triphosphates, from Sigma. Sequences for glyceraldehyde-3-phosphate dehydrogenase ( G3PDH ), IL-8,and TNF-a were amplified with primer...This was accomplished by normalizing all samples to the mRNA for the moderately expressed housekeeping function glyceraldehyde-3 -phosphate...without and with isolation of cells before reverse transcription and PCR. G3PDH mRNA target amplifies at 983 base pairs. The 630 base pair band is the