Alternative Splicing in Plant Genes: A Means of Regulating the Environmental Fitness of Plants.

PubMed

Shang, Xudong; Cao, Ying; Ma, Ligeng

2017-02-20

Gene expression can be regulated through transcriptional and post-transcriptional mechanisms. Transcription in eukaryotes produces pre-mRNA molecules, which are processed and spliced post-transcriptionally to create translatable mRNAs. More than one mRNA may be produced from a single pre-mRNA by alternative splicing (AS); thus, AS serves to diversify an organism's transcriptome and proteome. Previous studies of gene expression in plants have focused on the role of transcriptional regulation in response to environmental changes. However, recent data suggest that post-transcriptional regulation, especially AS, is necessary for plants to adapt to a changing environment. In this review, we summarize recent advances in our understanding of AS during plant development in response to environmental changes. We suggest that alternative gene splicing is a novel means of regulating the environmental fitness of plants.

CstF-64 and 3'-UTR cis-element determine Star-PAP specificity for target mRNA selection by excluding PAPα.

PubMed

Kandala, Divya T; Mohan, Nimmy; A, Vivekanand; A P, Sudheesh; G, Reshmi; Laishram, Rakesh S

2016-01-29

Almost all eukaryotic mRNAs have a poly (A) tail at the 3'-end. Canonical PAPs (PAPα/γ) polyadenylate nuclear pre-mRNAs. The recent identification of the non-canonical Star-PAP revealed specificity of nuclear PAPs for pre-mRNAs, yet the mechanism how Star-PAP selects mRNA targets is still elusive. Moreover, how Star-PAP target mRNAs having canonical AAUAAA signal are not regulated by PAPα is unclear. We investigate specificity mechanisms of Star-PAP that selects pre-mRNA targets for polyadenylation. Star-PAP assembles distinct 3'-end processing complex and controls pre-mRNAs independent of PAPα. We identified a Star-PAP recognition nucleotide motif and showed that suboptimal DSE on Star-PAP target pre-mRNA 3'-UTRs inhibit CstF-64 binding, thus preventing PAPα recruitment onto it. Altering 3'-UTR cis-elements on a Star-PAP target pre-mRNA can switch the regulatory PAP from Star-PAP to PAPα. Our results suggest a mechanism of poly (A) site selection that has potential implication on the regulation of alternative polyadenylation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

CstF-64 and 3′-UTR cis-element determine Star-PAP specificity for target mRNA selection by excluding PAPα

PubMed Central

Kandala, Divya T.; Mohan, Nimmy; A, Vivekanand; AP, Sudheesh; G, Reshmi; Laishram, Rakesh S.

2016-01-01

Almost all eukaryotic mRNAs have a poly (A) tail at the 3′-end. Canonical PAPs (PAPα/γ) polyadenylate nuclear pre-mRNAs. The recent identification of the non-canonical Star-PAP revealed specificity of nuclear PAPs for pre-mRNAs, yet the mechanism how Star-PAP selects mRNA targets is still elusive. Moreover, how Star-PAP target mRNAs having canonical AAUAAA signal are not regulated by PAPα is unclear. We investigate specificity mechanisms of Star-PAP that selects pre-mRNA targets for polyadenylation. Star-PAP assembles distinct 3′-end processing complex and controls pre-mRNAs independent of PAPα. We identified a Star-PAP recognition nucleotide motif and showed that suboptimal DSE on Star-PAP target pre-mRNA 3′-UTRs inhibit CstF-64 binding, thus preventing PAPα recruitment onto it. Altering 3′-UTR cis-elements on a Star-PAP target pre-mRNA can switch the regulatory PAP from Star-PAP to PAPα. Our results suggest a mechanism of poly (A) site selection that has potential implication on the regulation of alternative polyadenylation. PMID:26496945

The cytoplasmic mRNA degradation factor Pat1 is required for rRNA processing

PubMed Central

Muppavarapu, Mridula; Huch, Susanne; Nissan, Tracy

2016-01-01

ABSTRACT Pat1 is a key cytoplasmic mRNA degradation factor, the loss of which severely increases mRNA half-lives. Several recent studies have shown that Pat1 can enter the nucleus and can shuttle between the nucleus and the cytoplasm. As a result, many nuclear roles have been proposed for Pat1. In this study, we analyzed four previously suggested nuclear roles of Pat1 and show that Pat1 is not required for efficient pre-mRNA splicing or pre-mRNA decay in yeast. However, lack of Pat1 results in accumulation of pre-rRNA processing intermediates. Intriguingly, we identified a novel genetic relationship between Pat1 and the rRNA decay machinery, specifically the exosome and the TRAMP complex. While the pre-rRNA processing intermediates that accumulate in the pat1 deletion mutant are, at least to some extent, recognized as aberrant by the rRNA degradation machinery, it is unlikely that these accumulations are the cause of their synthetic sick relationship. Here, we show that the dysregulation of the levels of mRNAs related to ribosome biogenesis could be the cause of the accumulation of the pre-rRNA processing intermediates. Although our results support a role for Pat1 in transcription, they nevertheless suggest that the primary cause of the dysregulated mRNA levels is most likely due to Pat1's role in mRNA decapping and mRNA degradation. PMID:26918764

The cytoplasmic mRNA degradation factor Pat1 is required for rRNA processing.

PubMed

Muppavarapu, Mridula; Huch, Susanne; Nissan, Tracy

2016-01-01

Pat1 is a key cytoplasmic mRNA degradation factor, the loss of which severely increases mRNA half-lives. Several recent studies have shown that Pat1 can enter the nucleus and can shuttle between the nucleus and the cytoplasm. As a result, many nuclear roles have been proposed for Pat1. In this study, we analyzed four previously suggested nuclear roles of Pat1 and show that Pat1 is not required for efficient pre-mRNA splicing or pre-mRNA decay in yeast. However, lack of Pat1 results in accumulation of pre-rRNA processing intermediates. Intriguingly, we identified a novel genetic relationship between Pat1 and the rRNA decay machinery, specifically the exosome and the TRAMP complex. While the pre-rRNA processing intermediates that accumulate in the pat1 deletion mutant are, at least to some extent, recognized as aberrant by the rRNA degradation machinery, it is unlikely that these accumulations are the cause of their synthetic sick relationship. Here, we show that the dysregulation of the levels of mRNAs related to ribosome biogenesis could be the cause of the accumulation of the pre-rRNA processing intermediates. Although our results support a role for Pat1 in transcription, they nevertheless suggest that the primary cause of the dysregulated mRNA levels is most likely due to Pat1's role in mRNA decapping and mRNA degradation.

Splicing of goose parvovirus pre-mRNA influences cytoplasmic translation of the processed mRNA

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

Li, Long; Pintel, David J., E-mail: pinteld@missouri.edu

2012-04-25

Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained inmore » unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins.« less

Nanoplasmonic probes of RNA folding and assembly during pre-mRNA splicing

NASA Astrophysics Data System (ADS)

Nguyen, Anh H.; Lee, Jong Uk; Sim, Sang Jun

2016-02-01

RNA splicing plays important roles in transcriptome and proteome diversity. Herein, we describe the use of a nanoplasmonic system that unveils RNA folding and assembly during pre-mRNA splicing wherein the quantification of mRNA splice variants is not taken into account. With a couple of SERS-probes and plasmonic probes binding at the boundary sites of exon-2/intron-2 and intron-2/exon-3 of the pre-mature RNA of the β-globin gene, the splicing process brings the probes into the plasmonic bands. For plasmonic probes, a plasmon shift increase of ~29 nm, corresponding to intron removal and exon-2 and exon-3 connection to form the mRNA molecule, is measured by plasmonic coupling. The increased scattering intensity and surface-enhanced Raman scattering (SERS) fingerprinting reveal the clear dynamics of pre-mRNA splicing. Moreover, a time-resolved experiment of individual RNA molecules exhibited a successful splicing and an inhibited splicing event by 33 μM biflavonoid isoginkgetin, a general inhibitor of RNA splicing. The results suggest that the RNA splicing is successfully monitored with the nanoplasmonic system. Thus, this platform can be useful for studying RNA nanotechnology, biomolecular folding, alternative splicing, and maturation of microRNA.

Effects of airborne particulate matter on alternative pre-mRNA splicing in colon cancer cells

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

Buggiano, Valeria; Petrillo, Ezequiel; Alló, Mariano

2015-07-15

Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5′ untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colonmore » cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing. - Highlights: • Airborne particulate matter (PM10) affects alternative splicing in colon cells. • PM10 upregulates one of the two mRNA variants of the growth factor BMP-4. • This variant has a longer 5′ unstranslated region and introduces an upstream AUG. • By regulating BMP-4 mRNA splicing PM10 inhibits total expression of BMP-4 protein. • BMP-4 downregulation was previously reported to be associated to colon cancer.« less

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

Genome-Wide Survey of Cold Stress Regulated Alternative Splicing in Arabidopsis thaliana with Tiling Microarray

PubMed Central

Leviatan, Noam; Alkan, Noam; Leshkowitz, Dena; Fluhr, Robert

2013-01-01

Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression. PMID:23776682

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

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.

SKIP Is a Component of the Spliceosome Linking Alternative Splicing and the Circadian Clock in Arabidopsis[W

PubMed Central

Wang, Xiaoxue; Wu, Fangming; Xie, Qiguang; Wang, Huamei; Wang, Ying; Yue, Yanling; Gahura, Ondrej; Ma, Shuangshuang; Liu, Lei; Cao, Ying; Jiao, Yuling; Puta, Frantisek; McClung, C. Robertson; Xu, Xiaodong; Ma, Ligeng

2012-01-01

Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5′ and 3′ splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level. PMID:22942380

Allele-Specific Alternative mRNA processing (ASARP) | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

A software pipeline for prediction of allele-specific alternative RNA processing events using single RNA-seq data. The current version focuses on prediction of alternative splicing and alternative polyadenylation modulated by genetic variants.

Effects of gestational hypertension and pre-eclampsia in mRNA expression of fibrinolysis genes in primary cultured human umbilical vein endothelial cells.

PubMed

Poblete-Naredo, Irais; Rodríguez-Yáñez, Yury; Corona-Núñez, Rogelio O; González-Monroy, Stuart; Salinas, Juan E; Albores, Arnulfo

2018-05-17

Hypertension disorders (HD) and pre-eclampsia (PRE) are leading causes of maternal deaths worldwide. PRE is associated with vascular endothelial dysfunction and with deregulation of the fibrinolysis pathway genes. Fibrinolysis is the fibrin clot hydrolysis process catalyzed by plasmin, a proteolytic enzyme formed from plasminogen. Plasminogen is cleaved by tissue-type (tPA) and urokinase-type (uPA) activators and inhibited by the plasminogen activator inhibitors type-1 (PAI-1) and type-2 (PAI-2). The whole process maintains blood hemostasis. This study aims to assess PAI-1, PAI-2, tPA and uPA mRNA expression in primary cultured human umbilical vein endothelial cells (HUVEC) isolated and cultured from healthy, HD and PRE women. Results show that PAI-1 and PAI-2 mRNA decreased in HD-HUVEC, whereas PAI-1 and uPA decreased in PRE-HUVEC cultures compared to control ones. Notably, the expression ratio between pro- and anti-fibrinolytic actors remained unchanged among the studied groups. It seems that newborn's hemostasis is maintained balanced probably by a compensatory mechanism that involves changes in the fibrinolysis gene expression profile. The real impact of these changes in mRNA expression is unknown, however, it is suggested that these changes could be associated with an increased predisposition to vascular disease development in the progeny. Copyright © 2018. Published by Elsevier Ltd.

Genome level analysis of rice mRNA 3′-end processing signals and alternative polyadenylation

PubMed Central

Shen, Yingjia; Ji, Guoli; Haas, Brian J.; Wu, Xiaohui; Zheng, Jianti; Reese, Greg J.; Li, Qingshun Quinn

2008-01-01

The position of a poly(A) site of eukaryotic mRNA is determined by sequence signals in pre-mRNA and a group of polyadenylation factors. To reveal rice poly(A) signals at a genome level, we constructed a dataset of 55 742 authenticated poly(A) sites and characterized the poly(A) signals. This resulted in identifying the typical tripartite cis-elements, including FUE, NUE and CE, as previously observed in Arabidopsis. The average size of the 3′-UTR was 289 nucleotides. When mapped to the genome, however, 15% of these poly(A) sites were found to be located in the currently annotated intergenic regions. Moreover, an extensive alternative polyadenylation profile was evident where 50% of the genes analyzed had more than one unique poly(A) site (excluding microheterogeneity sites), and 13% had four or more poly(A) sites. About 4% of the analyzed genes possessed alternative poly(A) sites at their introns, 5′-UTRs, or protein coding regions. The authenticity of these alternative poly(A) sites was partially confirmed using MPSS data. Analysis of nucleotide profile and signal patterns indicated that there may be a different set of poly(A) signals for those poly(A) sites found in the coding regions. Based on the features of rice poly(A) signals, an updated algorithm termed PASS-Rice was designed to predict poly(A) sites. PMID:18411206

Multiple cis-acting sequence elements are required for efficient splicing of simian virus 40 small-t antigen pre-mRNA.

PubMed Central

Fu, X Y; Colgan, J D; Manley, J L

1988-01-01

We have determined the effects of a number of mutations in the small-t antigen mRNA intron on the alternative splicing pattern of the simian virus 40 early transcript. Expansion of the distance separating the small-t pre-mRNA lariat branch point and the shared large T-small t 3' splice site from 18 to 29 nucleotides (nt) resulted in a relative enhancement of small-t splicing in vivo. This finding, coupled with the observation that large-T pre-RNA splicing in vitro was not affected by this expansion, suggests that small-t splicing is specifically constrained by a short branch point-3' splice site distance. Similarly, the distance separating the 5' splice site and branch point (48 nt) was found to be at or near a minimum for small-t splicing, because deletions in this region as small as 2 nt dramatically reduced the ratio of small-t to large-T mRNA that accumulated in transfected cells. Finally, a specific sequence within the small-t intron, encompassing the upstream branch sites used in large-T splicing, was found to be an important element in the cell-specific pattern of early alternative splicing. Substitutions within this region reduced the ratio of small-t to large-T mRNA produced in HeLa cells but had only minor effects in human 293 cells. Images PMID:2851720

Alternative exon definition events control the choice between nuclear retention and cytoplasmic export of U11/U12-65K mRNA.

PubMed

Verbeeren, Jens; Verma, Bhupendra; Niemelä, Elina H; Yap, Karen; Makeyev, Eugene V; Frilander, Mikko J

2017-05-01

Cellular homeostasis of the minor spliceosome is regulated by a negative feed-back loop that targets U11-48K and U11/U12-65K mRNAs encoding essential components of the U12-type intron-specific U11/U12 di-snRNP. This involves interaction of the U11 snRNP with an evolutionarily conserved splicing enhancer giving rise to unproductive mRNA isoforms. In the case of U11/U12-65K, this mechanism controls the length of the 3' untranslated region (3'UTR). We show that this process is dynamically regulated in developing neurons and some other cell types, and involves a binary switch between translation-competent mRNAs with a short 3'UTR to non-productive isoforms with a long 3'UTR that are retained in the nucleus or/and spliced to the downstream amylase locus. Importantly, the choice between these alternatives is determined by alternative terminal exon definition events regulated by conserved U12- and U2-type 5' splice sites as well as sequence signals used for pre-mRNA cleavage and polyadenylation. We additionally show that U11 snRNP binding to the U11/U12-65K mRNA species with a long 3'UTR is required for their nuclear retention. Together, our studies uncover an intricate molecular circuitry regulating the abundance of a key spliceosomal protein and shed new light on the mechanisms limiting the export of non-productively spliced mRNAs from the nucleus to the cytoplasm.

Cancer-Associated Perturbations in Alternative Pre-messenger RNA Splicing.

PubMed

Shkreta, Lulzim; Bell, Brendan; Revil, Timothée; Venables, Julian P; Prinos, Panagiotis; Elela, Sherif Abou; Chabot, Benoit

2013-01-01

For most of our 25,000 genes, the removal of introns by pre-messenger RNA (pre-mRNA) splicing represents an essential step toward the production of functional messenger RNAs (mRNAs). Alternative splicing of a single pre-mRNA results in the production of different mRNAs. Although complex organisms use alternative splicing to expand protein function and phenotypic diversity, patterns of alternative splicing are often altered in cancer cells. Alternative splicing contributes to tumorigenesis by producing splice isoforms that can stimulate cell proliferation and cell migration or induce resistance to apoptosis and anticancer agents. Cancer-specific changes in splicing profiles can occur through mutations that are affecting splice sites and splicing control elements, and also by alterations in the expression of proteins that control splicing decisions. Recent progress in global approaches that interrogate splicing diversity should help to obtain specific splicing signatures for cancer types. The development of innovative approaches for annotating and reprogramming splicing events will more fully establish the essential contribution of alternative splicing to the biology of cancer and will hopefully provide novel targets and anticancer strategies. Metazoan genes are usually made up of several exons interrupted by introns. The introns are removed from the pre-mRNA by RNA splicing. In conjunction with other maturation steps, such as capping and polyadenylation, the spliced mRNA is then transported to the cytoplasm to be translated into a functional protein. The basic mechanism of splicing requires accurate recognition of each extremity of each intron by the spliceosome. Introns are identified by the binding of U1 snRNP to the 5' splice site and the U2AF65/U2AF35 complex to the 3' splice site. Following these interactions, other proteins and snRNPs are recruited to generate the complete spliceosomal complex needed to excise the intron. While many introns are constitutively removed by the spliceosome, other splice junctions are not used systematically, generating the phenomenon of alternative splicing. Alternative splicing is therefore the process by which a single species of pre-mRNA can be matured to produce different mRNA molecules (Fig. 1). Depending on the number and types of alternative splicing events, a pre-mRNA can generate from two to several thousands different mRNAs leading to the production of a corresponding number of proteins. It is now believed that the expression of at least 70 % of human genes is subjected to alternative splicing, implying an enormous contribution to proteomic diversity, and by extension, to the development and the evolution of complex animals. Defects in splicing have been associated with human diseases (Caceres and Kornblihtt, Trends Genet 18(4):186-93, 2002, Cartegni et al., Nat Rev Genet 3(4):285-98, 2002, Pagani and Baralle, Nat Rev Genet 5(5):389-96, 2004), including cancer (Brinkman, Clin Biochem 37(7):584-94, 2004, Venables, Bioessays 28(4):378-86, 2006, Srebrow and Kornblihtt, J Cell Sci 119(Pt 13):2635-2641, 2006, Revil et al., Bull Cancer 93(9):909-919, 2006, Venables, Transworld Res Network, 2006, Pajares et al., Lancet Oncol 8(4):349-57, 2007, Skotheim and Nees, Int J Biochem Cell Biol 39:1432-1449, 2007). Numerous studies have now confirmed the existence of specific differences in the alternative splicing profiles between normal and cancer tissues. Although there are a few cases where specific mutations are the primary cause for these changes, global alterations in alternative splicing in cancer cells may be primarily derived from changes in the expression of RNA-binding proteins that control splice site selection. Overall, these cancer-specific differences in alternative splicing offer an immense potential to improve the diagnosis and the prognosis of cancer. This review will focus on the functional impact of cancer-associated alternative splicing variants, the molecular determinants that alter the splicing decisions in cancer cells, and future therapeutic strategies.

Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches

PubMed Central

Chen, Mo; Manley, James L.

2010-01-01

Alternative splicing of mRNA precursors provides an important means of genetic control and is a crucial step in the expression of most genes. Alternative splicing markedly affects human development, and its misregulation underlies many human diseases. Although the mechanisms of alternative splicing have been studied extensively, until the past few years we had not begun to realize fully the diversity and complexity of alternative splicing regulation by an intricate protein–RNA network. Great progress has been made by studying individual transcripts and through genome-wide approaches, which together provide a better picture of the mechanistic regulation of alternative pre-mRNA splicing. PMID:19773805

Effects of airborne particulate matter on alternative pre-mRNA splicing in colon cancer cells.

PubMed

Buggiano, Valeria; Petrillo, Ezequiel; Alló, Mariano; Lafaille, Celina; Redal, María Ana; Alghamdi, Mansour A; Khoder, Mamdouh I; Shamy, Magdy; Muñoz, Manuel J; Kornblihtt, Alberto R

2015-07-01

Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5' untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colon cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Alternative exon definition events control the choice between nuclear retention and cytoplasmic export of U11/U12-65K mRNA

PubMed Central

Verbeeren, Jens; Verma, Bhupendra

2017-01-01

Cellular homeostasis of the minor spliceosome is regulated by a negative feed-back loop that targets U11-48K and U11/U12-65K mRNAs encoding essential components of the U12-type intron-specific U11/U12 di-snRNP. This involves interaction of the U11 snRNP with an evolutionarily conserved splicing enhancer giving rise to unproductive mRNA isoforms. In the case of U11/U12-65K, this mechanism controls the length of the 3′ untranslated region (3′UTR). We show that this process is dynamically regulated in developing neurons and some other cell types, and involves a binary switch between translation-competent mRNAs with a short 3′UTR to non-productive isoforms with a long 3′UTR that are retained in the nucleus or/and spliced to the downstream amylase locus. Importantly, the choice between these alternatives is determined by alternative terminal exon definition events regulated by conserved U12- and U2-type 5′ splice sites as well as sequence signals used for pre-mRNA cleavage and polyadenylation. We additionally show that U11 snRNP binding to the U11/U12-65K mRNA species with a long 3′UTR is required for their nuclear retention. Together, our studies uncover an intricate molecular circuitry regulating the abundance of a key spliceosomal protein and shed new light on the mechanisms limiting the export of non-productively spliced mRNAs from the nucleus to the cytoplasm. PMID:28549066

Radiation-induced alternative transcripts as detected in total and polysome-bound mRNA.

PubMed

Wahba, Amy; Ryan, Michael C; Shankavaram, Uma T; Camphausen, Kevin; Tofilon, Philip J

2018-01-02

Alternative splicing is a critical event in the posttranscriptional regulation of gene expression. To investigate whether this process influences radiation-induced gene expression we defined the effects of ionizing radiation on the generation of alternative transcripts in total cellular mRNA (the transcriptome) and polysome-bound mRNA (the translatome) of the human glioblastoma stem-like cell line NSC11. For these studies, RNA-Seq profiles from control and irradiated cells were compared using the program SpliceSeq to identify transcripts and splice variations induced by radiation. As compared to the transcriptome (total RNA) of untreated cells, the radiation-induced transcriptome contained 92 splice events suggesting that radiation induced alternative splicing. As compared to the translatome (polysome-bound RNA) of untreated cells, the radiation-induced translatome contained 280 splice events of which only 24 were overlapping with the radiation-induced transcriptome. These results suggest that radiation not only modifies alternative splicing of precursor mRNA, but also results in the selective association of existing mRNA isoforms with polysomes. Comparison of radiation-induced alternative transcripts to radiation-induced gene expression in total RNA revealed little overlap (about 3%). In contrast, in the radiation-induced translatome, about 38% of the induced alternative transcripts corresponded to genes whose expression level was affected in the translatome. This study suggests that whereas radiation induces alternate splicing, the alternative transcripts present at the time of irradiation may play a role in the radiation-induced translational control of gene expression and thus cellular radioresponse.

Cleavage and polyadenylation: Ending the message expands gene regulation

PubMed Central

Neve, Jonathan

2017-01-01

ABSTRACT Cleavage and polyadenylation (pA) is a fundamental step that is required for the maturation of primary protein encoding transcripts into functional mRNAs that can be exported from the nucleus and translated in the cytoplasm. 3′end processing is dependent on the assembly of a multiprotein processing complex on the pA signals that reside in the pre-mRNAs. Most eukaryotic genes have multiple pA signals, resulting in alternative cleavage and polyadenylation (APA), a widespread phenomenon that is important to establish cell state and cell type specific transcriptomes. Here, we review how pA sites are recognized and comprehensively summarize how APA is regulated and creates mRNA isoform profiles that are characteristic for cell types, tissues, cellular states and disease. PMID:28453393

Comparative effects of low-level laser therapy pre- and post-injury on mRNA expression of MyoD, myogenin, and IL-6 during the skeletal muscle repair.

PubMed

Alves, Agnelo Neves; Ribeiro, Beatriz Guimarães; Fernandes, Kristianne Porta Santos; Souza, Nadhia Helena Costa; Rocha, Lília Alves; Nunes, Fabio Daumas; Bussadori, Sandra Kalil; Mesquita-Ferrari, Raquel Agnelli

2016-05-01

This study analyzed the effect of pre-injury and post-injury irradiation with low-level laser therapy (LLLT) on the mRNA expression of myogenic regulatory factors and interleukin 6 (IL-6) during the skeletal muscle repair. Male rats were divided into six groups: control group, sham group, LLLT group, injury group; pre-injury LLLT group, and post-injury LLLT group. LLLT was performed with a diode laser (wavelength 780 nm; output power 40 mW' and total energy 3.2 J). Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly onto the belly of the tibialis anterior (TA) muscle. After euthanasia, the TA muscle was removed for the isolation of total RNA and analysis of MyoD, myogenin, and IL-6 using real-time quantitative PCR. Significant increases were found in the expression of MyoD mRNA at 3 and 7 days as well as the expression of myogenin mRNA at 14 days in the post-injury LLLT group in comparison to injury group. A significant reduction was found in the expression of IL-6 mRNA at 3 and 7 days in the pre-injury LLLT and post-injury LLLT groups. A significant increase in IL-6 mRNA was found at 14 days in the post-injury LLLT group in comparison to the injury group. LLLT administered following muscle injury modulates the mRNA expression of MyoD and myogenin. Moreover, the both forms of LLLT administration were able to modulate the mRNA expression of IL-6 during the muscle repair process.

Evidence that Poly(A) Binding Protein C1 Binds Nuclear Pre-mRNA Poly(A) Tails

PubMed Central

Hosoda, Nao; Lejeune, Fabrice; Maquat, Lynne E.

2006-01-01

In mammalian cells, poly(A) binding protein C1 (PABP C1) has well-known roles in mRNA translation and decay in the cytoplasm. However, PABPC1 also shuttles in and out of the nucleus, and its nuclear function is unknown. Here, we show that PABPC1, like the major nuclear poly(A) binding protein PABPN1, associates with nuclear pre-mRNAs that are polyadenylated and intron containing. PABPC1 does not bind nonpolyadenylated histone mRNA, indicating that the interaction of PABPC1 with pre-mRNA requires a poly(A) tail. Consistent with this conclusion, UV cross-linking results obtained using intact cells reveal that PABPC1 binds directly to pre-mRNA poly(A) tails in vivo. We also show that PABPC1 immunopurifies with poly(A) polymerase, suggesting that PABPC1 is acquired by polyadenylated transcripts during poly(A) tail synthesis. Our findings demonstrate that PABPC1 associates with polyadenylated transcripts earlier in mammalian mRNA biogenesis than previously thought and offer insights into the mechanism by which PABPC1 is recruited to newly synthesized poly(A). Our results are discussed in the context of pre-mRNA processing and stability and mRNA trafficking and the pioneer round of translation. PMID:16581783

In vivo characterization of the Drosophila mRNA 3′ end processing core cleavage complex

PubMed Central

Michalski, Daniel; Steiniger, Mindy

2015-01-01

A core cleavage complex (CCC) consisting of CPSF73, CPSF100, and Symplekin is required for cotranscriptional 3′ end processing of all metazoan pre-mRNAs, yet little is known about the in vivo molecular interactions within this complex. The CCC is a component of two distinct complexes, the cleavage/polyadenylation complex and the complex that processes nonpolyadenylated histone pre-mRNAs. RNAi-depletion of CCC factors in Drosophila culture cells causes reduction of CCC processing activity on histone mRNAs, resulting in read through transcription. In contrast, RNAi-depletion of factors only required for histone mRNA processing allows use of downstream cryptic polyadenylation signals to produce polyadenylated histone mRNAs. We used Dmel-2 tissue culture cells stably expressing tagged CCC components to determine that amino acids 272–1080 of Symplekin and the C-terminal approximately 200 amino acids of both CPSF73 and CPSF100 are required for efficient CCC formation in vivo. Additional experiments reveal that the C-terminal 241 amino acids of CPSF100 are sufficient for histone mRNA processing indicating that the first 524 amino acids of CPSF100 are dispensable for both CCC formation and histone mRNA 3′ end processing. CCCs containing deletions of Symplekin lacking the first 271 amino acids resulted in dramatic increased use of downstream polyadenylation sites for histone mRNA 3′ end processing similar to RNAi-depletion of histone-specific 3′ end processing factors FLASH, SLBP, and U7 snRNA. We propose a model in which CCC formation is mediated by CPSF73, CPSF100, and Symplekin C-termini, and the N-terminal region of Symplekin facilitates cotranscriptional 3′ end processing of histone mRNAs. PMID:26081560

Alternative polyadenylation of mRNA precursors

PubMed Central

Tian, Bin; Manley, James L.

2017-01-01

Alternative polyadenylation (APA) is an RNA-processing mechanism that generates distinct 3′ termini on mRNAs and other RNA polymerase II transcripts. It is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. APA exhibits tissue specificity and is important for cell proliferation and differentiation. In this Review, we discuss the roles of APA in diverse cellular processes, including mRNA metabolism, protein diversification and protein localization, and more generally in gene regulation. We also discuss the molecular mechanisms underlying APA, such as variation in the concentration of core processing factors and RNA-binding proteins, as well as transcription-based regulation. PMID:27677860

Protein interactions and complexes in human microRNA biogenesis and function

PubMed Central

Perron, Marjorie P.; Provost, Patrick

2010-01-01

Encoded in the genome of most eukaryotes, microRNAs (miRNAs) have been proposed to regulate specifically up to 90% of human genes through a process known as miRNA-guided RNA silencing. The aim of this review is to present this process as the integration of a succession of specialized molecular machines exerting well defined functions. The nuclear microprocessor complex initially recognizes and processes its primary miRNA substrate into a miRNA precursor (pre-miRNA). This structure is then exported to the cytoplasm by the Exportin-5 complex where it is presented to the pre-miRNA processing complex. Following pre-miRNA conversion into a miRNA:miRNA* duplex, this complex is assembled into a miRNA-containing ribonucleoprotein (miRNP) complex, after which the miRNA strand is selected. The degree of complementarity of the miRNA for its messenger RNA (mRNA) target guides the recruitment of the miRNP complex. Initially repressing its translation, the miRNP-silenced mRNA is directed to the P-bodies, where the mRNA is either released from its inhibition upon a cellular signal and/or actively degraded. The potency and specificity of miRNA biogenesis and function rely on the distinct protein·protein, protein·RNA and RNA:RNA interactions found in different complexes, each of which fulfill a specific function in a well orchestrated process. PMID:17981733

Expression of REST4 in human gliomas in vivo and influence of pioglitazone on REST in vitro

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

Ren, Huan; Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha 410078; Gao, Zhangfeng

The repressor element-1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) has an irreplaceable role during the differentiation of neurons. REST has multiple splice variants which link to various types of cancer. Previous work had highlighted the role of REST in glioma, where the expression of REST is enhanced. But whether alternative splicing of REST is expressed in glioma has not been described. Here, we show that a specific isoform REST4 is expressed in glioma specimens, and will influence the mRNA level of REST in vivo. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have a role of antineoplastic in various tumor cells, which includingmore » glioma cells. Moreover, study indicated that PPARγ agonist pioglitazone can promote alternative splicing of REST pre-mRNA. In this study, we selected pioglitazone as a tool drug to explore whether the role of pioglitazone in anti-glioma is mediated by regulating REST expression or promoting alternative splicing of REST in glioma cells. Results show that pioglitazone can inhibit proliferation and induce apoptosis of glioma cell in vitro, which may be mediated by down-regulating REST mRNA level but not by inducing alternative splicing of REST pre-mRNA. Our study firstly reports the expression of REST4 in glioma tissue samples. And we recommend that pioglitazone, which can reduce the expression level of REST, represents a promising drug for therapy of glioma. - Highlights: • A specific isoform REST4 is expressed in glioma specimens in vivo. • REST4 will influence the mRNA level of REST in vivo. • Pioglitazone can inhibit proliferation and induce apoptosis of glioma cells. • The role of pioglitazone in anti-glioma may be mediated by down-regulating REST.« less

Analysis of Autoregulation at the Level of Pre-mRNA Splicing of the Suppressor-of-White-Apricot Gene in Drosophila

PubMed Central

Zachar, Z.; Chou, T. B.; Kramer, J.; Mims, I. P.; Bingham, P. M.

1994-01-01

The Drosophila suppressor-of-white-apricot [su(w(a))] protein regulates/modulates at least two somatic RNA processing events. It is a potent regulator of its own expression. We report here new studies of this autoregulatory circuit. Among other things, our studies show the following. First, new evidence that su(w(a)) expression is autoregulated at the level of pre-mRNA splicing is reported. su(w(a)) protein represses accumulation of the fully spliced su(w(a)) mRNA encoding it and promotes accumulation of high levels of incompletely spliced su(w(a)) pre-mRNA. Second, the fully spliced su(w(a)) mRNA is sufficient for all known su(w(a)) genetic functions indicating that it encodes the sole su(w(a)) protein. Third, the incompletely spliced su(w(a)) pre-mRNAs resulting from autoregulation are not translated (probably as a result of nuclear retention) and apparently represent nonfunctional by-products. Fourth, the special circumstances of su(w(a)) expression during oogenesis allows maternal deposition exclusively of fully spliced su(w(a)) mRNA. Fifth, su(w(a)) protein immunolocalizes to nuclei consistent with its being a direct regulator of pre-mRNA processing. We discuss the implications of our results for mechanisms of splicing regulation and for developmental control of su(w(a)) expression. PMID:8056305

RNA Binding Protein RBM38 Regulates Expression of the 11-Kilodalton Protein of Parvovirus B19, Which Facilitates Viral DNA Replication.

PubMed

Ganaie, Safder S; Chen, Aaron Yun; Huang, Chun; Xu, Peng; Kleiboeker, Steve; Du, Aifang; Qiu, Jianming

2018-04-15

Human parvovirus B19 (B19V) expresses a single precursor mRNA (pre-mRNA), which undergoes alternative splicing and alternative polyadenylation to generate 12 viral mRNA transcripts that encode two structural proteins (VP1 and VP2) and three nonstructural proteins (NS1, 7.5-kDa protein, and 11-kDa protein). Splicing at the second 5' donor site (D2 site) of the B19V pre-mRNA is essential for the expression of VP2 and the 11-kDa protein. We previously identified that cis -acting intronic splicing enhancer 2 (ISE2) that lies immediately after the D2 site facilitates the recognition of the D2 donor for its efficient splicing. In this study, we report that ISE2 is critical for the expression of the 11-kDa viral nonstructural protein. We found that ISE2 harbors a consensus RNA binding motif protein 38 (RBM38) binding sequence, 5'-UGUGUG-3'. RBM38 is expressed during the middle stage of erythropoiesis. We first confirmed that RBM38 binds specifically with the ISE2 element in vitro The knockdown of RBM38 significantly decreases the level of spliced mRNA at D2 that encodes the 11-kDa protein but not that of the D2-spliced mRNA that encodes VP2. Importantly, we found that the 11-kDa protein enhances viral DNA replication and virion release. Accordingly, the knockdown of RBM38 decreases virus replication via downregulating 11-kDa protein expression. Taken together, these results suggest that the 11-kDa protein facilitates B19V DNA replication and that RBM38 is an essential host factor for B19V pre-mRNA splicing and for the expression of the 11-kDa protein. IMPORTANCE B19V is a human pathogen that can cause fifth disease, arthropathy, anemia in immunocompromised patients and sickle cell disease patients, myocarditis, and hydrops fetalis in pregnant women. Human erythroid progenitor cells (EPCs) are most susceptible to B19V infection and fully support viral DNA replication. The exclusive tropism of B19V for erythroid-lineage cells is dependent not only on the expression of viral receptors and coreceptors on the cell surface but also on the intracellular host factors that support B19V replication. Our present study shows that B19V uses a host factor, RNA binding motif protein 38 (RBM38), for the processing of its pre-mRNA during virus replication. Specifically, RBM38 interacts with the intronic splicing enhancer 2 (ISE2) element of B19V pre-mRNA and promotes 11-kDa protein expression, thereby regulating the 11-kDa protein-mediated augmentation of B19V replication. The identification of this novel host-pathogen interaction will provide mechanistic insights into B19V replication and aid in finding new targets for anti-B19V therapeutics. Copyright © 2018 American Society for Microbiology.

Mechanisms and Regulation of Alternative Pre-mRNA Splicing

PubMed Central

Lee, Yeon

2015-01-01

Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA–protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA–RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions. PMID:25784052

A Novel Intra-U1 snRNP Cross-Regulation Mechanism: Alternative Splicing Switch Links U1C and U1-70K Expression

PubMed Central

Rösel-Hillgärtner, Tanja Dorothe; Hung, Lee-Hsueh; Khrameeva, Ekaterina; Le Querrec, Patrick; Gelfand, Mikhail S.; Bindereif, Albrecht

2013-01-01

The U1 small nuclear ribonucleoprotein (snRNP)-specific U1C protein participates in 5′ splice site recognition and regulation of pre-mRNA splicing. Based on an RNA-Seq analysis in HeLa cells after U1C knockdown, we found a conserved, intra-U1 snRNP cross-regulation that links U1C and U1-70K expression through alternative splicing and U1 snRNP assembly. To investigate the underlying regulatory mechanism, we combined mutational minigene analysis, in vivo splice-site blocking by antisense morpholinos, and in vitro binding experiments. Alternative splicing of U1-70K pre-mRNA creates the normal (exons 7–8) and a non-productive mRNA isoform, whose balance is determined by U1C protein levels. The non-productive isoform is generated through a U1C-dependent alternative 3′ splice site, which requires an adjacent cluster of regulatory 5′ splice sites and binding of intact U1 snRNPs. As a result of nonsense-mediated decay (NMD) of the non-productive isoform, U1-70K mRNA and protein levels are down-regulated, and U1C incorporation into the U1 snRNP is impaired. U1-70K/U1C-deficient particles are assembled, shifting the alternative splicing balance back towards productive U1-70K splicing, and restoring assembly of intact U1 snRNPs. Taken together, we established a novel feedback regulation that controls U1-70K/U1C homeostasis and ensures correct U1 snRNP assembly and function. PMID:24146627

Intergenic mRNA molecules resulting from trans-splicing.

PubMed

Finta, Csaba; Zaphiropoulos, Peter G

2002-02-22

Accumulated recent evidence is indicating that alternative splicing represents a generalized process that increases the complexity of human gene expression. Here we show that mRNA production may not necessarily be limited to single genes, as human liver also has the potential to produce a variety of hybrid cytochrome P450 3A mRNA molecules. The four known cytochrome P450 3A genes in humans, CYP3A4, CYP3A5, CYP3A7, and CYP3A43, share a high degree of similarity, consist of 13 exons with conserved exon-intron boundaries, and form a cluster on chromosome 7. The chimeric CYP3A mRNA molecules described herein are characterized by CYP3A43 exon 1 joined at canonical splice sites to distinct sets of CYP3A4 or CYP3A5 exons. Because the CYP3A43 gene is in a head-to-head orientation with the CYP3A4 and CYP3A5 genes, bypassing transcriptional termination can not account for the formation of hybrid CYP3A mRNAs. Thus, the mechanism generating these molecules has to be an RNA processing event that joins exons of independent pre-mRNA molecules, i.e. trans-splicing. Using quantitative real-time polymerase chain reaction, the ratio of one CYP3A43/3A4 intergenic combination was estimated to be approximately 0.15% that of the CYP3A43 mRNAs. Moreover, trans-splicing has been found not to interfere with polyadenylation. Heterologous expression of the chimeric species composed of CYP3A43 exon 1 joined to exons 2-13 of CYP3A4 revealed catalytic activity toward testosterone.

Pre-acclimation to low ammonia improves ammonia handling in common carp (Cyprinus carpio) when exposed subsequently to high environmental ammonia.

PubMed

Shrivastava, Jyotsna; Sinha, Amit Kumar; Datta, Surjya Narayan; Blust, Ronny; De Boeck, Gudrun

2016-11-01

We tested whether exposing fish to low ammonia concentrations induced acclimation processes and helped fish to tolerate subsequent (sub)lethal ammonia exposure by activating ammonia excretory pathways. Common carp (Cyprinus carpio) were pre-exposed to 0.27mM ammonia (∼10% 96h LC 50 ) for 3, 7 and 14days. Thereafter, each of these pre-exposed and parallel naïve groups were exposed to 1.35mM high environmental ammonia (HEA, ∼50% 96h LC 50 ) for 12h and 48h to assess the occurrence of ammonia acclimation based on sub-lethal end-points, and to lethal ammonia concentrations (2.7mM, 96h LC 50 ) in order to assess improved survival time. Results show that fish pre-exposed to ammonia for 3 and 7days had a longer survival time than the ammonia naïve fish. However, this effect disappeared after prolonged (14days) pre-exposure. Ammonia excretion rate (J amm ) was strongly inhibited (or even reversed) in the unacclimated groups during HEA. On the contrary, after 3days the pre-exposure fish maintained J amm while after 7days these pre-acclimated fish were able to increase J amm efficiently. Again, this effect disappeared after 14days of pre-acclimation. The efficient ammonia efflux in pre-acclimated fish was associated with the up-regulation of branchial mRNA expression of ammonia transporters and exchangers. Pre-exposure with ammonia for 3-7days stimulated an increment in the transcript level of gill Rhcg-a and Rhcg-b mRNA relative to the naïve control group and the up-regulation of these two Rhcg homologs was reinforced during subsequent HEA exposure. No effect of pre-exposure was noted for Rhbg. Relative to unacclimated fish, the transcript level of Na + /H + exchangers (NHE-3) was raised in 3-7days pre-acclimated fish and remained higher during the subsequent HEA exposure while gill H + -ATPase activities and mRNA levels were not affected by pre-acclimation episodes. Likewise, ammonia pre-acclimated fish with or without HEA exposure displayed pronounced up-regulation in Na + /K + -ATPase activity and mRNA expression relative to the corresponding ammonia naïve groups. Overall, these data suggest that ammonia acclimation was evident for both lethal and the sub-lethal endpoints through priming mechanisms in ammonia excretory transcriptional processes, but these acclimation effects were transient and disappeared after prolonged pre-exposure. Copyright © 2016 Elsevier B.V. All rights reserved.

Mutations That Affect the Efficiency of Translation of mRNA for the cII Gene of Coliphage Lambda

PubMed Central

Dul, Ed; Mahoney, Michael E.; Wulff, Daniel L.

1987-01-01

Starting with the λ pRE- strain λctr1 cy3008, which forms clear plaques, we have isolated two mutant strains, λdya2 ctr1 cy3008 and λ dya3 ctr1 cy3008, that form plaques with very slightly turbid centers. The dya2 and dya3 mutations lie in the region of overlap between the PRE promoter and the ribosome recognition region of the cII gene, and have nucleotide alterations at positions -1 and +5 of pRE, and alterations of cII mRNA at -16 and -21 nucleotides before the initial AUG codon of the gene. Both mutations destabilize a stem structure that may be formed by cII mRNA, and dya2 also changes the sequence on cII mRNA that is complementary to the 3'-end of 16 S rRNA from 5'-UAAGGA-3' to 5'-UGAGGA-3'.—The dya2 and dya3 mutations, along with the ctr1 mutation, which destabilizes either of two alternate stem structures which may be formed by cII mRNA (these being more stable stem structures than the one affected by dya2 and dya3), were tested for their ability to reverse two cII- mutations that are characterized by inefficient translation of cII mRNA. These are cII3088, an A → G mutation four bases before the initial AUG codon, and cII3059 , a GUU → GAU (Val2 → Asp) second codon mutation. It was found that ctr1 completely reverses the translation defects of these two mutations, while dya2 partially reverses these translation defects. The dya3 mutation has no effect on translation efficiency under any condition tested. However neither the ctr1 mutation nor the dya2 mutation has much effect on translation efficiency in an otherwise cII+ background, indicating that other factors must limit the rate of translation of cII mRNA under these conditions. PMID:2953647

L-Dopa decarboxylase expression profile in human cancer cells.

PubMed

Chalatsa, Ioanna; Nikolouzou, Eleftheria; Fragoulis, Emmanuel G; Vassilacopoulou, Dido

2011-02-01

L-Dopa decarboxylase (DDC) catalyses the decarboxylation of L-Dopa. It has been shown that the DDC gene undergoes alternative splicing within its 5'-untranslated region (UTR), in a tissue-specific manner, generating identical protein products. The employment of two alternative 5'UTRs is thought to be responsible for tissue-specific expression of the human DDC mRNA. In this study, we focused on the investigation of the nature of the mRNA expression in human cell lines of neural and non-neural origin. Our results show the expression of a neural-type DDC mRNA splice variant, lacking exon 3 in all cell lines studied. Co-expression of the full length non-neural DDC mRNA and the neural-type DDC splice variant lacking exon 3 was detected in all cell lines. The alternative DDC protein isoform, Alt-DDC, was detected in SH-SY5Y and HeLa cells. Our findings suggest that the human DDC gene undergoes complex processing, leading to the formation of multiple mRNA isoforms. The study of the significance of this phenomenon of multiple DDC mRNA isoforms could provide us with new information leading to the elucidation of the complex biological pathways that the human enzyme is involved in.

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

PubMed Central

Davis, Ryan; Shi, Yongsheng

2014-01-01

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

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

PubMed Central

Brogna, S

1999-01-01

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

Cellular RNA binding proteins NS1-BP and hnRNP K regulate influenza A virus RNA splicing.

PubMed

Tsai, Pei-Ling; Chiou, Ni-Ting; Kuss, Sharon; García-Sastre, Adolfo; Lynch, Kristen W; Fontoura, Beatriz M A

2013-01-01

Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA3, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.

Alternative mRNA polyadenylation in eukaryotes: an effective regulator of gene expression

PubMed Central

Lutz, Carol S.; Moreira, Alexandra

2010-01-01

Alternative RNA processing mechanisms, including alternative splicing and alternative polyadenylation, are increasingly recognized as important regulators of gene expression. This article will focus on what has recently been described about alternative polyadenylation in development, differentiation, and disease in higher eukaryotes. We will also describe how the evolving global methodologies for examining the cellular transcriptome, both experimental and bioinformatic, are revealing new details about the complex nature of alternative 3′ end formation, as well as interactions with other RNA-mediated and RNA processing mechanisms. PMID:21278855

Architecture of eukaryotic mRNA 3′-end processing machinery

PubMed Central

Hill, Chris H.; Easter, Ashley D.; Emsley, Paul; Degliesposti, Gianluca; Gordiyenko, Yuliya; Santhanam, Balaji; Wolf, Jana; Wiederhold, Katrin; Dornan, Gillian L.; Skehel, Mark; Robinson, Carol V.; Passmore, Lori A.

2018-01-01

Newly transcribed eukaryotic precursor messenger RNAs (pre-mRNAs) are processed at their 3′ ends by the ~1-megadalton multiprotein cleavage and polyadenylation factor (CPF). CPF cleaves pre-mRNAs, adds a polyadenylate tail, and triggers transcription termination, but it is unclear how its various enzymes are coordinated and assembled. Here, we show that the nuclease, polymerase, and phosphatase activities of yeast CPF are organized into three modules. Using electron cryomicroscopy, we determined a 3.5-angstrom-resolution structure of the ~200-kilodalton polymerase module. This revealed four β propellers, in an assembly markedly similar to those of other protein complexes that bind nucleic acid. Combined with in vitro reconstitution experiments, our data show that the polymerase module brings together factors required for specific and efficient polyadenylation, to help coordinate mRNA 3′-end processing. PMID:29074584

Regulation of gene expression in mammalian nervous system through alternative pre-mRNA splicing coupled with RNA quality control mechanisms.

PubMed

Yap, Karen; Makeyev, Eugene V

2013-09-01

Eukaryotic gene expression is orchestrated on a genome-wide scale through several post-transcriptional mechanisms. Of these, alternative pre-mRNA splicing expands the proteome diversity and modulates mRNA stability through downstream RNA quality control (QC) pathways including nonsense-mediated decay (NMD) of mRNAs containing premature termination codons and nuclear retention and elimination (NRE) of intron-containing transcripts. Although originally identified as mechanisms for eliminating aberrant transcripts, a growing body of evidence suggests that NMD and NRE coupled with deliberate changes in pre-mRNA splicing patterns are also used in a number of biological contexts for deterministic control of gene expression. Here we review recent studies elucidating molecular mechanisms and biological significance of these gene regulation strategies with a specific focus on their roles in nervous system development and physiology. This article is part of a Special Issue entitled 'RNA and splicing regulation in neurodegeneration'. Copyright © 2013 Elsevier Inc. All rights reserved.

RNA editing in nascent RNA affects pre-mRNA splicing

PubMed Central

Hsiao, Yun-Hua Esther; Bahn, Jae Hoon; Yang, Yun; Lin, Xianzhi; Tran, Stephen; Yang, Ei-Wen; Quinones-Valdez, Giovanni

2018-01-01

In eukaryotes, nascent RNA transcripts undergo an intricate series of RNA processing steps to achieve mRNA maturation. RNA editing and alternative splicing are two major RNA processing steps that can introduce significant modifications to the final gene products. By tackling these processes in isolation, recent studies have enabled substantial progress in understanding their global RNA targets and regulatory pathways. However, the interplay between individual steps of RNA processing, an essential aspect of gene regulation, remains poorly understood. By sequencing the RNA of different subcellular fractions, we examined the timing of adenosine-to-inosine (A-to-I) RNA editing and its impact on alternative splicing. We observed that >95% A-to-I RNA editing events occurred in the chromatin-associated RNA prior to polyadenylation. We report about 500 editing sites in the 3′ acceptor sequences that can alter splicing of the associated exons. These exons are highly conserved during evolution and reside in genes with important cellular function. Furthermore, we identified a second class of exons whose splicing is likely modulated by RNA secondary structures that are recognized by the RNA editing machinery. The genome-wide analyses, supported by experimental validations, revealed remarkable interplay between RNA editing and splicing and expanded the repertoire of functional RNA editing sites. PMID:29724793

RNA editing in nascent RNA affects pre-mRNA splicing.

PubMed

Hsiao, Yun-Hua Esther; Bahn, Jae Hoon; Yang, Yun; Lin, Xianzhi; Tran, Stephen; Yang, Ei-Wen; Quinones-Valdez, Giovanni; Xiao, Xinshu

2018-06-01

In eukaryotes, nascent RNA transcripts undergo an intricate series of RNA processing steps to achieve mRNA maturation. RNA editing and alternative splicing are two major RNA processing steps that can introduce significant modifications to the final gene products. By tackling these processes in isolation, recent studies have enabled substantial progress in understanding their global RNA targets and regulatory pathways. However, the interplay between individual steps of RNA processing, an essential aspect of gene regulation, remains poorly understood. By sequencing the RNA of different subcellular fractions, we examined the timing of adenosine-to-inosine (A-to-I) RNA editing and its impact on alternative splicing. We observed that >95% A-to-I RNA editing events occurred in the chromatin-associated RNA prior to polyadenylation. We report about 500 editing sites in the 3' acceptor sequences that can alter splicing of the associated exons. These exons are highly conserved during evolution and reside in genes with important cellular function. Furthermore, we identified a second class of exons whose splicing is likely modulated by RNA secondary structures that are recognized by the RNA editing machinery. The genome-wide analyses, supported by experimental validations, revealed remarkable interplay between RNA editing and splicing and expanded the repertoire of functional RNA editing sites. © 2018 Hsiao et al.; Published by Cold Spring Harbor Laboratory Press.

Effects of RNAi-Mediated Knockdown of Histone Methyltransferases on the Sex-Specific mRNA Expression of Imp in the Silkworm Bombyx mori

PubMed Central

Suzuki, Masataka G.; Ito, Haruka; Aoki, Fugaku

2014-01-01

Sexual differentiation in Bombyx mori is controlled by sex-specific splicing of Bmdsx, which results in the omission of exons 3 and 4 in a male-specific manner. In B. mori, insulin-like growth factor II mRNA-binding protein (Imp) is a male-specific factor involved in male-specific splicing of Bmdsx. Male-specific Imp mRNA results from the male-specific inclusion of exon 8. To verify the link between histone methylation and alternative RNA processing in Imp, we examined the effects of RNAi-mediated knockdown of several histone methyltransferases on the sex-specific mRNA expression of Imp. As a result, male-specific expression of Imp mRNA was completely abolished when expression of the H3K79 methyltransferase DOT1L was repressed to <10% of that in control males. Chromatin immunoprecipitation-quantitative PCR analysis revealed a higher distribution of H3K79me2 in normal males than in normal females across Imp. RNA polymerase II (RNAP II) processivity assays indicated that RNAi knockdown of DOT1L in males caused a twofold decrease in RNAP II processivity compared to that in control males, with almost equivalent levels to those observed in normal females. Inhibition of RNAP II-mediated elongation in male cells repressed the male-specific splicing of Imp. Our data suggest the possibility that H3K79me2 accumulation along Imp is associated with the male-specific alternative processing of Imp mRNA that results from increased RNAP II processivity. PMID:24758924

Antisense oligonucleotide-induced alternative splicing of the APOB mRNA generates a novel isoform of APOB.

PubMed

Khoo, Bernard; Roca, Xavier; Chew, Shern L; Krainer, Adrian R

2007-01-17

Apolipoprotein B (APOB) is an integral part of the LDL, VLDL, IDL, Lp(a) and chylomicron lipoprotein particles. The APOB pre-mRNA consists of 29 constitutively-spliced exons. APOB exists as two natural isoforms: the full-length APOB100 isoform, assembled into LDL, VLDL, IDL and Lp(a) and secreted by the liver in humans; and the C-terminally truncated APOB48, assembled into chylomicrons and secreted by the intestine in humans. Down-regulation of APOB100 is a potential therapy to lower circulating LDL and cholesterol levels. We investigated the ability of 2'O-methyl RNA antisense oligonucleotides (ASOs) to induce the skipping of exon 27 in endogenous APOB mRNA in HepG2 cells. These ASOs are directed towards the 5' and 3' splice-sites of exon 27, the branch-point sequence (BPS) of intron 26-27 and several predicted exonic splicing enhancers within exon 27. ASOs targeting either the 5' or 3' splice-site, in combination with the BPS, are the most effective. The splicing of other alternatively spliced genes are not influenced by these ASOs, suggesting that the effects seen are not due to non-specific changes in alternative splicing. The skip 27 mRNA is translated into a truncated isoform, APOB87SKIP27. The induction of APOB87SKIP27 expression in vivo should lead to decreased LDL and cholesterol levels, by analogy to patients with hypobetalipoproteinemia. As intestinal APOB mRNA editing and APOB48 expression rely on sequences within exon 26, exon 27 skipping should not affect APOB48 expression unlike other methods of down-regulating APOB100 expression which also down-regulate APOB48.

mRNA Cap Methyltransferase, RNMT-RAM, Promotes RNA Pol II-Dependent Transcription.

PubMed

Varshney, Dhaval; Lombardi, Olivia; Schweikert, Gabriele; Dunn, Sianadh; Suska, Olga; Cowling, Victoria H

2018-05-01

mRNA cap addition occurs early during RNA Pol II-dependent transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

A case of cervical cancer expressed three mRNA variant of Hyaluronan-mediated motility receptor

PubMed Central

Villegas-Ruíz, Vanessa; Salcedo, Mauricio; Zentella-Dehesa, Alejandro; de Oca, Edén V Montes; Román-Basaure, Edgar; Mantilla-Morales, Alejandra; Dávila-Borja, Víctor M; Juárez-Méndez, Sergio

2014-01-01

Cervical cancer is the second malignancy in Mexico, little is known about the prognostic factors associated with this disease. Several cellular components are important in their transformation and progression. Alternative mRNA splice is an important mechanism for generating protein diversity, nevertheless, in cancer unknown mRNA diversity is expressed. Hyaluronan-mediated motility receptor (HMMR, RHAMM, CD168) is a family member of proteins, hyaluronan acid dependent, and has been associated with different malignant processes such as: angiogenesis, cell invasiveness, proliferation, metastasis and poor outcome in some tumors. In the present study we identified expression of HMMR in cervical cancer by means of RT-PCR and sequencing. Our results indicate co-expression of two HMMR variants in all samples, and one case expressed three alternative HMMR splice transcripts. These results showed the heterogeneity of mRNA transcripts of HMMR that could express in cancer and the expression of HMMR could be marker of malignancy in CC. PMID:24966934

The multiple roles of TDP-43 in pre-mRNA processing and gene expression regulation.

PubMed

Buratti, Emanuele; Baralle, Francisco Ernesto

2010-01-01

Heterogeneous ribonucleoproteins (hnRNPs) are multifunctional RNA-binding proteins (RBPs) involved in many cellular processes. They participate in most gene expression pathways, from DNA replication and repair to mRNA translation. Among this class of proteins, TDP-43 (and more recently FUS/TLS) have received considerable attention due to their involvement in several neurodegenerative diseases. This finding has prompted many research groups to focus on the gene expression pathways that are regulated by these proteins. The results have uncovered a considerable complexity of TDP-43 and FUS/TLS functions due to the many independent mechanisms by which they may act to influence various cellular processes (such as DNA transcription, pre-mRNA splicing, mRNA export/import). The aim of this chapter will be to review especially some of the novel functions that have been uncovered, such as role in miRNA synthesis, regulation of transcript levels, and potential autoregulatory mechanisms in order to provide the basis for further investigations.

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 .

THE M-RNA, EXPRESSION OF SERCA2 AND NCX1 IN THE PROCESS OF PHARMACOLOGICAL CELL PROTECTION IN EXPERIMENTAL ACUTE PANCREATITIS INDUCED BY TAUROCHOLATE.

PubMed

Vasques, Enio Rodrigues; Cunha, José Eduardo Monteiro; Kubrusly, Marcia Saldanha; Coelho, Ana Maria; Sanpietri, Sandra N; Nader, Helena B; Tersariol, Ivarne L S; Lima, Marcelo A; Chaib, Eleazar; D'Albuquerque, Luiz Augusto Carneiro

2018-06-21

Intracellular calcium overload is known to be a precipitating factor of pancreatic cell injury in acute pancreatitis (AP). Intracellular calcium homeostasis depends of Plasmatic Membrane Calcium ATPase (PMCA), Sarcoplasmic Endothelial Reticulum Calcium ATPase 2 (SERCA 2) and the Sodium Calcium Exchanger (NCX1). The antioxidant melatonin (Mel) and Trisulfate Disaccharide (TD) that accelerates NCX1 action could reduce the cell damage determined by the AP. To evaluate m-RNA expressions of SERCA2 and NCX1 in acute pancreatitis induced by sodium taurocholate in Wistar rats pre-treated with melatonin and/or TD. Wistar rats were divided in groups: 1) without AP; 2) AP without pre-treatment; 3) AP and Melatonin; 4) AP and TD; 5) AP and Melatonin associated to TD. Pancreatic tissue samples were collected for detection of SERCA2 and NCX1 m-R NA levels by polymerase chain reaction (PCR). Increased m-RNA expression of SERCA2 in the melatonin treated group, without increase of m-RNA expression of the NCX1. The TD did not affect levels of SERCA2 and NCX1 m-RNA expressions. The combined melatonin and TD treatment reduced the m-RNA expression of SERCA2. The effect of melatonin is restricted to increased m-RNA expression of SERCA2. Although TD does not affect gene expression, its action in accelerating calcium exchanger function can explain the slightest expression of SERCA2 m-RNA when associated with Melatonin, perhaps by a joint action of drugs with different and but possibly complementary mechanisms.

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

The Role of CYP3A4 mRNA Transcript with Shortened 3′-Untranslated Region in Hepatocyte Differentiation, Liver Development, and Response to Drug InductionS⃞

PubMed Central

Li, Dan; Gaedigk, Roger; Hart, Steven N.; Leeder, J. Steven

2012-01-01

Cytochrome P450 3A4 (CYP3A4) metabolizes more than 50% of prescribed drugs. The expression of CYP3A4 changes during liver development and may be affected by the administration of some drugs. Alternative mRNA transcripts occur in more than 90% of human genes and are frequently observed in cells responding to developmental and environmental signals. Different mRNA transcripts may encode functionally distinct proteins or contribute to variability of mRNA stability or protein translation efficiency. The purpose of this study was to examine expression of alternative CYP3A4 mRNA transcripts in hepatocytes in response to developmental signals and drugs. cDNA cloning and RNA sequencing (RNA-Seq) were used to identify CYP3A4 mRNA transcripts. Three transcripts were found in HepaRG cells and liver tissues: one represented a canonical mRNA with full-length 3′-untranslated region (UTR), one had a shorter 3′-UTR, and one contained partial intron-6 retention. The alternative mRNA transcripts were validated by either rapid amplification of cDNA 3′-end or endpoint polymerase chain reaction (PCR). Quantification of the transcripts by RNA-Seq and real time quantitative PCR revealed that the CYP3A4 transcript with shorter 3′-UTR was preferentially expressed in developed livers, differentiated hepatocytes, and in rifampicin- and phenobarbital-induced hepatocytes. The CYP3A4 transcript with shorter 3′-UTR was more stable and produced more protein compared with the CYP3A4 transcript with canonical 3′-UTR. We conclude that the 3′-end processing of CYP3A4 contributes to the quantitative regulation of CYP3A4 gene expression through alternative polyadenylation, which may serve as a regulatory mechanism explaining changes of CYP3A4 expression and activity during hepatocyte differentiation and liver development and in response to drug induction. PMID:21998292

Notch and Delta mRNAs in early-stage and mid-stage Drosophila embryos exhibit complementary patterns of protein producing potentials

PubMed Central

Shepherd, Andrew; Wesley, Uma; Wesley, Cedric

2010-01-01

Notch and Delta proteins generate Notch signaling that specifies cell fates during animal development. There is an intriguing phenomenon in Drosophila embryogenesis that has not received much attention and whose significance to embryogenesis is unknown. Notch and Delta mRNAs expressed in early-stage embryos are shorter than their counterparts in mid-stage embryos. We show here that the difference in sizes is due to mRNA 3′ processing at alternate polyadenylation sites. While the early-stage Notch mRNA has a lower protein-producing potential than the mid-stage Notch mRNA, the early-stage Delta mRNA has a higher protein-producing potential than the mid-stage Delta mRNA. Our data can explain the complementary patterns of Notch and Delta protein levels in early-stage and mid-stage embryos. Our data also raise the possibility that the manner and regulation of Notch signaling change in the course of embryogenesis and that this change is effected by 3′ UTR and mRNA 3′ processing factors. PMID:20201103

A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18

PubMed Central

Chi, Binkai; Wang, Ke; Du, Yanhua; Gui, Bin; Chang, Xingya; Wang, Lantian; Fan, Jing; Chen, She; Wu, Xudong; Li, Guohui; Cheng, Hong

2014-01-01

Viral RNA elements that facilitate mRNA export are useful tools for identifying cellular RNA export factors. Here we show that hepatitis B virus post-transcriptional element (PRE) is one such element, and using PRE several new cellular mRNA export factors were identified. We found that PRE drastically enhances the cytoplasmic accumulation of cDNA transcripts independent of any viral protein. Systematic deletion analysis revealed the existence of a 116 nt functional Sub-Element of PRE (SEP1). The RNP that forms on the SEP1 RNA was affinity purified, in which TREX components as well as several other proteins were identified. TREX components and the SEP1-associating protein ZC3H18 are required for SEP1-mediated mRNA export. Significantly, ZC3H18 directly binds to the SEP1 RNA, interacts with TREX and is required for stable association of TREX with the SEP1-containing mRNA. Requirements for SEP1-mediated mRNA export are similar to those for splicing-dependent mRNA export. Consistent with these similarities, several SEP1-interacting proteins, including ZC3H18, ARS2, Acinus and Brr2, are required for efficient nuclear export of polyA RNAs. Together, our data indicate that SEP1 enhances mRNA export by recruiting TREX via ZC3H18. The new mRNA export factors that we identified might be involved in cap- and splicing-dependent TREX recruitment to cellular mRNAs. PMID:24782531

Melanoma Differentiation-associated Gene 7/IL-24 Exerts Cytotoxic Effects by Altering the Alternative Splicing of Bcl-x Pre-mRNA via the SRC/PKCδ Signaling Axis*

PubMed Central

Shapiro, Brian A.; Vu, Ngoc T.; Shultz, Michael D.; Shultz, Jacqueline C.; Mietla, Jennifer A.; Gouda, Mazen M.; Yacoub, Adly; Dent, Paul; Fisher, Paul B.; Park, Margaret A.; Chalfant, Charles E.

2016-01-01

Melanoma differentiation-associated gene 7 (MDA-7/IL-24) exhibits cytotoxic effects on tumor cells while sparing untransformed cells, and Bcl-x(L) is reported to efficiently block the induction of cell death by MDA-7/IL-24. The expression of Bcl-x(L) is regulated at the level of RNA splicing via alternative 5′ splice site selection within exon 2 to produce either the pro-apoptotic Bcl-x(s) or the anti-apoptotic Bcl-x(L). Our laboratory previously reported that Bcl-x RNA splicing is dysregulated in a large percentage of human non-small cell lung cancer (NSCLC) tumors. Therefore, we investigated whether the alternative RNA splicing of Bcl-x pre-mRNA was modulated by MDA-7/IL-24, which would suggest that specific NSCLC tumors are valid targets for this cytokine therapy. Adenovirus-delivered MDA-7/IL-24 (Ad.mda-7) reduced the viability of NSCLC cells of varying oncogenotypes, which was preceded by a decrease in the ratio of Bcl-x(L)/Bcl-x(s) mRNA and Bcl-x(L) protein expression. Importantly, both the expression of Bcl-x(L) and the loss of cell viability were “rescued” in Ad.mda-7-treated cells incubated with Bcl-x(s) siRNA. In addition, NSCLC cells ectopically expressing Bcl-x(s) exhibited significantly reduced Bcl-x(L) expression, which was again restored by Bcl-x(s) siRNA, suggesting the existence of a novel mechanism by which Bcl-x(s) mRNA restrains the expression of Bcl-x(L). In additional mechanistic studies, inhibition of SRC and PKCδ completely ablated the ability of MDA-7/IL-24 to reduce the Bcl-x(L)/(s) mRNA ratio and cell viability. These findings show that Bcl-x(s) expression is an important mediator of MDA-7/IL-24-induced cytotoxicity requiring the SRC/PKCδ signaling axis in NSCLC cells. PMID:27519412

Interrelations between translation and general mRNA degradation in yeast

PubMed Central

Huch, Susanne; Nissan, Tracy

2014-01-01

Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system. How to cite this article: WIREs RNA 2014, 5:747–763. doi: 10.1002/wrna.1244 PMID:24944158

Interrelations between translation and general mRNA degradation in yeast.

PubMed

Huch, Susanne; Nissan, Tracy

2014-01-01

Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system. © 2014 The Authors. WIREs RNA published by John Wiley & Sons, Ltd.

CPSF30 at the Interface of Alternative Polyadenylation and Cellular Signaling in Plants

PubMed Central

Chakrabarti, Manohar; Hunt, Arthur G.

2015-01-01

Post-transcriptional processing, involving cleavage of precursor messenger RNA (pre mRNA), and further incorporation of poly(A) tail to the 3' end is a key step in the expression of genetic information. Alternative polyadenylation (APA) serves as an important check point for the regulation of gene expression. Recent studies have shown widespread prevalence of APA in diverse systems. A considerable amount of research has been done in characterizing different subunits of so-called Cleavage and Polyadenylation Specificity Factor (CPSF). In plants, CPSF30, an ortholog of the 30 kD subunit of mammalian CPSF is a key polyadenylation factor. CPSF30 in the model plant Arabidopsis thaliana was reported to possess unique biochemical properties. It was also demonstrated that poly(A) site choice in a vast majority of genes in Arabidopsis are CPSF30 dependent, suggesting a pivotal role of this gene in APA and subsequent regulation of gene expression. There are also indications of this gene being involved in oxidative stress and defense responses and in cellular signaling, suggesting a role of CPSF30 in connecting physiological processes and APA. This review will summarize the biochemical features of CPSF30, its role in regulating APA, and possible links with cellular signaling and stress response modules. PMID:26061761

Transcriptome analyses reveal SR45 to be a neutral splicing regulator and a suppressor of innate immunity in Arabidopsis thaliana.

PubMed

Zhang, Xiao-Ning; Shi, Yifei; Powers, Jordan J; Gowda, Nikhil B; Zhang, Chong; Ibrahim, Heba M M; Ball, Hannah B; Chen, Samuel L; Lu, Hua; Mount, Stephen M

2017-10-11

Regulation of pre-mRNA splicing diversifies protein products and affects many biological processes. Arabidopsis thaliana Serine/Arginine-rich 45 (SR45), regulates pre-mRNA splicing by interacting with other regulatory proteins and spliceosomal subunits. Although SR45 has orthologs in diverse eukaryotes, including human RNPS1, the sr45-1 null mutant is viable. Narrow flower petals and reduced seed formation suggest that SR45 regulates genes involved in diverse processes, including reproduction. To understand how SR45 is involved in the regulation of reproductive processes, we studied mRNA from the wild-type and sr45-1 inflorescences using RNA-seq, and identified SR45-bound RNAs by immunoprecipitation. Using a variety of bioinformatics tools, we identified a total of 358 SR45 differentially regulated (SDR) genes, 542 SR45-dependent alternative splicing (SAS) events, and 1812 SR45-associated RNAs (SARs). There is little overlap between SDR genes and SAS genes, and neither set of genes is enriched for flower or seed development. However, transcripts from reproductive process genes are significantly overrepresented in SARs. In exploring the fate of SARs, we found that a total of 81 SARs are subject to alternative splicing, while 14 of them are known Nonsense-Mediated Decay (NMD) targets. Motifs related to GGNGG are enriched both in SARs and near different types of SAS events, suggesting that SR45 recognizes this motif directly. Genes involved in plant defense are significantly over-represented among genes whose expression is suppressed by SR45, and sr45-1 plants do indeed show enhanced immunity. We find that SR45 is a suppressor of innate immunity. We find that a single motif (GGNGG) is highly enriched in both RNAs bound by SR45 and in sequences near SR45- dependent alternative splicing events in inflorescence tissue. We find that the alternative splicing events regulated by SR45 are enriched for this motif whether the effect of SR45 is activation or repression of the particular event. Thus, our data suggests that SR45 acts to control splice site choice in a way that defies simple categorization as an activator or repressor of splicing.

Alternative polyadenylation of tumor suppressor genes in small intestinal neuroendocrine tumors.

PubMed

Rehfeld, Anders; Plass, Mireya; Døssing, Kristina; Knigge, Ulrich; Kjær, Andreas; Krogh, Anders; Friis-Hansen, Lennart

2014-01-01

The tumorigenesis of small intestinal neuroendocrine tumors (SI-NETs) is poorly understood. Recent studies have associated alternative polyadenylation (APA) with proliferation, cell transformation, and cancer. Polyadenylation is the process in which the pre-messenger RNA is cleaved at a polyA site and a polyA tail is added. Genes with two or more polyA sites can undergo APA. This produces two or more distinct mRNA isoforms with different 3' untranslated regions. Additionally, APA can also produce mRNAs containing different 3'-terminal coding regions. Therefore, APA alters both the repertoire and the expression level of proteins. Here, we used high-throughput sequencing data to map polyA sites and characterize polyadenylation genome-wide in three SI-NETs and a reference sample. In the tumors, 16 genes showed significant changes of APA pattern, which lead to either the 3' truncation of mRNA coding regions or 3' untranslated regions. Among these, 11 genes had been previously associated with cancer, with 4 genes being known tumor suppressors: DCC, PDZD2, MAGI1, and DACT2. We validated the APA in three out of three cases with quantitative real-time-PCR. Our findings suggest that changes of APA pattern in these 16 genes could be involved in the tumorigenesis of SI-NETs. Furthermore, they also point to APA as a new target for both diagnostic and treatment of SI-NETs. The identified genes with APA specific to the SI-NETs could be further tested as diagnostic markers and drug targets for disease prevention and treatment.

Alternative Polyadenylation of Tumor Suppressor Genes in Small Intestinal Neuroendocrine Tumors

PubMed Central

Rehfeld, Anders; Plass, Mireya; Døssing, Kristina; Knigge, Ulrich; Kjær, Andreas; Krogh, Anders; Friis-Hansen, Lennart

2014-01-01

The tumorigenesis of small intestinal neuroendocrine tumors (SI-NETs) is poorly understood. Recent studies have associated alternative polyadenylation (APA) with proliferation, cell transformation, and cancer. Polyadenylation is the process in which the pre-messenger RNA is cleaved at a polyA site and a polyA tail is added. Genes with two or more polyA sites can undergo APA. This produces two or more distinct mRNA isoforms with different 3′ untranslated regions. Additionally, APA can also produce mRNAs containing different 3′-terminal coding regions. Therefore, APA alters both the repertoire and the expression level of proteins. Here, we used high-throughput sequencing data to map polyA sites and characterize polyadenylation genome-wide in three SI-NETs and a reference sample. In the tumors, 16 genes showed significant changes of APA pattern, which lead to either the 3′ truncation of mRNA coding regions or 3′ untranslated regions. Among these, 11 genes had been previously associated with cancer, with 4 genes being known tumor suppressors: DCC, PDZD2, MAGI1, and DACT2. We validated the APA in three out of three cases with quantitative real-time-PCR. Our findings suggest that changes of APA pattern in these 16 genes could be involved in the tumorigenesis of SI-NETs. Furthermore, they also point to APA as a new target for both diagnostic and treatment of SI-NETs. The identified genes with APA specific to the SI-NETs could be further tested as diagnostic markers and drug targets for disease prevention and treatment. PMID:24782827

The role of alternative splicing coupled to nonsense-mediated mRNA decay in human disease.

PubMed

da Costa, Paulo J; Menezes, Juliane; Romão, Luísa

2017-10-01

Alternative pre-mRNA splicing (AS) affects gene expression as it generates proteome diversity. Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature translation-termination codons (PTCs), preventing the production of truncated proteins that could result in disease. Several studies have also implicated NMD in the regulation of steady-state levels of physiological mRNAs. In addition, it is known that several regulated AS events do not lead to generation of protein products, as they lead to transcripts that carry PTCs and thus, they are committed to NMD. Indeed, an estimated one-third of naturally occurring, alternatively spliced mRNAs is targeted for NMD, being AS coupled to NMD (AS-NMD) an efficient strategy to regulate gene expression. In this review, we will focus on how AS mechanism operates and how can be coupled to NMD to fine-tune gene expression levels. Furthermore, we will demonstrate the physiological significance of the interplay among AS and NMD in human disease, such as cancer and neurological disorders. The understanding of how AS-NMD orchestrates expression of vital genes is of utmost importance for the advance in diagnosis, prognosis and treatment of many human disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Alvarez, Enrique, E-mail: ealvarez@cbm.uam.es; Castello, Alfredo; Carrasco, Luis

Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of proteasemore » fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.« less

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

PubMed

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

2011-03-01

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

Control of calcitonin/calcitonin gene-related peptide pre-mRNA processing by constitutive intron and exon elements.

PubMed Central

Yeakley, J M; Hedjran, F; Morfin, J P; Merillat, N; Rosenfeld, M G; Emeson, R B

1993-01-01

The calcitonin/calcitonin gene-related peptide (CGRP) primary transcript is alternatively spliced in thyroid C cells and neurons, resulting in the tissue-specific production of calcitonin and CGRP mRNAs. Analyses of mutated calcitonin/CGRP transcription units in permanently transfected cell lines have indicated that alternative splicing is regulated by a differential capacity to utilize the calcitonin-specific splice acceptor. The analysis of an extensive series of mutations suggests that tissue-specific regulation of calcitonin mRNA production does not depend on the presence of a single, unique cis-active element but instead appears to be a consequence of suboptimal constitutive splicing signals. While only those mutations that altered constitutive splicing signals affected splice choices, the action of multiple regulatory sequences cannot be formally excluded. Further, we have identified a 13-nucleotide purine-rich element from a constitutive exon that, when placed in exon 4, entirely switches splice site usage in CGRP-producing cells. These data suggest that specific exon recruitment sequences, in combination with other constitutive elements, serve an important function in exon recognition. These results are consistent with the hypothesis that tissue-specific alternative splicing of the calcitonin/CGRP primary transcript is mediated by cell-specific differences in components of the constitutive splicing machinery. Images PMID:8413203

RNA therapeutics: Beyond RNA interference and antisense oligonucleotides

PubMed Central

Kole, Ryszard; Krainer, Adrian R.; Altman, Sidney

2016-01-01

Here we discuss three RNA therapeutic technologies exploiting various oligonucleotides that bind RNA by base-pairing in a sequence-specific manner yet have different mechanisms of action and effects. RNA interference and antisense oligonucleotides downregulate gene expression by enzyme-dependent degradation of targeted mRNA. Steric blocking oligonucleotides block access of cellular machinery to pre-mRNA and mRNA without degrading the RNA. Through this mechanism, blocking oligonucleotides can redirect alternative splicing, repair defective RNA, restore protein production or also downregulate gene expression. Moreover, they can be extensively chemically modified, resulting in more drug-like properties. The ability of RNA blocking oligonucleotides to restore gene function makes them suited for treatment of genetic disorders. Positive results from clinical trials for the treatment of Duchenne muscular dystrophy show that this technology is close to realizing its clinical potential. PMID:22262036

Crystal Structure of the HEAT Domain from the Pre-mRNA Processing Factor Symplekin

PubMed Central

Kennedy, Sarah A.; Frazier, Monica L.; Steiniger, Mindy; Mast, Ann M.; Marzluff, William F.; Redinbo, Matthew R.

2009-01-01

The majority of eukaryotic pre-mRNAs are processed by 3′-end cleavage and polyadenylation, although in metazoa the replication-dependant histone mRNAs are processed by 3′-end cleavage but not polyadenylation. The macromolecular complex responsible for processing both canonical and histone pre-mRNAs contains the ~1,160-residue protein Symplekin. Secondary structural prediction algorithms identified putative HEAT domains in the 300 N-terminal residues of all Symplekins of known sequence. The structure and dynamics of this domain were investigated to begin elucidating the role Symplekin plays in mRNA maturation. The crystal structure of the Drosophila melanogaster Symplekin HEAT domain was determined to 2.4 Å resolution using SAD phasing methods. The structure exhibits 5 canonical HEAT repeats along with an extended 31 amino acid loop (loop 8) between the fourth and fifth repeat that is conserved within closely related Symplekin sequences. Molecular dynamics simulations of this domain show that the presence of loop 8 dampens correlated and anticorrelated motion in the HEAT domain, therefore providing a neutral surface for potential protein-protein interactions. HEAT domains are often employed for such macromolecular contacts. The Symplekin HEAT region not only structurally aligns with several established scaffolding proteins, but also has been reported to contact proteins essential for regulating 3′-end processing. Taken together, these data support the conclusion that the Symplekin HEAT domain serves as a scaffold for protein-protein interactions essential to the mRNA maturation process. PMID:19576221

Increased alternate splicing of Htr2c in a mouse model for Prader-Willi syndrome leads disruption of 5HT2C receptor mediated appetite.

PubMed

Garfield, Alastair S; Davies, Jennifer R; Burke, Luke K; Furby, Hannah V; Wilkinson, Lawrence S; Heisler, Lora K; Isles, Anthony R

2016-12-08

Alternate splicing of serotonin (5-hydroxytryptamine; 5-HT) 2C receptor (5-HT 2C R) pre-RNA is negatively regulated by the small nucleolar RNA, Snord115, loss of which is observed in nearly all individuals with Prader-Willi Syndrome (PWS), a multigenic disorder characterised by hyperphagia and obesity. Given the role of the 5-HT 2C R in the regulation of ingestive behaviour we investigated the pathophysiological implications of Snord115 deficiency on 5-HT 2C R regulated appetite in a genotypically relevant PWS mouse model (PWS-IC). Specifically, we demonstrate that loss of Snord115 expression is associated with increased levels of hypothalamic truncated 5-HT 2C R pre-mRNA. The 5-HT 2C R promotes appetite suppression via engagement of the central melanocortin system. Pro-opiomelancortin (Pomc) mRNA levels within the arcuate nucleus of the hypothalamus (ARC) were reduced in PWS-IC mice. We then went on to assess the functional consequences of these molecular changes, demonstrating that PWS-IC mice are unresponsive to an anorectic doses of a 5-HT 2C R agonist and that this is associated with attenuated activation of POMC neurons within the ARC. These data provide new insight into the significance of Htr2c pre-mRNA processing to the physiological regulation of appetite and potentially the pathological manifestation of hyperphagia in PWS. Furthermore, these findings have translational relevance for individuals with PWS who may seek to control appetite with another 5-HT 2C R agonist, the new obesity treatment lorcaserin.

Identification of human short introns

PubMed Central

Abebrese, Emmanuel L.; Arnold, Zachary R.; Armstrong, Katharine; Burns, Lindsay; Day, R. Thomas; Hsu, Daniel G.; Jarrell, Katherine; Luo, Yi; Mugayo, Daphine

2017-01-01

Canonical pre-mRNA splicing requires snRNPs and associated splicing factors to excise conserved intronic sequences, with a minimum intron length required for efficient splicing. Non-canonical splicing–intron excision without the spliceosome–has been documented; most notably, some tRNAs and the XBP1 mRNA contain short introns that are not removed by the spliceosome. There have been some efforts to identify additional short introns, but little is known about how many short introns are processed from mRNAs. Here, we report an approach to identify RNA short introns from RNA-Seq data, discriminating against small genomic deletions. We identify hundreds of short introns conserved among multiple human cell lines. These short introns are often alternatively spliced and are found in a variety of RNAs–both mRNAs and lncRNAs. Short intron splicing efficiency is increased by secondary structure, and we detect both canonical and non-canonical short introns. In many cases, splicing of these short introns from mRNAs is predicted to alter the reading frame and change protein output. Our findings imply that standard gene prediction models which often assume a lower limit for intron size fail to predict short introns effectively. We conclude that short introns are abundant in the human transcriptome, and short intron splicing represents an added layer to mRNA regulation. PMID:28520720

The Polyadenosine RNA-binding Protein, Zinc Finger Cys3His Protein 14 (ZC3H14), Regulates the Pre-mRNA Processing of a Key ATP Synthase Subunit mRNA*

PubMed Central

Wigington, Callie P.; Morris, Kevin J.; Newman, Laura E.; Corbett, Anita H.

2016-01-01

Polyadenosine RNA-binding proteins (Pabs) regulate multiple steps in gene expression. This protein family includes the well studied Pabs, PABPN1 and PABPC1, as well as the newly characterized Pab, zinc finger CCCH-type containing protein 14 (ZC3H14). Mutations in ZC3H14 are linked to a form of intellectual disability. To probe the function of ZC3H14, we performed a transcriptome-wide analysis of cells depleted of either ZC3H14 or the control Pab, PABPN1. Depletion of PABPN1 affected ∼17% of expressed transcripts, whereas ZC3H14 affected only ∼1% of expressed transcripts. To assess the function of ZC3H14 in modulating target mRNAs, we selected the gene encoding the ATP synthase F0 subunit C (ATP5G1) transcript. Knockdown of ZC3H14 significantly reduced ATP5G1 steady-state mRNA levels. Consistent with results suggesting that ATP5G1 turnover increases upon depletion of ZC3H14, double knockdown of ZC3H14 and the nonsense-mediated decay factor, UPF1, rescues ATP5G1 transcript levels. Furthermore, fractionation reveals an increase in the amount of ATP5G1 pre-mRNA that reaches the cytoplasm when ZC3H14 is depleted and that ZC3H14 binds to ATP5G1 pre-mRNA in the nucleus. These data support a role for ZC3H14 in ensuring proper nuclear processing and retention of ATP5G1 pre-mRNA. Consistent with the observation that ATP5G1 is a rate-limiting component for ATP synthase activity, knockdown of ZC3H14 decreases cellular ATP levels and causes mitochondrial fragmentation. These data suggest that ZC3H14 modulates pre-mRNA processing of select mRNA transcripts and plays a critical role in regulating cellular energy levels, observations that have broad implications for proper neuronal function. PMID:27563065

Inflammatory airway responses by nasal inoculation of suspended particulate matter in NC/Nga mice.

PubMed

Ogino, Keiki; Takahashi, Noriko; Kubo, Masayuki; Takeuchi, Akihito; Nakagiri, Motoharu; Fujikura, Yoshihisa

2014-06-01

To evaluate the allergic effect of airborne particulate matter (PM) on the airway, separated soluble supernatant (Sup) and insoluble precipitate (Pre) in suspended PM were inoculated into NC/Nga mice with a high sensitivity for mite allergens. Sup, Pre, or both Sup and Pre with or without pronase treatment were inoculated via the nasal route five times for sensitization and a challenge inoculation on the 11th day in NC/Nga mice. On the 14th day, mice were examined for airway hyperresponsiveness (AHR), bronchoalveolar lavage fluid (BALF) cell count, mRNA expression of Th1 and Th2 cytokines in the lung tissue, and histopathology. Synergistic effects of Sup and Pre were observed as increases in AHR and a histopathological change of Periodic acid-Schiff (PAS) staining. Increases in neutrophils, macrophages, and lymphocytes of BALF cells were dependent on Pre. The expression of IL-4 mRNA was increased by Sup, and those of IL-5 mRNA and Il-13 mRNA was increased by Sup and Pre. Augmented AHR, mRNA expression of IL-4, peribronchial inflammation, and PAS staining by Sup plus Pre were attenuated by treatment of Sup with pronase to digest proteins. These results suggest that some proteins of ambient PM may be important environmental factors for AHR and airway inflammation with the aid of insoluble particulates, although some soluble factors such as endotoxins cannot be ruled out. Copyright © 2012 Wiley Periodicals, Inc.

[Alternative splicing regulation: implications in cancer diagnosis and treatment].

PubMed

Martínez-Montiel, Nancy; Rosas-Murrieta, Nora; Martínez-Contreras, Rebeca

2015-04-08

The accurate expression of the genetic information is regulated by processes like mRNA splicing, proposed after the discoveries of Phil Sharp and Richard Roberts, who demonstrated the existence of intronic sequences, present in almost every structural eukaryotic gene, which should be precisely removed. This intron removal is called "splicing", which generates different proteins from a single mRNA, with different or even antagonistic functions. We currently know that alternative splicing is the most important source of protein diversity, given that 70% of the human genes undergo splicing and that mutations causing defects in this process could originate up to 50% of genetic diseases, including cancer. When these defects occur in genes involved in cell adhesion, proliferation and cell cycle regulation, there is an impact on cancer progression, rising the opportunity to diagnose and treat some types of cancer according to a particular splicing profile. Copyright © 2013 Elsevier España, S.L.U. All rights reserved.

The expression analysis of Sfrs10 and Celf4 during mouse retinal development

PubMed Central

Karunakaran, Devi Krishna Priya; Congdon, Sean; Guerrette, Thomas; Banday, Abdul Rouf; Lemoine, Christopher; Chhaya, Nisarg; Kanadia, Rahul

2013-01-01

Processing of mRNAs including, alternative splicing (AS), mRNA transport and translation regulation are crucial to eukaryotic gene expression. For example, >90% of the gene in the human genome are known to undergo alternative splicing thereby expanding the proteome production capacity of a limited number of genes. Similarly, mRNA export and translation regulation plays a vital role in regulating protein production. Thus, it is important to understand how these RNA binding proteins including alternative splicing factors (ASFs) and mRNA transport and translation factors regulate these processes. Here we report the expression of an ASF, Serine-arginine rich splicing factor 10 (Sfrs10) and a mRNA translation regulation factor, CUGBP, elav like family member 4 (Celf4) in the developing mouse retina. Sfrs10 was expressed throughout postnatal (P) retinal development and was observed progressively in newly differentiating neurons. Immunofluorescence (IF) showed Sfrs10 in retinal ganglion cells (RGCs) at P0, followed by amacrine and bipolar cells, and at P8 it was enriched in red/green cone photoreceptor cells. By P22, Sfrs10 was observed in rod photoreceptors in a peri-nuclear pattern. Like Sfrs10, Celf4 was also observed in the developing retina, but with two distinct retinal isoforms. In situ hybridization (ISH) showed progressive expression of Celf4 in differentiating neurons, which was confirmed by IF that showed a dynamic shift in Celf4 localization. Early in development Celf4 expression was restricted to the nuclei of newly differentiating RGCs and later (E16 onwards) it was observed in the initial segments of RGC axons. Later, during postnatal development, Celf4 was observed in amacrine and bipolar cells, but here it was predominantly cytoplasmic and enriched in the two synaptic layers. Specifically, at P14, Celf4 was observed in the synaptic boutons of rod bipolar cells marked by Pkc-α. Thus, Celf4 might be regulating AS early in development besides its known role of regulating mRNA localization/translation. In all, our data suggests an important role for AS and mRNA localization/translation in retinal neuron differentiation. PMID:23932931

Epigenetic control of alternative mRNA processing at the imprinted Herc3/Nap1l5 locus

PubMed Central

Cowley, Michael; Wood, Andrew J.; Böhm, Sabrina; Schulz, Reiner; Oakey, Rebecca J.

2012-01-01

Alternative polyadenylation increases transcriptome diversity by generating multiple transcript isoforms from a single gene. It is thought that this process can be subject to epigenetic regulation, but few specific examples of this have been reported. We previously showed that the Mcts2/H13 locus is subject to genomic imprinting and that alternative polyadenylation of H13 transcripts occurs in an allele-specific manner, regulated by epigenetic mechanisms. Here, we demonstrate that allele-specific polyadenylation occurs at another imprinted locus with similar features. Nap1l5 is a retrogene expressed from the paternally inherited allele, is situated within an intron of a ‘host’ gene Herc3, and overlaps a CpG island that is differentially methylated between the parental alleles. In mouse brain, internal Herc3 polyadenylation sites upstream of Nap1l5 are used on the paternally derived chromosome, from which Nap1l5 is expressed, whereas a downstream site is used more frequently on the maternally derived chromosome. Ablating DNA methylation on the maternal allele at the Nap1l5 promoter increases the use of an internal Herc3 polyadenylation site and alters exon splicing. These changes demonstrate the influence of epigenetic mechanisms in regulating Herc3 alternative mRNA processing. Internal Herc3 polyadenylation correlates with expression levels of Nap1l5, suggesting a possible role for transcriptional interference. Similar mechanisms may regulate alternative polyadenylation elsewhere in the genome. PMID:22790983

Sequencing of mRNA identifies re-expression of fetal splice variants in cardiac hypertrophy

PubMed Central

Ames, EG; Lawson, MJ; Mackey, AJ; Holmes, JW

2013-01-01

Cardiac hypertrophy has been well-characterized at the level of transcription. During cardiac hypertrophy, genes normally expressed primarily during fetal heart development are reexpressed, and this fetal gene program is believed to be a critical component of the hypertrophic process. Recently, alternative splicing of mRNA transcripts has been shown to be temporally regulated during heart development, leading us to consider whether fetal patterns of splicing also reappear during hypertrophy. We hypothesized that patterns of alternative splicing occurring during heart development are recapitulated during cardiac hypertrophy. Here we present a study of isoform expression during pressure-overload cardiac hypertrophy induced by 10 days of transverse aortic constriction (TAC) in rats and in developing fetal rat hearts compared to sham-operated adult rat hearts, using high-throughput sequencing of poly(A) tail mRNA. We find a striking degree of overlap between the isoforms expressed differentially in fetal and pressure-overloaded hearts compared to control: forty-four percent of the isoforms with significantly altered expression in TAC hearts are also expressed at significantly different levels in fetal hearts compared to control (P < 0.001). The isoforms that are shared between hypertrophy and fetal heart development are significantly enriched for genes involved in cytoskeletal organization, RNA processing, developmental processes, and metabolic enzymes. Our data strongly support the concept that mRNA splicing patterns normally associated with heart development recur as part of the hypertrophic response to pressure overload. These findings suggest that cardiac hypertrophy shares post-transcriptional as well as transcriptional regulatory mechanisms with fetal heart development. PMID:23688780

Multilayer regulatory mechanisms control cleavage factor I proteins in filamentous fungi

PubMed Central

Rodríguez-Romero, J.; Franceschetti, M.; Bueno, E.; Sesma, A.

2015-01-01

Cleavage factor I (CFI) proteins are core components of the polyadenylation machinery that can regulate several steps of mRNA life cycle, including alternative polyadenylation, splicing, export and decay. Here, we describe the regulatory mechanisms that control two fungal CFI protein classes in Magnaporthe oryzae: Rbp35/CfI25 complex and Hrp1. Using mutational, genetic and biochemical studies we demonstrate that cellular concentration of CFI mRNAs is a limited indicator of their protein abundance. Our results suggest that several post-transcriptional mechanisms regulate Rbp35/CfI25 complex and Hrp1 in the rice blast fungus, some of which are also conserved in other ascomycetes. With respect to Rbp35, these include C-terminal processing, RGG-dependent localization and cleavage, C-terminal autoregulatory domain and regulation by an upstream open reading frame of Rbp35-dependent TOR signalling pathway. Our proteomic analyses suggest that Rbp35 regulates the levels of proteins involved in melanin and phenylpropanoids synthesis, among others. The drastic reduction of fungal CFI proteins in carbon-starved cells suggests that the pre-mRNA processing pathway is altered. Our findings uncover broad and multilayer regulatory mechanisms controlling fungal polyadenylation factors, which have profound implications in pre-mRNA maturation. This area of research offers new avenues for fungicide design by targeting fungal-specific proteins that globally affect thousands of mRNAs. PMID:25514925

An RNA electrophoretic mobility shift and mutational analysis of rnp-4f 5′-UTR intron splicing regulatory proteins in Drosophila reveals a novel new role for a dADAR protein isoform

PubMed Central

Lakshmi, G. Girija; Ghosh, Sushmita; Jones, Gabriel P.; Parikh, Roshni; Rawlins, Bridgette A.; Vaughn, Jack C.

2014-01-01

Alternative splicing greatly enhances the diversity of proteins encoded by eukaryotic genomes, and is also important in gene expression control. In contrast to the great depth of knowledge as to molecular mechanisms in the splicing pathway itself, relatively little is known about the regulatory events behind this process. The 5′-UTR and 3′-UTR in pre-mRNAs play a variety of roles in controlling eukaryotic gene expression, including translational modulation, and nearly 4,000 of the roughly 14,000 protein coding genes in Drosophila contain introns of unknown functional significance in their 5′-UTR. Here we report the results of an RNA electrophoretic mobility shift analysis of Drosophila rnp-4f 5′-UTR intron 0 splicing regulatory proteins. The pre-mRNA potential regulatory element consists of an evolutionarily-conserved 177-nt stem-loop arising from pairing of intron 0 with part of adjacent exon 2. Incubation of in vitro transcribed probe with embryo protein extract is shown to result in two shifted RNA-protein bands, and protein extract from a dADAR null mutant fly line results in only one shifted band. A mutated stem-loop in which the conserved exon 2 primary sequence is changed but secondary structure maintained by introducing compensatory base changes results in diminished band shifts. To test the hypothesis that dADAR plays a role in intron splicing regulation in vivo, levels of unspliced rnp-4f mRNA in dADAR mutant were compared to wild-type via real-time qRT-PCR. The results show that during embryogenesis unspliced rnp-4f mRNA levels fall by up to 85% in the mutant, in support of the hypothesis. Taken together, these results demonstrate a novel role for dADAR protein in rnp-4f 5′-UTR alternative intron splicing regulation which is consistent with a previously proposed model. PMID:23026215

Influence of Pre-Training Predator Stress on the Expression of c-fos mRNA in the Hippocampus, Amygdala, and Striatum Following Long-Term Spatial Memory Retrieval

PubMed Central

VanElzakker, Michael B.; Zoladz, Phillip R.; Thompson, Vanessa M.; Park, Collin R.; Halonen, Joshua D.; Spencer, Robert L.; Diamond, David M.

2011-01-01

We have studied the influence of pre-training psychological stress on the expression of c-fos mRNA following long-term spatial memory retrieval. Rats were trained to learn the location of a hidden escape platform in the radial-arm water maze, and then their memory for the platform location was assessed 24 h later. Rat brains were extracted 30 min after the 24-h memory test trial for analysis of c-fos mRNA. Four groups were tested: (1) Rats given standard training (Standard); (2) Rats given cat exposure (Predator Stress) 30 min prior to training (Pre-Training Stress); (3) Rats given water exposure only (Water Yoked); and (4) Rats given no water exposure (Home Cage). The Standard trained group exhibited excellent 24 h memory which was accompanied by increased c-fos mRNA in the dorsal hippocampus and basolateral amygdala (BLA). The Water Yoked group exhibited no increase in c-fos mRNA in any brain region. Rats in the Pre-Training Stress group were classified into two subgroups: good and bad memory performers. Neither of the two Pre-Training Stress subgroups exhibited a significant change in c-fos mRNA expression in the dorsal hippocampus or BLA. Instead, stressed rats with good memory exhibited significantly greater c-fos mRNA expression in the dorsolateral striatum (DLS) compared to stressed rats with bad memory. This finding suggests that stressed rats with good memory used their DLS to generate a non-spatial (cue-based) strategy to learn and subsequently retrieve the memory of the platform location. Collectively, these findings provide evidence at a molecular level for the involvement of the hippocampus and BLA in the retrieval of spatial memory and contribute novel observations on the influence of pre-training stress in activating the DLS in response to long-term memory retrieval. PMID:21738501

Molecular analysis of globin gene expression in different thalassaemia disorders: individual variation of β(E) pre-mRNA splicing determine disease severity.

PubMed

Tubsuwan, Alisa; Munkongdee, Thongperm; Jearawiriyapaisarn, Natee; Boonchoy, Chanikarn; Winichagoon, Pranee; Fucharoen, Suthat; Svasti, Saovaros

2011-09-01

Thalassaemia is characterized by the reduced or absent production of globins in the haemoglobin molecule leading to imbalanced α-globin/non α-globin chains. HbE, the result of a G to A mutation in codon 26 of the HBB (β-globin) gene, activates a cryptic 5' splice site in codon 25 leading to a reduction of correctly spliced β(E) -globin (HBB:c.79G>A) mRNA and consequently β(+) -thalassaemia. A wide range of clinical severities in bothα- and β-thalassaemia syndromes, from nearly asymptomatic to transfusion-dependent, has been observed. The correlation between clinical heterogeneity in various genotypes of thalassaemia and the levels of globin gene expression and β(E) -globin pre-mRNA splicing were examined using multiplex quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) and allele-specific RT-qPCR. The α-globin/non α-globin mRNA ratio was demonstrated to be a good indicator for disease severity among different thalassaemia disorders. However, the α-globin/non α-globin mRNA ratio ranged widely in β-thalassaemia/HbE patients, with no significant difference between mild and severe phenotypes. Interestingly, the correctly to aberrantly spliced β(E) -globin mRNA ratio in 30% of mild β-thalassaemia/HbE patients was higher than that of the severe patients. The splicing process of β(E) -globin pre-mRNA differs among β-thalassaemia/HbE patients and serves as one of the modifying factors for disease severity. © 2011 Blackwell Publishing Ltd.

The Arabidopsis THO/TREX component TEX1 functionally interacts with MOS11 and modulates mRNA export and alternative splicing events.

PubMed

Sørensen, Brian B; Ehrnsberger, Hans F; Esposito, Silvia; Pfab, Alexander; Bruckmann, Astrid; Hauptmann, Judith; Meister, Gunter; Merkl, Rainer; Schubert, Thomas; Längst, Gernot; Melzer, Michael; Grasser, Marion; Grasser, Klaus D

2017-02-01

We identify proteins that associate with the THO core complex, and show that the TEX1 and MOS11 components functionally interact, affecting mRNA export and splicing as well as plant development. TREX (TRanscription-EXport) is a multiprotein complex that plays a central role in the coordination of synthesis, processing and nuclear export of mRNAs. Using targeted proteomics, we identified proteins that associate with the THO core complex of Arabidopsis TREX. In addition to the RNA helicase UAP56 and the mRNA export factors ALY2-4 and MOS11 we detected interactions with the mRNA export complex TREX-2 and multiple spliceosomal components. Plants defective in the THO component TEX1 or in the mRNA export factor MOS11 (orthologue of human CIP29) are mildly affected. However, tex1 mos11 double-mutant plants show marked defects in vegetative and reproductive development. In tex1 plants, the levels of tasiRNAs are reduced, while miR173 levels are decreased in mos11 mutants. In nuclei of mos11 cells increased mRNA accumulation was observed, while no mRNA export defect was detected with tex1 cells. Nevertheless, in tex1 mos11 double-mutants, the mRNA export defect was clearly enhanced relative to mos11. The subnuclear distribution of TEX1 substantially overlaps with that of splicing-related SR proteins and in tex1 plants the ratio of certain alternative splicing events is altered. Our results demonstrate that Arabidopsis TEX1 and MOS11 are involved in distinct steps of the biogenesis of mRNAs and small RNAs, and that they interact regarding some aspects, but act independently in others.

Non-FG mediated transport of the large pre-ribosomal subunit through the nuclear pore complex by the mRNA export factor Gle2

PubMed Central

Occhipinti, Laura; Chang, Yiming; Altvater, Martin; Menet, Anna M.; Kemmler, Stefan; Panse, Vikram G.

2013-01-01

Multiple export receptors passage bound pre-ribosomes through nuclear pore complexes (NPCs) by transiently interacting with the Phe-Gly (FG) meshwork of their transport channels. Here, we reveal how the non-FG interacting yeast mRNA export factor Gly-Leu-FG lethal 2 (Gle2) functions in the export of the large pre-ribosomal subunit (pre-60S). Structure-guided studies uncovered conserved platforms used by Gle2 to export pre-60S: an uncharacterized basic patch required to bind pre-60S, and a second surface that makes non-FG contacts with the nucleoporin Nup116. A basic patch mutant of Gle2 is able to function in mRNA export, but not pre-60S export. Thus, Gle2 provides a distinct interaction platform to transport pre-60S to the cytoplasm. Notably, Gle2’s interaction platforms become crucial for pre-60S export when FG-interacting receptors are either not recruited to pre-60S or are impaired. We propose that large complex cargos rely on non-FG as well as FG-interactions for their efficient translocation through the nuclear pore complex channel. PMID:23907389

Alternative Splicing of hTERT Pre-mRNA: A Potential Strategy for the Regulation of Telomerase Activity.

PubMed

Liu, Xuewen; Wang, Yuchuan; Chang, Guangming; Wang, Feng; Wang, Fei; Geng, Xin

2017-03-07

The activation of telomerase is one of the key events in the malignant transition of cells, and the expression of human telomerase reverse transcriptase (hTERT) is indispensable in the process of activating telomerase. The pre-mRNA alternative splicing of hTERT at the post-transcriptional level is one of the mechanisms for the regulation of telomerase activity. Shifts in splicing patterns occur in the development, tumorigenesis, and response to diverse stimuli in a tissue-specific and cell type-specific manner. Despite the regulation of telomerase activity, the alternative splicing of hTERT pre-mRNA may play a role in other cellular functions. Modulating the mode of hTERT pre-mRNA splicing is providing a new precept of therapy for cancer and aging-related diseases. This review focuses on the patterns of hTERT pre-mRNA alternative splicing and their biological functions, describes the potential association between the alternative splicing of hTERT pre-mRNA and telomerase activity, and discusses the possible significance of the alternative splicing of the hTERT pre-mRNA in the diagnosis, therapy, and prognosis of cancer and aging-related diseases.

RNA Polymerase II cluster dynamics predict mRNA output in living cells

PubMed Central

Cho, Won-Ki; Jayanth, Namrata; English, Brian P; Inoue, Takuma; Andrews, J Owen; Conway, William; Grimm, Jonathan B; Spille, Jan-Hendrik; Lavis, Luke D; Lionnet, Timothée; Cisse, Ibrahim I

2016-01-01

Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output. DOI: http://dx.doi.org/10.7554/eLife.13617.001 PMID:27138339

RNA Flow Cytometry Using the Branched DNA Technique.

PubMed

Soh, Kah Teong; Wallace, Paul K

2018-01-01

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

An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

PubMed

Hayakawa-Yano, Yoshika; Suyama, Satoshi; Nogami, Masahiro; Yugami, Masato; Koya, Ikuko; Furukawa, Takako; Zhou, Li; Abe, Manabu; Sakimura, Kenji; Takebayashi, Hirohide; Nakanishi, Atsushi; Okano, Hideyuki; Yano, Masato

2017-09-15

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states. © 2017 Hayakawa-Yano et al.; Published by Cold Spring Harbor Laboratory Press.

Localization of the putative precursor of Alzheimer's disease-specific amyloid at nuclear envelopes of adult human muscle.

PubMed Central

Zimmermann, K; Herget, T; Salbaum, J M; Schubert, W; Hilbich, C; Cramer, M; Masters, C L; Multhaup, G; Kang, J; Lemaire, H G

1988-01-01

Cloning and sequence analysis revealed the putative amyloid A4 precursor (pre-A4) of Alzheimer's disease to have characteristics of a membrane-spanning glycoprotein. In addition to brain, pre-A4 mRNA was found in adult human muscle and other tissues. We demonstrate by in situ hybridization that pre-A4 mRNA is present in adult human muscle, in cultured human myoblasts and myotubes. Immunofluorescence with antipeptide antibodies shows the putative pre-A4 protein to be expressed in adult human muscle and associated with some but not all nuclear envelopes. Despite high levels of a single 3.5-kb pre-A4 mRNA species in cultured myoblasts and myotubes, the presence of putative pre-A4 protein could not be detected by immunofluorescence. This suggests that putative pre-A4 protein is stabilized and therefore functioning in the innervated muscle tissue but not in developing, i.e. non-innervated cultured muscle cells. The selective localization of the protein on distinct nuclear envelopes could reflect an interaction with motor endplates. Images PMID:2896589

Alternative Polyadenylation in Triple-Negative Breast Tumors Allows NRAS and c-JUN to Bypass PUMILIO Posttranscriptional Regulation

PubMed Central

Miles, Wayne O.; Lembo, Antonio; Volorio, Angela; Brachtel, Elena; Tian, Bin; Sgroi, Dennis; Provero, Paolo; Dyson, Nicholas

2017-01-01

Alternative polyadenylation (APA) is a process that changes the posttranscriptional regulation and translation potential of mRNAs via addition or deletion of 3′ untranslated region (3′ UTR) sequences. To identify posttranscriptional-regulatory events affected by APA in breast tumors, tumor datasets were analyzed for recurrent APA events. Motif mapping of the changed 3′ UTR regions found that APA-mediated removal of Pumilio regulatory elements (PRE) was unusually common. Breast tumor subtype–specific APA profiling identified triple-negative breast tumors as having the highest levels of APA. To determine the frequency of these events, an independent cohort of triple-negative breast tumors and normal breast tissue was analyzed for APA. APA-mediated shortening of NRAS and c-JUN was seen frequently, and this correlated with changes in the expression of downstream targets. mRNA stability and luciferase assays demonstrated APA-dependent alterations in RNA and protein levels of affected candidate genes. Examination of clinical parameters of these tumors found those with APA of NRAS and c-JUN to be smaller and less proliferative, but more invasive than non-APA tumors. RT-PCR profiling identified elevated levels of polyadenylation factor CSTF3 in tumors with APA. Overexpression of CSTF3 was common in triple-negative breast cancer cell lines, and elevated CSTF3 levels were sufficient to induce APA of NRAS and c-JUN. Our results support the hypothesis that PRE-containing mRNAs are disproportionately affected by APA, primarily due to high sequence similarity in the motifs utilized by polyadenylation machinery and the PUM complex. PMID:27758885

Evolutionary Insights into RNA trans-Splicing in Vertebrates

PubMed Central

Lei, Quan; Li, Cong; Zuo, Zhixiang; Huang, Chunhua; Cheng, Hanhua; Zhou, Rongjia

2016-01-01

Pre-RNA splicing is an essential step in generating mature mRNA. RNA trans-splicing combines two separate pre-mRNA molecules to form a chimeric non-co-linear RNA, which may exert a function distinct from its original molecules. Trans-spliced RNAs may encode novel proteins or serve as noncoding or regulatory RNAs. These novel RNAs not only increase the complexity of the proteome but also provide new regulatory mechanisms for gene expression. An increasing amount of evidence indicates that trans-splicing occurs frequently in both physiological and pathological processes. In addition, mRNA reprogramming based on trans-splicing has been successfully applied in RNA-based therapies for human genetic diseases. Nevertheless, clarifying the extent and evolution of trans-splicing in vertebrates and developing detection methods for trans-splicing remain challenging. In this review, we summarize previous research, highlight recent advances in trans-splicing, and discuss possible splicing mechanisms and functions from an evolutionary viewpoint. PMID:26966239

G-quadruplex in animal development: Contribution to gene expression and genomic heterogeneity.

PubMed

Armas, Pablo; Calcaterra, Nora Beatriz

2018-05-18

During animal development, gene expression is orchestrated by specific and highly evolutionarily conserved mechanisms that take place accurately, both at spatial and temporal levels. The last decades have provided compelling evidence showing that chromatin state plays essential roles in orchestrating most of the stages of development. The DNA molecule can adopt alternative structures different from the helical duplex architecture. G-rich DNA sequences can fold as intrastrand quadruple helix structures called G-quadruplexes or G4-DNA. G4 can also be formed in RNA molecules, such as mRNA, lncRNA and pre-miRNA. Emerging evidences suggest that G4s have crucial roles in a variety of biological processes, including transcription, recombination, replication, translation and chromosome stability. In this review, we have collected recent information gathered by various laboratories showing the important role of G4 DNA and RNA structures in several steps of animal development. Copyright © 2018 Elsevier B.V. All rights reserved.

A comparative analysis of serpin genes in the silkworm genome

PubMed Central

Zou, Zhen; Picheng, Zhao; Weng, Hua; Mita, Kazuei; Jiang, Haobo

2009-01-01

Serine protease inhibitors (serpins) are a superfamily of proteins, most of which control protease-mediated processes by inhibiting their cognate enzymes. Sequencing of the silkworm genome provides an opportunity to investigate serpin structure, function, and evolution at the genome level. There are thirty-four serpin genes in Bombyx mori. Six are highly similar to their Manduca sexta orthologs that regulate innate immunity. Three alternative exons in serpin1 gene and four in serpin28 encode a variable region including the reactive site loop. Splicing of serpin2 pre-mRNA yields variations in serpin2A, 2A′ and 2B. Sequence similarity and intron positions reveal the evolutionary pathway of seven serpin genes in group C. RT-PCR indicates an increase in the mRNA levels of serpin1, 3, 5, 6, 9, 12, 13, 25, 27, 32 and 34 in fat body and hemocytes of larvae injected with bacteria. These results suggest that the silkworm serpins play regulatory roles in defense responses. PMID:19150649

Inactivation of a Single Copy of Crebbp Selectively Alters Pre-mRNA Processing in Mouse Hematopoietic Stem Cells

PubMed Central

Lemieux, Madeleine E.; Cheng, Ziming; Zhou, Qing; White, Ruth; Cornell, John; Kung, Andrew L.; Rebel, Vivienne I.

2011-01-01

Global expression analysis of fetal liver hematopoietic stem cells (FL HSCs) revealed the presence of unspliced pre-mRNA for a number of genes in normal FL HSCs. In a subset of these genes, Crebbp+/− FL HSCs had less unprocessed pre-mRNA without a corresponding reduction in total mRNA levels. Among the genes thus identified were the key regulators of HSC function Itga4, Msi2 and Tcf4. A similar but much weaker effect was apparent in Ep300+/− FL HSCs, indicating that, in this context as in others, the two paralogs are not interchangeable. As a group, the down-regulated intronic probe sets could discriminate adult HSCs from more mature cell types, suggesting that the underlying mechanism is regulated with differentiation stage and is active in both fetal and adult hematopoiesis. Consistent with increased myelopoiesis in Crebbp hemizygous mice, targeted reduction of CREBBP abundance by shRNA in the multipotent EML cell line triggered spontaneous myeloid differentiation in the absence of the normally required inductive signals. In addition, differences in protein levels between phenotypically distinct EML subpopulations were better predicted by taking into account not only the total mRNA signal but also the amount of unspliced message present. CREBBP thus appears to selectively influence the timing and degree of pre-mRNA processing of genes essential for HSC regulation and thereby has the potential to alter subsequent cell fate decisions in HSCs. PMID:21901164

Biomaterials for mRNA Delivery

PubMed Central

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

2015-01-01

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

Chemotherapy induces alternative transcription and splicing: Facts and hopes for cancer treatment.

PubMed

Lambert, Charles A; Garbacki, Nancy; Colige, Alain C

2017-10-01

Alternative promoter usage, alternative splicing and alternative cleavage/polyadenylation (referred here as to alternative transcription and splicing) are main instruments to diversify the transcriptome from a limited set of genes. There is a good deal of evidence that chemotherapeutic drugs affect these processes, but the therapeutic incidence of these effects is poorly documented. The scope of this study is to review the impact of chemotherapy on alternative transcription and splicing and to discuss potential implications in cancer therapy. A literature survey identified >2200 events induced by chemotherapeutic drugs. The molecular pathways involved in these regulations are briefly discussed. The GO terms associated with the alternative transcripts are mainly related to cell cycle/division, mRNA processing, DNA repair, macromolecules catabolism and chromatin. A large fraction (43%) of transcripts are also related to the new hallmarks of cancer, mostly genetic instability and replicative immortality. Finally, we ask the question of the impact of alternative transcription and splicing on drug efficacy and of the possible curative benefit of combining chemotherapy and pharmaceutical regulation of this process. Copyright © 2017 Elsevier Ltd. All rights reserved.

CK2 is responsible for phosphorylation of human La protein serine-366 and can modulate rpL37 5'-terminal oligopyrimidine mRNA metabolism.

PubMed

Schwartz, Elena I; Intine, Robert V; Maraia, Richard J

2004-11-01

La protein binds precursors to 5S rRNA, tRNAs, and other transcripts that contain 3' UUU-OH and also promotes their maturation in the nucleus. Separate from this function, human La has been shown to positively modulate the translation of mRNAs that contain complex 5' regulatory motifs that direct internal initiation of translation. Nonphosphorylated La (npLa) inhibits pre-tRNA processing, while phosphorylation of human La serine-366 (S(366)) promotes pre-tRNA processing. npLa was found specifically associated with a class of mRNAs that have unusually short 5' untranslated regions comprised of terminal oligopyrimidine (5'TOP) tracts and that encode ribosomal proteins and translation elongation factors. Although La S(366) represents a CK2 phosphorylation site, there was no evidence that CK2 phosphorylates it in vivo. We used the CK2-specific inhibitor, 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), and antisense-mediated knockdown to demonstrate that CK2 is responsible for La S(366) phosphorylation in vivo. Hypophosphorylation was not associated with significant change in total La levels or proteolytic cleavage. Quantitative reverse transcription-PCR revealed increased association of the 5'TOP-mRNA encoding ribosomal protein L37 (rpL37) with La after TBB treatment. Transfection revealed more rpL37 mRNA associated with nonphosphorylatable La A(366) than with La S(366), concomitant with La A(366)-specific shift of a fraction of L37 mRNA off polysomes. The data indicate that CK2 phosphorylates La S(366) in vivo, that this limits 5'TOP mRNA binding, and that increasing npLa leads to greater association with potentially negative effects on TOP mRNA translation. Consistent with data that indicate that phosphorylation reverses negative effects of npLa on tRNA production, the present data suggest that CK2 phosphorylation of La can affect production of the translational machinery.

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.

Regulation of insulin preRNA splicing by glucose

PubMed Central

Wang, Juehu; Shen, Luping; Najafi, Habiba; Kolberg, Janice; Matschinsky, Franz M.; Urdea, Mickey; German, Michael

1997-01-01

Glucose tightly regulates the synthesis and secretion of insulin by β cells in the pancreatic islets of Langerhans. To investigate whether glucose regulates insulin synthesis at the level of insulin RNA splicing, we developed a method to detect and quantify a small amount of RNA by using the branched DNA (bDNA) signal-amplification technique. This assay is both sensitive and highly specific: mouse insulin II mRNA can be detected from a single β cell (βTC3 cells or mouse islets), whereas 1 million non-insulin-producing α cells (αTC1.6 cells) give no signal. By using intron and exon sequences, oligonucleotide probes were designed to distinguish the various unspliced and partially spliced insulin preRNAs from mature insulin mRNA. Insulin RNA splicing rates were estimated from the rate of disappearance of insulin preRNA signal from β cells treated with actinomycin D to block transcription. We found that the two introns in mouse insulin II are not spliced with the same efficiency. Intron 2 is spliced out more efficiently than intron 1. As a result, some mRNA retaining intron 1 enters the cytoplasm, making up ≈2-10% of insulin mRNA in the cell. This partially spliced cytoplasmic mRNA is quite stable, with a half-life similar to the completely spliced form. When islets grown in high glucose are shifted to low glucose medium, the level of insulin preRNA and the rate of splicing fall significantly. We conclude that glucose stimulates insulin gene transcription and insulin preRNA splicing. Previous estimates of insulin transcription rates based on insulin preRNA levels that did not consider the rate of splicing may have underestimated the effect of glucose on insulin gene transcription. PMID:9113994

Fip1 regulates mRNA alternative polyadenylation to promote stem cell self-renewal

PubMed Central

Lackford, Brad; Yao, Chengguo; Charles, Georgette M; Weng, Lingjie; Zheng, Xiaofeng; Choi, Eun-A; Xie, Xiaohui; Wan, Ji; Xing, Yi; Freudenberg, Johannes M; Yang, Pengyi; Jothi, Raja; Hu, Guang; Shi, Yongsheng

2014-01-01

mRNA alternative polyadenylation (APA) plays a critical role in post-transcriptional gene control and is highly regulated during development and disease. However, the regulatory mechanisms and functional consequences of APA remain poorly understood. Here, we show that an mRNA 3′ processing factor, Fip1, is essential for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Fip1 promotes stem cell maintenance, in part, by activating the ESC-specific APA profiles to ensure the optimal expression of a specific set of genes, including critical self-renewal factors. Fip1 expression and the Fip1-dependent APA program change during ESC differentiation and are restored to an ESC-like state during somatic reprogramming. Mechanistically, we provide evidence that the specificity of Fip1-mediated APA regulation depends on multiple factors, including Fip1-RNA interactions and the distance between APA sites. Together, our data highlight the role for post-transcriptional control in stem cell self-renewal, provide mechanistic insight on APA regulation in development, and establish an important function for APA in cell fate specification. PMID:24596251

MIR846 and MIR842 comprise a cistronic MIRNA pair that is regulated by abscisic acid by alternative splicing in roots of Arabidopsis

PubMed Central

Jia, Fan; Rock, Christopher D.

2013-01-01

MicroRNAs (miRNAs) are ~21-nucleotide long endogenous small RNAs that regulate gene expression through post-transcriptional or transcriptional gene silencing (PTGS/TGS) and/or translational inhibition. miRNAs can arise from the “exon” of a MIRNA gene, from an intron (e.g. mirtrons in animals), or from the antisense strand of a protein coding gene (natural antisense microRNAs, nat-miRNAs). Here we demonstrate that two functionally related miRNAs, miR842 and miR846, arise from the same transcription unit but from alternate splicing isoforms. miR846 is expressed only from Isoform1 while in Isoforms2 and -3, a part of pre-miR846 containing the miRNA* sequence is included in the intron. The splicing of the intron truncates the pre-MIRNA and disrupts the expression of the mature miR846.. We name this novel phenomenon splicing-regulated miRNA. Abscisic acid (ABA) is shown to mediate the alternative splicing event by reducing the functional Isoform1 and increasing the non-functional Isoform3, thus repressing the expression of miR846 concomitant with accumulation of an ABA-inducible target jacalin At5g28520 mRNA, whose cleavage was shown by modified 5′-RACE. This regulation shows the functional importance of splicing-regulated miRNA and suggests possible mechanisms for altered ABA response phenotypes of miRNA biogenesis mutants. A. lyrata-MIR842 and Aly-MIR846 have conserved genomic arrangements with A. thaliana and candidate target jacalins, similar primary transcript structures and intron processing, and better miRNA-miRNA* pairings, suggesting that the interactions between ABA, MIR842, MIR846 and jacalins are similar in A. lyrata. Together, splicing-regulated miRNAs, nat-miRNAs/inc-miRNAs and mirtrons illustrate the complexity of MIRNA genes, the importance of introns in the biogenesis and regulation of miRNAs, and raise questions about the processes and molecular mechanisms that drive MIRNA evolution. PMID:23341152

Drosophila stem loop binding protein coordinates accumulation of mature histone mRNA with cell cycle progression

PubMed Central

Sullivan, Eileen; Santiago, Carlos; Parker, Emily D.; Dominski, Zbigniew; Yang, Xiaocui; Lanzotti, David J.; Ingledue, Tom C.; Marzluff, William F.; Duronio, Robert J.

2001-01-01

Replication-associated histone genes encode the only metazoan mRNAs that lack polyA tails, ending instead in a conserved 26-nt sequence that forms a stem–loop. Most of the regulation of mammalian histone mRNA is posttranscriptional and mediated by this unique 3′ end. Stem–loop–binding protein (SLBP) binds to the histone mRNA 3′ end and is thought to participate in all aspects of histone mRNA metabolism, including cell cycle regulation. To examine SLBP function genetically, we have cloned the gene encoding Drosophila SLBP (dSLBP) by a yeast three-hybrid method and have isolated mutations in dSLBP. dSLBP function is required both zygotically and maternally. Strong dSLBP alleles cause zygotic lethality late in development and result in production of stable histone mRNA that accumulates in nonreplicating cells. These histone mRNAs are cytoplasmic and have polyadenylated 3′ ends like other polymerase II transcripts. Hypomorphic dSLBP alleles support zygotic development but cause female sterility. Eggs from these females contain dramatically reduced levels of histone mRNA, and mutant embryos are not able to complete the syncytial embryonic cycles. This is in part because of a failure of chromosome condensation at mitosis that blocks normal anaphase. These data demonstrate that dSLBP is required in vivo for 3′ end processing of histone pre-mRNA, and that this is an essential function for development. Moreover, dSLBP-dependent processing plays an important role in coupling histone mRNA production with the cell cycle. PMID:11157774

Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

PubMed

Marsollier, Anne-Charlotte; Ciszewski, Lukasz; Mariot, Virginie; Popplewell, Linda; Voit, Thomas; Dickson, George; Dumonceaux, Julie

2016-04-15

Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Context-dependent modulation of Pol II CTD phosphatase SSUP-72 regulates alternative polyadenylation in neuronal development

PubMed Central

Chen, Fei; Zhou, Yu; Qi, Yingchuan B.; Khivansara, Vishal; Li, Hairi; Chun, Sang Young; Kim, John K.; Fu, Xiang-Dong; Jin, Yishi

2015-01-01

Alternative polyadenylation (APA) is widespread in neuronal development and activity-mediated neural plasticity. However, the underlying molecular mechanisms are largely unknown. We used systematic genetic studies and genome-wide surveys of the transcriptional landscape to identify a context-dependent regulatory pathway controlling APA in the Caenorhabditis elegans nervous system. Loss of function in ssup-72, a Ser5 phosphatase for the RNA polymerase II (Pol II) C-terminal domain (CTD), dampens transcription termination at a strong intronic polyadenylation site (PAS) in unc-44/ankyrin yet promotes termination at the weak intronic PAS of the MAP kinase dlk-1. A nuclear protein, SYDN-1, which regulates neuronal development, antagonizes the function of SSUP-72 and several nuclear polyadenylation factors. This regulatory pathway allows the production of a neuron-specific isoform of unc-44 and an inhibitory isoform of dlk-1. Dysregulation of the unc-44 and dlk-1 mRNA isoforms in sydn-1 mutants impairs neuronal development. Deleting the intronic PAS of unc-44 results in increased pre-mRNA processing of neuronal ankyrin and suppresses sydn-1 mutants. These results reveal a mechanism by which regulation of CTD phosphorylation controls coding region APA in the nervous system. PMID:26588990

Mutually Exclusive Splicing of the Insect Dscam Pre-mRNA Directed by Competing Intronic RNA Secondary Structures

PubMed Central

Graveley, Brenton R.

2008-01-01

Summary Drosophila Dscam encodes 38,016 distinct axon guidance receptors through the mutually exclusive alternative splicing of 95 variable exons. Importantly, known mechanisms that ensure the mutually exclusive splicing of pairs of exons cannot explain this phenomenon in Dscam. I have identified two classes of conserved elements in the Dscam exon 6 cluster, which contains 48 alternative exons—the docking site, located in the intron downstream of constitutive exon 5, and the selector sequences, which are located upstream of each exon 6 variant. Strikingly, each selector sequence is complementary to a portion of the docking site, and this pairing juxtaposes one, and only one, alternative exon to the upstream constitutive exon. The mutually exclusive nature of the docking site:selector sequence interactions suggests that the formation of these competing RNA structures is a central component of the mechanism guaranteeing that only one exon 6 variant is included in each Dscam mRNA. PMID:16213213

A natural allele of Nxf1/TAP supresses retrovirus insertional mutations

PubMed Central

Floyd, Jennifer A.; Gold, David A.; Concepcion, Dorothy; Poon, Tiffany H.; Wang, Xiaobo; Keithley, Elizabeth; Chen, Dan; Ward, Erica J.; Chinn, Steven B.; Friedman, Rick A.; Yu, Hon-Tsen; Moriwaki, Kazuo; Shiroishi, Toshihiko; Hamilton, Bruce A.

2009-01-01

Endogenous retroviruses have shaped the evolution of mammalian genomes. Host genes that control the effects of retrovirus insertions are therefore of great interest. The Modifier-of-vibrator-1 locus controls level of correctly processed mRNA from genes mutated by endogenous retrovirus insertions into introns, including the pitpnvb tremor mutation and the Eya1BOR model of human branchiootorenal syndrome. Positional complementation cloning identifies Mvb1 as the nuclear export factor Nxf1, providing an unexpected link between mRNA export receptor and pre-mRNA processing. Population structure of the suppressing allele in wild M. m. castaneus suggests selective advantage. A congenic Mvb1CAST allele is a useful tool for modifying gene expression from existing mutations and could be used to manipulate engineered mutations containing retroviral elements. PMID:14517553

Temperature regulates splicing efficiency of the cold-inducible RNA-binding protein gene Cirbp

PubMed Central

Gotic, Ivana; Omidi, Saeed; Fleury-Olela, Fabienne; Molina, Nacho; Naef, Felix; Schibler, Ueli

2016-01-01

In mammals, body temperature fluctuates diurnally around a mean value of 36°C–37°C. Despite the small differences between minimal and maximal values, body temperature rhythms can drive robust cycles in gene expression in cultured cells and, likely, animals. Here we studied the mechanisms responsible for the temperature-dependent expression of cold-inducible RNA-binding protein (CIRBP). In NIH3T3 fibroblasts exposed to simulated mouse body temperature cycles, Cirbp mRNA oscillates about threefold in abundance, as it does in mouse livers. This daily mRNA accumulation cycle is directly controlled by temperature oscillations and does not depend on the cells’ circadian clocks. Here we show that the temperature-dependent accumulation of Cirbp mRNA is controlled primarily by the regulation of splicing efficiency, defined as the fraction of Cirbp pre-mRNA processed into mature mRNA. As revealed by genome-wide “approach to steady-state” kinetics, this post-transcriptional mechanism is widespread in the temperature-dependent control of gene expression. PMID:27633015

Identification of Novel Androgen-Regulated Pathways and mRNA Isoforms through Genome-Wide Exon-Specific Profiling of the LNCaP Transcriptome

PubMed Central

Carling, Phillippa J.; Buist, Thomas; Zhang, Chaolin; Grellscheid, Sushma N.; Armstrong, Kelly; Stockley, Jacqueline; Simillion, Cedric; Gaughan, Luke; Kalna, Gabriela; Zhang, Michael Q.; Robson, Craig N.; Leung, Hing Y.; Elliott, David J.

2011-01-01

Androgens drive the onset and progression of prostate cancer (PCa) by modulating androgen receptor (AR) transcriptional activity. Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome. While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes. Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin) pathway, which is commonly dysregulated in cancer. We also identified 1279 putative androgen-regulated alternative events, of which 325 (∼25%) mapped to known alternative splicing events or alternative first/last exons. We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators. Of seven positively-validating events (∼23%), five events involved transcripts derived from alternative promoters of known AR gene targets. In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition. We confirmed that expression of this alternative TSC2 mRNA isoform was directly regulated by androgens, and chromatin immunoprecipitation indicated recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts. Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical PCa. PMID:22194994

A novel point mutation (G[sup [minus]1] to T) in a 5[prime] splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker Muscular Dystrophy

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

Hagiwara, Yoko; Nishio, Hisahide; Kitoh, Yoshihiko

1994-01-01

The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. The authors now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5[prime] splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophinmore » lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5[prime] splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G[sup [minus]1]-to-T mutation at the 5[prime] splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. 48 refs., 5 figs.« less

Alternative pre-mRNA splicing of Toll-like receptor signaling components in peripheral blood mononuclear cells from patients with ARDS.

PubMed

Blumhagen, Rachel Z; Hedin, Brenna R; Malcolm, Kenneth C; Burnham, Ellen L; Moss, Marc; Abraham, Edward; Huie, Tristan J; Nick, Jerry A; Fingerlin, Tasha E; Alper, Scott

2017-11-01

A key physiological feature of acute respiratory distress syndrome (ARDS) is inflammation. Toll-like receptor (TLR) signaling is required to combat the infection that underlies many ARDS cases but also contributes to pathological inflammation. Several TLR signaling pathway genes encoding positive effectors of inflammation also produce alternatively spliced mRNAs encoding negative regulators of inflammation. An imbalance between these isoforms could contribute to pathological inflammation and disease severity. To determine whether splicing in TLR pathways is altered in patients with ARDS, we monitored alternative splicing of MyD88 and IRAK1 , two genes that function in multiple TLR pathways. The MyD88 and IRAK1 genes produce long proinflammatory mRNAs (MyD88 L and IRAK1) and shorter anti-inflammatory mRNAs (MyD88 S and IRAK1c). We quantified mRNA encoding inflammatory cytokines and MyD88 and IRAK1 isoforms in peripheral blood mononuclear cells (PBMCs) from 104 patients with ARDS and 30 healthy control subjects. We found that MyD88 pre-mRNA splicing is altered in patients with ARDS in a proinflammatory direction. We also observed altered MyD88 isoform levels in a second critically ill patient cohort, suggesting that these changes may not be unique to ARDS. Early in ARDS, PBMC IRAK1c levels were associated with patient survival. Despite the similarities in MyD88 and IRAK1 alternative splicing observed in previous in vitro studies, there were differences in how MyD88 and IRAK1 alternative splicing was altered in patients with ARDS. We conclude that pre-mRNA splicing of TLR signaling genes is altered in patients with ARDS, and further investigation of altered splicing may lead to novel prognostic and therapeutic approaches. Copyright © 2017 the American Physiological Society.

Extraction and purification of high added value compounds from by-products of the winemaking chain using alternative/nonconventional processes/technologies.

PubMed

Yammine, Sami; Brianceau, Sylène; Manteau, Sébastien; Turk, Mohammad; Ghidossi, Rémy; Vorobiev, Eugène; Mietton-Peuchot, Martine

2018-05-24

Grape byproducts are today considered as a cheap source of valuable compounds since existent technologies allow the recovery of target compounds and their recycling. The goal of the current article is to explore the different recovery stages used by both conventional and alternative techniques and processes. Alternative pre-treatments techniques reviewed are: ultrasounds, pulsed electric fields and high voltage discharges. In addition, nonconventional solvent extraction under high pressure, specifically, supercritical fluid extraction and subcritical water extraction are discussed. Finally alternative purification technologies, for example membrane processing were also examined. The intent is to describe the mechanisms involved by these alternative technologies and to summarize the work done on the improvement of the extraction process of phenolic compounds from winery by-products. With a focus on the developmental stage of each technology, highlighting the research need and challenges to be overcome for an industrial implementation of these unitary operations in the overall extraction process. A critical comparison of conventional and alternative techniques will be reviewed for ethe pre-treatment of raw material, the diffusion of polyphenols and the purification of these high added value compounds. This review intends to give the reader some key answers (costs, advantages, drawbacks) to help in the choice of alternative technologies for extraction purposes.

Opposite Influence of Perceptual Memory on Initial and Prolonged Perception of Sensory Ambiguity

PubMed Central

de Jong, Maartje Cathelijne; Knapen, Tomas; van Ee, Raymond

2012-01-01

Observers continually make unconscious inferences about the state of the world based on ambiguous sensory information. This process of perceptual decision-making may be optimized by learning from experience. We investigated the influence of previous perceptual experience on the interpretation of ambiguous visual information. Observers were pre-exposed to a perceptually stabilized sequence of an ambiguous structure-from-motion stimulus by means of intermittent presentation. At the subsequent re-appearance of the same ambiguous stimulus perception was initially biased toward the previously stabilized perceptual interpretation. However, prolonged viewing revealed a bias toward the alternative perceptual interpretation. The prevalence of the alternative percept during ongoing viewing was largely due to increased durations of this percept, as there was no reliable decrease in the durations of the pre-exposed percept. Moreover, the duration of the alternative percept was modulated by the specific characteristics of the pre-exposure, whereas the durations of the pre-exposed percept were not. The increase in duration of the alternative percept was larger when the pre-exposure had lasted longer and was larger after ambiguous pre-exposure than after unambiguous pre-exposure. Using a binocular rivalry stimulus we found analogous perceptual biases, while pre-exposure did not affect eye-bias. We conclude that previously perceived interpretations dominate at the onset of ambiguous sensory information, whereas alternative interpretations dominate prolonged viewing. Thus, at first instance ambiguous information seems to be judged using familiar percepts, while re-evaluation later on allows for alternative interpretations. PMID:22295095

Widespread Use of Non-productive Alternative Splice Sites in Saccharomyces cerevisiae

PubMed Central

Kawashima, Tadashi; Douglass, Stephen; Gabunilas, Jason; Pellegrini, Matteo; Chanfreau, Guillaume F.

2014-01-01

Saccharomyces cerevisiae has been used as a model system to investigate the mechanisms of pre-mRNA splicing but only a few examples of alternative splice site usage have been described in this organism. Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many S. cerevisiae intron-containing genes exhibit usage of alternative splice sites, but many transcripts generated by splicing at these sites are non-functional because they introduce premature termination codons, leading to degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3′ splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3′-splice site in GCR1. The use of non-productive alternative splice sites can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results show that alternative splicing is frequent in S. cerevisiae but masked by RNA degradation and that the use of alternative splice sites in this organism is mostly aimed at controlling transcript levels rather than increasing proteome diversity. PMID:24722551

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.

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

PubMed Central

Mitrovich, Quinn M.; Anderson, Philip

2000-01-01

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

High-throughput sequence analysis of Ciona intestinalis SL trans-spliced mRNAs: alternative expression modes and gene function correlates.

PubMed

Matsumoto, Jun; Dewar, Ken; Wasserscheid, Jessica; Wiley, Graham B; Macmil, Simone L; Roe, Bruce A; Zeller, Robert W; Satou, Yutaka; Hastings, Kenneth E M

2010-05-01

Pre-mRNA 5' spliced-leader (SL) trans-splicing occurs in some metazoan groups but not in others. Genome-wide characterization of the trans-spliced mRNA subpopulation has not yet been reported for any metazoan. We carried out a high-throughput analysis of the SL trans-spliced mRNA population of the ascidian tunicate Ciona intestinalis by 454 Life Sciences (Roche) pyrosequencing of SL-PCR-amplified random-primed reverse transcripts of tailbud embryo RNA. We obtained approximately 250,000 high-quality reads corresponding to 8790 genes, approximately 58% of the Ciona total gene number. The great depth of this data revealed new aspects of trans-splicing, including the existence of a significant class of "infrequently trans-spliced" genes, accounting for approximately 28% of represented genes, that generate largely non-trans-spliced mRNAs, but also produce trans-spliced mRNAs, in part through alternative promoter use. Thus, the conventional qualitative dichotomy of trans-spliced versus non-trans-spliced genes should be supplanted by a more accurate quantitative view recognizing frequently and infrequently trans-spliced gene categories. Our data include reads representing approximately 80% of Ciona frequently trans-spliced genes. Our analysis also revealed significant use of closely spaced alternative trans-splice acceptor sites which further underscores the mechanistic similarity of cis- and trans-splicing and indicates that the prevalence of +/-3-nt alternative splicing events at tandem acceptor sites, NAGNAG, is driven by spliceosomal mechanisms, and not nonsense-mediated decay, or selection at the protein level. The breadth of gene representation data enabled us to find new correlations between trans-splicing status and gene function, namely the overrepresentation in the frequently trans-spliced gene class of genes associated with plasma/endomembrane system, Ca(2+) homeostasis, and actin cytoskeleton.

Rationally designed small molecules targeting the RNA that causes myotonic dystrophy type 1 are potently bioactive.

PubMed

Childs-Disney, Jessica L; Hoskins, Jason; Rzuczek, Suzanne G; Thornton, Charles A; Disney, Matthew D

2012-05-18

RNA is an important drug target, but it is difficult to design or discover small molecules that modulate RNA function. In the present study, we report that rationally designed, modularly assembled small molecules that bind the RNA that causes myotonic dystrophy type 1 (DM1) are potently bioactive in cell culture models. DM1 is caused when an expansion of r(CUG) repeats, or r(CUG)(exp), is present in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA. r(CUG)(exp) folds into a hairpin with regularly repeating 5'CUG/3'GUC motifs and sequesters muscleblind-like 1 protein (MBNL1). A variety of defects are associated with DM1, including (i) formation of nuclear foci, (ii) decreased translation of DMPK mRNA due to its nuclear retention, and (iii) pre-mRNA splicing defects due to inactivation of MBNL1, which controls the alternative splicing of various pre-mRNAs. Previously, modularly assembled ligands targeting r(CUG)(exp) were designed using information in an RNA motif-ligand database. These studies showed that a bis-benzimidazole (H) binds the 5'CUG/3'GUC motif in r(CUG)(exp.) Therefore, we designed multivalent ligands to bind simultaneously multiple copies of this motif in r(CUG)(exp). Herein, we report that the designed compounds improve DM1-associated defects including improvement of translational and pre-mRNA splicing defects and the disruption of nuclear foci. These studies may establish a foundation to exploit other RNA targets in genomic sequence.

Proteasome-Mediated Proteolysis of SRSF5 Splicing Factor Intriguingly Co-occurs with SRSF5 mRNA Upregulation during Late Erythroid Differentiation

PubMed Central

Breig, Osman; Baklouti, Faouzi

2013-01-01

SR proteins exhibit diverse functions ranging from their role in constitutive and alternative splicing, to virtually all aspects of mRNA metabolism. These findings have attracted growing interest in deciphering the regulatory mechanisms that control the tissue-specific expression of these SR proteins. In this study, we show that SRSF5 protein decreases drastically during erythroid cell differentiation, contrasting with a concomitant upregulation of SRSF5 mRNA level. Proteasome chemical inhibition provided strong evidence that endogenous SRSF5 protein, as well as protein deriving from stably transfected SRSF5 cDNA, are both targeted to proteolysis as the cells undergo terminal differentiation. Consistently, functional experiments show that overexpression of SRSF5 enhances a specific endogenous pre-mRNA splicing event in proliferating cells, but not in differentiating cells, due to proteasome-mediated targeting of both endogenous and transfection-derived SRSF5. Further investigation of the relationship between SRSF5 structure and its post-translation regulation and function, suggested that the RNA recognition motifs of SRSF5 are sufficient to activate pre-mRNA splicing, whereas proteasome-mediated proteolysis of SRSF5 requires the presence of the C-terminal RS domain of the protein. Phosphorylation of SR proteins is a key post-translation regulation that promotes their activity and subcellular availability. We here show that inhibition of the CDC2-like kinase (CLK) family and mutation of the AKT phosphorylation site Ser86 on SRSF5, have no effect on SRSF5 stability. We reasoned that at least AKT and CLK signaling pathways are not involved in proteasome-induced turnover of SRSF5 during late erythroid development. PMID:23536862

Connecting the dots: chromatin and alternative splicing in EMT.

PubMed

Warns, Jessica A; Davie, James R; Dhasarathy, Archana

2016-02-01

Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.

Alternative splicing of natriuretic peptide A and B receptor transcripts in the rat brain.

PubMed

Francoeur, F; Gossard, F; Hamet, P; Tremblay, J

1995-12-01

1. In the present study we searched for variants of alternative splicing of guanylyl cyclase A and B mRNA in rats in vivo. 2. Guanylyl cyclase A2 and guanylyl cyclase B2 isoforms of guanylyl cyclase produced by alternative splicing leading to the deletion of exon 9 of both transcripts were quantified in several rat organs. 3. Only one alternative splicing was found in the regulatory domain, encoded by exons 8-15. 4. Quantification of the guanylyl cyclase B2 isoform in different rat organs and in cultured aortic smooth muscle cells showed that this alternative splicing was tissue-specific and occurred predominantly in the central nervous system where the alternatively spliced variant represented more than 50% of the guanylyl cyclase B mRNA. 5. The same alternative splicing existed for guanylyl cyclase A mRNA but at very low levels in the organs studied. 6. Alternative splicing of guanylyl cyclase B exon 9 in the brain may play an important role in signal transduction, since the expressed protein possesses a constitutionally active guanylyl cyclase acting independently of C-type natriuretic peptide regulation.

Circadian clock-dependent and -independent posttranscriptional regulation underlies temporal mRNA accumulation in mouse liver

PubMed Central

Wang, Jingkui; Yeung, Jake; Gobet, Cédric; Sobel, Jonathan; Lück, Sarah; Molina, Nacho; Naef, Felix

2018-01-01

The mammalian circadian clock coordinates physiology with environmental cycles through the regulation of daily oscillations of gene expression. Thousands of transcripts exhibit rhythmic accumulations across mouse tissues, as determined by the balance of their synthesis and degradation. While diurnally rhythmic transcription regulation is well studied and often thought to be the main factor generating rhythmic mRNA accumulation, the extent of rhythmic posttranscriptional regulation is debated, and the kinetic parameters (e.g., half-lives), as well as the underlying regulators (e.g., mRNA-binding proteins) are relatively unexplored. Here, we developed a quantitative model for cyclic accumulations of pre-mRNA and mRNA from total RNA-seq data, and applied it to mouse liver. This allowed us to identify that about 20% of mRNA rhythms were driven by rhythmic mRNA degradation, and another 15% of mRNAs regulated by both rhythmic transcription and mRNA degradation. The method could also estimate mRNA half-lives and processing times in intact mouse liver. We then showed that, depending on mRNA half-life, rhythmic mRNA degradation can either amplify or tune phases of mRNA rhythms. By comparing mRNA rhythms in wild-type and Bmal1−/− animals, we found that the rhythmic degradation of many transcripts did not depend on a functional BMAL1. Interestingly clock-dependent and -independent degradation rhythms peaked at distinct times of day. We further predicted mRNA-binding proteins (mRBPs) that were implicated in the posttranscriptional regulation of mRNAs, either through stabilizing or destabilizing activities. Together, our results demonstrate how posttranscriptional regulation temporally shapes rhythmic mRNA accumulation in mouse liver. PMID:29432155

Coordinating cell cycle-regulated histone gene expression through assembly and function of the Histone Locus Body

PubMed Central

Duronio, Robert J.; Marzluff, William F.

2017-01-01

ABSTRACT Metazoan replication-dependent (RD) histone genes encode the only known cellular mRNAs that are not polyadenylated. These mRNAs end instead in a conserved stem-loop, which is formed by an endonucleolytic cleavage of the pre-mRNA. The genes for all 5 histone proteins are clustered in all metazoans and coordinately regulated with high levels of expression during S phase. Production of histone mRNAs occurs in a nuclear body called the Histone Locus Body (HLB), a subdomain of the nucleus defined by a concentration of factors necessary for histone gene transcription and pre-mRNA processing. These factors include the scaffolding protein NPAT, essential for histone gene transcription, and FLASH and U7 snRNP, both essential for histone pre-mRNA processing. Histone gene expression is activated by Cyclin E/Cdk2-mediated phosphorylation of NPAT at the G1-S transition. The concentration of factors within the HLB couples transcription with pre-mRNA processing, enhancing the efficiency of histone mRNA biosynthesis. PMID:28059623

High-risk human papillomavirus (hrHPV) E6/E7 mRNA testing by PreTect HPV-Proofer for detection of cervical high-grade intraepithelial neoplasia and cancer among hrHPV DNA-positive women with normal cytology.

PubMed

Rijkaart, D C; Heideman, D A M; Coupe, V M H; Brink, A A T P; Verheijen, R H M; Skomedal, H; Karlsen, F; Morland, E; Snijders, P J F; Meijer, C J L M

2012-07-01

Our aim was to investigate whether high-risk HPV (hrHPV) mRNA detection by PreTect HPV-Proofer can be used to stratify hrHPV DNA-positive women of different cytology classes for risk of high-grade cervical intraepithelial neoplasia or worse (cervical precancer or cancer, i.e., cervical intraepithelial neoplasia grade 2 or higher [≥ CIN2]). A total of 375 women participating in population-based screening, with a GP5+/6+-PCR hrHPV DNA-positive cervical scrape with normal cytology (n = 202), borderline or mild dyskaryosis (BMD) (n = 88), or moderate dyskaryosis or worse (>BMD) (n = 85), were enrolled. Cervical scrapes were additionally subjected to HPV16/18/31/33/45 E6/E7 mRNA analysis by PreTect HPV-Proofer (mRNA test). Referral and follow-up policies were based on cytology, hrHPV DNA, and mRNA testing. The primary study endpoint was the number of ≥CIN2 detected within 3 years of follow-up. The mRNA positivity increased with the severity of cytological abnormality, ranging from 32% (64/202) in hrHPV DNA-positive women with normal cytology to 47% (41/88) in BMD and 68% (58/85) in >BMD groups (P < 0.01). Women with ≥ CIN2 were more likely to test positive by mRNA test (63%) than women without evidence of ≥ CIN2 (32%; P < 0.01). A positive mRNA test result conferred an increased ≥ CIN2 risk in hrHPV DNA-positive women with normal cytology, i.e., 0.55 (95% confidence interval [95% CI], 0.34 to 0.76) in mRNA-positive versus 0.20 (95% CI, 0.07 to 0.33) in mRNA-negative women. In hrHPV DNA-positive women with BMD or >BMD, the result of the mRNA test did not influence the ≥ CIN2 risk. In conclusion, mRNA testing by PreTect HPV-Proofer might be of value to select hrHPV DNA-positive women with normal cytology in need of immediate referral for colposcopy.

High-Risk Human Papillomavirus (hrHPV) E6/E7 mRNA Testing by PreTect HPV-Proofer for Detection of Cervical High-Grade Intraepithelial Neoplasia and Cancer among hrHPV DNA-Positive Women with Normal Cytology

PubMed Central

Rijkaart, D. C.; Heideman, D. A. M.; Coupe, V. M. H.; Brink, A. A. T. P.; Verheijen, R. H. M.; Skomedal, H.; Karlsen, F.; Morland, E.; Snijders, P. J. F.

2012-01-01

Our aim was to investigate whether high-risk HPV (hrHPV) mRNA detection by PreTect HPV-Proofer can be used to stratify hrHPV DNA-positive women of different cytology classes for risk of high-grade cervical intraepithelial neoplasia or worse (cervical precancer or cancer, i.e., cervical intraepithelial neoplasia grade 2 or higher [≥CIN2]). A total of 375 women participating in population-based screening, with a GP5+/6+-PCR hrHPV DNA-positive cervical scrape with normal cytology (n = 202), borderline or mild dyskaryosis (BMD) (n = 88), or moderate dyskaryosis or worse (>BMD) (n = 85), were enrolled. Cervical scrapes were additionally subjected to HPV16/18/31/33/45 E6/E7 mRNA analysis by PreTect HPV-Proofer (mRNA test). Referral and follow-up policies were based on cytology, hrHPV DNA, and mRNA testing. The primary study endpoint was the number of ≥CIN2 detected within 3 years of follow-up. The mRNA positivity increased with the severity of cytological abnormality, ranging from 32% (64/202) in hrHPV DNA-positive women with normal cytology to 47% (41/88) in BMD and 68% (58/85) in >BMD groups (P < 0.01). Women with ≥CIN2 were more likely to test positive by mRNA test (63%) than women without evidence of ≥CIN2 (32%; P < 0.01). A positive mRNA test result conferred an increased ≥CIN2 risk in hrHPV DNA-positive women with normal cytology, i.e., 0.55 (95% confidence interval [95% CI], 0.34 to 0.76) in mRNA-positive versus 0.20 (95% CI, 0.07 to 0.33) in mRNA-negative women. In hrHPV DNA-positive women with BMD or >BMD, the result of the mRNA test did not influence the ≥CIN2 risk. In conclusion, mRNA testing by PreTect HPV-Proofer might be of value to select hrHPV DNA-positive women with normal cytology in need of immediate referral for colposcopy. PMID:22553244

A Circadian Clock-Regulated Toggle Switch Explains AtGRP7 and AtGRP8 Oscillations in Arabidopsis thaliana

PubMed Central

Schmal, Christoph; Reimann, Peter; Staiger, Dorothee

2013-01-01

The circadian clock controls many physiological processes in higher plants and causes a large fraction of the genome to be expressed with a 24h rhythm. The transcripts encoding the RNA-binding proteins AtGRP7 (Arabidopsis thaliana Glycine Rich Protein 7) and AtGRP8 oscillate with evening peaks. The circadian clock components CCA1 and LHY negatively affect AtGRP7 expression at the level of transcription. AtGRP7 and AtGRP8, in turn, negatively auto-regulate and reciprocally cross-regulate post-transcriptionally: high protein levels promote the generation of an alternative splice form that is rapidly degraded. This clock-regulated feedback loop has been proposed to act as a molecular slave oscillator in clock output. While mathematical models describing the circadian core oscillator in Arabidopsis thaliana were introduced recently, we propose here the first model of a circadian slave oscillator. We define the slave oscillator in terms of ordinary differential equations and identify the model's parameters by an optimization procedure based on experimental results. The model successfully reproduces the pertinent experimental findings such as waveforms, phases, and half-lives of the time-dependent concentrations. Furthermore, we obtain insights into possible mechanisms underlying the observed experimental dynamics: the negative auto-regulation and reciprocal cross-regulation via alternative splicing could be responsible for the sharply peaking waveforms of the AtGRP7 and AtGRP8 mRNA. Moreover, our results suggest that the AtGRP8 transcript oscillations are subordinated to those of AtGRP7 due to a higher impact of AtGRP7 protein on alternative splicing of its own and of the AtGRP8 pre-mRNA compared to the impact of AtGRP8 protein. Importantly, a bifurcation analysis provides theoretical evidence that the slave oscillator could be a toggle switch, arising from the reciprocal cross-regulation at the post-transcriptional level. In view of this, transcriptional repression of AtGRP7 and AtGRP8 by LHY and CCA1 induces oscillations of the toggle switch, leading to the observed high-amplitude oscillations of AtGRP7 mRNA. PMID:23555221

Seed Dormancy in Arabidopsis Is Controlled by Alternative Polyadenylation of DOG1.

PubMed

Cyrek, Malgorzata; Fedak, Halina; Ciesielski, Arkadiusz; Guo, Yanwu; Sliwa, Aleksandra; Brzezniak, Lien; Krzyczmonik, Katarzyna; Pietras, Zbigniew; Kaczanowski, Szymon; Liu, Fuquan; Swiezewski, Szymon

2016-02-01

DOG1 (Delay of Germination 1) is a key regulator of seed dormancy in Arabidopsis (Arabidopsis thaliana) and other plants. Interestingly, the C terminus of DOG1 is either absent or not conserved in many plant species. Here, we show that in Arabidopsis, DOG1 transcript is subject to alternative polyadenylation. In line with this, mutants in RNA 3' processing complex display weakened seed dormancy in parallel with defects in DOG1 proximal polyadenylation site selection, suggesting that the short DOG1 transcript is functional. This is corroborated by the finding that the proximally polyadenylated short DOG1 mRNA is translated in vivo and complements the dog1 mutant. In summary, our findings indicate that the short DOG1 protein isoform produced from the proximally polyadenylated DOG1 mRNA is a key player in the establishment of seed dormancy in Arabidopsis and characterizes a set of mutants in RNA 3' processing complex required for production of proximally polyadenylated functional DOG1 transcript. © 2016 American Society of Plant Biologists. All Rights Reserved.

Impact of food restriction on ovarian development, RFamide-related peptide-3 and the hypothalamic-pituitary-ovarian axis in pre-pubertal ewes.

PubMed

Li, H; Song, H; Huang, M; Nie, H; Wang, Z; Wang, F

2014-10-01

RFamide-related peptide-3 (RFRP-3), the mammalian ortholog of gonadotropin-inhibiting hormone, has been implicated as a mediator between reproduction and energy balance. This study aimed to investigate the physiological effects of RFRP-3 on the process of ovarian development in food-restricted pre-pubertal ewes. The results showed that food restriction significantly inhibited the ovarian development and follicular growth. The data of qPCR in the hypothalamic-pituitary-ovarian (HPO) axis showed that food restriction not only upregulated RFRP-3 mRNA expression but also downregulated the mRNA expression of gonadotropin-releasing-hormone receptor, follicle-stimulating hormone receptor and luteinizing hormone receptor (LHR). Immunohistochemistry of RFRP-3 in the ovaries suggested that RFRP-3 may regulate the follicular development. These results suggested that the changes of RFRP-3 in response to food restriction might influence the HPO axis and inhibit ovarian development. © 2014 Blackwell Verlag GmbH.

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

PubMed

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

2017-03-28

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

Coordinated Post-transcriptional Regulation of Hsp70.3 Gene Expression by MicroRNA and Alternative Polyadenylation*

PubMed Central

Tranter, Michael; Helsley, Robert N.; Paulding, Waltke R.; McGuinness, Michael; Brokamp, Cole; Haar, Lauren; Liu, Yong; Ren, Xiaoping; Jones, W. Keith

2011-01-01

Heat shock protein 70 (Hsp70) is well documented to possess general cytoprotective properties in protecting the cell against stressful and noxious stimuli. We have recently shown that expression of the stress-inducible Hsp70.3 gene in the myocardium in response to ischemic preconditioning is NF-κB-dependent and necessary for the resulting late phase cardioprotection against a subsequent ischemia/reperfusion injury. Here we show that the Hsp70.3 gene product is subject to post-transcriptional regulation through parallel regulatory processes involving microRNAs and alternative polyadenylation of the mRNA transcript. First, we show that cardiac ischemic preconditioning of the in vivo mouse heart results in decreased levels of two Hsp70.3-targeting microRNAs: miR-378* and miR-711. Furthermore, an ischemic or heat shock stimulus induces alternative polyadenylation of the expressed Hsp70.3 transcript that results in the accumulation of transcripts with a shortened 3′-UTR. This shortening of the 3′-UTR results in the loss of the binding site for the suppressive miR-378* and thus renders the alternatively polyadenylated transcript insusceptible to miR-378*-mediated suppression. Results also suggest that the alternative polyadenylation-mediated shortening of the Hsp70.3 3′-UTR relieves translational suppression observed in the long 3′-UTR variant, allowing for a more robust increase in protein expression. These results demonstrate alternative polyadenylation of Hsp70.3 in parallel with ischemic or heat shock-induced up-regulation of mRNA levels and implicate the importance of this process in post-transcriptional control of Hsp70.3 expression. PMID:21757701

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.

Resveratrol, by modulating RNA processing factor levels, can influence the alternative splicing of pre-mRNAs.

PubMed

Markus, M Andrea; Marques, Francine Z; Morris, Brian J

2011-01-01

Alternative pre-mRNA splicing defects can contribute to, or result from, various diseases, including cancer. Aberrant mRNAs, splicing factors and other RNA processing factors have therefore become targets for new therapeutic interventions. Here we report that the natural polyphenol resveratrol can modulate alternative splicing in a target-specific manner. We transfected minigenes of several alternatively spliceable primary mRNAs into HEK293 cells in the presence or absence of 1, 5, 20 and 50 µM resveratrol and measured exon levels by semi-quantitative PCR after separation by agarose gel electrophoresis. We found that 20 µg/ml and 50 µg/ml of resveratrol affected exon inclusion of SRp20 and SMN2 pre-mRNAs, but not CD44v5 or tau pre-mRNAs. By Western blotting and immunofluorescence we showed that this effect may be due to the ability of resveratrol to change the protein level but not the localization of several RNA processing factors. The processing factors that increased significantly were ASF/SF2, hnRNPA1 and HuR, but resveratrol did not change the levels of RBM4, PTBP1 and U2AF35. By means of siRNA-mediated knockdown we depleted cells of SIRT1, regarded as a major target of resveratrol, and showed that the effect on splicing was not dependent on SIRT1. Our results suggest that resveratrol might be an attractive small molecule to treat diseases in which aberrant splicing has been implicated, and justify more extensive research on the effects of resveratrol on the splicing machinery.

APP processing and the APP-KPI domain involvement in the amyloid cascade.

PubMed

Menéndez-González, M; Pérez-Pinera, P; Martínez-Rivera, M; Calatayud, M T; Blázquez Menes, B

2005-01-01

Alternative APP mRNA splicing can generate isoforms of APP containing a Kunitz protease inhibitor (KPI) domain. KPI is one of the main serine protease inhibitors. Protein and mRNA KPI(+)APP levels are elevated in Alzheimer's disease (AD) brain and are associated with increased amyloid beta deposition. In the last years increasing evidence on multiple points in the amyloid cascade where KPI(+)APP is involved has been accumulated, admitting an outstanding position in the pathogenesis of AD to the KPI domain. This review focuses on the APP processing, the molecular activity of KPI and its physiological and pathological roles and the KPI involvement in the amyloid cascade through the nerve growth factor, the lipoprotein receptor-related protein, the tumor necrosis factor-alpha converting enzyme and the Notch1 protein.

CK2 Is Responsible for Phosphorylation of Human La Protein Serine-366 and Can Modulate rpL37 5′-Terminal Oligopyrimidine mRNA Metabolism

PubMed Central

Schwartz, Elena I.; Intine, Robert V.; Maraia, Richard J.

2004-01-01

La protein binds precursors to 5S rRNA, tRNAs, and other transcripts that contain 3′ UUU-OH and also promotes their maturation in the nucleus. Separate from this function, human La has been shown to positively modulate the translation of mRNAs that contain complex 5′ regulatory motifs that direct internal initiation of translation. Nonphosphorylated La (npLa) inhibits pre-tRNA processing, while phosphorylation of human La serine-366 (S366) promotes pre-tRNA processing. npLa was found specifically associated with a class of mRNAs that have unusually short 5′ untranslated regions comprised of terminal oligopyrimidine (5′TOP) tracts and that encode ribosomal proteins and translation elongation factors. Although La S366 represents a CK2 phosphorylation site, there was no evidence that CK2 phosphorylates it in vivo. We used the CK2-specific inhibitor, 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), and antisense-mediated knockdown to demonstrate that CK2 is responsible for La S366 phosphorylation in vivo. Hypophosphorylation was not associated with significant change in total La levels or proteolytic cleavage. Quantitative reverse transcription-PCR revealed increased association of the 5′TOP-mRNA encoding ribosomal protein L37 (rpL37) with La after TBB treatment. Transfection revealed more rpL37 mRNA associated with nonphosphorylatable La A366 than with La S366, concomitant with La A366-specific shift of a fraction of L37 mRNA off polysomes. The data indicate that CK2 phosphorylates La S366 in vivo, that this limits 5′TOP mRNA binding, and that increasing npLa leads to greater association with potentially negative effects on TOP mRNA translation. Consistent with data that indicate that phosphorylation reverses negative effects of npLa on tRNA production, the present data suggest that CK2 phosphorylation of La can affect production of the translational machinery. PMID:15485924

Developmental changes in the hypothalamic mRNA expression levels of brain-derived neurotrophic factor and serum leptin levels: Their responses to fasting in male and female rats.

PubMed

Iwasa, Takeshi; Matsuzaki, Toshiya; Yano, Kiyohito; Munkhzaya, Munkhsaikhan; Tungalagsuvd, Altankhuu; Yiliyasi, Maira; Kuwahara, Akira; Irahara, Minoru

2016-11-01

The actions and responses of hypothalamic appetite regulatory factors change markedly during the neonatal to pre-pubertal period in order to maintain appropriate metabolic and nutritional conditions. In this study, we examined the developmental changes in the hypothalamic mRNA levels of brain-derived neurotrophic factor (BDNF), which is a potent anorectic factor and the changes in the sensitivity of the hypothalamic expression of this factor to fasting during the neonatal to pre-pubertal period. Under fed conditions, hypothalamic BDNF mRNA expression decreased during development in both male and female rats. Similarly, the serum levels of leptin, which is a positive regulator of hypothalamic BDNF expression, also tended to fall during the developmental period. The serum leptin level and the hypothalamic BDNF mRNA level were found to be positively correlated in both sexes under the fed conditions. Hypothalamic BDNF mRNA expression was decreased by 24h fasting (separating the rats from their mothers) in the early neonatal period (postnatal day 10) in both males and females, but no such changes were seen at postnatal day 20. Twenty-four hours' fasting (food deprivation) did not affect hypothalamic BDNF mRNA expression in the pre-pubertal period (postnatal day 30). On the other hand, the rats' serum leptin levels were decreased by 24h fasting (separating the rats from their mothers at postnatal day 10 and 20, and food deprivation at postnatal day 30) throughout the early neonatal to pre-pubertal period. The correlation between serum leptin and hypothalamic BDNF mRNA levels was not significant under the fasted conditions. It can be speculated that leptin partially regulates hypothalamic BDNF mRNA levels, but only in fed conditions. Such changes in hypothalamic BDNF expression might play a role in maintaining appropriate metabolic and nutritional conditions and promoting normal physical development. In addition, because maternal separation induces a negative energy balance and short- and long-term stress responses, it is also possible that reductions in hypothalamic BDNF mRNA levels in the early neonatal period (postnatal day 10) may be partially induced by stress responses of the maternal deprivation. Copyright © 2016 ISDN. Published by Elsevier Ltd. All rights reserved.

Bioinspired Nanocomplex for Spatiotemporal Imaging of Sequential mRNA Expression in Differentiating Neural Stem Cells

PubMed Central

2015-01-01

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions. PMID:25494492

Bioinspired nanocomplex for spatiotemporal imaging of sequential mRNA expression in differentiating neural stem cells.

PubMed

Wang, Zhe; Zhang, Ruili; Wang, Zhongliang; Wang, He-Fang; Wang, Yu; Zhao, Jun; Wang, Fu; Li, Weitao; Niu, Gang; Kiesewetter, Dale O; Chen, Xiaoyuan

2014-12-23

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions.

Modulation of p53β and p53γ expression by regulating the alternative splicing of TP53 gene modifies cellular response

PubMed Central

Marcel, V; Fernandes, K; Terrier, O; Lane, D P; Bourdon, J-C

2014-01-01

In addition to the tumor suppressor p53 protein, also termed p53α, the TP53 gene produces p53β and p53γ through alternative splicing of exons 9β and 9γ located within TP53 intron 9. Here we report that both TG003, a specific inhibitor of Cdc2-like kinases (Clk) that regulates the alternative splicing pre-mRNA pathway, and knockdown of SFRS1 increase expression of endogenous p53β and p53γ at mRNA and protein levels. Development of a TP53 intron 9 minigene shows that TG003 treatment and knockdown of SFRS1 promote inclusion of TP53 exons 9β/9γ. In a series of 85 primary breast tumors, a significant association was observed between expression of SFRS1 and α variant, supporting our experimental data. Using siRNA specifically targeting exons 9β/9γ, we demonstrate that cell growth can be driven by modulating p53β and p53γ expression in an opposite manner, depending on the cellular context. In MCF7 cells, p53β and p53γ promote apoptosis, thus inhibiting cell growth. By transient transfection, we show that p53β enhanced p53α transcriptional activity on the p21 and Bax promoters, while p53γ increased p53α transcriptional activity on the Bax promoter only. Moreover, p53β and p53γ co-immunoprecipitate with p53α only in the presence of p53-responsive promoter. Interestingly, although p53β and p53γ promote apoptosis in MCF7 cells, p53β and p53γ maintain cell growth in response to TG003 in a p53α-dependent manner. The dual activities of p53β and p53γ isoforms observed in non-treated and TG003-treated cells may result from the impact of TG003 on both expression and activities of p53 isoforms. Overall, our data suggest that p53β and p53γ regulate cellular response to modulation of alternative splicing pre-mRNA pathway by a small drug inhibitor. The development of novel drugs targeting alternative splicing process could be used as a novel therapeutic approach in human cancers. PMID:24926616

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.

Can the HIV-1 splicing machinery be targeted for drug discovery?

PubMed Central

Dlamini, Zodwa; Hull, Rodney

2017-01-01

HIV-1 is able to express multiple protein types and isoforms from a single 9 kb mRNA transcript. These proteins are also expressed at particular stages of viral development, and this is achieved through the control of alternative splicing and the export of these transcripts from the nucleus. The nuclear export is controlled by the HIV protein Rev being required to transport incompletely spliced and partially spliced mRNA from the nucleus where they are normally retained. This implies a close relationship between the control of alternate splicing and the nuclear export of mRNA in the control of HIV-1 viral proliferation. This review discusses both the processes. The specificity and regulation of splicing in HIV-1 is controlled by the use of specific splice sites as well as exonic splicing enhancer and exonic splicing silencer sequences. The use of these silencer and enhancer sequences is dependent on the serine arginine family of proteins as well as the heterogeneous nuclear ribonucleoprotein family of proteins that bind to these sequences and increase or decrease splicing. Since alternative splicing is such a critical factor in viral development, it presents itself as a promising drug target. This review aims to discuss the inhibition of splicing, which would stall viral development, as an anti-HIV therapeutic strategy. In this review, the most recent knowledge of splicing in human immunodeficiency viral development and the latest therapeutic strategies targeting human immunodeficiency viral splicing are discussed. PMID:28331370

Connecting the dots: chromatin and alternative splicing in EMT

PubMed Central

Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

2015-01-01

Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

PABPN1-Dependent mRNA Processing Induces Muscle Wasting

PubMed Central

Raz, Yotam; van Putten, Maaike; Paniagua-Soriano, Guillem; Krom, Yvonne D.; Florea, Bogdan I.; Raz, Vered

2016-01-01

Poly(A) Binding Protein Nuclear 1 (PABPN1) is a multifunctional regulator of mRNA processing, and its expression levels specifically decline in aging muscles. An expansion mutation in PABPN1 is the genetic cause of oculopharyngeal muscle dystrophy (OPMD), a late onset and rare myopathy. Moreover, reduced PABPN1 expression correlates with symptom manifestation in OPMD. PABPN1 regulates alternative polyadenylation site (PAS) utilization. However, the impact of PAS utilization on cell and tissue function is poorly understood. We hypothesized that altered PABPN1 expression levels is an underlying cause of muscle wasting. To test this, we stably down-regulated PABPN1 in mouse tibialis anterior (TA) muscles by localized injection of adeno-associated viruses expressing shRNA to PABPN1 (shPab). We found that a mild reduction in PABPN1 levels causes muscle pathology including myofiber atrophy, thickening of extracellular matrix and myofiber-type transition. Moreover, reduced PABPN1 levels caused a consistent decline in distal PAS utilization in the 3’-UTR of a subset of OPMD-dysregulated genes. This alternative PAS utilization led to up-regulation of Atrogin-1, a key muscle atrophy regulator, but down regulation of proteasomal genes. Additionally reduced PABPN1 levels caused a reduction in proteasomal activity, and transition in MyHC isotope expression pattern in myofibers. We suggest that PABPN1-mediated alternative PAS utilization plays a central role in aging-associated muscle wasting. PMID:27152426

From General Aberrant Alternative Splicing in Cancers and Its Therapeutic Application to the Discovery of an Oncogenic DMTF1 Isoform

PubMed Central

Tian, Na; Li, Jialiang; Shi, Jinming; Sui, Guangchao

2017-01-01

Alternative pre-mRNA splicing is a crucial process that allows the generation of diversified RNA and protein products from a multi-exon gene. In tumor cells, this mechanism can facilitate cancer development and progression through both creating oncogenic isoforms and reducing the expression of normal or controllable protein species. We recently demonstrated that an alternative cyclin D-binding myb-like transcription factor 1 (DMTF1) pre-mRNA splicing isoform, DMTF1β, is increasingly expressed in breast cancer and promotes mammary tumorigenesis in a transgenic mouse model. Aberrant pre-mRNA splicing is a typical event occurring for many cancer-related functional proteins. In this review, we introduce general aberrant pre-mRNA splicing in cancers and discuss its therapeutic application using our recent discovery of the oncogenic DMTF1 isoform as an example. We also summarize new insights in designing novel targeting strategies of cancer therapies based on the understanding of deregulated pre-mRNA splicing mechanisms. PMID:28257090

A non-inflammatory form of immune competence prevails in acute pre-pubescent malnutrition: new evidence based on critical mRNA transcripts in the mouse.

PubMed

Monk, Jennifer M; Richard, Cynthia L; Woodward, Bill

2012-05-01

The declining inflammatory immune competence of acute (i.e. wasting) pre-pubescent protein-energy malnutrition has been regarded as reflecting an unregulated immunological disintegration. Recent evidence, however, suggests that malnutrition stimulates a regulated immunological reconfiguration to achieve a non-inflammatory form of competence, perhaps offering protection against autoimmune reactions - the 'Tolerance Model'. Our objective was to determine the influence of acute pre-pubescent malnutrition on the expression of genes critical to tolerogenic regulation. Male and female C57BL/6J mice, initially 19 d old, consumed a complete purified diet either ad libitum (age-matched controls) or in restricted daily quantities (mimicking marasmus), or consumed an isoenergetic low-protein diet ad libitum (mimicking incipient kwashiorkor) for 14 d (six animals per dietary group). Gene expression in the spleen, typically an inflammatory organ, and in the small intestine, a site designed for non-inflammatory defence, was assessed by real-time quantitative RT-PCR, and normalised to β-actin. In the spleen of the malnourished groups, both IL-10 and transforming growth factor-β1 mRNA expression increased compared with controls (P < 0.05), whereas mRNA expression of IL-12p40 decreased (P < 0.05). Conversely, malnutrition exerted no influence on the expression of mRNA for these cytokines in the small intestine (P>0.05). Moreover, forkhead box P3 mRNA expression, indicative of cell-based tolerogenic potential, was sustained in both the spleen and intestine of the malnourished groups (P>0.05). Thus, despite limited supplies of energy and substrates, the spleen shifted towards a non-inflammatory character and the intestine was sustained in this mode in advanced pre-pubescent weight loss. These findings provide the first support for the Tolerance Model at the level of mRNA transcript expression.

Tumor suppressive microRNA-1 mediated novel apoptosis pathways through direct inhibition of splicing factor serine/arginine-rich 9 (SRSF9/SRp30c) in bladder cancer

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

Yoshino, Hirofumi; Enokida, Hideki, E-mail: enokida@m.kufm.kagoshima-u.ac.jp; Chiyomaru, Takeshi

2012-01-06

Highlights: Black-Right-Pointing-Pointer Tumor suppressive miRNA-1 directly inhibits splicing factor serine/arginine-rich 9 (SRSF9). Black-Right-Pointing-Pointer SRSF9 mRNA expression was up-regulated in bladder cancer specimens compared to normal tissues. Black-Right-Pointing-Pointer Cell viability (proliferation, migration, and invasion) was reduced in SRSF9 knockdown cells. Black-Right-Pointing-Pointer SRSF9 knockdown by miR-1 induced cell apoptosis through caspase-3/7 activation in BC cell lines. -- Abstract: We have previously found that restoration of tumor suppressive microRNA-1 (miR-1), induced cell apoptosis in bladder cancer (BC) cell lines. However, the apoptosis mechanism induced by miR-1 was not fully elucidated. Alternative splicing of mRNA precursors provides cancer cells with opportunities to translate manymore » oncogenic protein variants, which promote cell proliferation and survival under unpreferable condition for cancer development. Serine/arginine-rich (SR) protein family, which involved in alternative pre-mRNA splicing, plays a critical role for regulating apoptosis by splicing apoptosis-related genes. However, transcriptional regulation of SR proteins, themselves, has not been elucidated. In this study, we focused on splicing factor serine/arginine-rich 9 (SRSF9/SRp30c) on the basis of our previous genome-wide gene expression analysis using miR-1-transfected BC cell lines because putative target sites of miR-1 are existed in 3 Prime -untranslated region (UTR) of SRSF9 mRNA. The expression levels of mRNA of SRSF9 were extremely reduced in the miR-1 transfectants. A luciferase activity significantly decreased in the transfectants suggesting that actual binding occurred between miR-1 and 3 Prime UTR of SRSF9 mRNA. Loss-of-function assays demonstrated that significant inhibitions of cell proliferation, migration, and invasion were observed in the si-SRSF9 transfectants. Apoptosis assays demonstrated that cell apoptosis fraction increased and that caspase-3/7 was activated in the si-SRSF9 transfectants. Our data indicated that tumor suppressive miR-1 induces apoptosis through direct inhibition of SRSF9 in BC. The identification of molecular mechanisms between miRNAs and SR proteins could provide novel apoptosis pathways and their epigenetic regulations and offer new strategies for BC treatment.« less

Differential expression of decorin and biglycan genes during mouse tooth development

NASA Technical Reports Server (NTRS)

Matsuura, T.; Duarte, W. R.; Cheng, H.; Uzawa, K.; Yamauchi, M.

2001-01-01

Small leucine-rich proteoglycans (SLRPs) have a number of biological functions and some of them are thought to regulate collagen mineralizaton in bone and tooth. We have previously identified and immunolocalized two members of the SLRPs family, decorin and biglycan, in bovine tooth/periodontium. To investigate their potential roles in tooth development, we examined the mRNA expression patterns of decorin, biglycan and type I collagen in newborn (day 19) mice tooth germs by in situ hybridization. At this developmental stage, the first maxillary and mandibular molars include stages before and after secretion of the predentin matrix, respectively. The expression of decorin mRNA coincided with that of type I collagen mRNA and was mostly observed in secretory odontoblasts, while the biglycan mRNA was expressed throughout the tooth germ, including pre-secretory odontoblasts/ameloblasts, dental papilla and stellate reticulum. However, its signal in secretory odontoblasts was not as evident as that of decorin. In mandibular incisors, where a significant amount of predentin matrix and a small amount of enamel matrix were already secreted, a similar differential expression pattern was observed. In secretory ameloblasts the biglycan mRNA expression was apparent, while that of decorin was not. These differential expression patterns suggest the distinct roles of biglycan and decorin in the process of tooth development.

Mechanisms and consequences of alternative polyadenylation

PubMed Central

Di Giammartino, Dafne Campigli; Nishida, Kensei; Manley, James L.

2011-01-01

Summary Alternative polyadenylation (APA) is emerging as a widespread mechanism used to control gene expression. Like alternative splicing, usage of alternative poly(A) sites allows a single gene to encode multiple mRNA transcripts. In some cases, this changes the mRNA coding potential; in other cases, the code remains unchanged but the 3’UTR length is altered, influencing the fate of mRNAs in several ways, for example, by altering the availability of RNA binding protein sites and microRNA binding sites. The mechansims governing both global and gene-specific APA are only starting to be deciphered. Here we review what is known about these mechanisms and the functional consequences of alternative polyadenlyation. PMID:21925375

Evolution and Biological Roles of Alternative 3'UTRs.

PubMed

Mayr, Christine

2016-03-01

More than half of human genes use alternative cleavage and polyadenylation to generate alternative 3' untranslated region (3'UTR) isoforms. Most efforts have focused on transcriptome-wide mapping of alternative 3'UTRs and on the question of how 3'UTR isoform ratios may be regulated. However, it remains less clear why alternative 3'UTRs have evolved and what biological roles they play. This review summarizes our current knowledge of the functional roles of alternative 3'UTRs, including mRNA localization, mRNA stability, and translational efficiency. Recent work suggests that alternative 3'UTRs may also enable the formation of protein-protein interactions to regulate protein localization or to diversify protein functions. These recent findings open an exciting research direction for the investigation of new biological roles of alternative 3'UTRs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Whole genome mRNA transcriptomics analysis reveals different modes of action of the diarrheic shellfish poisons okadaic acid and dinophysis toxin-1 versus azaspiracid-1 in Caco-2 cells.

PubMed

Bodero, Marcia; Hoogenboom, Ron L A P; Bovee, Toine F H; Portier, Liza; de Haan, Laura; Peijnenburg, Ad; Hendriksen, Peter J M

2018-02-01

A study with DNA microarrays was performed to investigate the effects of two diarrhetic and one azaspiracid shellfish poison, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and azaspiracid-1 (AZA-1) respectively, on the whole-genome mRNA expression of undifferentiated intestinal Caco-2 cells. Previously, the most responding genes were used to develop a dedicated array tube test to screen shellfish samples on the presence of these toxins. In the present study the whole genome mRNA expression was analyzed in order to reveal modes of action and obtain hints on potential biomarkers suitable to be used in alternative bioassays. Effects on key genes in the most affected pathways and processes were confirmed by qPCR. OA and DTX-1 induced almost identical effects on mRNA expression, which strongly indicates that OA and DTX-1induce similar toxic effects. Biological interpretation of the microarray data indicates that both compounds induce hypoxia related pathways/processes, the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. The gene expression profile of AZA-1 is different and shows increased mRNA expression of genes involved in cholesterol synthesis and glycolysis, suggesting a different mode of action for this toxin. Future studies should reveal whether identified pathways provide suitable biomarkers for rapid detection of DSPs in shellfish. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

The splicing activator DAZAP1 integrates splicing control into MEK/Erk-regulated cell proliferation and migration

NASA Astrophysics Data System (ADS)

Choudhury, Rajarshi; Roy, Sreerupa Ghose; Tsai, Yihsuan S.; Tripathy, Ashutosh; Graves, Lee M.; Wang, Zefeng

2014-01-01

Alternative splicing of pre-messenger RNA (mRNA) is a critical stage of gene regulation in response to environmental stimuli. Here we show that DAZAP1, an RNA-binding protein involved in mammalian development and spermatogenesis, promotes inclusion of weak exons through specific recognition of diverse cis-elements. The carboxy-terminal proline-rich domain of DAZAP1 interacts with and neutralizes general splicing inhibitors, and is sufficient to activate splicing when recruited to pre-mRNA. This domain is phosphorylated by the MEK/Erk (extracellular signal-regulated protein kinase) pathway and this modification is essential for the splicing regulatory activity and the nuclear/cytoplasmic translocation of DAZAP1. Using mRNA-seq, we identify endogenous splicing events regulated by DAZAP1, many of which are involved in maintaining cell growth. Knockdown or over-expression of DAZAP1 causes a cell proliferation defect. Taken together, these studies reveal a molecular mechanism that integrates splicing control into MEK/Erk-regulated cell proliferation.

Identification of a functionally distinct truncated BDNF mRNA splice variant and protein in Trachemys scripta elegans.

PubMed

Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

2013-01-01

Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein.

Identification of a Functionally Distinct Truncated BDNF mRNA Splice Variant and Protein in Trachemys scripta elegans

PubMed Central

Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

2013-01-01

Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein. PMID:23825634

Disturbed expression of type 1 iodothyronine deiodinase splice variants in human renal cancer.

PubMed

Piekielko-Witkowska, Agnieszka; Master, Adam; Wojcicka, Anna; Boguslawska, Joanna; Brozda, Izabela; Tanski, Zbigniew; Nauman, Alicja

2009-10-01

Alternative splicing, one of the sources of protein diversity, is often disturbed in cancer. Type 1 iodothyronine deiodinase (DIO1) catalyzes deiodination of thyroxine generating triiodothyronine, an important regulator of cell proliferation and differentiation. The expression of DIO1 is disturbed in different types of cancer. The aim of the study was to analyze the alternative splicing of DIO1 and its possible disturbance in renal cancer. Using real-time PCR, we analyzed 19 tissue samples (T) of renal cancer and 19 matched control samples (C) of the opposite pole of the kidney, not infiltrated by tumor, and 6 control samples (N) (nonneoplastic kidney abnormalities). Cloning of DIO1 mRNA isoforms revealed 11 different transcripts, among them 7 new splice variants, not previously reported. The expression of all variants of DIO1 was dramatically (>90%) and significantly (p < or = 0.0003) lowered in samples T compared to control samples C. The ratio of mRNA isoforms encoding DIO1 protein variants possessing or lacking the active center was lowered in samples T compared with control samples C, suggesting disturbed alternative splicing of DIO1. The expression of mRNA of splicing factors SF2/ASF (splicing factor-2/alternative-splicing factor) and hnRNPA1 (heterogeneous ribonucleoprotein A1), regulating 5'-splice site selection, was significantly but not proportionally lowered in samples T compared to samples C. The mRNA ratio of splicing factors SF2/ASF and hnRNPA1 correlated with the ratio of mRNA isoforms encoding DIO1 protein variants possessing or lacking the active center in controls C but not in samples T. Our results show that the expression and alternative splicing of DIO1 mRNA is disturbed in renal cancer, possibly due to changes in expression of splicing factors SF2/ASF and hnRNPA1.

Diverse environmental stresses elicit distinct responses at the level of pre-mRNA processing in yeast

PubMed Central

Bergkessel, Megan; Whitworth, Gregg B.; Guthrie, Christine

2011-01-01

Gene expression in eukaryotic cells is profoundly influenced by the post-transcriptional processing of mRNAs, including the splicing of introns in the nucleus and both nuclear and cytoplasmic degradation pathways. These processes have the potential to affect both the steady-state levels and the kinetics of changes to levels of intron-containing transcripts. Here we report the use of a splicing isoform-specific microarray platform to investigate the effects of diverse stress conditions on pre-mRNA processing. Interestingly, we find that diverse stresses cause distinct patterns of changes at this level. The responses we observed are most dramatic for the RPGs and can be categorized into three major classes. The first is characterized by accumulation of RPG pre-mRNA and is seen in multiple types of amino acid starvation regimes; the magnitude of splicing inhibition correlates with the severity of the stress. The second class is characterized by a rapid decrease in both pre- and mature RPG mRNA and is seen in many stresses that inactivate the TORC1 kinase complex. These decreases depend on nuclear turnover of the intron-containing pre-RNAs. The third class is characterized by a decrease in RPG pre-mRNA, with only a modest reduction in the mature species; this response is observed in hyperosmotic and cation-toxic stresses. We show that casein kinase 2 (CK2) makes important contributions to the changes in pre-mRNA processing, particularly for the first two classes of stress responses. In total, our data suggest that complex post-transcriptional programs cooperate to fine-tune expression of intron-containing transcripts in budding yeast. PMID:21697354

Diverse environmental stresses elicit distinct responses at the level of pre-mRNA processing in yeast.

PubMed

Bergkessel, Megan; Whitworth, Gregg B; Guthrie, Christine

2011-08-01

Gene expression in eukaryotic cells is profoundly influenced by the post-transcriptional processing of mRNAs, including the splicing of introns in the nucleus and both nuclear and cytoplasmic degradation pathways. These processes have the potential to affect both the steady-state levels and the kinetics of changes to levels of intron-containing transcripts. Here we report the use of a splicing isoform-specific microarray platform to investigate the effects of diverse stress conditions on pre-mRNA processing. Interestingly, we find that diverse stresses cause distinct patterns of changes at this level. The responses we observed are most dramatic for the RPGs and can be categorized into three major classes. The first is characterized by accumulation of RPG pre-mRNA and is seen in multiple types of amino acid starvation regimes; the magnitude of splicing inhibition correlates with the severity of the stress. The second class is characterized by a rapid decrease in both pre- and mature RPG mRNA and is seen in many stresses that inactivate the TORC1 kinase complex. These decreases depend on nuclear turnover of the intron-containing pre-RNAs. The third class is characterized by a decrease in RPG pre-mRNA, with only a modest reduction in the mature species; this response is observed in hyperosmotic and cation-toxic stresses. We show that casein kinase 2 (CK2) makes important contributions to the changes in pre-mRNA processing, particularly for the first two classes of stress responses. In total, our data suggest that complex post-transcriptional programs cooperate to fine-tune expression of intron-containing transcripts in budding yeast.

Identification of a chemical inhibitor for nuclear speckle formation: Implications for the function of nuclear speckles in regulation of alternative pre-mRNA splicing

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

Kurogi, Yutaro; Matsuo, Yota; Mihara, Yuki

2014-03-28

Highlights: • We identified tubercidin as a compound inducing aberrant formation of the speckles. • Tubercidin causes delocalization of poly (A){sup +}RNAs from nuclear speckles. • Tubercidin induces dispersion of splicing factors from nuclear speckles. • Tubercidin affects alternative pre-mRNA splicing. • Nuclear speckles play a role in regulation of alternative pre-mRNA splicing. - Abstract: Nuclear speckles are subnuclear structures enriched with RNA processing factors and poly (A){sup +} RNAs comprising mRNAs and poly (A){sup +} non-coding RNAs (ncRNAs). Nuclear speckles are thought to be involved in post-transcriptional regulation of gene expression, such as pre-mRNA splicing. By screening 3585 culturemore » extracts of actinomycetes with in situ hybridization using an oligo dT probe, we identified tubercidin, an analogue of adenosine, as an inhibitor of speckle formation, which induces the delocalization of poly (A){sup +} RNA and dispersion of splicing factor SRSF1/SF2 from nuclear speckles in HeLa cells. Treatment with tubercidin also decreased steady-state MALAT1 long ncRNA, thought to be involved in the retention of SRSF1/SF2 in nuclear speckles. In addition, we found that tubercidin treatment promoted exon skipping in the alternative splicing of Clk1 pre-mRNA. These results suggest that nuclear speckles play a role in modulating the concentration of splicing factors in the nucleoplasm to regulate alternative pre-mRNA splicing.« less

Simultaneous Quantification of Multiple Alternatively Spliced mRNA Transcripts Using Droplet Digital PCR.

PubMed

Sun, Bing; Zheng, Yun-Ling

2018-01-01

Currently there is no sensitive, precise, and reproducible method to quantitate alternative splicing of mRNA transcripts. Droplet digital™ PCR (ddPCR™) analysis allows for accurate digital counting for quantification of gene expression. Human telomerase reverse transcriptase (hTERT) is one of the essential components required for telomerase activity and for the maintenance of telomeres. Several alternatively spliced forms of hTERT mRNA in human primary and tumor cells have been reported in the literature. Using one pair of primers and two probes for hTERT, four alternatively spliced forms of hTERT (α-/β+, α+/β- single deletions, α-/β- double deletion, and nondeletion α+/β+) were accurately quantified through a novel analysis method via data collected from a single ddPCR reaction. In this chapter, we describe this ddPCR method that enables direct quantitative comparison of four alternatively spliced forms of the hTERT messenger RNA without the need for internal standards or multiple pairs of primers specific for each variant, eliminating the technical variation due to differential PCR amplification efficiency for different amplicons and the challenges of quantification using standard curves. This simple and straightforward method should have general utility for quantifying alternatively spliced gene transcripts.

Altered nuclear tRNA metabolism in La-deleted Schizosaccharomyces pombe is accompanied by a nutritional stress response involving Atf1p and Pcr1p that is suppressible by Xpo-t/Los1p.

PubMed

Cherkasova, Vera; Maury, Luis Lopez; Bacikova, Dagmar; Pridham, Kevin; Bähler, Jürg; Maraia, Richard J

2012-02-01

Deletion of the sla1(+) gene, which encodes a homologue of the human RNA-binding protein La in Schizosaccharomyces pombe, causes irregularities in tRNA processing, with altered distribution of pre-tRNA intermediates. We show, using mRNA profiling, that cells lacking sla1(+) have increased mRNAs from amino acid metabolism (AAM) genes and, furthermore, exhibit slow growth in Edinburgh minimal medium. A subset of these AAM genes is under control of the AP-1-like, stress-responsive transcription factors Atf1p and Pcr1p. Although S. pombe growth is resistant to rapamycin, sla1-Δ cells are sensitive, consistent with deficiency of leucine uptake, hypersensitivity to NH4, and genetic links to the target of rapamycin (TOR) pathway. Considering that perturbed intranuclear pre-tRNA metabolism and apparent deficiency in tRNA nuclear export in sla1-Δ cells may trigger the AAM response, we show that modest overexpression of S. pombe los1(+) (also known as Xpo-t), encoding the nuclear exportin for tRNA, suppresses the reduction in pre-tRNA levels, AAM gene up-regulation, and slow growth of sla1-Δ cells. The conclusion that emerges is that sla1(+) regulates AAM mRNA production in S. pombe through its effects on nuclear tRNA processing and probably nuclear export. Finally, the results are discussed in the context of stress response programs in Saccharomyces cerevisiae.

Altered nuclear tRNA metabolism in La-deleted Schizosaccharomyces pombe is accompanied by a nutritional stress response involving Atf1p and Pcr1p that is suppressible by Xpo-t/Los1p

PubMed Central

Cherkasova, Vera; Lopez Maury, Luis; Bacikova, Dagmar; Pridham, Kevin; Bähler, Jürg; Maraia, Richard J.

2012-01-01

Deletion of the sla1+ gene, which encodes a homologue of the human RNA-binding protein La in Schizosaccharomyces pombe, causes irregularities in tRNA processing, with altered distribution of pre-tRNA intermediates. We show, using mRNA profiling, that cells lacking sla1+ have increased mRNAs from amino acid metabolism (AAM) genes and, furthermore, exhibit slow growth in Edinburgh minimal medium. A subset of these AAM genes is under control of the AP-1–like, stress-responsive transcription factors Atf1p and Pcr1p. Although S. pombe growth is resistant to rapamycin, sla1-Δ cells are sensitive, consistent with deficiency of leucine uptake, hypersensitivity to NH4, and genetic links to the target of rapamycin (TOR) pathway. Considering that perturbed intranuclear pre-tRNA metabolism and apparent deficiency in tRNA nuclear export in sla1-Δ cells may trigger the AAM response, we show that modest overexpression of S. pombe los1+ (also known as Xpo-t), encoding the nuclear exportin for tRNA, suppresses the reduction in pre-tRNA levels, AAM gene up-regulation, and slow growth of sla1-Δ cells. The conclusion that emerges is that sla1+ regulates AAM mRNA production in S. pombe through its effects on nuclear tRNA processing and probably nuclear export. Finally, the results are discussed in the context of stress response programs in Saccharomyces cerevisiae. PMID:22160596

Ovarian Expression, Localization, and Function of Tissue Inhibitor of Metalloproteinase 3 (TIMP3) During the Periovulatory Period of the Human Menstrual Cycle1

PubMed Central

Rosewell, Katherine L.; Li, Feixue; Puttabyatappa, Muraly; Akin, James W.; Brännström, Mats; Curry, Thomas E.

2013-01-01

ABSTRACT Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability. PMID:24048576

Ovarian expression, localization, and function of tissue inhibitor of metalloproteinase 3 (TIMP3) during the periovulatory period of the human menstrual cycle.

PubMed

Rosewell, Katherine L; Li, Feixue; Puttabyatappa, Muraly; Akin, James W; Brännström, Mats; Curry, Thomas E

2013-11-01

Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability.

Interactome Analysis of NS1 Protein Encoded by Influenza A H7N9 Virus Reveals an Inhibitory Role of NS1 in Host mRNA Maturation.

PubMed

Kuo, Rei-Lin; Chen, Chi-Jene; Tam, Ee-Hong; Huang, Chung-Guei; Li, Li-Hsin; Li, Zong-Hua; Su, Pei-Chia; Liu, Hao-Ping; Wu, Chih-Ching

2018-04-06

Influenza A virus infections can result in severe respiratory diseases. The H7N9 subtype of avian influenza A virus has been transmitted to humans and caused severe disease and death. Nonstructural protein 1 (NS1) of influenza A virus is a virulence determinant during viral infection. To elucidate the functions of the NS1 encoded by influenza A H7N9 virus (H7N9 NS1), interaction partners of H7N9 NS1 in human cells were identified with immunoprecipitation followed by SDS-PAGE coupled with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). We identified 36 cellular proteins as the interacting partners of the H7N9 NS1, and they are involved in RNA processing, mRNA splicing via spliceosome, and the mRNA surveillance pathway. Two of the interacting partners, cleavage and polyadenylation specificity factor subunit 2 (CPSF2) and CPSF7, were confirmed to interact with H7N9 NS1 using coimmunoprecipitation and immunoblotting based on the previous finding that the two proteins are involved in pre-mRNA polyadenylation machinery. Furthermore, we illustrate that overexpression of H7N9 NS1, as well as infection by the influenza A H7N9 virus, interfered with pre-mRNA polyadenylation in host cells. This study comprehensively profiled the interactome of H7N9 NS1 in host cells, and the results demonstrate a novel endotype for H7N9 NS1 in inhibiting host mRNA maturation.

Characterization of 16S rRNA Processing with Pre-30S Subunit Assembly Intermediates from E. coli.

PubMed

Smith, Brian A; Gupta, Neha; Denny, Kevin; Culver, Gloria M

2018-06-08

Ribosomal RNA (rRNA) is a major component of ribosomes and is fundamental to the process of translation. In bacteria, 16S rRNA is a component of the small ribosomal subunit and plays a critical role in mRNA decoding. rRNA maturation entails the removal of intervening spacer sequences contained within the pre-rRNA transcript by nucleolytic enzymes. Enzymatic activities involved in maturation of the 5'-end of 16S rRNA have been identified, but those involved in 3'-end maturation of 16S rRNA are more enigmatic. Here, we investigate molecular details of 16S rRNA maturation using purified in vivo-formed small subunit (SSU) assembly intermediates (pre-SSUs) from wild-type Escherichia coli that contain precursor 16S rRNA (17S rRNA). Upon incubation of pre-SSUs with E. coli S100 cell extracts or purified enzymes implicated in 16S rRNA processing, the 17S rRNA is processed into additional intermediates and mature 16S rRNA. These results illustrate that exonucleases RNase R, RNase II, PNPase, and RNase PH can process the 3'-end of pre-SSUs in vitro. However, the endonuclease YbeY did not exhibit nucleolytic activity with pre-SSUs under these conditions. Furthermore, these data demonstrate that multiple pathways facilitate 16S rRNA maturation with pre-SSUs in vitro, with the dominant pathways entailing complete processing of the 5'-end of 17S rRNA prior to 3'-end maturation or partial processing of the 5'-end with concomitant processing of the 3'-end. These results reveal the multifaceted nature of SSU biogenesis and suggest that E. coli may be able to escape inactivation of any one enzyme by using an existing complementary pathway. Copyright © 2018 Elsevier Ltd. All rights reserved.

Alternative Polyadenylation of mRNAs: 3′-Untranslated Region Matters in Gene Expression

PubMed Central

Yeh, Hsin-Sung; Yong, Jeongsik

2016-01-01

Almost all of eukaryotic mRNAs are subjected to polyadenylation during mRNA processing. Recent discoveries showed that many of these mRNAs contain more than one polyadenylation sites in their 3′ untranslated regions (UTR) and that alternative polyadenylation (APA) is prevalent among these genes. Many biological processes such as differentiation, proliferation, and tumorigenesis have been correlated to global APA events in the 3′ UTR of mRNAs, suggesting that these APA events are tightly regulated and may play important physiological roles. In this review, recent discoveries in the physiological roles of APA events, as well as the known and proposed mechanisms are summarized. Perspective for future directions is also discussed. PMID:26912084

Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export

PubMed Central

Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

2016-01-01

The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms. PMID:26872259

Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export.

PubMed

Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

2016-01-01

The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.

Structure of a human cap-dependent 48S translation pre-initiation complex

PubMed Central

Eliseev, Boris; Yeramala, Lahari; Leitner, Alexander; Karuppasamy, Manikandan; Raimondeau, Etienne; Huard, Karine; Alkalaeva, Elena; Aebersold, Ruedi

2018-01-01

Abstract Eukaryotic translation initiation is tightly regulated, requiring a set of conserved initiation factors (eIFs). Translation of a capped mRNA depends on the trimeric eIF4F complex and eIF4B to load the mRNA onto the 43S pre-initiation complex comprising 40S and initiation factors 1, 1A, 2, 3 and 5 as well as initiator-tRNA. Binding of the mRNA is followed by mRNA scanning in the 48S pre-initiation complex, until a start codon is recognised. Here, we use a reconstituted system to prepare human 48S complexes assembled on capped mRNA in the presence of eIF4B and eIF4F. The highly purified h-48S complexes are used for cross-linking/mass spectrometry, revealing the protein interaction network in this complex. We report the electron cryo-microscopy structure of the h-48S complex at 6.3 Å resolution. While the majority of eIF4B and eIF4F appear to be flexible with respect to the ribosome, additional density is detected at the entrance of the 40S mRNA channel which we attribute to the RNA-recognition motif of eIF4B. The eight core subunits of eIF3 are bound at the 40S solvent-exposed side, as well as the subunits eIF3d, eIF3b and eIF3i. elF2 and initiator-tRNA bound to the start codon are present at the 40S intersubunit side. This cryo-EM structure represents a molecular snap-shot revealing the h-48S complex following start codon recognition. PMID:29401259

Alternative Fuels Data Center: Buying and Selling Pre-Owned Alternative

Science.gov Websites

Alternative Fuels Data Center: Buying and Selling Pre-Owned Alternative Fuel and Advanced Vehicles to someone by E-mail Share Alternative Fuels Data Center: Buying and Selling Pre-Owned Alternative Fuel and Advanced Vehicles on Facebook Tweet about Alternative Fuels Data Center: Buying and Selling Pre-Owned

Tandem alternative polyadenylation events of genes in non-eosinophilic nasal polyp tissue identified by high-throughput sequencing analysis

PubMed Central

TIAN, PENG; LI, JIE; LIU, XIANG; LI, YUXI; CHEN, MEIHENG; MA, YUN; ZHENG, YI QING; FU, YONGGUI; ZOU, HUA

2014-01-01

Nasal polyps (NP) is highly associated with the disorder of immune cells. Alternative polyadenylation (APA) produces mRNA isoforms with different length of 3′-untranslated region (UTR) and regulates gene expression. It has been proven that this APA-mediated regulation of 3′UTR length is an immune-associated phenomenon. The aim of this study was to investigate the genome-wide alternative tandem 3′UTR length switching events in non-eosinophilic nasal polyp tissue. Thirteen patients diagnosed as having non-eosinophilic nasal polyps were included in this study. Nasal polyp tissue and control mucosa were collected during surgery. The 3′ end library of cDNA was constructed. The recovered libraries were sequenced with second sequencing technology, and the sequencing data were analyzed by an in-house bioinformatics pipeline. Tandem 3′UTR length switching between samples was detected by a test of linear trend alternative to independence. We found a significant alteration in the tandem 3′UTR length in 1,920 genes in nasal polyp samples. Functional annotation results showed that several gene ontology (GO) terms were enriched in the list of genes with switched APA sites, including regulation of transcription, macromolecule catabolic localization and mRNA processing. The results suggested that APA-mediated alternative 3′UTR regulation plays an important role in the post-transcriptional regulation of gene expression in non-eosinophilic nasal polyps. PMID:24715051

AMPK does not play a requisite role in regulation of PPARGC1A gene expression via the alternative promoter in endurance-trained human skeletal muscle.

PubMed

Popov, Daniil V; Lysenko, Evgeny A; Butkov, Alexey D; Vepkhvadze, Tatiana F; Perfilov, Dmitriy V; Vinogradova, Olga L

2017-03-01

What is the central question of this study? This study was designed to investigate the role of AMPK in the regulation of PGC-1α gene expression via the alternative promoter through a cAMP response element-binding protein-1-dependent mechanism in human skeletal muscle. What is the main finding and its importance? Low-intensity exercise markedly increased the expression of PGC-1α mRNA via the alternative promoter, without increases in ACC Ser79/222 (a marker of AMPK activation) and AMPK Thr172 phosphorylation. A single dose of the AMPK activator metformin indicated that AMPK was not involved in regulating PGC-1α mRNA expression via the alternative promoter in endurance-trained human skeletal muscle. In human skeletal muscle, PGC-1α is constitutively expressed via the canonical promoter. In contrast, the expression of PGC-1α mRNA via the alternative promoter was found to be highly dependent on the intensity of exercise and to contribute largely to the postexercise increase of total PGC-1α mRNA. This study investigated the role of AMPK in regulating PGC-1α gene expression via the alternative promoter through a cAMP response element-binding protein-1-dependent mechanism in human skeletal muscle. AMPK activation and PGC-1α gene expression were assayed in skeletal muscle of nine endurance-trained men before and after low-intensity exercise (38% of maximal oxygen uptake) and with or without administration of a single dose (2 g) of the AMPK activator metformin. Low-intensity exercise markedly and significantly increased (∼100-fold, P < 0.05) the expression of PGC-1α mRNA via the alternative promoter, without increasing ACC Ser79/222 (a marker of AMPK activation) and AMPK Thr172 phosphorylation. Moreover, in contrast to placebo, metformin increased the level of ACC Ser79/222 phosphorylation immediately after exercise (2.6-fold, P < 0.05). However postexercise expression of PGC-1α gene via the alternative promoter was not affected. This study was unable to confirm that AMPK plays a role in regulating PGC-1α gene expression via the alternative promoter in endurance-trained human skeletal muscle. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

United States Department of Agriculture-Agricultural Research Service research on alternatives to methyl bromide: pre-plant and post-harvest.

PubMed

Schneider, Sally M; Rosskopf, Erin N; Leesch, James G; Chellemi, Daniel O; Bull, Carolee T; Mazzola, Mark

2003-01-01

Methyl bromide is a widely used fumigant for both pre-plant and post-harvest pest and pathogen control. The Montreal Protocol and the US Clean Air Act mandate a phase-out of the import and manufacture of methyl bromide, beginning in 2001 and culminating with a complete ban, except for quarantine and certain pre-shipment uses and exempted critical uses, in January 2005. In 1995, ARS built on its existing programs in soil-borne plant pathology and post-harvest entomology and plant pathology to initiate a national research program to develop alternatives to methyl bromide. The focus has been on strawberry, pepper, tomato, perennial and nursery cropping systems for pre-plant methyl bromide use and fresh and durable commodities for post-harvest use. Recently the program has been expanded to include research on alternatives for the ornamental and cut flower cropping systems. An overview of the national research program is presented. Results from four specific research trials are presented, ranging from organic to conventional systems. Good progress on short-term alternatives is being made. These will be used as the foundation of integrated management systems which begin with pre-plant management decisions and continue through post-harvest processing.

Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing.

PubMed

Huertas, César S; Carrascosa, L G; Bonnal, S; Valcárcel, J; Lechuga, L M

2016-04-15

Alternative splicing of mRNA precursors enables cells to generate different protein outputs from the same gene depending on their developmental or homeostatic status. Its deregulation is strongly linked to disease onset and progression. Current methodologies for monitoring alternative splicing demand elaborate procedures and often present difficulties in discerning between closely related isoforms, e.g. due to cross-hybridization during their detection. Herein, we report a general methodology using a Surface Plasmon Resonance (SPR) biosensor for label-free monitoring of alternative splicing events in real-time, without any cDNA synthesis or PCR amplification requirements. We applied this methodology to RNA isolated from HeLa cells for the quantification of alternatively spliced isoforms of the Fas gene, involved in cancer progression through regulation of programmed cell death. We demonstrate that our methodology is isoform-specific, with virtually no cross-hybridization, achieving limits of detection (LODs) in the picoMolar (pM) range. Similar results were obtained for the detection of the BCL-X gene mRNA isoforms. The results were independently validated by RT-qPCR, with excellent concordance in the determination of isoform ratios. The simplicity and robustness of this biosensor technology can greatly facilitate the exploration of alternative splicing biomarkers in disease diagnosis and therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Posttranscriptional regulation of retroviral gene expression: primary RNA transcripts play three roles as pre-mRNA, mRNA, and genomic RNA

PubMed Central

LeBlanc, Jason; Weil, Jason; Beemon, Karen

2013-01-01

After reverse transcription of the retroviral RNA genome and integration of the DNA provirus into the host genome, host machinery is used for viral gene expression along with viral proteins and RNA regulatory elements. Here, we discuss co-transcriptional and posttranscriptional regulation of retroviral gene expression, comparing simple and complex retroviruses. Cellular RNA polymerase II synthesizes full-length viral primary RNA transcripts that are capped and polyadenylated. All retroviruses generate a singly spliced env mRNA from this primary transcript, which encodes the viral glycoproteins. In addition, complex viral RNAs are alternatively spliced to generate accessory proteins, such as Rev, which is involved in posttranscriptional regulation of HIV-1 RNA. Importantly, the splicing of all retroviruses is incomplete; they must maintain and export a fraction of their primary RNA transcripts. This unspliced RNA functions both as the major mRNA for Gag and Pol proteins and as the packaged genomic RNA. Different retroviruses export their unspliced viral RNA from the nucleus to the cytoplasm by either Tap-dependent or Rev/CRM1-dependent routes. Translation of the unspliced mRNA involves frame-shifting or termination codon suppression so that the Gag proteins, which make up the capsid, are expressed more abundantly than the Pol proteins, which are the viral enzymes. After the viral polyproteins assemble into viral particles and bud from the cell membrane, a viral encoded protease cleaves them. Some retroviruses have evolved mechanisms to protect their unspliced RNA from decay by nonsense-mediated RNA decay and to prevent genome editing by the cellular APOBEC deaminases. PMID:23754689

Polyoma virus small tumor antigen pre-mRNA splicing requires cooperation between two 3' splice sites.

PubMed Central

Ge, H; Noble, J; Colgan, J; Manley, J L

1990-01-01

We have studied splicing of the polyoma virus early region pre-mRNA in vitro. This RNA is alternatively spliced in vivo to produce mRNA encoding the large, middle-sized (MTAg), and small (StAg) tumor antigens. Our primary interest was to learn how the 48-nucleotide StAg intron is excised, because the length of this intron is significantly less than the apparent minimum established for mammalian introns. Although the products of all three splices are detected in vitro, characterization of the pathway and sequence requirements of StAg splicing suggests that splicing factors interact with the precursor RNA in an unexpected way to catalyze removal of this intron. Specifically, StAg splicing uses either of two lariat branch points, one of which is located only 4 nucleotides from the 3' splice site. Furthermore, the StAg splice absolutely requires that the alternative MTAg 3' splice site, located 14 nucleotides downstream of the StAg 3' splice site, be intact. Insertion mutations that increase or decrease the quality of the MTAg pyrimidine stretch enhance or repress StAg as well as MTAg splicing, and a single-base change in the MTAg AG splice acceptor totally blocks both splices. These results demonstrate the ability of two 3' splice sites to cooperate with each other to bring about removal of a single intron. Images PMID:2159146

Complexity of the Alternative Splicing Landscape in Plants[C][W][OPEN

PubMed Central

Reddy, Anireddy S.N.; Marquez, Yamile; Kalyna, Maria; Barta, Andrea

2013-01-01

Alternative splicing (AS) of precursor mRNAs (pre-mRNAs) from multiexon genes allows organisms to increase their coding potential and regulate gene expression through multiple mechanisms. Recent transcriptome-wide analysis of AS using RNA sequencing has revealed that AS is highly pervasive in plants. Pre-mRNAs from over 60% of intron-containing genes undergo AS to produce a vast repertoire of mRNA isoforms. The functions of most splice variants are unknown. However, emerging evidence indicates that splice variants increase the functional diversity of proteins. Furthermore, AS is coupled to transcript stability and translation through nonsense-mediated decay and microRNA-mediated gene regulation. Widespread changes in AS in response to developmental cues and stresses suggest a role for regulated splicing in plant development and stress responses. Here, we review recent progress in uncovering the extent and complexity of the AS landscape in plants, its regulation, and the roles of AS in gene regulation. The prevalence of AS in plants has raised many new questions that require additional studies. New tools based on recent technological advances are allowing genome-wide analysis of RNA elements in transcripts and of chromatin modifications that regulate AS. Application of these tools in plants will provide significant new insights into AS regulation and crosstalk between AS and other layers of gene regulation. PMID:24179125

Alternative splicing of c-fos pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species.

PubMed

Jurado, Juan; Fuentes-Almagro, Carlos A; Prieto-Alamo, María J; Pueyo, Carmen

2007-09-21

Alternative splicing is a widespread mechanism of gene expression regulation. Previous analyses based on conventional RT-PCR reported the presence of an unspliced c-fos transcript in several mammalian systems. Compared to the well-defined knowledge on the alternative splicing of fosB, the physiological relevance of the unspliced c-fos transcript in regulating c-fos expression remains largely unknown. This work aimed to investigate the functional significance of the alternative splicing c-fos pre-mRNA. A set of primers was designed to demonstrate that, whereas introns 1 and 2 are regularly spliced from primary c-fos transcript, intron 3 remains unspliced in part of total transcript molecules. Here, the two species are referred to as c-fos-2 (+ intron 3) and spliced c-fos (- intron 3) transcripts. Then, we used a quantitatively rigorous approach based on real-time PCR to provide, for the first time, the actual steady-state copy numbers of the two c-fos transcripts. We tested how the mouse-organ context and mouse-gestational age, the synthesis and turnover rates of the investigated transcripts, and the serum stimulation of quiescent cells modulate their absolute-expression profiles. Intron 3 generates an in-frame premature termination codon that predicts the synthesis of a truncated c-Fos protein. This prediction was evaluated by immunoaffinity chromatography purification of c-Fos proteins. We demonstrate that: (i) The c-fos-2 transcript is ubiquitously synthesized either in vivo or in vitro, in amounts that are higher or similar to those of mRNAs coding for other Fos family members, like FosB, DeltaFosB, Fra-1 or Fra-2. (ii) Intron 3 confers to c-fos-2 an outstanding destabilizing effect of about 6-fold. (iii) Major determinant of c-fos-2 steady-state levels in cultured cells is its remarkably high rate of synthesis. (iv) Rapid changes in the synthesis and/or degradation rates of both c-fos transcripts in serum-stimulated cells give rise to rapid and transient changes in their relative proportions. Taken as a whole, these findings suggest a co-ordinated fine-tune of the two c-fos transcript species, supporting the notion that the alternative processing of the precursor mRNA might be physiologically relevant. Moreover, we detected a c-Fos immunoreactive species corresponding in mobility to the predicted truncated variant.

Allelic imbalance modulates surface expression of the tolerance-inducing HLA-G molecule on primary trophoblast cells.

PubMed

Djurisic, S; Teiblum, S; Tolstrup, C K; Christiansen, O B; Hviid, T V F

2015-03-01

The HLA-G molecule is expressed on trophoblast cells at the feto-maternal interface, where it interacts with local immune cells, and upholds tolerance against the semi-allogeneic fetus. Aberrant HLA-G expression in the placenta and reduced soluble HLA-G levels are observed in pregnancy complications, partly explained by HLA-G polymorphisms which are associated with differences in the alternative splicing pattern and of the stability of HLA-G mRNA. Of special importance is a 14 bp insertion/deletion polymorphism located in the 3'-untranslated region of the HLA-G gene. In the current study, we present novel evidence for allelic imbalance of the 14 bp insertion/deletion polymorphism, using a very accurate and sensitive Digital droplet PCR technique. Allelic imbalance in heterozygous samples was observed as differential expression levels of 14 bp insertion/deletion allele-specific mRNA transcripts, which was further associated with low levels of HLA-G surface expression on primary trophoblast cells. Full gene sequencing of HLA-G allowed us to study correlations between HLA-G extended haplotypes and single-nucleotide polymorphisms and HLA-G surface expression. We found that a 1:1 expression (allelic balance) of the 14 bp insertion/deletion mRNA alleles was associated with high surface expression of HLA-G and with a specific HLA-G extended haplotype. The 14 bp del/del genotype was associated with a significantly lower abundance of the G1 mRNA isoform, and a higher abundance of the G3 mRNA isoform. Overall, the present study provides original evidence for allelic imbalance of the 14 bp insertion/deletion polymorphism, which influences HLA-G surface expression on primary trophoblast cells, considered to be important in the pathogenesis of pre-eclampsia and other pregnancy complications. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

RNA G-quadruplexes: emerging mechanisms in disease

PubMed Central

Cammas, Anne

2017-01-01

Abstract RNA G-quadruplexes (G4s) are formed by G-rich RNA sequences in protein-coding (mRNA) and non-coding (ncRNA) transcripts that fold into a four-stranded conformation. Experimental studies and bioinformatic predictions support the view that these structures are involved in different cellular functions associated to both DNA processes (telomere elongation, recombination and transcription) and RNA post-transcriptional mechanisms (including pre-mRNA processing, mRNA turnover, targeting and translation). An increasing number of different diseases have been associated with the inappropriate regulation of RNA G4s exemplifying the potential importance of these structures on human health. Here, we review the different molecular mechanisms underlying the link between RNA G4s and human diseases by proposing several overlapping models of deregulation emerging from recent research, including (i) sequestration of RNA-binding proteins, (ii) aberrant expression or localization of RNA G4-binding proteins, (iii) repeat associated non-AUG (RAN) translation, (iv) mRNA translational blockade and (v) disabling of protein–RNA G4 complexes. This review also provides a comprehensive survey of the functional RNA G4 and their mechanisms of action. Finally, we highlight future directions for research aimed at improving our understanding on RNA G4-mediated regulatory mechanisms linked to diseases. PMID:28013268

Pre-mRNA mis-splicing of sarcomeric genes in heart failure.

PubMed

Zhu, Chaoqun; Chen, Zhilong; Guo, Wei

2017-08-01

Pre-mRNA splicing is an important biological process that allows production of multiple proteins from a single gene in the genome, and mainly contributes to protein diversity in eukaryotic organisms. Alternative splicing is commonly governed by RNA binding proteins to meet the ever-changing demands of the cell. However, the mis-splicing may lead to human diseases. In the heart of human, mis-regulation of alternative splicing has been associated with heart failure. In this short review, we focus on alternative splicing of sarcomeric genes and review mis-splicing related heart failure with relatively well studied Sarcomeric genes and splicing mechanisms with identified regulatory factors. The perspective of alternative splicing based therapeutic strategies in heart failure has also been discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

24 CFR 7.5 - EEO Alternative Dispute Resolution Program.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Statement regarding Alternative Dispute Resolution (ADR) located on the Department's website and 29 CFR 1614.102(b)(2), the Department shall establish and maintain an ADR program that addresses, at a minimum, EEO matters at the pre-complaint and formal complaint stages of the EEO process. ADR is a non...

Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

PubMed Central

Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

2013-01-01

DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

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.

Selective probing of mRNA expression levels within a living cell.

PubMed

Nawarathna, D; Turan, T; Wickramasinghe, H Kumar

2009-08-24

We report on a selective and nondestructive measurement of mRNA (messenger ribonucleic acid) expression levels within a living cell. We first modify an atomic force microscope tip to create a tapered nanoscale coaxial cable. Application of an ac (alternating potential) between the inner and outer electrodes of this cable creates a dielectrophoretic force attracting mRNA molecules toward the tip-end which is pretreated with gene specific primers. We selectively extracted and analyzed both high ( approximately 2500) and extremely low (11 0) copy number mRNA from a living cell mRNA in less than 10 s.

Mammalian Peptidylglycine α-Amidating Monooxygenase mRNA Expression Can Be Modulated by the La Autoantigen

PubMed Central

Brenet, Fabienne; Dussault, Nadège; Borch, Jonas; Ferracci, Géraldine; Delfino, Christine; Roepstorff, Peter; Miquelis, Raymond; Ouafik, L'Houcine

2005-01-01

Peptidylglycine α-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal α-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3′ untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3′ UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3′ UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3′ UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules. PMID:16107699

The yeast RNA1 gene product necessary for RNA processing is located in the cytosol and apparently excluded from the nucleus

PubMed Central

1990-01-01

The yeast RNA1 gene is required for RNA processing and nuclear transport of RNA. The rna1-1 mutation of this locus causes defects in pre-tRNA splicing, processing of the primary pre-rRNA transcript, production of mRNA and export of RNA from the nucleus to the cytosol. To understand how this gene product can pleiotropically affect these processes, we sought to determine the intracellular location of the RNA1 protein. As determined by indirect immunofluorescence localization and organelle fractionation, the RNA1 antigen is found exclusively or primarily in the cytoplasm. Only a tiny fraction of the endogenous protein could be localized to and functional in the nucleus. Furthermore, the RNA1 antigen does not localize differently under stress conditions. These findings suggest that the RNA1 protein is not directly involved in RNA processing but may modify nuclear proteins or otherwise transmit a signal from the cytosol to the nucleus or play a role in maintaining the integrity of the nucleus. PMID:2116418

C/EBPβ-LAP*/LAP Expression Is Mediated by RSK/eIF4B-Dependent Signalling and Boosted by Increased Protein Stability in Models of Monocytic Differentiation

PubMed Central

Christmann, Martin; Friesenhagen, Judith; Westphal, Andreas; Pietsch, Daniel; Brand, Korbinian

2015-01-01

The transcription factor C/EBPβ plays a key role in monocytic differentiation and inflammation. Its small isoform LIP is associated with proliferation at early premonocytic developmental stages and regulated via mTOR-dependent signalling. During later stages of (pre)monocytic differentiation there is a considerable increase in the large C/EBPβ isoforms LAP*/LAP which inhibit proliferation thus supporting terminal differentiation. Here, we showed in different models of monocytic differentiation that this dramatic increase in the LAP*/LAP protein and LAP/LIP ratio was accompanied by an only modest/retarded mRNA increase suggesting an important role for (post)translational mechanisms. We found that LAP*/LAP formation was induced via MEK/RSK-dependent cascades, whereas mTOR/S6K1 were not involved. Remarkably, LAP*/LAP expression was dependent on phosphorylated eIF4B, an acceleratory protein of RNA helicase eIF4A. PKR inhibition reduced the expression of eIF4B and C/EBPβ in an eIF2α-independent manner. Furthermore, under our conditions a marked stabilisation of LAP*/LAP protein occurred, accompanied by reduced chymotrypsin-like proteasome/calpain activities and increased calpastatin levels. Our study elucidates new signalling pathways inducing LAP*/LAP expression and indicates new alternative PKR functions in monocytes. The switch from mTOR- to RSK-mediated signalling to orchestrate eIF4B-dependent LAP*/LAP translation, accompanied by increased protein stability but only small mRNA changes, may be a prototypical example for the regulation of protein expression during selected processes of differentiation/proliferation. PMID:26646662

CELFish ways to modulate mRNA decay

PubMed Central

St. Louis, Irina Vlasova; Dickson, Alexa M.; Bohjanen, Paul R.; Wilusz, Carol J.

2013-01-01

The CELF family of RNA-binding proteins regulates many steps of mRNA metabolism. Although their best characterized function is in pre-mRNA splice site choice, CELF family members are also powerful modulators of mRNA decay. In this review we focus on the different modes of regulation that CELF proteins employ to mediate mRNA decay by binding to GU-rich elements. After starting with an overview of the importance of CELF proteins during development and disease pathogenesis, we then review the mRNA networks and cellular pathways these proteins regulate and the mechanisms by which they influence mRNA decay. Finally, we discuss how CELF protein activity is modulated during development and in response to cellular signals. We conclude by highlighting the priorities for new experiments in this field. PMID:23328451

The Effect of Two Amino acid Residue Substitutions via RNA Editing on Dark-operative Protochlorophyllide Oxidoreductase in the Black Pine Chloroplasts.

PubMed

Yamamoto, Haruki; Kusumi, Junko; Yamakawa, Hisanori; Fujita, Yuichi

2017-05-24

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a key enzyme to produce chlorophyll in the dark. Among photosynthetic eukaryotes, all three subunits chlL, chlN, and chlB are encoded by plastid genomes. In some gymnosperms, two codons of chlB mRNA are changed by RNA editing to codons encoding evolutionarily conserved amino acid residues. However, the effect of these substitutions on DPOR activity remains unknown. We first prepared cyanobacterial ChlB variants with amino acid substitution(s) to mimic ChlB translated from pre-edited mRNA. Their activities were evaluated by measuring chlorophyll content of dark-grown transformants of a chlB-lacking mutant of the cyanobacterium Leptolyngbya boryana that was complemented with pre-edited mimic chlB variants. The chlorophyll content of the transformant cells expressing the ChlB variant from the fully pre-edited mRNA was only one-fourth of the control cells. Co-purification experiments of ChlB with Strep-ChlN suggested that a stable complex with ChlN is greatly impaired in the substituted ChlB variant. We then confirmed that RNA editing efficiency was markedly greater in the dark than in the light in cotyledons of the black pine Pinus thunbergii. These results indicate that RNA editing on chlB mRNA is important to maintain appropriate DPOR activity in black pine chloroplasts.

Folding of a transcriptionally acting PreQ1 riboswitch

PubMed Central

Rieder, Ulrike; Kreutz, Christoph; Micura, Ronald

2010-01-01

7-Aminomethyl-7-deazaguanine (preQ1) sensitive mRNA domains belong to the smallest riboswitches known to date. Although recent efforts have revealed the three-dimensional architecture of the ligand–aptamer complex less is known about the molecular details of the ligand-induced response mechanism that modulates gene expression. We present an in vitro investigation on the ligand-induced folding process of the preQ1 responsive RNA element from Fusobacterium nucleatum using biophysical methods, including fluorescence and NMR spectroscopy of site-specifically labeled riboswitch variants. We provide evidence that the full-length riboswitch domain adopts two different coexisting stem-loop structures in the expression platform. Upon addition of preQ1, the equilibrium of the competing hairpins is significantly shifted. This system therefore, represents a finely tunable antiterminator/terminator interplay that impacts the in vivo cellular response mechanism. A model is presented how a riboswitch that provides no obvious overlap between aptamer and terminator stem-loop solves this communication problem by involving bistable sequence determinants. PMID:20534493

RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators

PubMed Central

Redfern, Andrew D.; Colley, Shane M.; Beveridge, Dianne J.; Ikeda, Naoya; Epis, Michael R.; Li, Xia; Foulds, Charles E.; Stuart, Lisa M.; Barker, Andrew; Russell, Victoria J.; Ramsay, Kerry; Kobelke, Simon J.; Li, Xiaotao; Hatchell, Esme C.; Payne, Christine; Giles, Keith M.; Messineo, Adriana; Gatignol, Anne; Lanz, Rainer B.; O’Malley, Bert W.; Leedman, Peter J.

2013-01-01

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing. PMID:23550157

RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators.

PubMed

Redfern, Andrew D; Colley, Shane M; Beveridge, Dianne J; Ikeda, Naoya; Epis, Michael R; Li, Xia; Foulds, Charles E; Stuart, Lisa M; Barker, Andrew; Russell, Victoria J; Ramsay, Kerry; Kobelke, Simon J; Li, Xiaotao; Hatchell, Esme C; Payne, Christine; Giles, Keith M; Messineo, Adriana; Gatignol, Anne; Lanz, Rainer B; O'Malley, Bert W; Leedman, Peter J

2013-04-16

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.

RNA splicing and splicing regulator changes in prostate cancer pathology.

PubMed

Munkley, Jennifer; Livermore, Karen; Rajan, Prabhakar; Elliott, David J

2017-09-01

Changes in mRNA splice patterns have been associated with key pathological mechanisms in prostate cancer progression. The androgen receptor (abbreviated AR) transcription factor is a major driver of prostate cancer pathology and activated by androgen steroid hormones. Selection of alternative promoters by the activated AR can critically alter gene function by switching mRNA isoform production, including creating a pro-oncogenic isoform of the normally tumour suppressor gene TSC2. A number of androgen-regulated genes generate alternatively spliced mRNA isoforms, including a prostate-specific splice isoform of ST6GALNAC1 mRNA. ST6GALNAC1 encodes a sialyltransferase that catalyses the synthesis of the cancer-associated sTn antigen important for cell mobility. Genetic rearrangements occurring early in prostate cancer development place ERG oncogene expression under the control of the androgen-regulated TMPRSS2 promoter to hijack cell behaviour. This TMPRSS2-ERG fusion gene shows different patterns of alternative splicing in invasive versus localised prostate cancer. Alternative AR mRNA isoforms play a key role in the generation of prostate cancer drug resistance, by providing a mechanism through which prostate cancer cells can grow in limited serum androgen concentrations. A number of splicing regulator proteins change expression patterns in prostate cancer and may help drive key stages of disease progression. Up-regulation of SRRM4 establishes neuronal splicing patterns in neuroendocrine prostate cancer. The splicing regulators Sam68 and Tra2β increase expression in prostate cancer. The SR protein kinase SRPK1 that modulates the activity of SR proteins is up-regulated in prostate cancer and has already given encouraging results as a potential therapeutic target in mouse models.

Alternative splicing of iodothyronine deiodinases in pituitary adenomas. Regulation by oncoprotein SF2/ASF.

PubMed

Piekielko-Witkowska, Agnieszka; Kedzierska, Hanna; Poplawski, Piotr; Wojcicka, Anna; Rybicka, Beata; Maksymowicz, Maria; Grajkowska, Wieslawa; Matyja, Ewa; Mandat, Tomasz; Bonicki, Wieslaw; Nauman, Pawel

2013-06-01

Pituitary tumors belong to the group of most common neoplasms of the sellar region. Iodothyronine deiodinase types 1 (DIO1) and 2 (DIO2) are enzymes contributing to the levels of locally synthesized T3, a hormone regulating key physiological processes in the pituitary, including its development, cellular proliferation, and hormone secretion. Previous studies revealed that the expression of deiodinases in pituitary tumors is variable and, moreover, there is no correlation between mRNA and protein products of the particular gene, suggesting the potential role of posttranscriptional regulatory mechanisms. In this work we hypothesized that one of such mechanisms could be the alternative splicing. Therefore, we analyzed expression and sequences of DIO1 and DIO2 splicing variants in 30 pituitary adenomas and 9 non-tumorous pituitary samples. DIO2 mRNA was expressed as only two mRNA isoforms. In contrast, nine splice variants of DIO1 were identified. Among them, five were devoid of exon 3. In silico sequence analysis of DIO1 revealed multiple putative binding sites for splicing factor SF2/ASF, of which the top-ranked sites were located in exon 3. Silencing of SF2/ASF in pituitary tumor GH3 cells resulted in change of ratio between DIO1 isoforms with or without exon 3, favoring the expression of variants without exon 3. The expression of SF2/ASF mRNA in pituitary tumors was increased when compared with non-neoplastic control samples. In conclusion, we provide a new mechanism of posttranscriptional regulation of DIO1 and show deregulation of DIO1 expression in pituitary adenoma, possibly resulting from disturbed expression of SF2/ASF. Copyright © 2013 Elsevier B.V. All rights reserved.

Evolution of a tissue-specific splicing network

PubMed Central

Taliaferro, J. Matthew; Alvarez, Nehemiah; Green, Richard E.; Blanchette, Marco; Rio, Donald C.

2011-01-01

Alternative splicing of precursor mRNA (pre-mRNA) is a strategy employed by most eukaryotes to increase transcript and proteomic diversity. Many metazoan splicing factors are members of multigene families, with each member having different functions. How these highly related proteins evolve unique properties has been unclear. Here we characterize the evolution and function of a new Drosophila splicing factor, termed LS2 (Large Subunit 2), that arose from a gene duplication event of dU2AF50, the large subunit of the highly conserved heterodimeric general splicing factor U2AF (U2-associated factor). The quickly evolving LS2 gene has diverged from the splicing-promoting, ubiquitously expressed dU2AF50 such that it binds a markedly different RNA sequence, acts as a splicing repressor, and is preferentially expressed in testes. Target transcripts of LS2 are also enriched for performing testes-related functions. We therefore propose a path for the evolution of a new splicing factor in Drosophila that regulates specific pre-mRNAs and contributes to transcript diversity in a tissue-specific manner. PMID:21406555

Identification of an additional member of the protein-tyrosine-phosphatase family: evidence for alternative splicing in the tyrosine phosphatase domain.

PubMed Central

Matthews, R J; Cahir, E D; Thomas, M L

1990-01-01

Protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.13.48) have been implicated in the regulation of cell growth; however, to date few tyrosine phosphatases have been characterized. To identify additional family members, the cDNA for the human tyrosine phosphatase leukocyte common antigen (LCA; CD45) was used to screen, under low stringency, a mouse pre-B-cell cDNA library. Two cDNA clones were isolated and sequence analysis predicts a protein sequence of 793 amino acids. We have named the molecule LRP (LCA-related phosphatase). RNA transfer analysis indicates that the cDNAs were derived from a 3.2-kilobase mRNA. The LRP mRNA is transcribed in a wide variety of tissues. The predicted protein structure can be divided into the following structural features: a short 19-amino acid leader sequence, an exterior domain of 123 amino acids that is predicted to be highly glycosylated, a 24-amino acid membrane-spanning region, and a 627-amino acid cytoplasmic region. The cytoplasmic region contains two approximately 260-amino acid domains, each with homology to the tyrosine phosphatase family. One of the cDNA clones differed in that it had a 108-base-pair insertion that, while preserving the reading frame, would disrupt the first protein-tyrosine-phosphatase domain. Analysis of genomic DNA indicates that the insertion is due to an alternatively spliced exon. LRP appears to be evolutionarily conserved as a putative homologue has been identified in the invertebrate Styela plicata. Images PMID:2162042

Consequences of daily corticosteroid dosing with or without pre-treatment with quinidine on the in vivo cytochrome P450 2D (CYP2D) enzyme in rats: effect on O-demethylation activity of dextromethorphan and expression levels of CYP2D1 mRNA.

PubMed

Giri, Poonam; Delvadia, Prashant; Gupta, Laxmikant; Patel, Nirmal; Trivedi, Priyal; Lad, Krishna; Patel, Hiren M; Srinivas, Nuggehally R

2018-01-01

1. Present investigation was carried out in rats to study influence of corticosteroids after repeated dosing with/without pre-treatment with CYP2D inhibitor quinidine on the CYP2D1 mRNA levels and CYP2D enzyme activity using dextromethorphan as probe substrate. 2. CYP2D1 mRNA was measured in liver homogenate using quantitative real-time polymerase chain reaction [qRT-PCR] and enzymatic reaction was studied ex vivo in liver S-9 fractions of rats treated with oral 10 mg/kg dexamethasone or prednisolone for five days or pre-treated with quinidine and followed by treatment with oral 10 mg/kg corticosteroids for five days. 3. Five days repeat dosing of dexamethasone or prednisolone decreased the activity of the rat liver CYP2D by 37% and 34%, at 30 min incubation and decreased CYP2D1 mRNA levels by 62% and 61%, respectively. 4. Pre-treatment of quinidine decreased the enzymatic activity of rat CYP2D by 58% and did not potentiate CYP2D inhibition by corticosteroids. This observation was further complemented by qRT-PCR data. 5. Corticosteroids caused CYP2D inhibition in rats vs. literature evidence of CYP2D induction in human hepatocytes/pregnant humans demonstrating lack of concordance. In vivo inhibition should be factored for interpretation of pharmacokinetic data of CYP2D substrates when treated with corticosteroids in rats.

XRN2 is required for the degradation of target RNAs by RNase H1-dependent antisense oligonucleotides

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

Hori, Shin-Ichiro; Yamamoto, Tsuyoshi; Obika, Satoshi, E-mail: obika@phs.osaka-u.ac.jp

Antisense oligonucleotides (ASOs) can suppress the expression of a target gene by cleaving pre-mRNA and/or mature mRNA via RNase H1. Following the initial endonucleolytic cleavage by RNase H1, the target RNAs are degraded by a mechanism that is poorly understood. To better understand this degradation pathway, we depleted the expression of two major 5′ to 3′ exoribonucleases (XRNs), named XRN1 and XRN2, and analyzed the levels of 3′ fragments of the target RNAs in vitro. We found that the 3′ fragments of target pre-mRNA generated by ASO were almost completely degraded from their 5′ ends by nuclear XRN2 after RNase H1-mediatedmore » cleavage, whereas the 3′ fragments of mature mRNA were partially degraded by XRN2. In contrast to ASO, small interference RNA (siRNA) could reduce the expression level of only mature mRNA, and the 3′ fragment was degraded by cytoplasmic XRN1. Our findings indicate that the RNAs targeted by RNase H1-dependent ASO are rapidly degraded in the nucleus, contrary to the cytoplasmic degradation pathway mediated by siRNA. - Highlights: • We compared the degradation mechanism of the transcript targeted by ASO and siRNA. • We focused on two 5′ to 3′ exoribonucleases, cytoplasmic XRN1, and nuclear XRN2. • The 3′ fragment of target pre-mRNA generated by ASO was degraded by XRN2. • The 3′ fragment of target mRNA generated by ASO was partially degraded by XRN2. • XRN1 depletion promoted accumulation of the 3′ fragment of mRNA generated by siRNA.« less

Selective probing of mRNA expression levels within a living cell

PubMed Central

Nawarathna, D.; Turan, T.; Wickramasinghe, H. Kumar

2009-01-01

We report on a selective and nondestructive measurement of mRNA (messenger ribonucleic acid) expression levels within a living cell. We first modify an atomic force microscope tip to create a tapered nanoscale coaxial cable. Application of an ac (alternating potential) between the inner and outer electrodes of this cable creates a dielectrophoretic force attracting mRNA molecules toward the tip-end which is pretreated with gene specific primers. We selectively extracted and analyzed both high (∼2500) and extremely low (11¯0) copy number mRNA from a living cell mRNA in less than 10 s. PMID:19777090

mRNA vaccines — a new era in vaccinology

PubMed Central

Pardi, Norbert; Hogan, Michael J.; Porter, Frederick W.; Weissman, Drew

2018-01-01

mRNA vaccines represent a promising alternative to conventional vaccine approaches because of their high potency, capacity for rapid development and potential for low-cost manufacture and safe administration. However, their application has until recently been restricted by the instability and inefficient in vivo delivery of mRNA. Recent technological advances have now largely overcome these issues, and multiple mRNA vaccine platforms against infectious diseases and several types of cancer have demonstrated encouraging results in both animal models and humans. This Review provides a detailed overview of mRNA vaccines and considers future directions and challenges in advancing this promising vaccine platform to widespread therapeutic use. PMID:29326426

mRNA changes in nucleus accumbens related to methamphetamine addiction in mice

NASA Astrophysics Data System (ADS)

Zhu, Li; Li, Jiaqi; Dong, Nan; Guan, Fanglin; Liu, Yufeng; Ma, Dongliang; Goh, Eyleen L. K.; Chen, Teng

2016-11-01

Methamphetamine (METH) is a highly addictive psychostimulant that elicits aberrant changes in the expression of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) in the nucleus accumbens of mice, indicating a potential role of METH in post-transcriptional regulations. To decipher the potential consequences of these post-transcriptional regulations in response to METH, we performed strand-specific RNA sequencing (ssRNA-Seq) to identify alterations in mRNA expression and their alternative splicing in the nucleus accumbens of mice following exposure to METH. METH-mediated changes in mRNAs were analyzed and correlated with previously reported changes in non-coding RNAs (miRNAs and lncRNAs) to determine the potential functions of these mRNA changes observed here and how non-coding RNAs are involved. A total of 2171 mRNAs were differentially expressed in response to METH with functions involved in synaptic plasticity, mitochondrial energy metabolism and immune response. 309 and 589 of these mRNAs are potential targets of miRNAs and lncRNAs respectively. In addition, METH treatment decreases mRNA alternative splicing, and there are 818 METH-specific events not observed in saline-treated mice. Our results suggest that METH-mediated addiction could be attributed by changes in miRNAs and lncRNAs and consequently, changes in mRNA alternative splicing and expression. In conclusion, our study reported a methamphetamine-modified nucleus accumbens transcriptome and provided non-coding RNA-mRNA interaction networks possibly involved in METH addiction.

Effects of SARA on oxygen-glucose deprivation in PC12 cell line.

PubMed

Wang, Jiao-Qi; He, Jin-Ting; Du, Zhen-Wu; Li, Zong-Shu; Liu, Yong-Feng; Mang, Jing; Xu, Zhong-Xin

2013-05-01

Ischemic stroke is a major composition of cerebrovascular disease, seriously threatening to human health in the world. Activin A (ActA), belonging to transforming growth factor-beta (TGF-β) super family, plays an important role in the hypoxic-ischemic brain injury through ActA/Smads pathway. While as an essential phosphorylation assistor in TGF-β signaling, the functions and mechanisms of smad anchor for receptor activation (SARA) in ischemic brain injury remain poorly understood. To solve this problem and explore the pathological processes of ischemic stroke, we used an Oxygen-Glucose deprivation (OGD) model in nerve growth factor-induced differentiated rattus PC12 pheochromocytoma cells and down regulated the expressions of SARA by RNA interference technology. Our results showed that the repression of SARA before OGD exposure reduced the expressions of Smad2, 3, 4 mRNA and the phosphorylation rate of Smad2 protein, but it did not affect the mRNA expressions of Smad7. After OGD treatment, ActA/Smads pathway was activated and the expression of SARA in the SARA pre-repression group was significantly up-regulated. The pre-repression of SARA increased the sensitivities of nerve-like cells to OGD damage. Moreover, the mRNA expression of Smad7 which was supposed to participate in the negative feedback of ActA/Smads pathway was also elevated due to OGD injury. Taken together, these results suggest a positive role of SARA in assisting the phosphorylation of Smad2 and maintaining the neuron protective effect of ActA/Smads pathway.

A Practical Platform for Blood Biomarker Study by Using Global Gene Expression Profiling of Peripheral Whole Blood

PubMed Central

Schmid, Patrick; Yao, Hui; Galdzicki, Michal; Berger, Bonnie; Wu, Erxi; Kohane, Isaac S.

2009-01-01

Background Although microarray technology has become the most common method for studying global gene expression, a plethora of technical factors across the experiment contribute to the variable of genome gene expression profiling using peripheral whole blood. A practical platform needs to be established in order to obtain reliable and reproducible data to meet clinical requirements for biomarker study. Methods and Findings We applied peripheral whole blood samples with globin reduction and performed genome-wide transcriptome analysis using Illumina BeadChips. Real-time PCR was subsequently used to evaluate the quality of array data and elucidate the mode in which hemoglobin interferes in gene expression profiling. We demonstrated that, when applied in the context of standard microarray processing procedures, globin reduction results in a consistent and significant increase in the quality of beadarray data. When compared to their pre-globin reduction counterparts, post-globin reduction samples show improved detection statistics, lowered variance and increased sensitivity. More importantly, gender gene separation is remarkably clearer in post-globin reduction samples than in pre-globin reduction samples. Our study suggests that the poor data obtained from pre-globin reduction samples is the result of the high concentration of hemoglobin derived from red blood cells either interfering with target mRNA binding or giving the pseudo binding background signal. Conclusion We therefore recommend the combination of performing globin mRNA reduction in peripheral whole blood samples and hybridizing on Illumina BeadChips as the practical approach for biomarker study. PMID:19381341

mRNA quality control is bypassed for immediate export of stress-responsive transcripts.

PubMed

Zander, Gesa; Hackmann, Alexandra; Bender, Lysann; Becker, Daniel; Lingner, Thomas; Salinas, Gabriela; Krebber, Heike

2016-12-12

Cells grow well only in a narrow range of physiological conditions. Surviving extreme conditions requires the instantaneous expression of chaperones that help to overcome stressful situations. To ensure the preferential synthesis of these heat-shock proteins, cells inhibit transcription, pre-mRNA processing and nuclear export of non-heat-shock transcripts, while stress-specific mRNAs are exclusively exported and translated. How cells manage the selective retention of regular transcripts and the simultaneous rapid export of heat-shock mRNAs is largely unknown. In Saccharomyces cerevisiae, the shuttling RNA adaptor proteins Npl3, Gbp2, Hrb1 and Nab2 are loaded co-transcriptionally onto growing pre-mRNAs. For nuclear export, they recruit the export-receptor heterodimer Mex67-Mtr2 (TAP-p15 in humans). Here we show that cellular stress induces the dissociation of Mex67 and its adaptor proteins from regular mRNAs to prevent general mRNA export. At the same time, heat-shock mRNAs are rapidly exported in association with Mex67, without the need for adapters. The immediate co-transcriptional loading of Mex67 onto heat-shock mRNAs involves Hsf1, a heat-shock transcription factor that binds to heat-shock-promoter elements in stress-responsive genes. An important difference between the export modes is that adaptor-protein-bound mRNAs undergo quality control, whereas stress-specific transcripts do not. In fact, regular mRNAs are converted into uncontrolled stress-responsive transcripts if expressed under the control of a heat-shock promoter, suggesting that whether an mRNA undergoes quality control is encrypted therein. Under normal conditions, Mex67 adaptor proteins are recruited for RNA surveillance, with only quality-controlled mRNAs allowed to associate with Mex67 and leave the nucleus. Thus, at the cost of error-free mRNA formation, heat-shock mRNAs are exported and translated without delay, allowing cells to survive extreme situations.

Alternative Splicing in Neurogenesis and Brain Development.

PubMed

Su, Chun-Hao; D, Dhananjaya; Tarn, Woan-Yuh

2018-01-01

Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

Fluorescence Reporter-Based Genome-Wide RNA Interference Screening to Identify Alternative Splicing Regulators.

PubMed

Misra, Ashish; Green, Michael R

2017-01-01

Alternative splicing is a regulated process that leads to inclusion or exclusion of particular exons in a pre-mRNA transcript, resulting in multiple protein isoforms being encoded by a single gene. With more than 90 % of human genes known to undergo alternative splicing, it represents a major source for biological diversity inside cells. Although in vitro splicing assays have revealed insights into the mechanisms regulating individual alternative splicing events, our global understanding of alternative splicing regulation is still evolving. In recent years, genome-wide RNA interference (RNAi) screening has transformed biological research by enabling genome-scale loss-of-function screens in cultured cells and model organisms. In addition to resulting in the identification of new cellular pathways and potential drug targets, these screens have also uncovered many previously unknown mechanisms regulating alternative splicing. Here, we describe a method for the identification of alternative splicing regulators using genome-wide RNAi screening, as well as assays for further validation of the identified candidates. With modifications, this method can also be adapted to study the splicing regulation of pre-mRNAs that contain two or more splice isoforms.

Splicing predictions reliably classify different types of alternative splicing

PubMed Central

Busch, Anke; Hertel, Klemens J.

2015-01-01

Alternative splicing is a key player in the creation of complex mammalian transcriptomes and its misregulation is associated with many human diseases. Multiple mRNA isoforms are generated from most human genes, a process mediated by the interplay of various RNA signature elements and trans-acting factors that guide spliceosomal assembly and intron removal. Here, we introduce a splicing predictor that evaluates hundreds of RNA features simultaneously to successfully differentiate between exons that are constitutively spliced, exons that undergo alternative 5′ or 3′ splice-site selection, and alternative cassette-type exons. Surprisingly, the splicing predictor did not feature strong discriminatory contributions from binding sites for known splicing regulators. Rather, the ability of an exon to be involved in one or multiple types of alternative splicing is dictated by its immediate sequence context, mainly driven by the identity of the exon's splice sites, the conservation around them, and its exon/intron architecture. Thus, the splicing behavior of human exons can be reliably predicted based on basic RNA sequence elements. PMID:25805853

Multiperspective smFRET reveals rate-determining late intermediates of ribosomal translocation.

PubMed

Wasserman, Michael R; Alejo, Jose L; Altman, Roger B; Blanchard, Scott C

2016-04-01

Directional translocation of the ribosome through the mRNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of tRNA and mRNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations revealed direct evidence of structurally and kinetically distinct late intermediates during substrate movement, whose resolution determines the rate of translocation. These steps involve intramolecular events within the EF-G-GDP-bound ribosome, including exaggerated, reversible fluctuations of the small-subunit head domain, which ultimately facilitate peptidyl-tRNA's movement into its final post-translocation position.

Detection of alternative splice variants at the proteome level in Aspergillus flavus.

PubMed

Chang, Kung-Yen; Georgianna, D Ryan; Heber, Steffen; Payne, Gary A; Muddiman, David C

2010-03-05

Identification of proteins from proteolytic peptides or intact proteins plays an essential role in proteomics. Researchers use search engines to match the acquired peptide sequences to the target proteins. However, search engines depend on protein databases to provide candidates for consideration. Alternative splicing (AS), the mechanism where the exon of pre-mRNAs can be spliced and rearranged to generate distinct mRNA and therefore protein variants, enable higher eukaryotic organisms, with only a limited number of genes, to have the requisite complexity and diversity at the proteome level. Multiple alternative isoforms from one gene often share common segments of sequences. However, many protein databases only include a limited number of isoforms to keep minimal redundancy. As a result, the database search might not identify a target protein even with high quality tandem MS data and accurate intact precursor ion mass. We computationally predicted an exhaustive list of putative isoforms of Aspergillus flavus proteins from 20 371 expressed sequence tags to investigate whether an alternative splicing protein database can assign a greater proportion of mass spectrometry data. The newly constructed AS database provided 9807 new alternatively spliced variants in addition to 12 832 previously annotated proteins. The searches of the existing tandem MS spectra data set using the AS database identified 29 new proteins encoded by 26 genes. Nine fungal genes appeared to have multiple protein isoforms. In addition to the discovery of splice variants, AS database also showed potential to improve genome annotation. In summary, the introduction of an alternative splicing database helps identify more proteins and unveils more information about a proteome.

La Deletion from Mouse Brain Alters Pre-tRNA Metabolism and Accumulation of Pre-5.8S rRNA, with Neuron Death and Reactive Astrocytosis

PubMed Central

Blewett, Nathan H.; Iben, James R.; Gaidamakov, Sergei

2017-01-01

ABSTRACT Human La antigen (Sjögren's syndrome antigen B [SSB]) is an abundant multifunctional RNA-binding protein. In the nucleoplasm, La binds to and protects from 3′ exonucleases, the ends of precursor tRNAs, and other transcripts synthesized by RNA polymerase III and facilitates their maturation, while a nucleolar isoform has been implicated in rRNA biogenesis by multiple independent lines of evidence. We showed previously that conditional La knockout (La cKO) from mouse cortex neurons results in defective tRNA processing, although the pathway(s) involved in neuronal loss thereafter was unknown. Here, we demonstrate that La is stably associated with a spliced pre-tRNA intermediate. Microscopic evidence of aberrant nuclear accumulation of 5.8S rRNA in La cKO is supported by a 10-fold increase in a pre-5.8S rRNA intermediate. To identify pathways involved in subsequent neurodegeneration and loss of brain mass in the cKO cortex, we employed mRNA sequencing (mRNA-Seq), immunohistochemistry, and other approaches. This revealed robust enrichment of immune and astrocyte reactivity in La cKO cortex. Immunohistochemistry, including temporal analyses, demonstrated neurodegeneration, followed by astrocyte invasion associated with immune response and decreasing cKO cortex size over time. Thus, deletion of La from postmitotic neurons results in defective pre-tRNA and pre-rRNA processing and progressive neurodegeneration with loss of cortical brain mass. PMID:28223366

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

Exploring the Digital Natives among Pre-Service Teachers in Turkey: A Cross-Cultural Validation of the Digital Native Assessment Scale

ERIC Educational Resources Information Center

Teo, Timothy; Kabakçi Yurdakul, Isil; Ursavas, Ömer Faruk

2016-01-01

This study aims to explore the digital natives among a sample of pre-service teachers and in the process, examine the validity of a Turkish adaptation of the digital native assessment scale (DNAS) [Teo, T., & Fan, X. (2013). "Coefficient alpha and beyond: Issues and alternatives for educational research." "The Asia-Pacific…

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.

The RtcB RNA ligase is an essential component of the metazoan unfolded protein response.

PubMed

Kosmaczewski, Sara Guckian; Edwards, Tyson James; Han, Sung Min; Eckwahl, Matthew J; Meyer, Benjamin Isaiah; Peach, Sally; Hesselberth, Jay R; Wolin, Sandra L; Hammarlund, Marc

2014-12-01

RNA ligation can regulate RNA function by altering RNA sequence, structure and coding potential. For example, the function of XBP1 in mediating the unfolded protein response requires RNA ligation, as does the maturation of some tRNAs. Here, we describe a novel in vivo model in Caenorhabditis elegans for the conserved RNA ligase RtcB and show that RtcB ligates the xbp-1 mRNA during the IRE-1 branch of the unfolded protein response. Without RtcB, protein stress results in the accumulation of unligated xbp-1 mRNA fragments, defects in the unfolded protein response, and decreased lifespan. RtcB also ligates endogenous pre-tRNA halves, and RtcB mutants have defects in growth and lifespan that can be bypassed by expression of pre-spliced tRNAs. In addition, animals that lack RtcB have defects that are independent of tRNA maturation and the unfolded protein response. Thus, RNA ligation by RtcB is required for the function of multiple endogenous target RNAs including both xbp-1 and tRNAs. RtcB is uniquely capable of performing these ligation functions, and RNA ligation by RtcB mediates multiple essential processes in vivo. © 2014 The Authors.

The RtcB RNA ligase is an essential component of the metazoan unfolded protein response

PubMed Central

Kosmaczewski, Sara Guckian; Edwards, Tyson James; Han, Sung Min; Eckwahl, Matthew J; Meyer, Benjamin Isaiah; Peach, Sally; Hesselberth, Jay R; Wolin, Sandra L; Hammarlund, Marc

2014-01-01

RNA ligation can regulate RNA function by altering RNA sequence, structure and coding potential. For example, the function of XBP1 in mediating the unfolded protein response requires RNA ligation, as does the maturation of some tRNAs. Here, we describe a novel in vivo model in Caenorhabditis elegans for the conserved RNA ligase RtcB and show that RtcB ligates the xbp-1 mRNA during the IRE-1 branch of the unfolded protein response. Without RtcB, protein stress results in the accumulation of unligated xbp-1 mRNA fragments, defects in the unfolded protein response, and decreased lifespan. RtcB also ligates endogenous pre-tRNA halves, and RtcB mutants have defects in growth and lifespan that can be bypassed by expression of pre-spliced tRNAs. In addition, animals that lack RtcB have defects that are independent of tRNA maturation and the unfolded protein response. Thus, RNA ligation by RtcB is required for the function of multiple endogenous target RNAs including both xbp-1 and tRNAs. RtcB is uniquely capable of performing these ligation functions, and RNA ligation by RtcB mediates multiple essential processes in vivo. Subject Categories Protein Biosynthesis & Quality Control; RNA Biology PMID:25366321

Comparative Analysis of mRNA Isoform Expression in Cardiac Hypertrophy and Development Reveals Multiple Post-Transcriptional Regulatory Modules

PubMed Central

Park, Ji Yeon; Li, Wencheng; Zheng, Dinghai; Zhai, Peiyong; Zhao, Yun; Matsuda, Takahisa; Vatner, Stephen F.; Sadoshima, Junichi; Tian, Bin

2011-01-01

Cardiac hypertrophy is enlargement of the heart in response to physiological or pathological stimuli, chiefly involving growth of myocytes in size rather than in number. Previous studies have shown that the expression pattern of a group of genes in hypertrophied heart induced by pressure overload resembles that at the embryonic stage of heart development, a phenomenon known as activation of the “fetal gene program”. Here, using a genome-wide approach we systematically defined genes and pathways regulated in short- and long-term cardiac hypertrophy conditions using mice with transverse aortic constriction (TAC), and compared them with those regulated at different stages of embryonic and postnatal development. In addition, exon-level analysis revealed widespread mRNA isoform changes during cardiac hypertrophy resulting from alternative usage of terminal or internal exons, some of which are also developmentally regulated and may be attributable to decreased expression of Fox-1 protein in cardiac hypertrophy. Genes with functions in certain pathways, such as cell adhesion and cell morphology, are more likely to be regulated by alternative splicing. Moreover, we found 3′UTRs of mRNAs were generally shortened through alternative cleavage and polyadenylation in hypertrophy, and microRNA target genes were generally de-repressed, suggesting coordinated mechanisms to increase mRNA stability and protein production during hypertrophy. Taken together, our results comprehensively delineated gene and mRNA isoform regulation events in cardiac hypertrophy and revealed their relations to those in development, and suggested that modulation of mRNA isoform expression plays an importance role in heart remodeling under pressure overload. PMID:21799842

Partial androgen insensitivity syndrome caused by a deep intronic mutation creating an alternative splice acceptor site of the AR gene.

PubMed

Ono, Hiroyuki; Saitsu, Hirotomo; Horikawa, Reiko; Nakashima, Shinichi; Ohkubo, Yumiko; Yanagi, Kumiko; Nakabayashi, Kazuhiko; Fukami, Maki; Fujisawa, Yasuko; Ogata, Tsutomu

2018-02-02

Although partial androgen insensitivity syndrome (PAIS) is caused by attenuated responsiveness to androgens, androgen receptor gene (AR) mutations on the coding regions and their splice sites have been identified only in <25% of patients with a diagnosis of PAIS. We performed extensive molecular studies including whole exome sequencing in a Japanese family with PAIS, identifying a deep intronic variant beyond the branch site at intron 6 of AR (NM_000044.4:c.2450-42 G > A). This variant created the splice acceptor motif that was accompanied by pyrimidine-rich sequence and two candidate branch sites. Consistent with this, reverse transcriptase (RT)-PCR experiments for cycloheximide-treated lymphoblastoid cell lines revealed a relatively large amount of aberrant mRNA produced by the newly created splice acceptor site and a relatively small amount of wildtype mRNA produced by the normal splice acceptor site. Furthermore, most of the aberrant mRNA was shown to undergo nonsense mediated decay (NMD) and, if a small amount of aberrant mRNA may have escaped NMD, such mRNA was predicted to generate a truncated AR protein missing some functional domains. These findings imply that the deep intronic mutation creating an alternative splice acceptor site resulted in the production of a relatively small amount of wildtype AR mRNA, leading to PAIS.

Detection and Quantification of the Fragile X Mental Retardation Protein 1 (FMRP).

PubMed

LaFauci, Giuseppe; Adayev, Tatyana; Kascsak, Richard; Brown, W Ted

2016-12-09

The final product of FMR1 gene transcription, Fragile X Mental Retardation Protein 1 (FMRP), is an RNA binding protein that acts as a repressor of translation. FMRP is expressed in several tissues and plays important roles in neurogenesis, synaptic plasticity, and ovarian functions and has been implicated in a number of neuropsychological disorders. The loss of FMRP causes Fragile X Syndrome (FXS). In most cases, FXS is due to large expansions of a CGG repeat in FMR1 -normally containing 6-54 repeats-to over 200 CGGs and identified as full mutation (FM). Hypermethylation of the repeat induces FMR1 silencing and lack of FMRP expression in FM male. Mosaic FM males express low levels of FMRP and present a less severe phenotype that inversely correlates with FMRP levels. Carriers of pre-mutations (55-200 CGG) show increased mRNA, and normal to reduced FMRP levels. Alternative splicing of FMR1 mRNA results in 24 FMRP predicted isoforms whose expression are tissues and developmentally regulated. Here, we summarize the approaches used by several laboratories including our own to (a) detect and estimate the amount of FMRP in different tissues, developmental stages and various pathologies; and (b) to accurately quantifying FMRP for a direct diagnosis of FXS in adults and newborns.

Detection and Quantification of the Fragile X Mental Retardation Protein 1 (FMRP)

PubMed Central

LaFauci, Giuseppe; Adayev, Tatyana; Kascsak, Richard; Brown, W. Ted

2016-01-01

The final product of FMR1 gene transcription, Fragile X Mental Retardation Protein 1 (FMRP), is an RNA binding protein that acts as a repressor of translation. FMRP is expressed in several tissues and plays important roles in neurogenesis, synaptic plasticity, and ovarian functions and has been implicated in a number of neuropsychological disorders. The loss of FMRP causes Fragile X Syndrome (FXS). In most cases, FXS is due to large expansions of a CGG repeat in FMR1—normally containing 6–54 repeats—to over 200 CGGs and identified as full mutation (FM). Hypermethylation of the repeat induces FMR1 silencing and lack of FMRP expression in FM male. Mosaic FM males express low levels of FMRP and present a less severe phenotype that inversely correlates with FMRP levels. Carriers of pre-mutations (55–200 CGG) show increased mRNA, and normal to reduced FMRP levels. Alternative splicing of FMR1 mRNA results in 24 FMRP predicted isoforms whose expression are tissues and developmentally regulated. Here, we summarize the approaches used by several laboratories including our own to (a) detect and estimate the amount of FMRP in different tissues, developmental stages and various pathologies; and (b) to accurately quantifying FMRP for a direct diagnosis of FXS in adults and newborns. PMID:27941672

The stress-induced heat shock protein 70.3 expression is regulated by a dual-component mechanism involving alternative polyadenylation and HuR.

PubMed

Kraynik, Stephen M; Gabanic, Andrew; Anthony, Sarah R; Kelley, Melissa; Paulding, Waltke R; Roessler, Anne; McGuinness, Michael; Tranter, Michael

2015-06-01

Heat shock protein 70.3 (Hsp70.3) expression increases in response to cellular stress and plays a cytoprotective role. We have previously shown that Hsp70.3 expression is controlled through coordinated post-transcriptional regulation by miRNAs and alternative polyadenylation (APA), and APA-mediated shortening of the Hsp70.3 3'-UTR facilitates increased protein expression. A stress-induced increase in Hsp70.3 mRNA and protein expression is accompanied by alternative polyadenylation (APA)-mediated truncation of the 3'UTR of the Hsp70.3 mRNA transcript. However, the role that APA plays in stress-induced expression of Hsp70.3 remains unclear. Our results show that APA-mediated truncation of the Hsp70.3 3'UTR increases protein expression through enhanced polyribosome loading. Additionally, we demonstrate that the RNA binding protein HuR, which has been previously shown to play a role in mediating APA, is necessary for heat shock mediated increase in Hsp70.3 mRNA and protein. However, it is somewhat surprising to note that HuR does not play a role in APA of the Hsp70.3 mRNA, and these two regulatory events appear to be mutually exclusive regulators of Hsp70.3 expression. These results not only provide important insight to the regulation of stress response genes following heat shock, but also contribute an enhanced understanding of how alternative polyadenylation contributes to gene regulation. Copyright © 2015 Elsevier B.V. All rights reserved.

Cyclic AMP-dependent modification of gonad-selective TAF(II)105 in a human ovarian granulosa cell line.

PubMed

Wu, Yimin; Lu, Yunzhe; Hu, Yanfen; Li, Rong

2005-11-01

In response to gonadotropins, the elevated level of intracellular-cyclic AMP (cAMP) in ovarian granulosa cells triggers an ordered activation of multiple ovarian genes, which in turn promotes various ovarian functions including folliculogenesis and steroidogenesis. Identification and characterization of transcription factors that control ovarian gene expression are pivotal to the understanding of the molecular basis of the tissue-specific gene regulation programs. The recent discovery of the mouse TATA binding protein (TBP)-associated factor 105 (TAF(II)105) as a gonad-selective transcriptional co-activator strongly suggests that general transcription factors such as TFIID may play a key role in regulating tissue-specific gene expression. Here we show that the human TAF(II)105 protein is preferentially expressed in ovarian granulosa cells. We also identified a novel TAF(II)105 mRNA isoform that results from alternative exon inclusion and is predicted to encode a dominant negative mutant of TAF(II)105. Following stimulation by the adenylyl cyclase activator forskolin, TAF(II)105 in granulosa cells undergoes rapid and transient phosphorylation that is dependent upon protein kinase A (PKA). Thus, our work suggests that pre-mRNA processing and post-translational modification represent two important regulatory steps for the gonad-specific functions of human TAF(II)105. Copyright 2005 Wiley-Liss, Inc.

Small Molecule Modulators of Pre-mRNA Splicing in Cancer Therapy.

PubMed

Salton, Maayan; Misteli, Tom

2016-01-01

Pre-mRNA splicing is a fundamental process in mammalian gene expression and alternative RNA splicing plays a considerable role in generating protein diversity. RNA splicing events are also key to the pathology of numerous diseases, particularly cancers. Some tumors are molecularly addicted to specific RNA splicing isoforms making interference with pre-mRNA processing a viable therapeutic strategy. Several RNA splicing modulators have recently been characterized, some showing promise in preclinical studies. While the targets of most splicing modulators are constitutive RNA processing components, possibly leading to undesirable side effects, selectivity for individual splicing events has been observed. Given the high prevalence of splicing defects in cancer, small molecule modulators of RNA processing represent a potentially promising novel therapeutic strategy in cancer treatment. Here, we review their reported effects, mechanisms, and limitations. Published by Elsevier Ltd.

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.

Effects of Coriolus versicolor polysaccharide B on monocyte chemoattractant protein 1 gene expression in rat.

PubMed

Song, Lie-Chang; Chen, Hai-Sheng; Lou, Ning; Song, Chang; Zeng, Jun; Fu, Ting-Huan

2002-06-01

To investigate the effect of Coriolus versicolor polysaccharide B (CVPS-B), a new water-soluble component of polysaccharides from the fungus Coriolus versicolor (Fr) L on monocyte chemoattractant protein-1 (MCP-1) gene expression in rat splenocytes. Expression of MCP-1 mRNA in rat splenocytes was examined by reverse transcription-polymerase chain reaction (RT-PCR) with beta- actin as an internal standard. Sequencing of RT-PCR products was performed to confirm their specificity in MCP-1 gene composition. (1) Without pre-treatment of lipopolysaccharide (LPS), the relative MCP-1 mRNA expression ratios (MCP-1/beta-actin) for the saline control group and for CVPS-B groups in 3 different doses (10, 20, and 30 mg . kg-1 . d-1, ip, for 4 d) were 1.4 +/- 0.3, 1.6 +/- 0.4, 1.7 +/- 0.5, and 1.5 +/- 0.4, respectively (P > 0.05); (2) LPS (10 microg . kg-1, ip) enhanced the expression of MPC-1 mRNA by the ratio of 114 %; (3) pre-treatment with CVPS-B of 4 different doses (5, 10, 30, and 50 mg . kg-1 . d-1, ip, for 4 d) decreased the LPS induced expression of MPC-1 mRNA by the ratios of 51 %, 70 %, 84 %, and 99 %, respectively (n = 6). In a dose-related fashion, CVPS-B inhibited the expression of MCP-1 mRNA induced by LPS in the rat splenocytes, but did not significantly affect the expression of MPC-1 mRNA in the normal rat.

Heart failure-associated changes in RNA splicing of sarcomere genes.

PubMed

Kong, Sek Won; Hu, Yong Wu; Ho, Joshua W K; Ikeda, Sadakatsu; Polster, Sean; John, Ranjit; Hall, Jennifer L; Bisping, Egbert; Pieske, Burkert; dos Remedios, Cristobal G; Pu, William T

2010-04-01

Alternative mRNA splicing is an important mechanism for regulation of gene expression. Altered mRNA splicing occurs in association with several types of cancer, and a small number of disease-associated changes in splicing have been reported in heart disease. However, genome-wide approaches have not been used to study splicing changes in heart disease. We hypothesized that mRNA splicing is different in diseased hearts compared with control hearts. We used the Affymetrix Exon array to globally evaluate mRNA splicing in left ventricular myocardial RNA from controls (n=15) and patients with ischemic cardiomyopathy (n=15). We observed a broad and significant decrease in mRNA splicing efficiency in heart failure, which affected some introns to a greater extent than others. The profile of mRNA splicing separately clustered ischemic cardiomyopathy and control samples, suggesting distinct changes in mRNA splicing between groups. Reverse transcription-polymerase chain reaction validated 9 previously unreported alternative splicing events. Furthermore, we demonstrated that splicing of 4 key sarcomere genes, cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin heavy chain 7 (MYH7), and filamin C, gamma (FLNC), was significantly altered in ischemic cardiomyopathy and in dilated cardiomyopathy and aortic stenosis. In aortic stenosis samples, these differences preceded the onset of heart failure. Remarkably, the ratio of minor to major splice variants of TNNT2, MYH7, and FLNC classified independent test samples as control or disease with >98% accuracy. Our data indicate that mRNA splicing is broadly altered in human heart disease and that patterns of aberrant RNA splicing accurately assign samples to control or disease classes.

The involvement of mRNA processing factors TIA-1, TIAR, and PABP-1 during mammalian hibernation.

PubMed

Tessier, Shannon N; Audas, Timothy E; Wu, Cheng-Wei; Lee, Stephen; Storey, Kenneth B

2014-11-01

Mammalian hibernators survive low body temperatures, ischemia-reperfusion, and restricted nutritional resources via global reductions in energy-expensive cellular processes and selective increases in stress pathways. Consequently, studies that analyze hibernation uncover mechanisms which balance metabolism and support survival by enhancing stress tolerance. We hypothesized processing factors that influence messenger ribonucleic acid (mRNA) maturation and translation may play significant roles in hibernation. We characterized the amino acid sequences of three RNA processing proteins (T cell intracellular antigen 1 (TIA-1), TIA1-related (TIAR), and poly(A)-binding proteins (PABP-1)) from thirteen-lined ground squirrels (Ictidomys tridecemlineatus), which all displayed a high degree of sequence identity with other mammals. Alternate Tia-1 and TiaR gene variants were found in the liver with higher expression of isoform b versus a in both cases. The localization of RNA-binding proteins to subnuclear structures was assessed by immunohistochemistry and confirmed by subcellular fractionation; TIA-1 was identified as a major component of subnuclear structures with up to a sevenfold increase in relative protein levels in the nucleus during hibernation. By contrast, there was no significant difference in the relative protein levels of TIARa/TIARb in the nucleus, and a decrease was observed for TIAR isoforms in cytoplasmic fractions of torpid animals. Finally, we used solubility tests to analyze the formation of reversible aggregates that are associated with TIA-1/R function during stress; a shift towards the soluble fraction (TIA-1a, TIA-1b) was observed during hibernation suggesting enhanced protein aggregation was not present during torpor. The present study identifies novel posttranscriptional regulatory mechanisms that may play a role in reducing translational rates and/or mRNA processing under unfavorable environmental conditions.

How can Steganography BE AN Interpretation of the Redundancy in Pre-Mrna Ribbon?

NASA Astrophysics Data System (ADS)

Regoli, Massimo

2013-01-01

In the past years we have developed a new symmetric encryption algorithm based on a new interpretation of the biological phenomenon of the presence of redundant sequences inside pre-mRNA (the introns apparently junk DNA) from a `science of information' point of view. For the first, we have shown the flow of the algorithm by creating a parallel between the various biological aspects of the phenomenon of redundancy and the corresponding agents in our encryption algorithm. Then we set a strict mathematical terminology identifying spaces and mathematical operators for the correct application and interpretation of the algorithm. Finally, last year, we proved that our algorithm has excellent statistics behavior being able to exceed the standard static tests. This year we will try to add a new operator (agent) that is capable of allowing the introduction of a mechanisms like a steganographic sub message (sub ribbon of mRNA) inside the original message (mRNA ribbon).

Effects of interleukin-8 on estradiol and progesterone production by bovine granulosa cells from large follicles and progesterone production by luteinizing granulosa cells in culture.

PubMed

Shimizu, Takashi; Kaji, Ayami; Murayama, Chiaki; Magata, Fumie; Shirasuna, Koumei; Wakamiya, Kaori; Okuda, Kiyoshi; Miyamoto, Akio

2012-01-01

Interleukin 8 (IL-8) is a chemoattractant involved in the recruitment and activation of neutrophils and is associated with the ovulate process. We examined the possible role of IL-8 in steroid production by bovine granulosa cells before and after ovulation. The concentration of IL-8 in the follicular fluid of estrogen-active dominant (EAD) and pre-ovulatory follicles (POF) was higher than that of small follicles (SF). CXCR1 mRNA expression was higher in the granulosa cells of EAD and POF than that of SF. In contrast, CXCR2 mRNA expression was lower in granulosa cells of EAD and POF than in SF. IL-8 inhibited estradiol (E2) production in follicle-stimulating hormone (FSH)-treated granulosa cells at 48 h of culture. IL-8 also suppressed CYP19A1 mRNA expression in FSH-treated granulosa cells. IL-8 stimulated progesterone (P4) production in luteinizing hormone (LH)-treated granulosa cells at 48 h of culture. Although IL-8 did not alter the expression of genes associated with P4 production, it induced StAR protein expression in LH-treated granulosa cells. The expression of CXCR1 mRNA in corpus luteum (CL) did not change during the luteal phase. In contrast, the expression of CXCR2 mRNA in middle CL was significantly higher than in early and regression CL during the luteal phase. In luteinizing granulosa cells, an in vitro model of granulosa cell luteinization, CXCR2 mRNA expression was downregulated, whereas CXCR1 mRNA expression was unchanged. IL-8 also stimulated P4 production in luteinizing granulosa cells. These data provide evidence that IL-8 functions not only as a chemokine, but also act as a regulator of steroid synthesis in granulosa cells to promote luteinization after ovulation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Transcription boundaries of U1 small nuclear RNA.

PubMed Central

Kunkel, G R; Pederson, T

1985-01-01

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly. Images PMID:2942763

Alternative promoter usage generates novel shorter MAPT mRNA transcripts in Alzheimer's disease and progressive supranuclear palsy brains.

PubMed

Huin, Vincent; Buée, Luc; Behal, Hélène; Labreuche, Julien; Sablonnière, Bernard; Dhaenens, Claire-Marie

2017-10-03

Alternative promoter usage is an important mechanism for transcriptome diversity and the regulation of gene expression. Indeed, this alternative usage may influence tissue/subcellular specificity, protein translation and function of the proteins. The existence of an alternative promoter for MAPT gene was considered for a long time to explain differential tissue specificity and differential response to transcription and growth factors between mRNA transcripts. The alternative promoter usage could explain partly the different tau proteins expression patterns observed in tauopathies. Here, we report on our discovery of a functional alternative promoter for MAPT, located upstream of the gene's second exon (exon 1). By analyzing genome databases and brain tissue from control individuals and patients with Alzheimer's disease or progressive supranuclear palsy, we identified novel shorter transcripts derived from this alternative promoter. These transcripts are increased in patients' brain tissue as assessed by 5'RACE-PCR and qPCR. We suggest that these new MAPT isoforms can be translated into normal or amino-terminal-truncated tau proteins. We further suggest that activation of MAPT's alternative promoter under pathological conditions leads to the production of truncated proteins, changes in protein localization and function, and thus neurodegeneration.

[Molecular mechanisms of protein biosynthesis initiation--biochemical and biomedical implications of a new model of translation enhanced by the RNA hypoxia response element (rHRE)].

PubMed

Master, Adam; Nauman, Alicja

2014-01-01

Translation initiation is a key rate-limiting step in cellular protein synthesis. A cap-dependent initiation is the most effective mechanism of the translation. However, some physiological (mitosis) and pathological (oxidative stress) processes may switch the classic mechanism to an alternative one that is regulated by an mRNA element such as IRES, uORF, IRE, CPE, DICE, AURE or CITE. A recently discovered mechanism of RNA hypoxia response element (rHRE)-dependent translation initiation, may change the view of oxygen-regulated translation and give a new insight into unexplained biochemical processes. Hypoxia is one of the better-known factors that may trigger an alternative mechanism of the translation initiation. Temporal events of oxygen deficiency within tissues and organs may activate processes such as angiogenesis, myogenesis, regeneration, wound healing, and may promote an adaptive response in cardiovascular and neurodegenerative diseases. On the other hand, growth of solid tumors may be accompanied by cyclic hypoxia, allowing for synthesis of proteins required for further progression of cancer cells. This paper provides a review of current knowledge on translational control in the context of alternative models of translation initiation.

Coupling mRNA processing with transcription in time and space

PubMed Central

Bentley, David L.

2015-01-01

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

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.

Bio—Cryptography: A Possible Coding Role for RNA Redundancy

NASA Astrophysics Data System (ADS)

Regoli, M.

2009-03-01

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 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 behavior 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.

A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease.

PubMed

Chang, Mary Y; Tanino, Yoshinori; Vidova, Veronika; Kinsella, Michael G; Chan, Christina K; Johnson, Pamela Y; Wight, Thomas N; Frevert, Charles W

2014-02-01

The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4(-/-) mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection. Copyright © 2014 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

A Life Cycle Assessment (LCA) comparison of three management options for waste papers: bioethanol production, recycling and incineration with energy recovery.

PubMed

Wang, Lei; Templer, Richard; Murphy, Richard J

2012-09-01

This study uses Life Cycle Assessment (LCA) to assess the environmental profiles and greenhouse gas (GHG) emissions for bioethanol production from waste papers and to compare them with the alternative waste management options of recycling or incineration with energy recovery. Bioethanol production scenarios both with and without pre-treatments were conducted. It was found that an oxidative lime pre-treatment reduced GHG emissions and overall environmental burdens for a newspaper-to-bioethanol process whereas a dilute acid pre-treatment raised GHG emissions and overall environmental impacts for an office paper-to-bioethanol process. In the comparison of bioethanol production systems with alternative management of waste papers by different technologies, it was found that the environmental profiles of each system vary significantly and this variation affects the outcomes of the specific comparisons made. Overall, a number of configurations of bioethanol production from waste papers offer environmentally favourable or neutral profiles when compared with recycling or incineration. Copyright © 2012 Elsevier Ltd. All rights reserved.

Alternative intronic promoters in development and disease.

PubMed

Vacik, Tomas; Raska, Ivan

2017-05-01

Approximately 20,000 mammalian genes are estimated to encode between 250 thousand and 1 million different proteins. This enormous diversity of the mammalian proteome is caused by the ability of a single-gene locus to encode multiple protein isoforms. Protein isoforms encoded by one gene locus can be functionally distinct, and they can even have antagonistic functions. One of the mechanisms involved in creating this proteome complexity is alternative promoter usage. Alternative intronic promoters are located downstream from their canonical counterparts and drive the expression of alternative RNA isoforms that lack upstream exons. These upstream exons can encode some important functional domains, and proteins encoded by alternative mRNA isoforms can be thus functionally distinct from the full-length protein encoded by canonical mRNA isoforms. Since any misbalance of functionally distinct protein isoforms is likely to have detrimental consequences for the cell and the whole organism, their expression must be precisely regulated. Misregulation of alternative intronic promoters is frequently associated with various developmental defects and diseases including cancer, and it is becoming increasingly clear that this phenomenon deserves more attention.

Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

PubMed Central

Martins, Rute; Proença, Daniela; Silva, Bruno; Barbosa, Cristina; Silva, Ana Luísa; Faustino, Paula; Romão, Luísa

2012-01-01

Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 3′ untranslated region (UTR), along with exon seven. Therefore, this 3′ UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 3′-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements. PMID:22530027

Alternative splicing of the tyrosinase gene transcript in normal human melanocytes and lymphocytes.

PubMed

Fryer, J P; Oetting, W S; Brott, M J; King, R A

2001-11-01

We have identified and isolated ectopically expressed tyrosinase transcripts in normal human melanocytes and lymphocytes and in a human melanoma (MNT-1) cell line to establish a baseline for the expression pattern of this gene in normal tissue. Tyrosinase mRNA from human lymphoblastoid cell lines was reverse transcribed and amplified using specific "nested" primers. This amplification yielded eight identifiable transcripts; five that resulted from alternative splicing patterns arising from the utilization of normal and alternative splice sequences. Identical splicing patterns were found in transcripts from human primary melanocytes in culture and a melanoma cell line, indicating that lymphoblastoid cell lines provide an accurate reflection of transcript processing in melanocytes. Similar splicing patterns have also been found with murine melanocyte tyrosinase transcripts. Our results demonstrate that alternative splicing of human tyrosinase gene transcript produces a number of predictable and identifiable transcripts, and that human lymphoblastoid cell lines provide a source of ectopically expressed transcripts that can be used to study the biology of tyrosinase gene expression in humans.

A novel sodium bicarbonate cotransporter-like gene in an ancient duplicated region: SLC4A9 at 5q31

PubMed Central

Lipovich, Leonard; Lynch, Eric D; Lee, Ming K; King, Mary-Claire

2001-01-01

Background: Sodium bicarbonate cotransporter (NBC) genes encode proteins that execute coupled Na+ and HCO3- transport across epithelial cell membranes. We report the discovery, characterization, and genomic context of a novel human NBC-like gene, SLC4A9, on chromosome 5q31. Results: SLC4A9 was initially discovered by genomic sequence annotation and further characterized by sequencing of long-insert cDNA library clones. The predicted protein of 990 amino acids has 12 transmembrane domains and high sequence similarity to other NBCs. The 23-exon gene has 14 known mRNA isoforms. In three regions, mRNA sequence variation is generated by the inclusion or exclusion of portions of an exon. Noncoding SLC4A9 cDNAs were recovered multiple times from different libraries. The 3' untranslated region is fragmented into six alternatively spliced exons and contains expressed Alu, LINE and MER repeats. SLC4A9 has two alternative stop codons and six polyadenylation sites. Its expression is largely restricted to the kidney. In silico approaches were used to characterize two additional novel SLC4A genes and to place SLC4A9 within the context of multiple paralogous gene clusters containing members of the epidermal growth factor (EGF), ankyrin (ANK) and fibroblast growth factor (FGF) families. Seven human EGF-SLC4A-ANK-FGF clusters were found. Conclusion: The novel sodium bicarbonate cotransporter-like gene SLC4A9 demonstrates abundant alternative mRNA processing. It belongs to a growing class of functionally diverse genes characterized by inefficient highly variable splicing. The evolutionary history of the EGF-SLC4A-ANK-FGF gene clusters involves multiple rounds of duplication, apparently followed by large insertions and deletions at paralogous loci and genome-wide gene shuffling. PMID:11305939

Profiling Pre-MicroRNA and Mature MicroRNA Expressions Using a Single Microarray and Avoiding Separate Sample Preparation

PubMed Central

Gan, Lin; Denecke, Bernd

2013-01-01

Mature microRNA is a crucial component in the gene expression regulation network. At the same time, microRNA gene expression and procession is regulated in a precise and collaborated way. Pre-microRNAs mediate products during the microRNA transcription process, they can provide hints of microRNA gene expression regulation or can serve as alternative biomarkers. To date, little effort has been devoted to pre-microRNA expression profiling. In this study, three human and three mouse microRNA profile data sets, based on the Affymetrix miRNA 2.0 array, have been re-analyzed for both mature and pre-microRNA signals as a primary test of parallel mature/pre-microRNA expression profiling on a single platform. The results not only demonstrated a glimpse of pre-microRNA expression in human and mouse, but also the relationship of microRNA expressions between pre- and mature forms. The study also showed a possible application of currently available microRNA microarrays in profiling pre-microRNA expression in a time and cost effective manner. PMID:27605179

Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood.

PubMed

Zheng, Zhi; Luo, Yuling; McMaster, Gary K

2006-07-01

Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format. We performed single- and multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection. The single- and multiplex assays could quantitatively measure as few as 6000 and 24,000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 microL of whole blood. Both formats had CVs < 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar to those obtained by quantitative reverse transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAXgene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood. Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood.

Nuclear m6A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development.

PubMed

Kasowitz, Seth D; Ma, Jun; Anderson, Stephen J; Leu, N Adrian; Xu, Yang; Gregory, Brian D; Schultz, Richard M; Wang, P Jeremy

2018-05-25

The N6-methyladenosine (m6A) modification is the most prevalent internal RNA modification in eukaryotes. The majority of m6A sites are found in the last exon and 3' UTRs. Here we show that the nuclear m6A reader YTHDC1 is essential for embryo viability and germline development in mouse. Specifically, YTHDC1 is required for spermatogonial development in males and for oocyte growth and maturation in females; Ythdc1-deficient oocytes are blocked at the primary follicle stage. Strikingly, loss of YTHDC1 leads to extensive alternative polyadenylation in oocytes, altering 3' UTR length. Furthermore, YTHDC1 deficiency causes massive alternative splicing defects in oocytes. The majority of splicing defects in mutant oocytes are rescued by introducing wild-type, but not m6A-binding-deficient, YTHDC1. YTHDC1 is associated with the pre-mRNA 3' end processing factors CPSF6, SRSF3, and SRSF7. Thus, YTHDC1 plays a critical role in processing of pre-mRNA transcripts in the oocyte nucleus and may have similar non-redundant roles throughout fetal development.

WNT2B2 mRNA, up-regulated in primary gastric cancer, is a positive regulator of the WNT- beta-catenin-TCF signaling pathway.

PubMed

Katoh, M; Kirikoshi, H; Terasaki, H; Shiokawa, K

2001-12-21

Genetic alterations of WNT signaling molecules lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway. We have previously cloned and characterized WNT2B/WNT13 gene on human chromosome 1p13, which is homologous to proto-oncogene WNT2 on human chromosome 7q31. WNT2B1 and WNT2B2 mRNAs, generated from the WNT2B gene due to alternative splicing of the alternative promoter type, encode almost identical polypeptides with divergence in the N-terminal region. WNT2B2 mRNA rather than WNT2B1 mRNA is preferentially expressed in NT2 cells with the potential of neuronal differentiation. Here, we describe our investigations of expression of WNT2B mRNAs in various types of human primary cancer. Matched tumor/normal expression array analysis revealed that WNT2B mRNAs were significantly up-regulated in 2 of 8 cases of primary gastric cancer. WNT2B2 mRNA rather than WNT2B1 mRNA was found to be preferentially up-regulated in a case of primary gastric cancer (signet ring cell carcinoma). Function of WNT2B1 mRNA and that of WNT2B2 mRNA were investigated by using Xenopus axis duplication assay. Injection of synthetic WNT2B1 mRNA into the ventral marginal zone of fertilized Xenopus eggs at the 4-cell stage did not induce axis duplication. In contrast, ventral injection of synthetic WNT2B2 mRNA induced axis duplication in 90% of embryos (complete axis duplication, 24%). These results strongly suggest that WNT2B2 up-regulation in some cases of gastric cancer might lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway.

Cloning and Analysis of the Alternative Oxidase Gene of Neurospora Crassa

PubMed Central

Li, Q.; Ritzel, R. G.; McLean, LLT.; McIntosh, L.; Ko, T.; Bertrand, H.; Nargang, F. E.

1996-01-01

Mitochondria of Neurospora crassa contain a cyanide-resistant alternative respiratory pathway in addition to the cytochrome pathway. The alternative oxidase is present only when electron flow through the cytochrome chain is restricted. Both genomic and cDNA copies for the alternative oxidase gene have been isolated and analyzed. The sequence of the predicted protein is homologous to that of other species. The mRNA for the alternative oxidase is scarce in wild-type cultures grown under normal conditions, but it is abundant in cultures grown in the presence of chloramphenicol, an inhibitor of mitochondrial protein synthesis, or in mutants deficient in mitochondrial cytochromes. Thus, induction of alternative oxidase appears to be at the transcriptional level. Restriction fragment length polymorphism mapping of the isolated gene demonstrated that it is located in a position corresponding to the aod-1 locus. Sequence analysis of mutant aod-1 alleles reveals mutations affecting the coding sequence of the alternative oxidase. The level of aod-1 mRNA in an aod-2 mutant strain that had been grown in the presence of chloramphenicol was reduced several fold relative to wild-type, supporting the hypothesis that the product of aod-2 is required for optimal expression of aod-1. PMID:8770590

Alternative Pre-mRNA Splicing in Mammals and Teleost Fish: A Effective Strategy for the Regulation of Immune Responses Against Pathogen Infection.

PubMed

Chang, Ming Xian; Zhang, Jie

2017-07-15

Pre-mRNA splicing is the process by which introns are removed and the protein coding elements assembled into mature mRNAs. Alternative pre-mRNA splicing provides an important source of transcriptome and proteome complexity through selectively joining different coding elements to form mRNAs, which encode proteins with similar or distinct functions. In mammals, previous studies have shown the role of alternative splicing in regulating the function of the immune system, especially in the regulation of T-cell activation and function. As lower vertebrates, teleost fish mainly rely on a large family of pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) from various invading pathogens. In this review, we summarize recent advances in our understanding of alternative splicing of piscine PRRs including peptidoglycan recognition proteins (PGRPs), nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) and their downstream signaling molecules, compared to splicing in mammals. We also discuss what is known and unknown about the function of splicing isoforms in the innate immune responses against pathogens infection in mammals and teleost fish. Finally, we highlight the consequences of alternative splicing in the innate immune system and give our view of important directions for future studies.

ELAV, a Drosophila neuron-specific protein, mediates the generation of an alternatively spliced neural protein isoform.

PubMed

Koushika, S P; Lisbin, M J; White, K

1996-12-01

Tissue-specific alternative pre-mRNA splicing is a widely used mechanism for gene regulation and the generation of different protein isoforms, but relatively little is known about the factors and mechanisms that mediate this process. Tissue-specific RNA-binding proteins could mediate alternative pre-mRNA splicing. In Drosophila melanogaster, the RNA-binding protein encoded by the elav (embryonic lethal abnormal visual system) gene is a candidate for such a role. The ELAV protein is expressed exclusively in neurons, and is important for the formation and maintenance of the nervous system. In this study, photoreceptor neurons genetically depleted of ELAV, and elav-null central nervous system neurons, were analyzed immunocytochemically for the expression of neural proteins. In both situations, the lack of ELAV corresponded with a decrease in the immunohistochemical signal of the neural-specific isoform of Neuroglian, which is generated by alternative splicing. Furthermore, when ELAV was expressed ectopically in cells that normally express only the non-neural isoform of Neuroglian, we observed the generation of the neural isoform of Neuroglian. Drosophila ELAV promotes the generation of the neuron-specific isoform of Neuroglian by the regulation of pre-mRNA splicing. The findings reported in this paper demonstrate that ELAV is necessary, and the ectopic expression of ELAV in imaginal disc cells is sufficient, to mediate neuron-specific alternative splicing.

Regulation of LPS-induced tissue factor expression in human monocytic THP-1 cells by curcumin

USDA-ARS?s Scientific Manuscript database

Tissue factor (TF) is a transmembrane receptor, which initiates thrombotic episodes associated with various diseases. In addition to membrane-bound TF, we have discovered an alternatively spliced form of human TF mRNA. It was later confirmed that this form of TF mRNA expresses a soluble protein circ...

KDM5 lysine demethylases are involved in maintenance of 3′UTR length

PubMed Central

Blair, Lauren P.; Liu, Zongzhi; Labitigan, Ramon Lorenzo D.; Wu, Lizhen; Zheng, Dinghai; Xia, Zheng; Pearson, Erica L.; Nazeer, Fathima I.; Cao, Jian; Lang, Sabine M.; Rines, Rachel J.; Mackintosh, Samuel G.; Moore, Claire L.; Li, Wei; Tian, Bin; Tackett, Alan J.; Yan, Qin

2016-01-01

The complexity by which cells regulate gene and protein expression is multifaceted and intricate. Regulation of 3′ untranslated region (UTR) processing of mRNA has been shown to play a critical role in development and disease. However, the process by which cells select alternative mRNA forms is not well understood. We discovered that the Saccharomyces cerevisiae lysine demethylase, Jhd2 (also known as KDM5), recruits 3′UTR processing machinery and promotes alteration of 3′UTR length for some genes in a demethylase-dependent manner. Interaction of Jhd2 with both chromatin and RNA suggests that Jhd2 affects selection of polyadenylation sites through a transcription-coupled mechanism. Furthermore, its mammalian homolog KDM5B (also known as JARID1B or PLU1), but not KDM5A (also known as JARID1A or RBP2), promotes shortening of CCND1 transcript in breast cancer cells. Consistent with these results, KDM5B expression correlates with shortened CCND1 in human breast tumor tissues. In contrast, both KDM5A and KDM5B are involved in the lengthening of DICER1. Our findings suggest both a novel role for this family of demethylases and a novel targetable mechanism for 3′UTR processing. PMID:28138513

Posttranscriptional mRNA processing as a mechanism for regulation of human A1 adenosine receptor expression.

PubMed Central

Ren, H; Stiles, G L

1994-01-01

The human A1 adenosine receptor gene contains six exons with exons 1, 2, 3, 4, and part of 5 representing 5' untranslated regions. Reverse transcription-PCR with exon-specific primers showed two distinct transcripts containing either exons 3, 5, and 6 or exons 4, 5, and 6, with exons 3 and 4 being mutually exclusive. No mature mRNAs containing exons 1 and 2 have been detected. All human tissues that express any A1 receptors contain mRNA with exons 4, 5, and 6. Tissues which express high levels of A1 receptors contain mRNA with exons 3, 5, and 6. Exon 4 contains two upstream ATG codons whereas exon 3 contains none. COS cells transfected with expression vectors containing exon 4 (exons 1-6, 3-6, or Ex4-6) express much lower levels of A1 receptors than vectors without exon 4 (exons 3, 5, and 6). Mutation of upstream ATG codons in exon 4 leads to 3- to 7-fold increased A1 receptor expression, up to the level seen with the construct containing exons 3, 5, and 6. Thus, in human tissues "basal" levels of A1 receptors can be expressed by use of mRNA containing exons 4, 5, and 6, but when high levels are needed, alternative transcripts with exons 3, 5, and 6 are produced. Images PMID:8197148

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

Headspace solid-phase microextraction (HS-SPME) and liquid-liquid extraction (LLE): comparison of the performance in classification of ecstasy tablets. Part 2.

PubMed

Bonadio, Federica; Margot, Pierre; Delémont, Olivier; Esseiva, Pierre

2008-11-20

Headspace solid-phase microextraction (HS-SPME) is assessed as an alternative to liquid-liquid extraction (LLE) currently used for 3,4-methylenedioxymethampethamine (MDMA) profiling. Both methods were compared evaluating their performance in discriminating and classifying samples. For this purpose 62 different seizures were analysed using both extraction techniques followed by gas chromatography-mass spectroscopy (GC-MS). A previously validated method provided data for HS-SPME, whereas LLE data were collected applying a harmonized methodology developed and used in the European project CHAMP. After suitable pre-treatment, similarities between sample pairs were studied using the Pearson correlation. Both methods enable to distinguish between samples coming from the same pre-tabletting batches and samples coming from different pre-tabletting batches. This finding emphasizes the use of HS-SPME as an effective alternative to LLE, with additional advantages such as sample preparation and a solvent-free process.

The helicase Ded1p controls use of near-cognate translation initiation codons in 5' UTRs.

PubMed

Guenther, Ulf-Peter; Weinberg, David E; Zubradt, Meghan M; Tedeschi, Frank A; Stawicki, Brittany N; Zagore, Leah L; Brar, Gloria A; Licatalosi, Donny D; Bartel, David P; Weissman, Jonathan S; Jankowsky, Eckhard

2018-06-27

The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation 1 . Mutations in DDX3 are linked to tumorigenesis 2-4 and intellectual disability 5 , and the enzyme is targeted by a range of viruses 6 . How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5' untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5' untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5' untranslated regions.

HNRNPLL stabilizes mRNAs for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells.

PubMed

Sakuma, Keiichiro; Sasaki, Eiichi; Kimura, Kenya; Komori, Koji; Shimizu, Yasuhiro; Yatabe, Yasushi; Aoki, Masahiro

2018-06-05

HNRNPLL (heterogeneous nuclear ribonucleoprotein L-like), an RNA-binding protein that regulates alternative splicing of pre-mRNAs, has been shown to regulate differentiation of lymphocytes, as well as metastasis of colorectal cancer cells. Here we show that HNRNPLL promotes cell cycle progression and hence proliferation of colorectal cancer cells. Functional annotation analysis of those genes whose expression levels were changed by three-fold or more in RNA sequencing analysis between SW480 cells overexpressing HNRNPLL and those knocked down for HNRNPLL revealed enrichment of DNA replication-related genes by HNRNPLL overexpression. Among 13 genes detected in the DNA replication pathway, PCNA, RFC3, and FEN1 showed reproducible upregulation by HNRNPLL overexpression both at mRNA and protein levels in SW480 and HT29 cells. Importantly, knockdown of any of these genes alone suppressed the proliferation promoting effect induced by HNRNPLL overexpression. RNA-immunoprecipitation assay presented a binding of FLAG-tagged HNRNPLL to mRNA of these genes, and HNRNPLL overexpression significantly suppressed the downregulation of these genes during 12 hours of actinomycin D treatment, suggesting a role of HNRNPLL in mRNA stability. Finally, analysis of a public RNA sequencing dataset of clinical samples suggested a link between overexpression of HNRNPLL and that of PCNA, RFC3, and FEN1. This link was further supported by immunohistochemistry of colorectal cancer clinical samples, whereas expression of CDKN1A, which is known to inhibit the cooperative function of PCNA, RFC3, and FEN1, was negatively associated with HNRNPLL expression. These results indicate that HNRNPLL stabilizes mRNAs encoding regulators of DNA replication and promotes colorectal cancer cell proliferation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Correct mRNA Processing at a Mutant TT Splice Donor in FANCC Ameliorates the Clinical Phenotype in Patients and Is Enhanced by Delivery of Suppressor U1 snRNAs

PubMed Central

Hartmann, Linda; Neveling, Kornelia; Borkens, Stephanie; Schneider, Hildegard; Freund, Marcel; Grassman, Elke; Theiss, Stephan; Wawer, Angela; Burdach, Stefan; Auerbach, Arleen D.; Schindler, Detlev; Hanenberg, Helmut; Schaal, Heiner

2010-01-01

The U1 small nuclear RNA (U1 snRNA) as a component of the major U2-dependent spliceosome recognizes 5′ splice sites (5′ss) containing GT as the canonical dinucleotide in the intronic positions +1 and +2. The c.165+1G>T germline mutation in the 5′ss of exon 2 of the Fanconi anemia C (FANCC) gene commonly predicted to prevent correct splicing was identified in nine FA patients from three pedigrees. RT-PCR analysis of the endogenous FANCC mRNA splicing pattern of patient-derived fibroblasts revealed aberrant mRNA processing, but surprisingly also correct splicing at the TT dinucleotide, albeit with lower efficiency. This consequently resulted in low levels of correctly spliced transcript and minute levels of normal posttranslationally processed FANCD2 protein, indicating that this naturally occurring TT splicing might contribute to the milder clinical manifestations of the disease in these patients. Functional analysis of this FANCC 5′ss within splicing reporters revealed that both the noncanonical TT dinucleotide and the genomic context of FANCC were required for the residual correct splicing at this mutant 5′ss. Finally, use of lentiviral vectors as a delivery system to introduce expression cassettes for TT-adapted U1 snRNAs into primary FANCC patient fibroblasts allowed the correction of the DNA-damage-induced G2 cell-cycle arrest in these cells, thus representing an alternative transcript-targeting approach for genetic therapy of inherited splice-site mutations. PMID:20869034

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.

IMP3 Stabilization of WNT5B mRNA Facilitates TAZ Activation in Breast Cancer.

PubMed

Samanta, Sanjoy; Guru, Santosh; Elaimy, Ameer L; Amante, John J; Ou, Jianhong; Yu, Jun; Zhu, Lihua J; Mercurio, Arthur M

2018-05-29

Insulin-like growth factor-2 mRNA-binding protein 3 (IMP3) is an oncofetal protein associated with many aggressive cancers and implicated in the function of breast cancer stem cells (CSCs). The mechanisms involved, however, are poorly understood. We observed that IMP3 facilitates the activation of TAZ, a transcriptional co-activator of Hippo signaling that is necessary for the function of breast CSCs. The mechanism by which IMP3 activates TAZ involves both mRNA stability and transcriptional regulation. IMP3 stabilizes the mRNA of an alternative WNT ligand (WNT5B) indirectly by repressing miR145-5p, which targets WNT5B, resulting in TAZ activation by alternative WNT signaling. IMP3 also facilitates the transcription of SLUG, which is necessary for TAZ nuclear localization and activation, by a mechanism that is also mediated by WNT5B. These results demonstrate that TAZ can be regulated by an mRNA-binding protein and that this regulation involves the integration of Hippo and alternative WNT-signaling pathways. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Identification by high-throughput imaging of the histone methyltransferase EHMT2 as an epigenetic regulator of VEGFA alternative splicing

PubMed Central

Salton, Maayan; Voss, Ty C.; Misteli, Tom

2014-01-01

Recent evidence points to a role of chromatin in regulation of alternative pre-mRNA splicing (AS). In order to identify novel chromatin regulators of AS, we screened an RNAi library of chromatin proteins using a cell-based high-throughput in vivo assay. We identified a set of chromatin proteins that regulate AS. Using simultaneous genome-wide expression and AS analysis, we demonstrate distinct and non-overlapping functions of these chromatin modifiers on transcription and AS. Detailed mechanistic characterization of one dual function chromatin modifier, the H3K9 methyltransferase EHMT2 (G9a), identified VEGFA as a major chromatin-mediated AS target. Silencing of EHMT2, or its heterodimer partner EHMT1, affects AS by promoting exclusion of VEGFA exon 6a, but does not alter total VEGFA mRNA levels. The epigenetic regulatory mechanism of AS by EHMT2 involves an adaptor system consisting of the chromatin modulator HP1γ, which binds methylated H3K9 and recruits splicing regulator SRSF1. The epigenetic regulation of VEGFA is physiologically relevant since EHMT2 is transcriptionally induced in response to hypoxia and triggers concomitant changes in AS of VEGFA. These results characterize a novel epigenetic regulatory mechanism of AS and they demonstrate separate roles of epigenetic modifiers in transcription and alternative splicing. PMID:25414343

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

PubMed

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

2016-05-05

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

A review of craniofacial disorders caused by spliceosomal defects.

PubMed

Lehalle, D; Wieczorek, D; Zechi-Ceide, R M; Passos-Bueno, M R; Lyonnet, S; Amiel, J; Gordon, C T

2015-11-01

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNA transcripts. Mutations in EFTUD2, encoding a component of the major spliceosome, have recently been identified as the cause of mandibulofacial dysostosis, Guion-Almeida type (MFDGA), characterized by mandibulofacial dysostosis, microcephaly, external ear malformations and intellectual disability. Mutations in several other genes involved in spliceosomal function or linked aspects of mRNA processing have also recently been identified in human disorders with specific craniofacial malformations: SF3B4 in Nager syndrome, an acrofacial dysostosis (AFD); SNRPB in cerebrocostomandibular syndrome, characterized by Robin sequence and rib defects; EIF4A3 in the AFD Richieri-Costa-Pereira syndrome, characterized by Robin sequence, median mandibular cleft and limb defects; and TXNL4A in Burn-McKeown syndrome, involving specific craniofacial dysmorphisms. Here, we review phenotypic and molecular aspects of these syndromes. Given the apparent sensitivity of craniofacial development to defects in mRNA processing, it is possible that mutations in other proteins involved in spliceosomal function will emerge in the future as causative for related human disorders. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Cloning and characterization of cDNAs encoding human gastrin-releasing peptide.

PubMed Central

Spindel, E R; Chin, W W; Price, J; Rees, L H; Besser, G M; Habener, J F

1984-01-01

We have prepared and cloned cDNAs derived from poly(A)+ RNA from a human pulmonary carcinoid tumor rich in immunoreactivity to gastrin-releasing peptide, a peptide closely related in structure to amphibian bombesin. Mixtures of synthetic oligodeoxyribonucleotides corresponding to amphibian bombesin were used as hybridization probes to screen a cDNA library prepared from the tumor RNA. Sequencing of the recombinant plasmids shows that human gastrin-releasing peptide (hGRP) mRNA encodes a precursor of 148 amino acids containing a typical signal sequence, hGRP consisting of 27 or 28 amino acids, and a carboxyl-terminal extension peptide. hGRP is flanked at its carboxyl terminus by two basic amino acids, following a glycine used for amidation of the carboxyl-terminal methionine. RNA blot analyses of tumor RNA show a major mRNA of 900 bases and a minor mRNA of 850 bases. Blot hybridization analyses using human genomic DNA are consistent with a single hGRP-encoding gene. The presence of two mRNAs encoding the hGRP precursor protein in the face of a single hGRP gene raises the possibility of alternative processing of the single RNA transcript. Images PMID:6207529

Remodeling of the pioneer translation initiation complex involves translation and the karyopherin importin β

PubMed Central

Sato, Hanae; Maquat, Lynne E.

2009-01-01

Mammalian mRNAs lose and acquire proteins throughout their life span while undergoing processing, transport, translation, and decay. How translation affects messenger RNA (mRNA)–protein interactions is largely unknown. The pioneer round of translation uses newly synthesized mRNA that is bound by cap-binding protein 80 (CBP80)–CBP20 (also known as the cap-binding complex [CBC]) at the cap, poly(A)-binding protein N1 (PABPN1) and PABPC1 at the poly(A) tail, and, provided biogenesis involves pre-mRNA splicing, exon junction complexes (EJCs) at exon–exon junctions. Subsequent rounds of translation engage mRNA that is bound by eukaryotic translation initiation factor 4E (eIF4E) at the cap and PABPC1 at the poly(A) tail, but that lacks detectable EJCs and PABPN1. Using the level of intracellular iron to regulate the translation of specific mRNAs, we show that translation promotes not only removal of EJC constituents, including the eIF4AIII anchor, but also replacement of PABPN1 by PABPC1. Remarkably, translation does not affect replacement of CBC by eIF4E. Instead, replacement of CBC by eIF4E is promoted by importin β (IMPβ): Inhibiting the binding of IMPβ to the complex of CBC–IMPα at an mRNA cap using the IMPα IBB (IMPβ-binding) domain or a RAN variant increases the amount of CBC-bound mRNA and decreases the amount of eIF4E-bound mRNA. Our studies uncover a previously unappreciated role for IMPβ and a novel paradigm for how newly synthesized messenger ribonucleoproteins (mRNPs) are matured. PMID:19884259

DjhnRNPA2/B1-like gene is required for planarian regeneration and tissue homeostasis.

PubMed

Dong, Zimei; Yang, Tong; Yang, Yibo; Dou, He; Chen, Guangwen

2017-10-30

The hnRNPs play important roles in physiological processes in eukaryotic organisms by regulation of pre-mRNA after transcription, including pre-mRNA splicing, mRNA stability, DNA replication and repair and telomere maintenance and so on. However, it remains unclear about the specific functions of these genes. In this study, the full-length cDNA sequence of hnRNPA2/B1-like was first cloned from Dugesia japonica, and its roles were investigated by WISH and RNAi. The results showed that: (1) DjhnRNPA2/B1-like was highly conserved during animal evolution; (2) DjhnRNPA2/B1-like mRNA was mainly distributed each side of the body in intact worms and regenerative blastemas, and its expression levels were up-regulated on days 0 and 5 after amputation; (3) the intact and regenerating worms gradually lysed or lost regeneration capacity after DjhnRNPA2/B1-like RNAi; and (4) DjhnRNPA2/B1-like expression is induced by temperature and heavy metal ion stress. The data suggests that DjhnRNPA2/B1-like is a multiple functional gene, it plays important roles in regeneration and homeostatic maintenance and it is also involved in stress responses in planarians. Our work provides basic data for the study of regenerative mechanism and stress responses in freshwater planarians. Copyright © 2017. Published by Elsevier B.V.

Existence of a microRNA pathway in anucleate platelets

PubMed Central

Landry, Patricia; Plante, Isabelle; Ouellet, Dominique L; Perron, Marjorie P; Rousseau, Guy; Provost, Patrick

2010-01-01

Platelets play a critical role in the maintenance of hemostasis as well as in thrombosis and vessel occlusion that underlie stroke and acute coronary syndromes. Anucleate platelets contain messenger RNAs (mRNAs) and are capable of protein synthesis, raising the issue of how these mRNAs are regulated. Here we show that human platelets harbor an abundant and diverse array of microRNAs (miRNAs), which are known as key regulators of mRNA translation. Further analyses revealed that platelets contain Dicer and Argonaute 2 (Ago2) complexes functional in exogenously supplied miRNA precursor (pre-miRNA) processing and the control of specific reporter transcripts, respectively. Detection of the receptor P2Y12 mRNA in Ago2 immunoprecipitates suggests that P2Y12 expression may be subjected to miRNA control in human platelets. Our study lends an additional level of complexity to the control of gene expression in these anucleate elements of the cardiovascular system. PMID:19668211

Post-transcriptional control of DGCR8 expression by the Microprocessor.

PubMed

Triboulet, Robinson; Chang, Hao-Ming; Lapierre, Robert J; Gregory, Richard I

2009-06-01

The Microprocessor, comprising the RNase III Drosha and the double-stranded RNA binding protein DGCR8, is essential for microRNA (miRNA) biogenesis. In the miRNA processing pathway certain hairpin structures within primary miRNA (pri-miRNA) transcripts are specifically cleaved by the Microprocessor to release approximately 60-70-nucleotide precursor miRNA (pre-miRNA) intermediates. Although both Drosha and DGCR8 are required for Microprocessor activity, the mechanisms regulating the expression of these proteins are unknown. Here we report that the Microprocessor negatively regulates DGCR8 expression. Using in vitro reconstitution and in vivo studies, we demonstrate that a hairpin, localized in the 5' untranslated region (5'UTR) of DGCR8 mRNA, is cleaved by the Microprocessor. Accordingly, knockdown of Drosha leads to an increase in DGCR8 mRNA and protein levels in cells. Furthermore, we found that the DGCR8 5'UTR confers Microprocessor-dependent repression of a luciferase reporter gene in vivo. Our results uncover a novel feedback loop that regulates DGCR8 levels.

Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

PubMed Central

Meyer, Katja; Koester, Tino; Staiger, Dorothee

2015-01-01

Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance. PMID:26213982

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

PubMed Central

Hu, Wenchao; Liu, Yuting; Yan, Jun

2014-01-01

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

Adaptive Gaussian mixture models for pre-screening in GPR data

NASA Astrophysics Data System (ADS)

Torrione, Peter; Morton, Kenneth, Jr.; Besaw, Lance E.

2011-06-01

Due to the large amount of data generated by vehicle-mounted ground penetrating radar (GPR) antennae arrays, advanced feature extraction and classification can only be performed on a small subset of data during real-time operation. As a result, most GPR based landmine detection systems implement "pre-screening" algorithms to processes all of the data generated by the antennae array and identify locations with anomalous signatures for more advanced processing. These pre-screening algorithms must be computationally efficient and obtain high probability of detection, but can permit a false alarm rate which might be higher than the total system requirements. Many approaches to prescreening have previously been proposed, including linear prediction coefficients, the LMS algorithm, and CFAR-based approaches. Similar pre-screening techniques have also been developed in the field of video processing to identify anomalous behavior or anomalous objects. One such algorithm, an online k-means approximation to an adaptive Gaussian mixture model (GMM), is particularly well-suited to application for pre-screening in GPR data due to its computational efficiency, non-linear nature, and relevance of the logic underlying the algorithm to GPR processing. In this work we explore the application of an adaptive GMM-based approach for anomaly detection from the video processing literature to pre-screening in GPR data. Results with the ARA Nemesis landmine detection system demonstrate significant pre-screening performance improvements compared to alternative approaches, and indicate that the proposed algorithm is a complimentary technique to existing methods.

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

PubMed

Seth, Puneet; Yeowell, Heather N

2010-04-01

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

Increased precursor microRNA-21 following status epilepticus can compete with mature microRNA-21 to alter translation

PubMed Central

Chak, Kayam; Roy-Chaudhuri, Biswajoy; Kim, Hak Kyun; Kemp, Kayla C; Kay, Mark A

2016-01-01

MicroRNA-21 (miR-21) is consistently up-regulated in various neurological disorders, including epilepsy. Here, we show that the biogenesis of miR-21 is altered following pilocarpine status epilepticus (SE) with an increase in precursor miR-21 (pre-miR-21) in rats. We demonstrate that pre-miR-21 has an energetically favorable site overlapping with the miR-21 binding site and competes with mature miR-21 for binding in the 3′UTR of TGFBR2 mRNA, but not NT-3 mRNA in vitro. This binding competition influences miR-21-mediated repression in vitro and correlates with the increase in TGFBR2 and decrease in NT-3 following SE. Polysome profiling reveals co-localization of pre-miR-21 in the ribosome fraction with translating mRNAs in U-87 cells. The current work suggests that pre-miR-21 may post-transcriptionally counteract miR-21-mediated suppression following SE and could potentially lead to prolonged TGF-β receptor expression impacting epileptogenesis. The study further supports that the ratio of the pre to mature miRNA may be important in determining the regulatory effects of a miRNA gene. PMID:27725160

Processing of intervening sequences: a new yeast mutant which fails to excise intervening sequences from precursor tRNAs.

PubMed

Hopper, A K; Schultz, L D; Shapiro, R A

1980-03-01

By using conditional loss of suppression an an assay, we have been successful in screening for a yeast mutant which is defective in tRNA processing. The los1-1 mutation causes an accumulation of a subset of precursor tRNAs at the nonpermissive temperature. These pre-tRNAs are like those which accumulate in the yeast mutant ts 136 (rna1) in that they have transcribed intervening sequences. The mutations at los1-1 and rna1 complement and segregate independently of each other. The los1-1 mutation affects the expression of all 8 tyrosine-inserting suppressor loci, but does not seem to affect rRNA or mRNA synthesis.

Structure of the human myelin/oligodendrocyte glycoprotein gene and multiple alternative spliced isoforms

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

Pham-Dinh, D.; Gaspera, D.B.; Dautigny, A.

1995-09-20

Myelin/oligodendrocyte glycoprotein (MOG), a special component of the central nervous system localization on the outermost lamellae of mature myelin, is a member of the immunoglobulin superfamily. We report here the organization of the human MOG gene, which spans approximately 17 kb, and the characterization of six MOG mRNA splicing variants. The intron/exon structure of the human MOG gene confirmed the splicing pattern, supporting the hypothesis that mRNA isoforms could arise by alternative splicing of a single gene. In addition to the eight exons coding for the major MOG isoform, the human MOG gene also contains 3` region, a previously unknownmore » alternatively spliced coding exon, VIA. Alternative utilization of two acceptor splicing sites for exon VIII could produce two different C-termini. The nucleotide sequences presented here may be a useful tool to study further possible involvement if the MOG gene in hereditary neurological disorders. 23 refs., 5 figs.« less

Single-Cell Analysis Reveals Early Manifestation of Cancerous Phenotype in Pre-Malignant Esophageal Cells

PubMed Central

Wang, Jiangxin; Shi, Xu; Johnson, Roger H.; Kelbauskas, Laimonas; Zhang, Weiwen; Meldrum, Deirdre R.

2013-01-01

Cellular heterogeneity plays a pivotal role in a variety of functional processes in vivo including carcinogenesis. However, our knowledge about cell-to-cell diversity and how differences in individual cells manifest in alterations at the population level remains very limited mainly due to the lack of appropriate tools enabling studies at the single-cell level. We present a study on changes in cellular heterogeneity in the context of pre-malignant progression in response to hypoxic stress. Utilizing pre-malignant progression of Barrett’s esophagus (BE) as a disease model system we studied molecular mechanisms underlying the progression from metaplastic to dysplastic (pre-cancerous) stage. We used newly developed methods enabling measurements of cell-to-cell differences in copy numbers of mitochondrial DNA, expression levels of a set of mitochondrial and nuclear genes involved in hypoxia response pathways, and mitochondrial membrane potential. In contrast to bulk cell studies reported earlier, our study shows significant differences between metaplastic and dysplastic BE cells in both average values and single-cell parameter distributions of mtDNA copy numbers, mitochondrial function, and mRNA expression levels of studied genes. Based on single-cell data analysis, we propose that mitochondria may be one of the key factors in pre-malignant progression in BE. PMID:24116039

Dietary abscisic acid ameliorates glucose tolerance and obesity-related inflammation in db/db mice fed high-fat diets.

PubMed

Guri, Amir J; Hontecillas, Raquel; Si, Hongwei; Liu, Dongmin; Bassaganya-Riera, Josep

2007-02-01

Despite their efficacy in improving insulin sensitivity, thiazolidinediones (TZDs) are associated with a number of side effects (i.e. weight gain, hepatotoxicity, congestive heart failure) that have limited their use by millions of diabetic patients. We have investigated whether abscisic acid (ABA), a naturally occurring phytochemical with structural similarities to TZDs, could be used as an alternative to TZDs to improve glucose homeostasis. We first examined whether ABA, similar to TZDs, activates PPARgamma in vitro. We next determined the lowest effective dose of dietary ABA (100 mg/kg) and assessed its effect on glucose tolerance, obesity-related inflammation, and mRNA expression of PPARgamma and its responsive genes in white adipose tissue (WAT) of db/db mice fed high-fat diets. We found that ABA induced transactivation of PPARgamma in 3T3-L1 pre-adipocytes in vitro. Dietary ABA-supplementation for 36 days decreased fasting blood glucose concentrations, ameliorated glucose tolerance, and increased mRNA expression of PPARgamma and its responsive genes (i.e., adiponectin, aP2, and CD36) in WAT. We also found that adipocyte hypertrophy, tumor necrosis factor-alpha (TNF-alpha) expression, and macrophage infiltration in WAT were significantly attenuated in ABA-fed mice. These findings suggest that ABA could be used as a nutritional intervention against type II diabetes and obesity-related inflammation.

PPARα, PPARγ and SREBP-1 pathways mediated waterborne iron (Fe)-induced reduction in hepatic lipid deposition of javelin goby Synechogobius hasta.

PubMed

Chen, Guang-Hui; Luo, Zhi; Chen, Feng; Shi, Xi; Song, Yu-Feng; You, Wen-Jing; Liu, Xu

2017-07-01

The 42-day experiment was conducted to investigate the effects and mechanism of waterborne Fe exposure influencing hepatic lipid deposition in Synechogobius hasta. For that purpose, S. hasta were exposed to four Fe concentrations (0 (control), 0.36, 0.72 and 1.07μM Fe) for 42days. On days 21 and 42, morphological parameters, hepatic lipid deposition and Fe contents, and activities and mRNA levels of enzymes and genes related to lipid metabolism, including lipogenic enzymes (6PGD, G6PD, ME, ICDH, FAS and ACC) and lipolytic enzymes (CPTI, HSL), were analyzed. With the increase of Fe concentration, hepatic Fe content tended to increase but HSI and lipid content tended to decrease. On day 21, Fe exposure down-regulated the lipogenic activities of 6PGD, G6PD, ICDH and FAS as well as the mRNA levels of G6PD, ACCa, FAS, SREBP-1 and PPARγ, but up-regulated CPT I, HSLa and PPARα mRNA levels. On day 42, Fe exposure down-regulated the lipogenic activities of 6PGD, G6PD, ICDH and FAS as well as the mRNA levels of 6PGD, ACCa, FAS and SREBP-1, but up-regulated CPT I, HSLa, PPARα and PPARγ mRNA levels. Using primary S. hasta hepatocytes, specific pathway inhibitors (GW6471 for PPARα and fatostatin for SREBP-1) and activator (troglitazone for PPARγ) were used to explore the signaling pathways of Fe reducing lipid deposition. The GW6471 attenuated the Fe-induced down-regulation of mRNA levels of 6PGD, G6PD, ME, FAS and ACCa, and attenuated the Fe-induced up-regulation of mRNA levels of CPT I, HSLa and PPARα. Compared with single Fe-incubated group, the mRNA levels of G6PD, ME, FAS, ACCa, ACCb and PPARγ were up-regulated while the CPT I mRNA levels were down-regulated after troglitazone pre-treatment; fatostatin pre-treatment down-regulated the mRNA levels of 6PGD, ME, FAS, ACCa, ACCb and SREBP-1, and increased the CPT I and HSLa mRNA levels. Based on these results above, our study indicated that Fe exposure reduced hepatic lipid deposition by down-regulating lipogenesis and up-regulating lipolysis, and PPARα, PPARγ and SREBP-1 pathways mediated the Fe-induced reduction of hepatic lipid deposition in S. hasta. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of Coffee Extracts with Different Roasting Degrees on Antioxidant and Anti-Inflammatory Systems in Mice.

PubMed

Choi, Sukyoung; Jung, Soohan; Ko, Kwang Suk

2018-03-16

Coffee roasting affects the taste, color, and aroma of coffee. The Maillard reaction, a major reaction during the roasting process, produces melanoidin, which affects the overall antioxidant capacity and anti-inflammatory effects of coffee. In this experiment, coffee roasting was divided into four degrees: Light, Medium, City, and French. To examine the in vivo antioxidant and anti-inflammatory effects of coffee extracts with different roasting degrees, we used 10-week-old male C57BL/6 mice. Mice were pre-treated with coffee extracts for 10 days by oral gavage (300 mg/Kg.B.W). After the last pre-treatment, lipopolysaccharide (LPS, 15 mg/Kg.B.W) was injected intraperitoneally for immune stimulation. Histopathological analysis showed that hepatic portal vein invasion and liver necrosis were severe in the LPS-treated group. However, these phenomena were greatly ameliorated when mice were pre-treated with Light- or Medium-roasted coffee extracts. Hepatic glutathione level was increased in the French group but decreased in the LPS-stimulated group. When mice were treated with LPS, mRNA expression level of tumor necrosis factor-alpha (TNF-α) was increased, whereas TNF-α expression was significantly reduced in the Light and Medium groups. Treatment with coffee extracts decreased the mRNA expression levels of interleukin 6 (IL-6) in mice stimulated by LPS, regardless of coffee roasting degrees. These effects decreased with the increasing coffee roasting degree. Results of luciferase reporter assay revealed that these effects of coffee extracts were transcriptionally regulated by the NF-κB pathway. Taken together, these results suggest that the roasting degree affects the antioxidant and anti-inflammatory effects of coffee extracts.

Discovery of precursor and mature microRNAs and their putative gene targets using high-throughput sequencing in pineapple (Ananas comosus var. comosus).

PubMed

Yusuf, Noor Hydayaty Md; Ong, Wen Dee; Redwan, Raimi Mohamed; Latip, Mariam Abd; Kumar, S Vijay

2015-10-15

MicroRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that negatively regulate gene expression, resulting in the silencing of target mRNA transcripts through mRNA cleavage or translational inhibition. MiRNAs play significant roles in various biological and physiological processes in plants. However, the miRNA-mediated gene regulatory network in pineapple, the model tropical non-climacteric fruit, remains largely unexplored. Here, we report a complete list of pineapple mature miRNAs obtained from high-throughput small RNA sequencing and precursor miRNAs (pre-miRNAs) obtained from ESTs. Two small RNA libraries were constructed from pineapple fruits and leaves, respectively, using Illumina's Solexa technology. Sequence similarity analysis using miRBase revealed 579,179 reads homologous to 153 miRNAs from 41 miRNA families. In addition, a pineapple fruit transcriptome library consisting of approximately 30,000 EST contigs constructed using Solexa sequencing was used for the discovery of pre-miRNAs. In all, four pre-miRNAs were identified (MIR156, MIR399, MIR444 and MIR2673). Furthermore, the same pineapple transcriptome was used to dissect the function of the miRNAs in pineapple by predicting their putative targets in conjunction with their regulatory networks. In total, 23 metabolic pathways were found to be regulated by miRNAs in pineapple. The use of high-throughput sequencing in pineapples to unveil the presence of miRNAs and their regulatory pathways provides insight into the repertoire of miRNA regulation used exclusively in this non-climacteric model plant. Copyright © 2015 Elsevier B.V. All rights reserved.

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

An in vivo model of epithelial to mesenchymal transition reveals a mitogenic switch

PubMed Central

Jahn, Stephan C.; Law, Mary E.; Corsino, Patrick E.; Parker, Nicole N.; Pham, Kien; Davis, Bradley J.; Lu, Jianrong; Law, Brian K.

2012-01-01

The epithelial to mesenchymal transition (EMT) is a process by which differentiated epithelial cells transition to a mesenchymal phenotype. EMT enables the escape of epithelial cells from the rigid structural constraints of the tissue architecture to a phenotype more amenable to cell migration and, therefore, invasion and metastasis. We characterized an in vivo model of EMT and discovered that marked changes in mitogenic signaling occurred during this process. DNA microarray analysis revealed that the expression of a number of genes varied significantly between post-EMT and pre-EMT breast cancer cells. Post-EMT cancer cells upregulated mRNA encoding c-Met and the PDGF and LPA receptors, and acquired increased responsiveness to HGF, PDGF, and LPA. This rendered the post-EMT cells responsive to the growth inhibitory effects of HGF, PDGF, and LPA receptor inhibitors/antagonists. Furthermore, post- EMT cells exhibited decreased basal Raf and Erk phosphorylation, and in comparison to pre-EMT cells, their proliferation was poorly inhibited by a MEK inhibitor. These studies suggest that therapies need to be designed to target both pre-EMT and post-EMT cancer cells and that signaling changes in post- EMT cells may allow them to take advantage of paracrine signaling from the stroma in vivo. PMID:22906417

Maternal Factors Are Associated with the Expression of Placental Genes Involved in Amino Acid Metabolism and Transport

PubMed Central

Day, Pricilla E.; Ntani, Georgia; Crozier, Sarah R.; Mahon, Pam A.; Inskip, Hazel M.; Cooper, Cyrus; Harvey, Nicholas C.; Godfrey, Keith M.; Hanson, Mark A.; Lewis, Rohan M.; Cleal, Jane K.

2015-01-01

Introduction Maternal environment and lifestyle factors may modify placental function to match the mother’s capacity to support the demands of fetal growth. Much remains to be understood about maternal influences on placental metabolic and amino acid transporter gene expression. We investigated the influences of maternal lifestyle and body composition (e.g. fat and muscle content) on a selection of metabolic and amino acid transporter genes and their associations with fetal growth. Methods RNA was extracted from 102 term Southampton Women’s Survey placental samples. Expression of nine metabolic, seven exchange, eight accumulative and three facilitated transporter genes was analyzed using quantitative real-time PCR. Results Increased placental LAT2 (p = 0.01), y + LAT2 (p = 0.03), aspartate aminotransferase 2 (p = 0.02) and decreased aspartate aminotransferase 1 (p = 0.04) mRNA expression associated with pre-pregnancy maternal smoking. Placental mRNA expression of TAT1 (p = 0.01), ASCT1 (p = 0.03), mitochondrial branched chain aminotransferase (p = 0.02) and glutamine synthetase (p = 0.05) was positively associated with maternal strenuous exercise. Increased glutamine synthetase mRNA expression (r = 0.20, p = 0.05) associated with higher maternal diet quality (prudent dietary pattern) pre-pregnancy. Lower LAT4 (r = -0.25, p = 0.05) and aspartate aminotransferase 2 mRNA expression (r = -0.28, p = 0.01) associated with higher early pregnancy diet quality. Lower placental ASCT1 mRNA expression associated with measures of increased maternal fat mass, including pre-pregnancy BMI (r = -0.26, p = 0.01). Lower placental mRNA expression of alanine aminotransferase 2 associated with greater neonatal adiposity, for example neonatal subscapular skinfold thickness (r = -0.33, p = 0.001). Conclusion A number of maternal influences have been linked with outcomes in childhood, independently of neonatal size; our finding of associations between placental expression of transporter and metabolic genes and maternal smoking, physical activity and diet raises the possibility that their effects are mediated in part through alterations in placental function. The observed changes in placental gene expression in relation to modifiable maternal factors are important as they could form part of interventions aimed at maintaining a healthy lifestyle for the mother and for optimal fetal development. PMID:26657885

Alternative Polyadenylation Directs Tissue-Specific miRNA Targeting in Caenorhabditis elegans Somatic Tissues

PubMed Central

Blazie, Stephen M.; Geissel, Heather C.; Wilky, Henry; Joshi, Rajan; Newbern, Jason; Mangone, Marco

2017-01-01

mRNA expression dynamics promote and maintain the identity of somatic tissues in living organisms; however, their impact in post-transcriptional gene regulation in these processes is not fully understood. Here, we applied the PAT-Seq approach to systematically isolate, sequence, and map tissue-specific mRNA from five highly studied Caenorhabditis elegans somatic tissues: GABAergic and NMDA neurons, arcade and intestinal valve cells, seam cells, and hypodermal tissues, and studied their mRNA expression dynamics. The integration of these datasets with previously profiled transcriptomes of intestine, pharynx, and body muscle tissues, precisely assigns tissue-specific expression dynamics for 60% of all annotated C. elegans protein-coding genes, providing an important resource for the scientific community. The mapping of 15,956 unique high-quality tissue-specific polyA sites in all eight somatic tissues reveals extensive tissue-specific 3′untranslated region (3′UTR) isoform switching through alternative polyadenylation (APA) . Almost all ubiquitously transcribed genes use APA and harbor miRNA targets in their 3′UTRs, which are commonly lost in a tissue-specific manner, suggesting widespread usage of post-transcriptional gene regulation modulated through APA to fine tune tissue-specific protein expression. Within this pool, the human disease gene C. elegans orthologs rack-1 and tct-1 use APA to switch to shorter 3′UTR isoforms in order to evade miRNA regulation in the body muscle tissue, resulting in increased protein expression needed for proper body muscle function. Our results highlight a major positive regulatory role for APA, allowing genes to counteract miRNA regulation on a tissue-specific basis. PMID:28348061

Alternative Polyadenylation Directs Tissue-Specific miRNA Targeting in Caenorhabditis elegans Somatic Tissues.

PubMed

Blazie, Stephen M; Geissel, Heather C; Wilky, Henry; Joshi, Rajan; Newbern, Jason; Mangone, Marco

2017-06-01

mRNA expression dynamics promote and maintain the identity of somatic tissues in living organisms; however, their impact in post-transcriptional gene regulation in these processes is not fully understood. Here, we applied the PAT-Seq approach to systematically isolate, sequence, and map tissue-specific mRNA from five highly studied Caenorhabditis elegans somatic tissues: GABAergic and NMDA neurons, arcade and intestinal valve cells, seam cells, and hypodermal tissues, and studied their mRNA expression dynamics. The integration of these datasets with previously profiled transcriptomes of intestine, pharynx, and body muscle tissues, precisely assigns tissue-specific expression dynamics for 60% of all annotated C. elegans protein-coding genes, providing an important resource for the scientific community. The mapping of 15,956 unique high-quality tissue-specific polyA sites in all eight somatic tissues reveals extensive tissue-specific 3'untranslated region (3'UTR) isoform switching through alternative polyadenylation (APA) . Almost all ubiquitously transcribed genes use APA and harbor miRNA targets in their 3'UTRs, which are commonly lost in a tissue-specific manner, suggesting widespread usage of post-transcriptional gene regulation modulated through APA to fine tune tissue-specific protein expression. Within this pool, the human disease gene C. elegans orthologs rack-1 and tct-1 use APA to switch to shorter 3'UTR isoforms in order to evade miRNA regulation in the body muscle tissue, resulting in increased protein expression needed for proper body muscle function. Our results highlight a major positive regulatory role for APA, allowing genes to counteract miRNA regulation on a tissue-specific basis. Copyright © 2017 Blazie et al.

Cytokinin-induced cell death is associated with elevated expression of alternative oxidase in tobacco BY-2 cells.

PubMed

Mlejnek, Petr

2013-10-01

N(6)-benzyladenine (BA) and N(6)-benzyladenosine ([9R]BA) induce massive production of reactive oxygen species (ROS) that is eventually followed by a loss of cell viability in tobacco BY-2 cells (Mlejnek et al. Plant Cell Environ 26:1723-1735, 2003, Plant Sci 168:389-395, 2005). Results presented in this work suggest that the main sources of ROS are likely mitochondria and that the maintenance of the mitochondrial transmembrane potential is crucial for ROS production in cytokinin-treaded BY-2 cells. Therefore, the possible involvement of alternative oxidase (AOX) in cell death process induced by BA and [9R]BA was studied. About three- to fourfold increase in mRNA levels of AOX1 was observed a few hours after the BA and [9R]BA addition into the growth medium. The elevated expression of AOX1 mRNA could be prevented by adding adenine and adenosine which simultaneously reduced the cytotoxic effects of BA and [9R]BA, respectively. N(6)-benzyladenine 7-β-D-glucoside ([7G]BA) which is a common non-toxic metabolite of BA and [9R]BA did not affect the AOX1 mRNA expression. Although AOX1 seemed to be involved in protection of BY-2 cells against the abiotic stress induced by BA and [9R]BA, the results do not support the idea that it protects cells from death exclusively by scavenging of reactive oxygen species. Indeed, N-propyl gallate, an inhibitor of AOX, decreased cell survival despite it concomitantly decreased the ROS production. This finding is in contrast to the effect of salicylhydroxamic acid, another well-known inhibitor of AOX, which also increased the number of dying cells while it increased the ROS production.

A family of splice variants of CstF-64 expressed in vertebrate nervous systems

PubMed Central

Shankarling, Ganesh S; Coates, Penelope W; Dass, Brinda; MacDonald, Clinton C

2009-01-01

Background Alternative splicing and polyadenylation are important mechanisms for creating the proteomic diversity necessary for the nervous system to fulfill its specialized functions. The contribution of alternative splicing to proteomic diversity in the nervous system has been well documented, whereas the role of alternative polyadenylation in this process is less well understood. Since the CstF-64 polyadenylation protein is known to be an important regulator of tissue-specific polyadenylation, we examined its expression in brain and other organs. Results We discovered several closely related splice variants of CstF-64 – collectively called βCstF-64 – that could potentially contribute to proteomic diversity in the nervous system. The βCstF-64 splice variants are found predominantly in the brains of several vertebrate species including mice and humans. The major βCstF-64 variant mRNA is generated by inclusion of two alternate exons (that we call exons 8.1 and 8.2) found between exons 8 and 9 of the CstF-64 gene, and contains an additional 147 nucleotides, encoding 49 additional amino acids. Some variants of βCstF-64 contain only the first alternate exon (exon 8.1) while other variants contain both alternate exons (8.1 and 8.2). In mice, the predominant form of βCstF-64 also contains a deletion of 78 nucleotides from exon 9, although that variant is not seen in any other species examined, including rats. Immunoblot and 2D-PAGE analyses of mouse nuclear extracts indicate that a protein corresponding to βCstF-64 is expressed in brain at approximately equal levels to CstF-64. Since βCstF-64 splice variant family members were found in the brains of all vertebrate species examined (including turtles and fish), this suggests that βCstF-64 has an evolutionarily conserved function in these animals. βCstF-64 was present in both pre- and post-natal mice and in different regions of the nervous system, suggesting an important role for βCstF-64 in neural gene expression throughout development. Finally, experiments in representative cell lines suggest that βCstF-64 is expressed in neurons but not glia. Conclusion This is the first report of a family of splice variants encoding a key polyadenylation protein that is expressed in a nervous system-specific manner. We propose that βCstF-64 contributes to proteomic diversity by regulating alternative polyadenylation of neural mRNAs. PMID:19284619

Dyrk1A-ASF-CaMKIIδ Signaling Is Involved in Valsartan Inhibition of Cardiac Hypertrophy in Renovascular Hypertensive Rats.

PubMed

Yao, Jian; Qin, Xiaotong; Zhu, Jianhua; Sheng, Hongzhuan

2016-01-01

It is known that the expression, activity and alternative splicing of Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) are dysregulated in the cardiac remodeling process. Recently, we found a further signaling pathway, by which dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) regulates the alternative splicing of CaMKIIδ via the alternative splicing factor (ASF), i.e., Dyrk1A-ASF-CaMKIIδ. In this study, we aimed to investigate whether Dyrk1A-ASF-CaMKIIδ signaling was involved in valsartan inhibition of cardiac hypertrophy in renovascular hypertensive rats. Rats were subjected to two kidney-one clip (2K1C) surgery and then treated with valsartan (30 mg/kg/day) for 8 weeks. Hypertrophic parameter analysis was then performed. Western blot analysis was used to determine the protein expression of Dyrk1A and ASF and RT-PCR was used to analyze the alternative splicing of CaMKIIδ in the left ventricular (LV) sample. Valsartan attenuated cardiac hypertrophy in 2K1C rats but without impairment of cardiac systolic function. Increased protein expression of Dyrk1A and decreased protein expression of ASF were observed in the LV sample of 2K1C rats. Treatment of 2K1C rats with valsartan reversed the changes in Dyrk1A and ASF expression in the LV sample. Valsartan adjusted the 2K1C-induced imbalance in alternative splicing of CaMKIIδ by upregulating the mRNA expression of CaMKIIδC and downregulating the mRNA expression of CaMKIIδA and CaMKIIδB. Valsartan inhibition of cardiac hypertrophy in renovascular hypertensive rats was mediated, at least partly, by Dyrk1A-ASF-CaMKIIδ signaling. © 2015 S. Karger AG, Basel.

Estradiol In Females May Negate Skeletal Muscle Myostatin Mrna Expression And Serum Myostatin Propeptide Levels After Eccentric Muscle Contractions

PubMed Central

Willoughby, Darryn S.; Wilborn, Colin D.

2006-01-01

Eccentric contractions produce a significant degree of inflammation and muscle injury that may increase the expression of myostatin. Due to its anti- oxidant and anti-flammatory effects, circulating 17-β estradiol (E2) may attenuate myostatin expression. Eight males and eight females performed 7 sets of 10 reps of eccentric contractions of the knee extensors at 150% 1-RM. Each female performed the eccentric exercise bout on a day that fell within her mid-luteal phase (d 21-23 of her 28-d cycle). Blood and muscle samples were obtained before and 6 and 24 h after exercise, while additional blood samples were obtained at 48 and 72 h after exercise. Serum E2 and myostatin LAP/propeptide (LAP/pro) levels were determined with ELISA, and myostatin mRNA expression determined using RT-PCR. Data were analyzed with two-way ANOVA and bivariate correlations (p < 0.05). Females had greater levels of serum E2 throughout the 72- h sampling period (p < 0.05). While males had greater body mass and fat-free mass, neither was correlated to the pre-exercise levels of myostatin mRNA and LAP/pro for either gender (p > 0.05). Compared to pre-exercise, males had significant increases (p < 0.05) in LAP/propetide and mRNA of 78% and 28%, respectively, at 24 h post-exercise, whereas females underwent respective decreases of 10% and 21%. E2 and LAP/propeptide were correlated at 6 h (r = -0.804, p = 0.016) and 24 h post- exercise (r = -0.841, p = 0.009) in males, whereas in females E2 levels were correlated to myostatin mRNA at 6 h (r =0.739, p = 0.036) and 24 h (r = 0.813, p = 0.014) post-exercise and LAP/propeptide at 6 h (r = 0.713, p = 0.047) and 24 h (r = 0.735, p = 0.038). In females, myostatin mRNA expression and serum LAP/propeptide levels do not appear to be significantly up-regulated following eccentric exercise, and may be due to higher levels of circulating E2. Key Points The pre-exercise levels of myostatin mRNA and propeptide were not significantly different between genders, and even though the total body mass and fat-free mass of males were significantly greater than females, neither was correlated to myostatin mRNA or LAP/propeptide. Myostatin mRNA expression in females is less than in males 24 h after a single bout of eccentric exercise. Myostatin LAP/propeptide levels in females are lower in females than in males 24 h after a single bout of eccentric exercise, thereby suggesting a gender-specific mechanism in which females may be less responsive to eccentric exercise than males. Myostatin mRNA expression in females is attenuated, possibly due to inhibition in myostatin signaling, and appears to be more related to the presence of a higher level of circulating E2 rather than body composition. Due to their higher level of E2, females seem to be less susceptible to the mechanism by which eccentric exercise apparently up-regulates myostatin mRNA expression in males. PMID:24357964

Sensitivity, Specificity, and Clinical Value of Human Papillomavirus (HPV) E6/E7 mRNA Assay as a Triage Test for Cervical Cytology and HPV DNA Test ▿

PubMed Central

Benevolo, Maria; Vocaturo, Amina; Caraceni, Donatella; French, Deborah; Rosini, Sandra; Zappacosta, Roberta; Terrenato, Irene; Ciccocioppo, Lucia; Frega, Antonio; Rossi, Paolo Giorgi

2011-01-01

There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of HPV DNA positive-mRNA negative cases. PMID:21525231

Nuclear m6A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development

PubMed Central

Leu, N. Adrian; Xu, Yang; Schultz, Richard M.

2018-01-01

The N6-methyladenosine (m6A) modification is the most prevalent internal RNA modification in eukaryotes. The majority of m6A sites are found in the last exon and 3’ UTRs. Here we show that the nuclear m6A reader YTHDC1 is essential for embryo viability and germline development in mouse. Specifically, YTHDC1 is required for spermatogonial development in males and for oocyte growth and maturation in females; Ythdc1-deficient oocytes are blocked at the primary follicle stage. Strikingly, loss of YTHDC1 leads to extensive alternative polyadenylation in oocytes, altering 3’ UTR length. Furthermore, YTHDC1 deficiency causes massive alternative splicing defects in oocytes. The majority of splicing defects in mutant oocytes are rescued by introducing wild-type, but not m6A-binding-deficient, YTHDC1. YTHDC1 is associated with the pre-mRNA 3’ end processing factors CPSF6, SRSF3, and SRSF7. Thus, YTHDC1 plays a critical role in processing of pre-mRNA transcripts in the oocyte nucleus and may have similar non-redundant roles throughout fetal development. PMID:29799838

High-pressure carbon dioxide/water pre-treatment of sugarcane bagasse and elephant grass: Assessment of the effect of biomass composition on process efficiency.

PubMed

Toscan, Andréia; Morais, Ana Rita C; Paixão, Susana M; Alves, Luís; Andreaus, Jürgen; Camassola, Marli; Dillon, Aldo José Pinheiro; Lukasik, Rafal M

2017-01-01

The performance of two lignocellulosic biomasses was studied in high-pressure carbon dioxide/water pre-treatment. Sugarcane bagasse and elephant grass were used to produce C 5 -sugars from hemicellulose and, simultaneously, to promote cellulose digestibility for enzymatic saccharification. Different pre-treatment conditions, with combined severity factor ranging from -1.17 to -0.04, were evaluated and maximal total xylan to xylose yields of 59.2wt.% (34.4wt.% xylooligomers) and 46.4wt.% (34.9wt.% xylooligomers) were attained for sugarcane bagasse and elephant grass, respectively. Furthermore, pre-treated biomasses were highly digestible, with glucan to glucose yields of 77.2mol% and 72.4mol% for sugarcane bagasse and elephant grass, respectively. High-pressure carbon dioxide/water pre-treatment provides high total C 5 -sugars and glucose recovery from both lignocellulosic biomasses; however it is highly influenced by composition and intrinsic features of each biomass. The obtained results confirm this approach as an effective and greener alternative to conventional pre-treatment processes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts.

PubMed

Sanford, Jeremy R; Wang, Xin; Mort, Matthew; Vanduyn, Natalia; Cooper, David N; Mooney, Sean D; Edenberg, Howard J; Liu, Yunlong

2009-03-01

Metazoan genes are encrypted with at least two superimposed codes: the genetic code to specify the primary structure of proteins and the splicing code to expand their proteomic output via alternative splicing. Here, we define the specificity of a central regulator of pre-mRNA splicing, the conserved, essential splicing factor SFRS1. Cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) identified 23,632 binding sites for SFRS1 in the transcriptome of cultured human embryonic kidney cells. SFRS1 was found to engage many different classes of functionally distinct transcripts including mRNA, miRNA, snoRNAs, ncRNAs, and conserved intergenic transcripts of unknown function. The majority of these diverse transcripts share a purine-rich consensus motif corresponding to the canonical SFRS1 binding site. The consensus site was not only enriched in exons cross-linked to SFRS1 in vivo, but was also enriched in close proximity to splice sites. mRNAs encoding RNA processing factors were significantly overrepresented, suggesting that SFRS1 may broadly influence the post-transcriptional control of gene expression in vivo. Finally, a search for the SFRS1 consensus motif within the Human Gene Mutation Database identified 181 mutations in 82 different genes that disrupt predicted SFRS1 binding sites. This comprehensive analysis substantially expands the known roles of human SR proteins in the regulation of a diverse array of RNA transcripts.

Autoregulatory mechanisms controlling the Microprocessor.

PubMed

Triboulet, Robinson; Gregory, Richard I

2010-01-01

The Microprocessor, comprising the ribonuclease Drosha and its essential cofactor, the double-stranded RNA-binding protein, DGCR8, is essential for the first step of the miRNA biogenesis pathway. It specifically cleaves double-stranded RNA within stem-loop structures of primary miRNA transcripts (pri-miRNAs) to generate precursor (pre-miRNA) intermediates. Pre-miRNAs are subsequently processed by Dicer to their mature 22 nt form. Thus, Microprocessor is essential for miRNA maturation, and pri-miRNA cleavage by this complex defines one end of the mature miRNA. Moreover, it is emerging that dysregulation of the Microprocessor is associated with various human diseases. It is therefore important to understand the mechanisms by which the expression of the subunits of the Microprocessor is regulated. Recent findings have uncovered a post-transcriptional mechanism that maintains the integrity of the Microprocessor. These studies revealed that the Microprocessor is involved in the processing of the messenger RNA (mRNA) that encodes DGCR8. This regulatory feedback loop, along with the reported role played by DGCR8 in the stabilization of Drosha protein, is part ofa newly identified regulatory mechanism controlling Microprocessor activity.

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.

Maternal BMI and gestational diabetes alter placental lipid transporters and fatty acid composition.

PubMed

Segura, Maria Teresa; Demmelmair, Hans; Krauss-Etschmann, Susanne; Nathan, Petra; Dehmel, Stefan; Padilla, Maria Carmen; Rueda, Ricardo; Koletzko, Berthold; Campoy, Cristina

2017-09-01

Placental fatty acid (FA) uptake and metabolism depend on maternal supply which may be altered when women have a high pre-pregnancy body mass index (BMI) or develop gestational diabetes (GDM). Consequently, an impaired FA transport to the fetus may negatively affect fetal development. While placental adaptation of maternal-fetal glucose transfer in mild GDM has been described, knowledge on placental FA acid metabolism and possible adaptations in response to maternal obesity or GDM is lacking. We aimed to analyze the FA composition and the expression of key genes involved in FA uptake and metabolism in placentas from women with pre-pregnancy normal weight (18.5 ≤ BMI<25 kg/m2), overweight (25 ≤ BMI<30 kg/m 2 ), obesity (BMI ≥ 30 kg/m 2 ), and lean pregnant women with GDM. Placental FA content was determined by gas liquid chromatography. Placental mRNA expression of FA transport proteins (FATP1, FATP4, FATP6), FA binding proteins (FABP3, FABP4, FABP7), FA translocase (FAT/CD36) and enzymes (Endothelial lipase (EL) and lipoprotein lipase (LPL)) were quantified by qRT-PCR. High pre-pregnancy BMI and GDM were associated with decreased placental FATP1, FATP4, EL and increased FAT/CD36 and FATP6 expressions. LPL mRNA levels and placental total FA content were similar among groups. Specific FA, including some long-chain polyunsaturated FA, were altered. Our results demonstrate that high pre-pregnancy BMI or GDM independently alter mRNA expression levels of genes involved in FA uptake and metabolism and the placental FA profile, which could affect fetal development and long-term health. Copyright © 2017. Published by Elsevier Ltd.

mRNA Cancer Vaccines-Messages that Prevail.

PubMed

Grunwitz, Christian; Kranz, Lena M

2017-01-01

During the last decade, mRNA became increasingly recognized as a versatile tool for the development of new innovative therapeutics. Especially for vaccine development, mRNA is of outstanding interest and numerous clinical trials have been initiated. Strikingly, all of these studies have proven that large-scale GMP production of mRNA is feasible and concordantly report a favorable safety profile of mRNA vaccines. Induction of T-cell immunity is a multi-faceted process comprising antigen acquisition, antigen processing and presentation, as well as immune stimulation. The effectiveness of mRNA vaccines is critically dependent on making the antigen(s) of interest available to professional antigen-presenting cells, especially DCs. Efficient delivery of mRNA into DCs in vivo remains a major challenge in the mRNA vaccine field. This review summarizes the principles of mRNA vaccines and highlights the importance of in vivo mRNA delivery and recent advances in harnessing their therapeutic potential.

Interplay between Alternative Splicing and Alternative Polyadenylation Defines the Expression Outcome of the Plant Unique OXIDATIVE TOLERANT-6 Gene.

PubMed

Li, Qingshun Q; Liu, Zhaoyang; Lu, Wenjia; Liu, Man

2017-05-17

Pre-mRNA alternative splicing and alternative polyadenylation have been implicated to play important roles during eukaryotic gene expression. However, much remains unknown regarding the regulatory mechanisms and the interactions of these two processes in plants. Here we focus on an Arabidopsis gene OXT6 (Oxidative Tolerant-6) that has been demonstrated to encode two proteins through alternative splicing and alternative polyadenylation. Specifically, alternative polyadenylation at Intron-2 of OXT6 produces a transcript coding for AtCPSF30, an Arabidopsis ortholog of 30 kDa subunit of the Cleavage and Polyadenylation Specificity Factor. On the other hand, alternative splicing of Intron-2 generates a longer transcript encoding a protein named AtC30Y, a polypeptide including most part of AtCPSF30 and a YT521B domain. To investigate the expression outcome of OXT6 in plants, a set of mutations were constructed to alter the splicing and polyadenylation patterns of OXT6. Analysis of transgenic plants bearing these mutations by quantitative RT-PCR revealed a competition relationship between these two processes. Moreover, when both splice sites and poly(A) signals were mutated, polyadenylation became the preferred mode of OXT6 processing. These results demonstrate the interplay between alternative splicing and alternative polyadenylation, and it is their concerted actions that define a gene's expression outcome.

Quantitative Single-Cell mRNA Analysis in Hydrogel Beads.

PubMed

Rakszewska, Agata; Stolper, Rosa J; Kolasa, Anna B; Piruska, Aigars; Huck, Wilhelm T S

2016-06-01

In recent years, technologies capable of analyzing single cells have emerged that are transforming many fields of biological research. Herein we report how DNA-functionalized hydrogel beads can serve as a matrix to capture mRNA from lysed single cells. mRNA quantification free of pre-amplification bias is ensured by using padlock probes and rolling circle amplification followed by hybridization with fluorescent probes. The number of transcripts in individual cells is assessed by simply counting fluorescent dots inside gel beads. The method extends the potential of existing techniques and provides a general platform for capturing molecules of interest from single cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Managing meat tenderness.

PubMed

Thompson, John

2002-11-01

This paper discusses the management of meat tenderness using a carcass grading scheme which utilizes the concept of total quality management of those factors which impact on beef palatability. The scheme called Meat Standards Australia (MSA) has identified the Critical Control Points (CCPs) from the production, pre-slaughter, processing and value adding sectors of the beef supply chain and quantified their relative importance using large-scale consumer testing. These CCPs have been used to manage beef palatability in two ways. Firstly, CCPs from the pre-slaughter and processing sectors have been used as mandatory criteria for carcasses to be graded. Secondly, other CCPs from the production and processing sectors have been incorporated into a model to predict palatability for individual muscles. The evidence for the importance of CCPs from the production (breed, growth path and HGP implants), pre-slaughter and processing (pH/temperature window, alternative carcass suspension, marbling and ageing) sectors are reviewed and the accuracy of the model to predict palatability for specific muscle×cooking techniques is presented.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

PubMed Central

Jablonski, Joseph; Clementz, Mark; Ryan, Kevin; Valente, Susana T.

2014-01-01

The 3’ end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages. Processing of the 3’ end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5’ cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3’ end processing of HIV-1 mRNAs. PMID:24835792

Nuclear Export of Messenger RNA

PubMed Central

Katahira, Jun

2015-01-01

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

46 CFR 160.027-7 - Pre-approval tests for alternate platform designs.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 6 2014-10-01 2014-10-01 false Pre-approval tests for alternate platform designs. 160... § 160.027-7 Pre-approval tests for alternate platform designs. (a) The tests in this section are for... deformation as a result of this test. (c) The float body must be supported so that the platform is suspended...

46 CFR 160.027-7 - Pre-approval tests for alternate platform designs.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 6 2011-10-01 2011-10-01 false Pre-approval tests for alternate platform designs. 160... § 160.027-7 Pre-approval tests for alternate platform designs. (a) The tests in this section are for... deformation as a result of this test. (c) The float body must be supported so that the platform is suspended...

46 CFR 160.027-7 - Pre-approval tests for alternate platform designs.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 6 2013-10-01 2013-10-01 false Pre-approval tests for alternate platform designs. 160... § 160.027-7 Pre-approval tests for alternate platform designs. (a) The tests in this section are for... deformation as a result of this test. (c) The float body must be supported so that the platform is suspended...

46 CFR 160.027-7 - Pre-approval tests for alternate platform designs.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 6 2010-10-01 2010-10-01 false Pre-approval tests for alternate platform designs. 160... § 160.027-7 Pre-approval tests for alternate platform designs. (a) The tests in this section are for... deformation as a result of this test. (c) The float body must be supported so that the platform is suspended...

46 CFR 160.027-7 - Pre-approval tests for alternate platform designs.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 6 2012-10-01 2012-10-01 false Pre-approval tests for alternate platform designs. 160... § 160.027-7 Pre-approval tests for alternate platform designs. (a) The tests in this section are for... deformation as a result of this test. (c) The float body must be supported so that the platform is suspended...

Department of Energy Technology Readiness Assessments - Process Guide and Training Plan

DTIC Science & Technology

2008-09-12

Hanford Waste Treatment and Immobilization Plant ( WTP ) Analytical Laboratory, Low Activity Waste (LAW) Facility and Balance of Facilities (3 TRAs... WTP High-Level Waste (HLW) Facility – WTP Pre-Treatment (PT) Facility – Hanford River Protection Project Low Activity Waste Treatment Alternatives

Cyclosporin A reduces matrix metalloproteinases and collagen expression in dermal fibroblasts from regenerative FOXN1 deficient (nude) mice

PubMed Central

2013-01-01

Background Cyclosporin A (CsA), an immunosuppressive agent modifies the wound healing process through an influence on extracellular matrix metabolism. We have compared the effects of CsA on dermal fibroblasts from nude (FOXN1 deficient) mice, a genetic model of skin scarless healing, and from control (C57BL/6 J (B6) mice to evaluate metabolic pathways that appear to have important roles in the process of scarless healing/regeneration. Results High levels of matrix metalloproteinases (MMPs) and collagen III expression in dermal fibroblasts from nude (regenerative) mice were down-regulated by CsA treatment to the levels observed in dermal fibroblasts from B6 (non-regenerative) mice. In contrast, dermal fibroblasts from control mice respond to CsA treatment with a minor reduction of Mmps mRNA and 2.5-fold increase expression of collagen I mRNA. An in vitro migratory assay revealed that CsA treatment profoundly delayed the migratory behavior of dermal fibroblasts from both nude and control mice. Conclusion The data suggest that by alternation of the accumulation of extracellular matrix components CsA treatment stimulates the transition from a scarless to a scar healing. PMID:23547542

Schistosoma japonicum ova maintains epithelial barrier function during experimental colitis.

PubMed

Xia, Chen-Mei; Zhao, Yuan; Jiang, Li; Jiang, Jie; Zhang, Shun-Cai

2011-11-21

To evaluate the impacts of Schistosoma japonicum (S. japonicum) ova on the tight junction barriers in a trinitrobenzenesulfonic acid (TNBS)-induced colitis model. Balb/c mice were randomly divided into three groups: control group; TNBS(+)ova(-) group and TNBS(+)ova(+) group. TNBS was used intracolonic to induce colitis and mice of the TNBS(+)ova(+) group were pre-exposed to S. japonicum ova as a prophylactic intervention. Colon inflammation was quantified using following variables: mouse mortality, weight loss, colon extent and microscopic inflammation score. Serum expression of tumor necrosis factor-α and interferon-γ were assessed to evaluate the systemic inflammatory response. NOD2 and its mRNA were also tested. Bacterial translocations were tested by culturing blood and several tissues. ZO-1 and occludin were chosen as the representations of tight junction proteins. Both the proteins and mRNA were assessed. Ova pre-treatment contributed to the relief of colitis and decreased the mortality of the models. NOD2 expression was significantly downregulated when pretreated with the ova. The TNBS injection caused a significant downregulation of ZO-1 and occludin mRNA together with their proteins in the colon; ova pre-exposure reversed these alterations. Treatment with S. japonicum ova in the colitis model caused lower intestinal bacterial translocation frequency. S. japonicum ova can maintain epithelial barrier function through increasing tight junction proteins, thus causing less exposure of NOD2 to the luminal antigens which may activate a series of inflammatory factors and induce colitis.

Sequence variants of KHDRBS1 as high penetrance susceptibility risks for primary ovarian insufficiency by mis-regulating mRNA alternative splicing.

PubMed

Wang, Binbin; Li, Lin; Zhu, Ying; Zhang, Wei; Wang, Xi; Chen, Beili; Li, Tengyan; Pan, Hong; Wang, Jing; Kee, Kehkooi; Cao, Yunxia

2017-10-01

Does a novel heterozygous KHDRBS1 variant, identified using whole-exome sequencing (WES) in two patients with primary ovarian insufficiency (POI) in a pedigree, cause defects in mRNA alternative splicing? The heterozygous variant of KHDRBS1 was confirmed to cause defects in alternative splicing of many genes involved in DNA replication and repair. Studies in mice revealed that Khdrbs1 deficient females are subfertile, which manifests as delayed sexual maturity and significantly reduced numbers of secondary and pre-antral follicles. No mutation of KHDRBS1, however, has been reported in patients with POI. This genetic and functional study used WES to find putative mutations in a POI pedigree. Altogether, 215 idiopathic POI patients and 400 healthy controls were screened for KHDRBS1 mutations. Two POI patients were subjected to WES to identify sequence variants. Mutational analysis of the KHDRBS1 gene in 215 idiopathic POI patients and 400 healthy controls were performed. RNA-sequencing was carried out to find the mis-regulation of gene expression due to KHDRBS1 mutation. Bioinformatics was used to analyze the change in alternative splicing events. We identified a heterozygous mutation (c.460A > G, p.M154V) in KHDRBS1 in two patients. Further mutational analysis of 215 idiopathic POI patients with the KHDRBS1 gene found one heterozygous mutation (c.263C > T, p.P88L). We failed to find these two mutations in 400 healthy control women. Using RNA-sequencing, we found that the KGN cells expressing the M154V KHDRBS1 mutant had different expression of 66 genes compared with wild-type (WT) cells. Furthermore, 145 genes were alternatively spliced in M154V cells, and these genes were enriched for DNA replication and repair function, revealing a potential underlying mechanism of the pathology that leads to POI. Although the in vitro assays demonstrated the effect of the KHDRBS1 variant on alternative splicing, further studies are needed to validate the in vivo effects on germ cell and follicle development. This finding provides researchers and clinicians a better understanding of the etiology and molecular mechanism of POI. This study was supported by the Ministry of Science and Technology of China (2012CB944704; 2012CB966702), National Research Institute for Family Planning (2017GJZ05), the National Natural Science Foundation of China (31171429) and Beijing Advanced Innovation Center for Structural Biology. The authors declare no conflict of interest. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

The determinants of alternative RNA splicing in human cells.

PubMed

Ramanouskaya, Tatsiana V; Grinev, Vasily V

2017-12-01

Alternative splicing represents an important level of the regulation of gene function in eukaryotic organisms. It plays a critical role in virtually every biological process within an organism, including regulation of cell division and cell death, differentiation of tissues in the embryo and the adult organism, as well as in cellular response to diverse environmental factors. In turn, studies of the last decade have shown that alternative splicing itself is controlled by different mechanisms. Unfortunately, there is no clear understanding of how these diverse mechanisms, or determinants, regulate and constrain the set of alternative RNA species produced from any particular gene in every cell of the human body. Here, we provide a consolidated overview of alternative splicing determinants including RNA-protein interactions, epigenetic regulation via chromatin remodeling, coupling of transcription-to-alternative splicing, effect of secondary structures in pre-RNA, and function of the RNA quality control systems. We also extensively and critically discuss some mechanistic insights on coordinated inclusion/exclusion of exons during the formation of mature RNA molecules. We conclude that the final structure of RNA is pre-determined by a complex interplay between cis- and trans-acting factors. Altogether, currently available empirical data significantly expand our understanding of the functioning of the alternative splicing machinery of cells in normal and pathological conditions. On the other hand, there are still many blind spots that require further deep investigations.

miR-200/375 control epithelial plasticity-associated alternative splicing by repressing the RNA-binding protein Quaking.

PubMed

Pillman, Katherine A; Phillips, Caroline A; Roslan, Suraya; Toubia, John; Dredge, B Kate; Bert, Andrew G; Lumb, Rachael; Neumann, Daniel P; Li, Xiaochun; Conn, Simon J; Liu, Dawei; Bracken, Cameron P; Lawrence, David M; Stylianou, Nataly; Schreiber, Andreas W; Tilley, Wayne D; Hollier, Brett G; Khew-Goodall, Yeesim; Selth, Luke A; Goodall, Gregory J; Gregory, Philip A

2018-06-05

Members of the miR-200 family are critical gatekeepers of the epithelial state, restraining expression of pro-mesenchymal genes that drive epithelial-mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR-200c and another epithelial-enriched miRNA, miR-375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA-binding protein Quaking (QKI). During EMT, QKI-5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI-5 is both necessary and sufficient to direct EMT-associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial-derived cancer types. Importantly, several actin cytoskeleton-associated genes are directly targeted by both QKI and miR-200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT These findings demonstrate the existence of a miR-200/miR-375/QKI axis that impacts cancer-associated epithelial cell plasticity through widespread control of alternative splicing. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Systematic variation in mRNA 3′-processing signals during mouse spermatogenesis

PubMed Central

Liu, Donglin; Brockman, J. Michael; Dass, Brinda; Hutchins, Lucie N.; Singh, Priyam; McCarrey, John R.; MacDonald, Clinton C.; Graber, Joel H.

2007-01-01

Gene expression and processing during mouse male germ cell maturation (spermatogenesis) is highly specialized. Previous reports have suggested that there is a high incidence of alternative 3′-processing in male germ cell mRNAs, including reduced usage of the canonical polyadenylation signal, AAUAAA. We used EST libraries generated from mouse testicular cells to identify 3′-processing sites used at various stages of spermatogenesis (spermatogonia, spermatocytes and round spermatids) and testicular somatic Sertoli cells. We assessed differences in 3′-processing characteristics in the testicular samples, compared to control sets of widely used 3′-processing sites. Using a new method for comparison of degenerate regulatory elements between sequence samples, we identified significant changes in the use of putative 3′-processing regulatory sequence elements in all spermatogenic cell types. In addition, we observed a trend towards truncated 3′-untranslated regions (3′-UTRs), with the most significant differences apparent in round spermatids. In contrast, Sertoli cells displayed a much smaller trend towards 3′-UTR truncation and no significant difference in 3′-processing regulatory sequences. Finally, we identified a number of genes encoding mRNAs that were specifically subject to alternative 3′-processing during meiosis and postmeiotic development. Our results highlight developmental differences in polyadenylation site choice and in the elements that likely control them during spermatogenesis. PMID:17158511

Improving Theory Application among Pre-Service Teachers

ERIC Educational Resources Information Center

Jones, Anne

2009-01-01

This article describes the process of implementing Inter-collegially Supported Learning (Tigchelaar and Melief, 1997) and reflection using the ALACT [action, looking back, awareness of essential aspects, creating alternative methods of action, and trial] model (Korthhagen, 1985, 1988) an Elementary Masters in Teaching Program. This study takes a…

Four weeks one-leg training and high fat diet does not alter PPARalpha protein or mRNA expression in human skeletal muscle.

PubMed

Helge, J W; Bentley, D; Schjerling, P; Willer, M; Gibala, M J; Franch, J; Tapia-Laliena, M A; Daugaard, J R; Andersen, J L

2007-09-01

Fatty acid metabolism is influenced by training and diet with exercise training mediating this through activation of nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in skeletal muscle. This study investigated the effect of training and high fat or normal diet on PPARalpha expression in human skeletal muscle. Thirteen men trained one leg (T) four weeks (31.5 h in total), while the other leg (UT) served as control. During the 4 weeks six subjects consumed high fat (FAT) diet and seven subjects maintained a normal (CHO) diet. Biopsies were obtained from vastus lateralis muscle in both legs before and after training. After the biopsy, one-leg extension exercise was performed in random order with both legs 30 min at 95% of workload max. A training effect was evident as citrate synthase activity increased (P < 0.05) by 15% in the trained, but not the control leg in both groups. During exercise respiratory exchange ratio was lower in FAT (0.86 +/- 0.01, 0.83 +/- 0.01, mean +/- SEM) than CHO (0.96 +/- 0.02, 0.94 +/- 0.03) and in UT than T legs, respectively. The PPARalpha protein (144 +/- 44, 104 +/- 28, 79 +/- 15, 79 +/- 14, % of pre level) and PPARalpha mRNA (69 +/- [2, 2], 78 +/- [7, 6], 92 +/- [22, 18], 106 +/- [21, 18], % of pre level, geometric mean +/- SEM) expression remained unchanged by diet and training in FAT (UT, T) and CHO (UT, T), respectively. After the training and diet CS, HAD, PPARalpha, UCP2, UCP3 and mFABP mRNA content remained unchanged, whereas GLUT4 mRNA was lower in both groups and LDHA mRNA was lower (P < 0.05) only in FAT. 4 weeks one leg knee extensor training did not affect PPARalpha protein or mRNA expression. Furthermore, higher fat oxidation during exercise after fat rich diet was not accompanied by an increased PPARalpha protein or mRNA expression after 4 weeks.

Cloning and expression of a sorghum gene with homology to maize vp1. Its potential involvement in pre-harvest sprouting resistance.

PubMed

Carrari, F; Perez-Flore, L; Lijavetzky, D; Enciso, S; Sanchez, R; Benech-Arnold, R; Iusem, N

2001-04-01

Pre-harvest sprouting (PHS) in sorghum is related to the lack of a normal dormancy level during seed development and maturation. Based on previous evidence that seed dormancy in maize is controlled by the vp1 gene, we used a PCR-based approach to isolate two Sorghum bicolor genomic and cDNA clones from two genotypes exhibiting different PHS behaviour and sensitivity to abscisic acid (ABA). The two 699 amino acid predicted protein sequences differ in two residues at positions 341 (Gly or Cys within the repression domain) and 448 (Pro or Ser) and show over 80, 70 and 60% homology to maize, rice and oat VP1 proteins respectively. Expression analysis of the sorghum vp1 gene in the two lines shows a slightly higher level of vp1 mRNA in the embryos susceptible to PHS than in those resistant to PHS during embryogenesis. However, timing of expression was different between these genotypes during this developmental process. Whereas for the former the main peak of expression was observed at 20 days after pollination (DAP), the peak in the latter was found at later developmental stages when seed maturation was almost complete. Under favourable germination conditions and in the presence of fluridone (an inhibitor of ABA biosynthesis), sorghum vp1 mRNA showed to be consistently correlated with sensitivity to ABA but not with ABA content and dormancy.

Improving performances of suboptimal greedy iterative biclustering heuristics via localization.

PubMed

Erten, Cesim; Sözdinler, Melih

2010-10-15

Biclustering gene expression data is the problem of extracting submatrices of genes and conditions exhibiting significant correlation across both the rows and the columns of a data matrix of expression values. Even the simplest versions of the problem are computationally hard. Most of the proposed solutions therefore employ greedy iterative heuristics that locally optimize a suitably assigned scoring function. We provide a fast and simple pre-processing algorithm called localization that reorders the rows and columns of the input data matrix in such a way as to group correlated entries in small local neighborhoods within the matrix. The proposed localization algorithm takes its roots from effective use of graph-theoretical methods applied to problems exhibiting a similar structure to that of biclustering. In order to evaluate the effectivenesss of the localization pre-processing algorithm, we focus on three representative greedy iterative heuristic methods. We show how the localization pre-processing can be incorporated into each representative algorithm to improve biclustering performance. Furthermore, we propose a simple biclustering algorithm, Random Extraction After Localization (REAL) that randomly extracts submatrices from the localization pre-processed data matrix, eliminates those with low similarity scores, and provides the rest as correlated structures representing biclusters. We compare the proposed localization pre-processing with another pre-processing alternative, non-negative matrix factorization. We show that our fast and simple localization procedure provides similar or even better results than the computationally heavy matrix factorization pre-processing with regards to H-value tests. We next demonstrate that the performances of the three representative greedy iterative heuristic methods improve with localization pre-processing when biological correlations in the form of functional enrichment and PPI verification constitute the main performance criteria. The fact that the random extraction method based on localization REAL performs better than the representative greedy heuristic methods under same criteria also confirms the effectiveness of the suggested pre-processing method. Supplementary material including code implementations in LEDA C++ library, experimental data, and the results are available at http://code.google.com/p/biclustering/ cesim@khas.edu.tr; melihsozdinler@boun.edu.tr Supplementary data are available at Bioinformatics online.

Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

PubMed Central

Aitken, Colin Echeverría; Beznosková, Petra; Vlčkova, Vladislava; Chiu, Wen-Ling; Zhou, Fujun; Valášek, Leoš Shivaya; Hinnebusch, Alan G; Lorsch, Jon R

2016-01-01

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA. DOI: http://dx.doi.org/10.7554/eLife.20934.001 PMID:27782884

Brief review: Preparation techniques of biomass based activated carbon monolith electrode for supercapacitor applications

NASA Astrophysics Data System (ADS)

Taer, Erman; Taslim, Rika

2018-02-01

The synthesis of activated carbon monolith electrode made from a biomass material using the hydrolytic pressure or the pelletization technique of pre-carbonized materials is one of standard reported methods. Several steps such as pre-carbonization, milling, chemical activation, hydraulic press, carbonization, physical activation, polishing and washing need to be accomplished in the production of electrodes by this method. This is relatively a long process that need to be simplified. In this paper we present the standard method and proceed with the introduction to several alternative methods in the synthesis of activated carbon monolith electrodes. The alternative methods were emphasized on the selection of suitable biomass materials. All of carbon electrodes prepared by different methods will be analyzed for physical and electrochemical properties. The density, degree of crystallinity, surface morphology are examples for physical study and specific capacitance was an electrochemical properties that has been analysed. This alternative method has offered a specific capacitance in the range of 10 to 171 F/g.

Alternative Splicing of a Novel Inducible Exon Diversifies the CASK Guanylate Kinase Domain

PubMed Central

Dembowski, Jill A.; An, Ping; Scoulos-Hanson, Maritsa; Yeo, Gene; Han, Joonhee; Fu, Xiang-Dong; Grabowski, Paula J.

2012-01-01

Alternative pre-mRNA splicing has a major impact on cellular functions and development with the potential to fine-tune cellular localization, posttranslational modification, interaction properties, and expression levels of cognate proteins. The plasticity of regulation sets the stage for cells to adjust the relative levels of spliced mRNA isoforms in response to stress or stimulation. As part of an exon profiling analysis of mouse cortical neurons stimulated with high KCl to induce membrane depolarization, we detected a previously unrecognized exon (E24a) of the CASK gene, which encodes for a conserved peptide insertion in the guanylate kinase interaction domain. Comparative sequence analysis shows that E24a appeared selectively in mammalian CASK genes as part of a >3,000 base pair intron insertion. We demonstrate that a combination of a naturally defective 5′ splice site and negative regulation by several splicing factors, including SC35 (SRSF2) and ASF/SF2 (SRSF1), drives E24a skipping in most cell types. However, this negative regulation is countered with an observed increase in E24a inclusion after neuronal stimulation and NMDA receptor signaling. Taken together, E24a is typically a skipped exon, which awakens during neuronal stimulation with the potential to diversify the protein interaction properties of the CASK polypeptide. PMID:23008758

Alternative splicing: a novel mechanism of regulation identified in the chorismate mutase gene of the potato cyst nematode Globodera rostochiensis.

PubMed

Lu, Shun-Wen; Tian, Duanhua; Borchardt-Wier, Harmony B; Wang, Xiaohong

2008-11-01

Chorismate mutase (CM) secreted from the stylet of plant-parasitic nematodes plays an important role in plant parasitism. We isolated and characterized a new nematode CM gene (Gr-cm-1) from the potato cyst nematode, Globodera rostochiensis. The Gr-cm-1 gene was found to exist in the nematode genome as a single-copy gene that has two different alleles, Gr-cm-1A and Gr-cm-1B, both of which could give rise to two different mRNA transcripts of Gr-cm-1 and Gr-cm-1-IRII. In situ mRNA hybridization showed that the Gr-cm-1 gene was exclusively expressed within the subventral oesophageal gland cells of the nematode. Gr-cm-1 was demonstrated to encode a functional CM (GR-CM-1) potentially having a dimeric structure as the secreted bacterial *AroQ CMs. Gr-cm-1-IRII, generated by retention of intron 2 of the Gr-cm-1 pre-mRNA through alternative splicing (AS), would encode a truncated protein (GR-CM-1t) lacking the CM domain with no CM activity. The quantitative real-time reverse transcription-PCR assay revealed that splicing of the Gr-cm-1 gene was developmentally regulated; Gr-cm-1 was up-regulated whereas Gr-cm-1-IRII was down-regulated in early nematode parasitic stages compared to the preparasitic juvenile stage. Low-temperature SDS-PAGE analysis revealed that GR-CM-1 could form homodimers when expressed in Escherichia coli and the dimerization domain was retained in the truncated GR-CM-1t protein. The specific interaction between the two proteins was demonstrated in yeast. Our data suggested that the novel splice variant might function as a dominant negative isoform through heterodimerization with the full-length GR-CM-1 protein and that AS may represent an important mechanism for regulating CM activity during nematode parasitism.

Specificity in endoplasmic reticulum-stress signaling in yeast entails a step-wise engagement of HAC1 mRNA to clusters of the stress sensor Ire1.

PubMed

van Anken, Eelco; Pincus, David; Coyle, Scott; Aragón, Tomás; Osman, Christof; Lari, Federica; Gómez Puerta, Silvia; Korennykh, Alexei V; Walter, Peter

2014-12-30

Insufficient protein-folding capacity in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR). In the ER lumen, accumulation of unfolded proteins activates the transmembrane ER-stress sensor Ire1 and drives its oligomerization. In the cytosol, Ire1 recruits HAC1 mRNA, mediating its non-conventional splicing. The spliced mRNA is translated into Hac1, the key transcription activator of UPR target genes that mitigate ER-stress. In this study, we report that oligomeric assembly of the ER-lumenal domain is sufficient to drive Ire1 clustering. Clustering facilitates Ire1's cytosolic oligomeric assembly and HAC1 mRNA docking onto a positively charged motif in Ire1's cytosolic linker domain that tethers the kinase/RNase to the transmembrane domain. By the use of a synthetic bypass, we demonstrate that mRNA docking per se is a pre-requisite for initiating Ire1's RNase activity and, hence, splicing. We posit that such step-wise engagement between Ire1 and its mRNA substrate contributes to selectivity and efficiency in UPR signaling.

mRNA: From a chemical blueprint for protein production to an off-the-shelf therapeutic.

PubMed

Van Lint, Sandra; Heirman, Carlo; Thielemans, Kris; Breckpot, Karine

2013-02-01

Two decades ago, mRNA became the focus of research in molecular medicine and was proposed as an active pharmaceutical ingredient for the therapy of cancer. In this regard, mRNA has been mainly used for ex vivo modification of antigen-presenting cells (APCs), such as dendritic cells (DCs). This vaccination strategy has proven to be safe, well tolerated and capable of inducing tumor antigen-specific immune responses. Recently, the direct application of mRNA for in situ modification of APCs, hence immunization was shown to be feasible and at least as effective as DC-based immunization in pre-clinical models. It is believed that application of mRNA as an off-the-shelf vaccine represents an important step in the development of future cancer immunotherapeutic strategies. Here, we will discuss the use of ex vivo mRNA-modified DCs and "naked mRNA" for cancer immunotherapy focusing on parameters such as the employed DC subtype, DC activation stimulus and route of immunization. In addition, we will provide an overview on the clinical trials published so far, trying to link their outcome to the aforementioned parameters.

Diverse alternative back-splicing and alternative splicing landscape of circular RNAs

PubMed Central

Zhang, Xiao-Ou; Dong, Rui; Zhang, Yang; Zhang, Jia-Lin; Luo, Zheng; Zhang, Jun; Chen, Ling-Ling; Yang, Li

2016-01-01

Circular RNAs (circRNAs) derived from back-spliced exons have been widely identified as being co-expressed with their linear counterparts. A single gene locus can produce multiple circRNAs through alternative back-splice site selection and/or alternative splice site selection; however, a detailed map of alternative back-splicing/splicing in circRNAs is lacking. Here, with the upgraded CIRCexplorer2 pipeline, we systematically annotated different types of alternative back-splicing and alternative splicing events in circRNAs from various cell lines. Compared with their linear cognate RNAs, circRNAs exhibited distinct patterns of alternative back-splicing and alternative splicing. Alternative back-splice site selection was correlated with the competition of putative RNA pairs across introns that bracket alternative back-splice sites. In addition, all four basic types of alternative splicing that have been identified in the (linear) mRNA process were found within circRNAs, and many exons were predominantly spliced in circRNAs. Unexpectedly, thousands of previously unannotated exons were detected in circRNAs from the examined cell lines. Although these novel exons had similar splice site strength, they were much less conserved than known exons in sequences. Finally, both alternative back-splicing and circRNA-predominant alternative splicing were highly diverse among the examined cell lines. All of the identified alternative back-splicing and alternative splicing in circRNAs are available in the CIRCpedia database (http://www.picb.ac.cn/rnomics/circpedia). Collectively, the annotation of alternative back-splicing and alternative splicing in circRNAs provides a valuable resource for depicting the complexity of circRNA biogenesis and for studying the potential functions of circRNAs in different cells. PMID:27365365

Hydrothermal pre-treatment of rapeseed straw.

PubMed

Díaz, Manuel J; Cara, Cristóbal; Ruiz, Encarnación; Romero, Inmaculada; Moya, Manuel; Castro, Eulogio

2010-04-01

As a first step for ethanol production from alternative raw materials, rapeseed straw was studied for fermentable sugar production. Liquid hot water was used as a pre-treatment method and the influence of the main pre-treatment variables was assessed. Experimental design and response surface methodology were applied using pre-treatment temperature and process time as factors. The pretreated solids were further submitted to enzymatic hydrolysis and the corresponding yields were used as pre-treatment performance evaluation. Liquid fractions obtained from pre-treatment were also characterized in terms of sugars and no-sugar composition. A mathematical model describing pre-treatment effects is proposed. Results show that enzymatic hydrolysis yields near to 100% based on pretreated materials can be achieved at 210-220 degrees C for 30-50 min, equivalent to near 70% of glucose present in the raw material. According to the mathematical model, a softer pre-treatment at 193 degrees C for 27 min results in 65% of glucose and 39% of xylose available for fermentation. Copyright 2009 Elsevier Ltd. All rights reserved.

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.

Drosophila Symplekin localizes dynamically to the histone locus body and tricellular junctions.

PubMed

Tatomer, Deirdre C; Rizzardi, Lindsay F; Curry, Kaitlin P; Witkowski, Alison M; Marzluff, William F; Duronio, Robert J

2014-01-01

The scaffolding protein Symplekin is part of multiple complexes involved in generating and modifying the 3' end of mRNAs, including cleavage-polyadenylation, histone pre-mRNA processing and cytoplasmic polyadenylation. To study these functions in vivo, we examined the localization of Symplekin during development and generated mutations of the Drosophila Symplekin gene. Mutations in Symplekin that reduce Symplekin protein levels alter the efficiency of both poly A(+) and histone mRNA 3' end formation resulting in lethality or sterility. Histone mRNA synthesis takes place at the histone locus body (HLB) and requires a complex composed of Symplekin and several polyadenylation factors that associates with the U7 snRNP. Symplekin is present in the HLB in the early embryo when Cyclin E/Cdk2 is active and histone genes are expressed and is absent from the HLB in cells that have exited the cell cycle. During oogenesis, Symplekin is preferentially localized to HLBs during S-phase in endoreduplicating follicle cells when histone mRNA is synthesized. After the completion of endoreplication, Symplekin accumulates in the cytoplasm, in addition to the nucleoplasm, and localizes to tricellular junctions of the follicle cell epithelium. This localization depends on the RNA binding protein ypsilon schachtel. CPSF-73 and a number of mRNAs are localized at this same site, suggesting that Symplekin participates in cytoplasmic polyadenylation at tricellular junctions.

The "periodic table" of the genetic code: A new way to look at the code and the decoding process.

PubMed

Komar, Anton A

2016-01-01

Henri Grosjean and Eric Westhof recently presented an information-rich, alternative view of the genetic code, which takes into account current knowledge of the decoding process, including the complex nature of interactions between mRNA, tRNA and rRNA that take place during protein synthesis on the ribosome, and it also better reflects the evolution of the code. The new asymmetrical circular genetic code has a number of advantages over the traditional codon table and the previous circular diagrams (with a symmetrical/clockwise arrangement of the U, C, A, G bases). Most importantly, all sequence co-variances can be visualized and explained based on the internal logic of the thermodynamics of codon-anticodon interactions.

Mapping contacts between gRNA and mRNA in trypanosome RNA editing.

PubMed

Leung, S S; Koslowsky, D J

1999-02-01

All guide RNAs (gRNAs) identified to date have defined 5' anchor sequences, guiding sequences and a non-encoded 3' uridylate tail. The 5' anchor is required for in vitro editing and is thought to be responsible for selection and binding to the pre-edited mRNA. Little is known, however, about how the gRNAs are used to direct RNA editing. Utilizing the photo-reactive crosslinking agent, azidophenacyl (APA), attached to the 5'- or 3'-terminus of the gRNA, we have begun to map the structural relationships between the different defined regions of the gRNA with the pre-edited mRNA. Analyses of crosslinked conjugates produced with a 5'-terminal APA group confirm that the anchor of the gRNA is correctly positioning the interacting molecules. 3' Crosslinks (X-linker placed at the 3'-end of a U10tail) have also been mapped for three different gRNA/mRNA pairs. In all cases, analyses indicate that the U-tail can interact with a range of nucleotides located upstream of the first edited site. It appears that the U-tail prefers purine-rich sites, close to the first few editing sites. These results suggest that the U-tail may act in concert with the anchor to melt out secondary structure in the mRNA in the immediate editing domain, possibly increasing the accessibility of the editing complex to the proper editing sites.

Tocotrienol-rich fraction prevents cellular aging by modulating cell proliferation signaling pathways.

PubMed

Khor, S C; Mohd Yusof, Y A; Wan Ngah, W Z; Makpol, S

Vitamin E has been suggested as nutritional intervention for the prevention of degenerative and age-related diseases. In this study, we aimed to elucidate the underlying mechanism of tocotrienol-rich fraction (TRF) in delaying cellular aging by targeting the proliferation signaling pathways in human diploid fibroblasts (HDFs). Tocotrienol-rich fraction was used to treat different stages of cellular aging of primary human diploid fibroblasts viz. young (passage 6), pre-senescent (passage 15) and senescent (passage 30). Several selected targets involved in the downstream of PI3K/AKT and RAF/MEK/ERK pathways were compared in total RNA and protein. Different transcriptional profiles were observed in young, pre-senescent and senescent HDFs, in which cellular aging increased AKT, FOXO3, CDKN1A and RSK1 mRNA expression level, but decreased ELK1, FOS and SIRT1 mRNA expression level. With tocotrienol-rich fraction treatment, gene expression of AKT, FOXO3, ERK and RSK1 mRNA was decreased in senescent cells, but not in young cells. The three down-regulated mRNA in cellular aging, ELK1, FOS and SIRT1, were increased with tocotrienol-rich fraction treatment. Expression of FOXO3 and P21Cip1 proteins showed up-regulation in senescent cells but tocotrienol-rich fraction only decreased P21Cip1 protein expression in senescent cells. Tocotrienol-rich fraction exerts gene modulating properties that might be responsible in promoting cell cycle progression during cellular aging.

Elevating serotonin pre-partum alters the Holstein dairy cow hepatic adaptation to lactation

PubMed Central

Weaver, Samantha R.; Prichard, Allan S.; Maerz, Noah L.; Prichard, Austin P.; Endres, Elizabeth L.; Hernández-Castellano, Lorenzo E.; Akins, Matthew S.; Bruckmaier, Rupert M.

2017-01-01

Serotonin is known to regulate energy and calcium homeostasis in several mammalian species. The objective of this study was to determine if pre-partum infusions of 5-hydroxytryptophan (5-HTP), the immediate precursor to serotonin synthesis, could modulate energy homeostasis at the level of the hepatocyte in post-partum Holstein and Jersey dairy cows. Twelve multiparous Holstein cows and twelve multiparous Jersey cows were intravenously infused daily for approximately 7 d pre-partum with either saline or 1 mg/kg bodyweight of 5-HTP. Blood was collected for 14 d post-partum and on d30 post-partum. Liver biopsies were taken on d1 and d7 post-partum. There were no changes in the circulating concentrations of glucose, insulin, glucagon, non-esterified fatty acids, or urea nitrogen in response to treatment, although there were decreased beta-hydroxybutyrate concentrations with 5-HTP treatment around d6 to d10 post-partum, particularly in Jersey cows. Cows infused with 5-HTP had increased hepatic serotonin content and increased mRNA expression of the serotonin 2B receptor on d1 and d7 post-partum. Minimal changes were seen in the hepatic mRNA expression of various gluconeogenic enzymes. There were no changes in the mRNA expression profile of cell-cycle progression marker cyclin-dependent kinase 4 or apoptotic marker caspase 3, although proliferating cell nuclear antigen expression tended to be increased in Holstein cows infused with 5-HTP on d1 post-partum. Immunofluorescence assays showed an increased number of CASP3- and Ki67-positive cells in Holstein cows infused with 5-HTP on d1 post-partum. Given the elevated hepatic serotonin content and increased mRNA abundance of 5HTR2B, 5-HTP infusions may be stimulating an autocrine-paracrine adaptation to lactation in the Holstein cow liver. PMID:28922379

Elevating serotonin pre-partum alters the Holstein dairy cow hepatic adaptation to lactation.

PubMed

Weaver, Samantha R; Prichard, Allan S; Maerz, Noah L; Prichard, Austin P; Endres, Elizabeth L; Hernández-Castellano, Lorenzo E; Akins, Matthew S; Bruckmaier, Rupert M; Hernandez, Laura L

2017-01-01

Serotonin is known to regulate energy and calcium homeostasis in several mammalian species. The objective of this study was to determine if pre-partum infusions of 5-hydroxytryptophan (5-HTP), the immediate precursor to serotonin synthesis, could modulate energy homeostasis at the level of the hepatocyte in post-partum Holstein and Jersey dairy cows. Twelve multiparous Holstein cows and twelve multiparous Jersey cows were intravenously infused daily for approximately 7 d pre-partum with either saline or 1 mg/kg bodyweight of 5-HTP. Blood was collected for 14 d post-partum and on d30 post-partum. Liver biopsies were taken on d1 and d7 post-partum. There were no changes in the circulating concentrations of glucose, insulin, glucagon, non-esterified fatty acids, or urea nitrogen in response to treatment, although there were decreased beta-hydroxybutyrate concentrations with 5-HTP treatment around d6 to d10 post-partum, particularly in Jersey cows. Cows infused with 5-HTP had increased hepatic serotonin content and increased mRNA expression of the serotonin 2B receptor on d1 and d7 post-partum. Minimal changes were seen in the hepatic mRNA expression of various gluconeogenic enzymes. There were no changes in the mRNA expression profile of cell-cycle progression marker cyclin-dependent kinase 4 or apoptotic marker caspase 3, although proliferating cell nuclear antigen expression tended to be increased in Holstein cows infused with 5-HTP on d1 post-partum. Immunofluorescence assays showed an increased number of CASP3- and Ki67-positive cells in Holstein cows infused with 5-HTP on d1 post-partum. Given the elevated hepatic serotonin content and increased mRNA abundance of 5HTR2B, 5-HTP infusions may be stimulating an autocrine-paracrine adaptation to lactation in the Holstein cow liver.

Vitamin D inhibits lipopolysaccharide-induced inflammatory response potentially through the Toll-like receptor 4 signalling pathway in the intestine and enterocytes of juvenile Jian carp (Cyprinus carpio var. Jian).

PubMed

Jiang, Jun; Shi, Dan; Zhou, Xiao-Qiu; Yin, Long; Feng, Lin; Jiang, Wei-Dan; Liu, Yang; Tang, Ling; Wu, Pei; Zhao, Ye

2015-11-28

The present study was conducted to investigate the anti-inflammatory effect of vitamin D both in juvenile Jian carp (Cyprinus carpio var. Jian) in vivo and in enterocytes in vitro. In primary enterocytes, exposure to 10 mg lipopolysaccharide (LPS)/l increased lactate dehydrogenase activity in the culture medium (P<0·05) and resulted in a significant loss of cell viability (P<0·05). LPS exposure increased (P<0·05) the mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8), which was decreased by pre-treatment with 1,25-dihydroxyvitamin D (1,25D3) in a dose-dependent manner (P<0·05). Further results showed that pre-treatment with 1,25D3 down-regulated Toll-like receptor 4 (TLR4), myeloid differentiation primary response gene 88 (Myd88) and NF-κB p65 mRNA expression (P<0·05), suggesting potential mechanisms against LPS-induced inflammatory response. In vivo, intraperitoneal injection of LPS significantly increased TNF-α, IL-1β, IL-6 and IL-8 mRNA expression in the intestine of carp (P<0·05). Pre-treatment of fish with vitamin D3 protected the fish intestine from the LPS-induced increase of TNF-α, IL-1β, IL-6 and IL-8 mainly by downregulating TLR4, Myd88 and NF-κB p65 mRNA expression (P<0·05). These observations suggest that vitamin D could inhibit LPS-induced inflammatory response in juvenile Jian carp in vivo and in enterocytes in vitro. The anti-inflammatory effect of vitamin D is mediated at least in part by TLR4-Myd88 signalling pathways in the intestine and enterocytes of juvenile Jian carp.

ADAM-17 and TIMP3 protein and mRNA expression in spinal cord white matter of rats with acute experimental autoimmune encephalomyelitis.

PubMed

Plumb, Jonnie; Cross, Alison K; Surr, Jessica; Haddock, Gail; Smith, Terence; Bunning, Rowena A D; Woodroofe, M Nicola

2005-07-01

Tumour necrosis factor (TNF) is a major immunomodulatory and proinflammatory cytokine implicated in the pathogenesis of multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE). ADAM-17 cleaves membrane-bound TNF into its soluble form. The distribution and level of ADAM-17 expression within spinal cords of Lewis rats with EAE was investigated. ADAM-17 was associated with endothelial cells in the naïve and pre-disease spinal cords. In peak disease astrocytic and inflammatory cells expressed ADAM-17. Upregulation of ADAM-17 mRNA expression was coupled with a decrease in mRNA levels of its inhibitor TIMP3 suggesting a role for ADAM-17 in EAE pathogenesis.

Leaching of Au, Ag, and Pd from waste printed circuit boards of mobile phone by iodide lixiviant after supercritical water pre-treatment.

PubMed

Xiu, Fu-Rong; Qi, Yingying; Zhang, Fu-Shen

2015-07-01

Precious metals are the most attractive resources in waste printed circuit boards (PCBs) of mobile phones. In this work, an alternative process for recovering Au, Ag, and Pd from waste PCBs of mobile phones by supercritical water oxidation (SCWO) pre-treatment combined with iodine-iodide leaching process was developed. In the process, the waste PCBs of mobile phones were pre-treated in supercritical water, then a diluted hydrochloric acid leaching (HL) process was used to recovery the Cu, whose leaching efficiency was approximately 100%, finally the resulting residue was subjected to the iodine-iodide leaching process for recovering the Au, Ag, and Pd. Experimental results indicated that SCWO pre-treatment temperature, time, and pressure had significant influence on the Au, Ag, and Pd leaching from (SCWO+HL)-treated waste PCBs. The optimal SCWO pre-treatment conditions were 420°C and 60min for Au and Pd, and 410°C and 30min for Ag. The optimum dissolution parameters for Au, Pd, and Ag in (SCWO+HL)-treated PCBs with iodine-iodide system were leaching time of 120min (90min for Ag), iodine/iodide mole ratio of 1:5 (1:6 for Ag), solid-to-liquid ratio (S/L) of 1:10g/mL (1:8g/mL for Ag), and pH of 9, respectively. It is believed that the process developed in this study is environment friendly for the recovery of Au, Ag, and Pd from waste PCBs of mobile phones by SCWO pre-treatment combined with iodine-iodide leaching process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Brain-Derived Neurotrophic Factor in Patients with Huntington's Disease

PubMed Central

Zuccato, Chiara; Mariotti, Caterina; Valenza, Marta; Lahiri, Nayana; Wild, Edward J.; Sassone, Jenny; Ciammola, Andrea; Bachoud-Lèvi, Anne Catherine; Tabrizi, Sarah J.; Di Donato, Stefano; Cattaneo, Elena

2011-01-01

Reduced Brain-Derived Neurotrophic Factor (BDNF) levels have been described in a number of patho-physiological conditions, most notably, in Huntington's disease (HD), a progressive neurodegenerative disorder. Since BDNF is also produced in blood, we have undertaken the measurement of its peripheral levels in the attempt to identify a possible link with HD prognosis and/or its progression. Here we evaluated BDNF level in 398 blood samples including 138 controls, 56 preHD, and 204 HD subjects. We found that BDNF protein levels were not reliably different between groups, whether measured in plasma (52 controls, 26 preHD, 105 HD) or serum (39 controls, 5 preHD, 29 HD). Our experience, and a re-analysis of the literature highlighted that intra-group variability and methodological aspects affect this measurement, especially in serum. We also assessed BDNF mRNA levels in blood samples from 47 controls, 25 preHD, and 70 HD subjects, and found no differences among the groups. We concluded that levels of BDNF in human blood were not informative (mRNA levels or plasma protein level) nor reliable (serum protein levels) as HD biomarkers. We also wish to warn the scientific community in interpreting the significance of changes measured in BDNF protein levels in serum from patients suffering from different conditions. PMID:21857974

A novel protein factor is required for use of distal alternative 5' splice sites in vitro.

PubMed Central

Harper, J E; Manley, J L

1991-01-01

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites. Images PMID:1658620

Electroacupuncture brain protection during ischemic stroke: A role for the parasympathetic nervous system.

PubMed

Chi, Laiting; Du, Kairong; Liu, Dongdong; Bo, Yulong; Li, Wenzhi

2018-03-01

The demand for using parasympathetic activation for stroke therapy is unmet. In the current study, we investigated whether the neuroprotection provided by electroacupuncture (EA) in an experimental stroke model was associated with activation of the parasympathetic nervous system (PNS). The results showed that parasympathetic dysfunction (PD), performed as unilateral vagotomy combined with peripheral atropine, attenuated both the functional benefits of EA and its effects in improving cerebral perfusion, reducing infarct volume, and hindering apoptosis, neuronal and peripheral inflammation, and oxidative stress. Most importantly, EA rats showed a dramatically less reduction in the mRNA level of choline acetyltransferase, five subtypes of muscarinic receptors and α7nAChR, suggesting the inhibition of the impairment of the central cholinergic system; EA also activated dorsal motor nucleus of the vagus, the largest source of parasympathetic pre-ganglionic neurons in the lower brainstem (detected by c-fos immunohistochemistry), and PD suppressed these changes. These findings indicated EA may serve as an alternative modality of PNS activation for stroke therapy.

Membrane-association of mRNA decapping factors is independent of stress in budding yeast

PubMed Central

Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

2016-01-01

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation. PMID:27146487

Membrane-association of mRNA decapping factors is independent of stress in budding yeast.

PubMed

Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

2016-05-05

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation.

The toxic influence of dibromoacetic acid on the hippocampus and pre-frontal cortex of rat: involvement of neuroinflammation response and oxidative stress.

PubMed

Jiang, Wenbo; Li, Bai; Chen, Yingying; Gao, Shuying

2017-12-01

Dibromoacetic acid (DBA) exsits in drinking water as a by-product of disinfection as a result of chlorination or ozonation processes. Hippocampus and pre-frontal cortex are the key structures in memory formation and weanling babies are more sensitive to environmental toxicant than adults, so this study was conducted to evaluate the potential neurotoxicity effects of DBA exposure when administered intragastrically for 4 weeks to weanling Sprague-Dawley rats, at concentration of 0, 20, 50, 125 mg/kg via the neurobehavioral and neurochemical effects. Results indicated that animals weight gain and food consumption were not significantly affected by DBA. However, morris water maze test showed varying degrees of changes between control and high-dose group. Additionally, the level of malondialdehyde (MDA) and generation of reactive oxygen species (ROS) in the hippocampus and pre-frontal cortex of rats increased significantly. The activities of total superoxide dismutase (SOD) and the glutathione (GSH) content in the hippocampus and pre-frontal cortex of rats decreased significantly after treatment with DBA. Treatment with DBA increased the protein and mRNA expression of Iba-1, NF-κB, TNF-α, IL-6, IL-1β and HO-1 in the hippocampus and pre-frontal cortex of rats. These data suggested that DBA had a toxic influence on the hippocampus and pre-frontal cortex of rats, and that the mechanism of toxicity might be associated with the neuroinflammation response and oxidative stress.

TEAC's Accreditation Process at a Glance, 2009-2011

ERIC Educational Resources Information Center

Teacher Education Accreditation Council, 2011

2011-01-01

The Teacher Education Accreditation Council (TEAC), founded in 1997, is dedicated to improving academic degree programs for professional educators--those who teach and lead in schools, pre-K through grade 12. TEAC accredits undergraduate and graduate programs, including alternate route programs, based on (1) the evidence they have that they…

Humans as holobionts: implications for prevention and therapy.

PubMed

van de Guchte, Maarten; Blottière, Hervé M; Doré, Joël

2018-05-01

The human gut microbiota is increasingly recognized for its important or even decisive role in health. As it becomes clear that microbiota and host mutually affect and depend on each other in an intimate relationship, a holistic view of the gut microbiota-host association imposes itself. Ideally, a stable state of equilibrium, homeostasis, is maintained and serves health, but signs are that perturbation of this equilibrium beyond the limits of resilience can propel the system into an alternative stable state, a pre-disease state, more susceptible to the development of chronic diseases. The microbiota-host equilibrium of a large and growing proportion of individuals in Western society may represent such a pre-disease state and explain the explosive development of chronic diseases such as inflammatory bowel disease, obesity, and other inflammatory diseases. These diseases themselves represent other alternative stable states again and are therefore hard to cure. The holistic view of the microbiota-host association where feedback loops between microbiota and host are thought to maintain the system in a stable state-be it a healthy, pre-disease, or disease state-implies that integrated approaches, addressing host processes and microbiota, should be used to treat or prevent (pre-)disease.

Polypyrimidine Tract Binding Protein Homologs from Arabidopsis Are Key Regulators of Alternative Splicing with Implications in Fundamental Developmental Processes[W

PubMed Central

Rühl, Christina; Stauffer, Eva; Kahles, André; Wagner, Gabriele; Drechsel, Gabriele; Rätsch, Gunnar; Wachter, Andreas

2012-01-01

Alternative splicing (AS) generates transcript variants by variable exon/intron definition and massively expands transcriptome diversity. Changes in AS patterns have been found to be linked to manifold biological processes, yet fundamental aspects, such as the regulation of AS and its functional implications, largely remain to be addressed. In this work, widespread AS regulation by Arabidopsis thaliana Polypyrimidine tract binding protein homologs (PTBs) was revealed. In total, 452 AS events derived from 307 distinct genes were found to be responsive to the levels of the splicing factors PTB1 and PTB2, which predominantly triggered splicing of regulated introns, inclusion of cassette exons, and usage of upstream 5′ splice sites. By contrast, no major AS regulatory function of the distantly related PTB3 was found. Dependent on their position within the mRNA, PTB-regulated events can both modify the untranslated regions and give rise to alternative protein products. We find that PTB-mediated AS events are connected to diverse biological processes, and the functional implications of selected instances were further elucidated. Specifically, PTB misexpression changes AS of PHYTOCHROME INTERACTING FACTOR6, coinciding with altered rates of abscisic acid–dependent seed germination. Furthermore, AS patterns as well as the expression of key flowering regulators were massively changed in a PTB1/2 level-dependent manner. PMID:23192226

Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays.

PubMed

Johnson, Jason M; Castle, John; Garrett-Engele, Philip; Kan, Zhengyan; Loerch, Patrick M; Armour, Christopher D; Santos, Ralph; Schadt, Eric E; Stoughton, Roland; Shoemaker, Daniel D

2003-12-19

Alternative pre-messenger RNA (pre-mRNA) splicing plays important roles in development, physiology, and disease, and more than half of human genes are alternatively spliced. To understand the biological roles and regulation of alternative splicing across different tissues and stages of development, systematic methods are needed. Here, we demonstrate the use of microarrays to monitor splicing at every exon-exon junction in more than 10,000 multi-exon human genes in 52 tissues and cell lines. These genome-wide data provide experimental evidence and tissue distributions for thousands of known and novel alternative splicing events. Adding to previous studies, the results indicate that at least 74% of human multi-exon genes are alternatively spliced.

SWI/SNF interacts with cleavage and polyadenylation factors and facilitates pre-mRNA 3' end processing.

PubMed

Yu, Simei; Jordán-Pla, Antonio; Gañez-Zapater, Antoni; Jain, Shruti; Rolicka, Anna; Östlund Farrants, Ann-Kristin; Visa, Neus

2018-05-31

SWI/SNF complexes associate with genes and regulate transcription by altering the chromatin at the promoter. It has recently been shown that these complexes play a role in pre-mRNA processing by associating at alternative splice sites. Here, we show that SWI/SNF complexes are involved also in pre-mRNA 3' end maturation by facilitating 3' end cleavage of specific pre-mRNAs. Comparative proteomics show that SWI/SNF ATPases interact physically with subunits of the cleavage and polyadenylation complexes in fly and human cells. In Drosophila melanogaster, the SWI/SNF ATPase Brahma (dBRM) interacts with the CPSF6 subunit of cleavage factor I. We have investigated the function of dBRM in 3' end formation in S2 cells by RNA interference, single-gene analysis and RNA sequencing. Our data show that dBRM facilitates pre-mRNA cleavage in two different ways: by promoting the association of CPSF6 to the cleavage region and by stabilizing positioned nucleosomes downstream of the cleavage site. These findings show that SWI/SNF complexes play a role also in the cleavage of specific pre-mRNAs in animal cells.

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae.

PubMed

Huch, Susanne; Müller, Maren; Muppavarapu, Mridula; Gommlich, Jessie; Balagopal, Vidya; Nissan, Tracy

2016-10-15

The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization. © 2016. Published by The Company of Biologists Ltd.

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae

PubMed Central

Huch, Susanne; Müller, Maren; Muppavarapu, Mridula; Gommlich, Jessie; Balagopal, Vidya; Nissan, Tracy

2016-01-01

ABSTRACT The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization. PMID:27543059

Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides

NASA Astrophysics Data System (ADS)

Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard

1996-11-01

In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

Regulation of estrogen receptor beta mRNA in the brain: opposite effects of 17beta-estradiol and the phytoestrogen, coumestrol.

PubMed

Patisaul, H B; Whitten, P L; Young, L J

1999-04-06

Estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) are differentially distributed in the brain and likely mediate different estrogen-dependent processes. ERbeta is abundant in the bed nucleus of the stria terminalis, medial preoptic nucleus, paraventricular nucleus of the hypothalamus and the amygdala of the rat. In the paraventricular nucleus, which is devoid of ERalpha, ERbeta is colocalized with the neuropeptides, oxytocin and vasopressin, suggesting a potential functional role for ERbeta in the regulation of these peptides. We examined the regulation of ERbeta mRNA expression in the rat brain by 17beta-estradiol and the phytoestrogen, coumestrol. 17beta-Estradiol treatment decreased ERbeta mRNA in situ hybridization signal by 44.5% in the paraventricular nucleus of the hypothalamus (PVN), but had no effect in the bed nucleus of the stria terminalis (BnST) or the medial preoptic nucleus (MPA). In contrast, dietary exposure to coumestrol increased ERbeta mRNA signal by 47.5% in the PVN but had no effect in the BnST or the MPA. These data demonstrate that like ERalpha, ERbeta is down regulated by estrogen in a region specific manner in the rat brain. Furthermore, exposure to coumestrol may modulate ERbeta-dependent processes by acting as an anti-estrogen at ERbeta. This data contradicts results from cell transfection assays which suggest an estrogenic activity of coumestrol on ERbeta, indicating that the mode of action may be tissue specific, or that metabolism of dietary coumestrol may alter its effects. Because the highest concentrations of phytoestrogens are found in legumes, vegetables and grains, they are most prevalent in vegetarian and traditional Asian diets. Understanding the neuroendocrine effects of phytoestrogens is particularly important now that they are being marketed as a natural alternative to estrogen replacement therapy and sold in highly concentrated pills and powders. Copyright 1999 Elsevier Science B.V.

Effect of neonatal handling on serotonin 1A sub-type receptors in the rat hippocampus.

PubMed

Stamatakis, A; Mantelas, A; Papaioannou, A; Pondiki, S; Fameli, M; Stylianopoulou, F

2006-06-19

Serotonin 1A sub-type receptors play an important role in the etiopathogenesis of depression, which is known to occur more often in females than males. Early experiences can be a predisposing factor for depression; however, the underlying cellular processes remain unknown. In an effort to address such issues, we employed neonatal handling, an experimental model of early experience, which has been previously shown to render females more vulnerable to display enhanced depression-like behavior in response to chronic stress, while it increases the ability of males to cope. In rat pre-pubertal (30 days of age) and adult (90 days) hippocampus, of both males and females, the effect of neonatal handling on serotonin 1A sub-type receptor mRNA and protein levels was determined by in situ hybridization and immunohistochemistry, respectively, while the number of binding sites was determined by in vitro autoradiography using [(3)H]8-hydroxy-2(di-n-propylamino)tetralin as the ligand. Our results revealed a significant sex difference in serotonin 1A sub-type receptor mRNA, protein and binding sites, with females having higher levels than males. Handling resulted in statistically significant decreased numbers of cells positive for serotonin 1A sub-type receptor mRNA or protein, as well as [(3)H]8-hydroxy-2(di-n-propylamino)tetralin binding sites in the area 4 of Ammon's horn and dentate gyrus of both pre-pubertal males and females. In adult animals the number of serotonin 1A sub-type receptor mRNA positive cells was increased as a result of handling in the area 1 of Ammon's horn, area 4 of Ammon's horn and dentate gyrus of males, while it was decreased only in the area 4 of Ammon's horn of females. Furthermore, the number of serotonin sub-type 1A receptor immunopositive cells, as well as [(3)H]8-hydroxy-2(di-n-propylamino)tetralin binding sites was increased in the area 1 of Ammon's horn, area 4 of Ammon's horn and dentate gyrus of handled males, whereas it was decreased in these same brain areas in the handled females. We can thus infer that neonatal handling results in alterations in postsynaptic serotonergic neurotransmission, which may contribute to the sex dimorphic effects of handling as to the vulnerability toward depression-like behavior in response to chronic stressful stimuli.

Functional Analyses of c.2268dup in Thyroid Peroxidase Gene Associated with Goitrous Congenital Hypothyroidism

PubMed Central

Harun, Fatimah; Jalaludin, Muhammad Yazid; Lim, Chor Yin; Ng, Khoon Leong

2014-01-01

The c.2268dup mutation in thyroid peroxidase (TPO) gene was reported to be a founder mutation in Taiwanese patients with dyshormonogenetic congenital hypothyroidism (CH). The functional impact of the mutation is not well documented. In this study, homozygous c.2268dup mutation was detected in two Malaysian-Chinese sisters with goitrous CH. Normal and alternatively spliced TPO mRNA transcripts were present in thyroid tissues of the two sisters. The abnormal transcript contained 34 nucleotides originating from intron 12. The c.2268dup is predicted to generate a premature termination codon (PTC) at position 757 (p.Glu757X). Instead of restoring the normal reading frame, the alternatively spliced transcript has led to another stop codon at position 740 (p.Asp739ValfsX740). The two PTCs are located at 116 and 201 nucleotides upstream of the exons 13/14 junction fulfilling the requirement for a nonsense-mediated mRNA decay (NMD). Quantitative RT-PCR revealed an abundance of unidentified transcripts believed to be associated with the NMD. TPO enzyme activity was not detected in both patients, even though a faint TPO band of about 80 kD was present. In conclusion, the c.2268dup mutation leads to the formation of normal and alternatively spliced TPO mRNA transcripts with a consequential loss of TPO enzymatic activity in Malaysian-Chinese patients with goitrous CH. PMID:24745015

Spliceosomal protein U1A is involved in alternative splicing and salt stress tolerance in Arabidopsis thaliana

PubMed Central

Gu, Jinbao; Xia, Zhiqiang; Luo, Yuehua; Jiang, Xingyu; Qian, Bilian; Xie, He; Zhu, Jian-Kang; Xiong, Liming; Zhu, Jianhua; Wang, Zhen-Yu

2018-01-01

Abstract Soil salinity is a significant threat to sustainable agricultural production worldwide. Plants must adjust their developmental and physiological processes to cope with salt stress. Although the capacity for adaptation ultimately depends on the genome, the exceptional versatility in gene regulation provided by the spliceosome-mediated alternative splicing (AS) is essential in these adaptive processes. However, the functions of the spliceosome in plant stress responses are poorly understood. Here, we report the in-depth characterization of a U1 spliceosomal protein, AtU1A, in controlling AS of pre-mRNAs under salt stress and salt stress tolerance in Arabidopsis thaliana. The atu1a mutant was hypersensitive to salt stress and accumulated more reactive oxygen species (ROS) than the wild-type under salt stress. RNA-seq analysis revealed that AtU1A regulates AS of many genes, presumably through modulating recognition of 5′ splice sites. We showed that AtU1A is associated with the pre-mRNA of the ROS detoxification-related gene ACO1 and is necessary for the regulation of ACO1 AS. ACO1 is important for salt tolerance because ectopic expression of ACO1 in the atu1a mutant can partially rescue its salt hypersensitive phenotype. Our findings highlight the critical role of AtU1A as a regulator of pre-mRNA processing and salt tolerance in plants. PMID:29228330

Dicer cleaves 5'-extended microRNA precursors originating from RNA polymerase II transcription start sites.

PubMed

Sheng, Peike; Fields, Christopher; Aadland, Kelsey; Wei, Tianqi; Kolaczkowski, Oralia; Gu, Tongjun; Kolaczkowski, Bryan; Xie, Mingyi

2018-05-09

MicroRNAs (miRNAs) are approximately 22 nucleotide (nt) long and play important roles in post-transcriptional regulation in both plants and animals. In animals, precursor (pre-) miRNAs are ∼70 nt hairpins produced by Drosha cleavage of long primary (pri-) miRNAs in the nucleus. Exportin-5 (XPO5) transports pre-miRNAs into the cytoplasm for Dicer processing. Alternatively, pre-miRNAs containing a 5' 7-methylguanine (m7G-) cap can be generated independently of Drosha and XPO5. Here we identify a class of m7G-capped pre-miRNAs with 5' extensions up to 39 nt long. The 5'-extended pre-miRNAs are transported by Exportin-1 (XPO1). Unexpectedly, a long 5' extension does not block Dicer processing. Rather, Dicer directly cleaves 5'-extended pre-miRNAs by recognizing its 3' end to produce mature 3p miRNA and extended 5p miRNA both in vivo and in vitro. The recognition of 5'-extended pre-miRNAs by the Dicer Platform-PAZ-Connector (PPC) domain can be traced back to ancestral animal Dicers, suggesting that this previously unrecognized Dicer reaction mode is evolutionarily conserved. Our work reveals additional genetic sources for small regulatory RNAs and substantiates Dicer's essential role in RNAi-based gene regulation.

Consumption of a high-fat, high-calorie meal is associated with an increase in intracellular co-localization of PPAR-γ mRNA and protein in monocytes.

PubMed

Henning, Andrea L; McFarlin, Brian K

2017-01-01

Acute and habitual dietary habits contribute to the onset and progression of many forms of cardiovascular disease. Circulating peripheral blood monocytes have been a target of pre-clinical research related to the risk of atherosclerosis. Specifically, when monocytes migrate into the subendothelial space and endocytosize modified LDL (i.e. acLDL or oxLDL) they phenotypically transform into foam cells. The endocytosis of modified LDL is mediated by the scavenger receptor CD36, whose expression is in tern regulated by the transcription factor PPAR-γ. In this report, we describe a novel technique for the simultaneous measurement of intracellular PPAR-γ mRNA and protein in peripheral blood monocytes collected from human subjects in fasted state or 3 and 5-h after consuming a high-calorie (65% of daily calorie needs), high-fat meal. Intracellular detection and co-localization of PPAR-γ was made possible using a combination of image-based flow cytometry (MilliporeSigma FlowSight) and an amplified mRNA FISH staining technique (Affymetrix/eBioscience PrimeFlow). Consumption of a high-calorie, high-fat meal increased the percentage of co-localization at both 3 and 5-h post prandial compared to pre-meal. No obvious difference in co-localization was observed when cells were treated by acLDL in vitro. More research is needed to determine how to best use this method to study pre-clinical risk of atherosclerosis. Copyright © 2016. Published by Elsevier Inc.

A novel snRNA-like transcript affects amyloidogenesis and cell cycle progression through perturbation of Fe65L1 (APBB2) alternative splicing.

PubMed

Penna, Ilaria; Vassallo, Irene; Nizzari, Mario; Russo, Debora; Costa, Delfina; Menichini, Paola; Poggi, Alessandro; Russo, Claudio; Dieci, Giorgio; Florio, Tullio; Cancedda, Ranieri; Pagano, Aldo

2013-06-01

FE65 proteins constitute a family of adaptors which modulates the processing of amyloid precursor protein and the consequent amyloid β production. Thus, they have been involved in the complex and partially unknown cascade of reactions at the base of Alzheimer's disease etiology. However, FE65 and FE65-like proteins may be linked to neurodegeneration through the regulation of cell cycle in post-mitotic neurons. In this work we disclose novel molecular mechanisms by which APBB2 can modulate APP processing. We show that APBB2 mRNA splicing, driven by the over-expression of a novel non-coding RNA named 45A, allow the generation of alternative protein forms endowed with differential effects on Aβ production, cell cycle control, and DNA damage response. 45A overexpression also favors cell transformation and tumorigenesis leading to a marked increase of malignancy of neuroblastoma cells. Therefore, our results highlight a novel regulatory pathway of considerable interest linking APP processing with cell cycle regulation and DNA-surveillance systems, that may represent a molecular mechanism to induce neurodegeneration in post-mitotic neurons. Copyright © 2013 Elsevier B.V. All rights reserved.

European Science Notes (ESN) Information Bulletin. Reports on Current European/Middle Eastern Science,

DTIC Science & Technology

1987-12-01

mRNA), lular viruses within a few hours in dif- and Sl-analysis showed that anti-IgM and ferent body fluids and may be used for phorbol esters...suppressed mRNA coding for general virus diagnosis. the secreted form of IgM, showing that Thiophilic adsorption for the puri- these additives affect...constructs were and can be an alternative method to pro- utilized containing the prokaryotic CAT - tein A affinity chromatography, especial- gene

SECONDARY WASTE/ETF (EFFLUENT TREATMENT FACILITY) PRELIMINARY PRE-CONCEPTUAL ENGINEERING STUDY

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

MAY TH; GEHNER PD; STEGEN GARY

2009-12-28

This pre-conceptual engineering study is intended to assist in supporting the critical decision (CD) 0 milestone by providing a basis for the justification of mission need (JMN) for the handling and disposal of liquid effluents. The ETF baseline strategy, to accommodate (WTP) requirements, calls for a solidification treatment unit (STU) to be added to the ETF to provide the needed additional processing capability. This STU is to process the ETF evaporator concentrate into a cement-based waste form. The cementitious waste will be cast into blocks for curing, storage, and disposal. Tis pre-conceptual engineering study explores this baseline strategy, in additionmore » to other potential alternatives, for meeting the ETF future mission needs. Within each reviewed case study, a technical and facility description is outlined, along with a preliminary cost analysis and the associated risks and benefits.« less

Building a bridge between neurobiology and mental illness.

PubMed

Costa, E

1992-10-01

GABA (gamma amino butyric acid) is the most abundant and important inhibitory transmitter in mammalian CNS. It counterbalances the glutamate mediated neuronal excitation. Abnormalities of the interaction of these two transmitters might change the mechanisms of neuronal group selection that according to Edelman [Neural Darwinism. Basic Books, New York] play a role in mediating several brain functions including cognition processes. Indeed imbalances in GABAergic functions were shown to elicit psychoses. They can be obtained by administration of drugs that affect synthesis, metabolism and uptake of GABA and thereby cause a persistent stimulation of GABAA receptors or perhaps by genetic abnormalities in DNA transcription, pre-mRNA splicing, mRNA translation and posttranslation modifications of GABAA receptor subunits. The complexities in the regulation of GABAA receptor subunit structure, synthesis, assembly and the brain location of specific mRNA encoding for these subunits are investigated with in situ mRNA hybridization specific for subunits of GABAA receptors. The role of the variability resulting from the complexities in the regulation of GABAA receptor allosteric modulation by drugs and putative endogenous allosteric modulators of GABA action at GABAA receptors is discussed. This discussion gives relevance to the possibility that genetic abnormalities in the expression of proteins participating in GABAergic function are to be considered as a possible target of the genetic defects operative in psychoses. In line with this thinking, it is suggested that partial allosteric modulators (partial agonists) of GABAA receptors and the phosphothioate or methylphosphonate analogs antisense to specific mRNA oligonucleotides that mediate the expression of genetic information concerning GABAA and glutamate receptor subunits may become valuable tools in psychiatric research. Perhaps in the future these studies might generate new ideas useful in the therapy of genetically determined psychiatric illness.

Circadian processes in the RNA life cycle.

PubMed

Torres, Manon; Becquet, Denis; Franc, Jean-Louis; François-Bellan, Anne-Marie

2018-05-01

The circadian clock drives daily rhythms of multiple physiological processes, allowing organisms to anticipate and adjust to periodic changes in environmental conditions. These physiological rhythms are associated with robust oscillations in the expression of at least 30% of expressed genes. While the ability for the endogenous timekeeping system to generate a 24-hr cycle is a cell-autonomous mechanism based on negative autoregulatory feedback loops of transcription and translation involving core-clock genes and their protein products, it is now increasingly evident that additional mechanisms also govern the circadian oscillations of clock-controlled genes. Such mechanisms can take place post-transcriptionally during the course of the RNA life cycle. It has been shown that many steps during RNA processing are regulated in a circadian manner, thus contributing to circadian gene expression. These steps include mRNA capping, alternative splicing, changes in splicing efficiency, and changes in RNA stability controlled by the tail length of polyadenylation or the use of alternative polyadenylation sites. RNA transport can also follow a circadian pattern, with a circadian nuclear retention driven by rhythmic expression within the nucleus of particular bodies (the paraspeckles) and circadian export to the cytoplasm driven by rhythmic proteins acting like cargo. Finally, RNA degradation may also follow a circadian pattern through the rhythmic involvement of miRNAs. In this review, we summarize the current knowledge of the post-transcriptional circadian mechanisms known to play a prominent role in shaping circadian gene expression in mammals. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing RNA Processing > RNA Editing and Modification RNA Export and Localization > Nuclear Export/Import. © 2018 Wiley Periodicals, Inc.

Risk Evaluation in the Pre-Phase A Conceptual Design of Spacecraft

NASA Technical Reports Server (NTRS)

Fabisinski, Leo L., III; Maples, Charlotte Dauphne

2010-01-01

Typically, the most important decisions in the design of a spacecraft are made in the earliest stages of its conceptual design the Pre-Phase A stages. It is in these stages that the greatest number of design alternatives is considered, and the greatest number of alternatives is rejected. The focus of Pre-Phase A conceptual development is on the evaluation and comparison of whole concepts and the larger-scale systems comprising those concepts. This comparison typically uses general Figures of Merit (FOMs) to quantify the comparative benefits of designs and alternative design features. Along with mass, performance, and cost, risk should be one of the major FOMs in evaluating design decisions during the conceptual design phases. However, risk is often given inadequate consideration in conceptual design practice. The reasons frequently given for this lack of attention to risk include: inadequate mission definition, lack of rigorous design requirements in early concept phases, lack of fidelity in risk assessment methods, and under-evaluation of risk as a viable FOM for design evaluation. In this paper, the role of risk evaluation in early conceptual design is discussed. The various requirements of a viable risk evaluation tool at the Pre-Phase A level are considered in light of the needs of a typical spacecraft design study. A technique for risk identification and evaluation is presented. The application of the risk identification and evaluation approach to the conceptual design process is discussed. Finally, a computational tool for risk profiling is presented and applied to assess the risk for an existing Pre-Phase A proposal. The resulting profile is compared to the risks identified for the proposal by other means.

Follicular thyroglobulin induces cathepsin H expression and activity in thyrocytes

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

Oda, Kenzaburo; Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aoba-cho, Higashimurayama, Tokyo 189-0002; Division of Diabetes, Metabolism and Endocrinology, Department of Internal Medicine, Toho University, 5-21-16 Omorinishi, Ota, Tokyo 143-8540

Thyroglobulin (Tg) stored in thyroid follicles exerts a potent negative-feedback effect on each step of pre-hormone biosynthesis, including Tg gene transcription and iodine uptake and organification, by suppressing the expression of specific transcription factors that regulate these steps. Pre-hormones are stored in the follicular colloid before being reabsorbed. Following lysosomal proteolysis of its precursor, thyroid hormone (TH) is released from thyroid follicles. Although the suppressive effects of follicular Tg on each step of pre-hormone biosynthesis have been extensively characterized, whether follicular Tg accumulation also affects hormone reabsorption, proteolysis, and secretion is unclear. In this study we explored whether follicular Tgmore » can regulate the expression and function of the lysosomal endopeptidases cathepsins. We found that in the rat thyroid cell line FRTL-5 follicular Tg induced cathepsin H mRNA and protein expression, as well as cathepsin H enzyme activity. Double immunofluorescence staining showed that Tg endocytosis promoted cathepsin H translocalization into lysosomes where it co-localized with internalized Tg. These results suggest that cathepsin H is an active participant in lysosome-mediated pre-hormone degradation, and that follicular Tg stimulates mobilization of pre-hormones by activating cathepsin H-associated proteolysis pathways. - Highlights: • Follicular Tg increases cathepsin H mRNA and protein levels in rat thyroid cells. • Follicular Tg increases cathepsin H enzyme activity in rat thyroid cells. • After Tg stimulation cathepsin H co-localizes to lysosomes with follicular Tg. • Cathepsin H promotes hormone secretion by lysosome-mediated mechanisms.« less

The Rtr1p CTD phosphatase autoregulates its mRNA through a degradation pathway involving the REX exonucleases

PubMed Central

Hodko, Domagoj; Ward, Taylor; Chanfreau, Guillaume

2016-01-01

Rtr1p is a phosphatase that impacts gene expression by modulating the phosphorylation status of the C-terminal domain of the large subunit of RNA polymerase II. Here, we show that Rtr1p is a component of a novel mRNA degradation pathway that promotes its autoregulation through turnover of its own mRNA. We show that the 3′UTR of the RTR1 mRNA contains a cis element that destabilizes this mRNA. RTR1 mRNA turnover is achieved through binding of Rtr1p to the RTR1 mRNP in a manner that is dependent on this cis element. Genetic evidence shows that Rtr1p-mediated decay of the RTR1 mRNA involves the 5′-3′ DExD/H-box RNA helicase Dhh1p and the 3′-5′ exonucleases Rex2p and Rex3p. Rtr1p and Rex3p are found associated with Dhh1p, suggesting a model for recruiting the REX exonucleases to the RTR1 mRNA for degradation. Rtr1p-mediated decay potentially impacts additional transcripts, including the unspliced BMH2 pre-mRNA. We propose that Rtr1p may imprint its RNA targets cotranscriptionally and determine their downstream degradation mechanism by directing these transcripts to a novel turnover pathway that involves Rtr1p, Dhh1p, and the REX family of exonucleases. PMID:26843527

Organ-specific effects of low-dose zinc pre-exposure on high-dose zinc induced mitochondrial dysfunction in large yellow croaker Pseudosciaena crocea.

PubMed

Zheng, Jia-Lang; Yuan, Shuang-Shuang; Shen, Bin; Wu, Chang-Wen

2017-04-01

The study was carried out to evaluate the effects of low-dose zinc (Zn) pre-exposure on survival rate, new Zn accumulation, and mitochondrial bioenergetics in the liver and spleen of large yellow croaker exposed to high-dose Zn. To the end, fish were pre-exposed to 0 and 2 mg L -1 Zn for 48 h and post-exposed to 0 and 12 mg L -1 Zn for 48 h. Twelve milligrams Zn per liter exposure alone reduced survival rate, but the effect did not appear in the 2 mg L -1 Zn pre-exposure groups. Two milligrams per liter Zn pre-exposure also ameliorated 12 mg Zn L -1 induced new Zn accumulation, reactive oxygen species (ROS) levels, and mitochondrial swelling in the liver. However, these effects did not appear in the spleen. In the liver, 2 mg L -1 Zn pre-exposure apparently relieved 12 mg L -1 Zn induced down-regulation of activities of ATP synthase (F-ATPase), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH). The mRNA levels of these genes remained relatively stable in fish exposed to 12 mg L -1 Zn alone, but increased in fish exposed to 12 mg L -1 Zn with 2 mg L -1 Zn pre-treatment. In the spleen, 2 mg Zn L -1 pre-exposure did not mitigate the down-regulation of mRNA levels of genes and activities of relative enzymes induced by 12 mg L -1 Zn. In conclusion, our study demonstrated low-dose zinc pre-exposure ameliorated high-dose zinc induced mitochondrial dysfunction in the liver but not in the spleen of large yellow croaker, indicating an organ-specific effect.

Autoregulatory mechanisms controlling the microprocessor.

PubMed

Triboulet, Robinson; Gregory, Richard I

2011-01-01

The Microprocessor, comprising the ribonuclease Drosha and its essential cofactor, the double-stranded RNA-binding protein, DGCR8, is essential for the first step of the miRNA biogenesis pathway. It specifically cleaves double-stranded RNA within stem-loop structures of primary miRNA transcripts (pri-miRNAs) to generate precursor (pre-miRNA) intermediates. Pre-miRNAs are subsequently processed by Dicer to their mature ∼22 nt form. Thus, Microprocessor is essential for miRNA maturation, and pri-miRNA cleavage by this complex defines one end of the mature miRNA. Moreover, it is emerging that dysregulation of the Microprocessor is associated with various human diseases. It is therefore important to understand the mechanisms by which the expression of the subunits of the Microprocessor is regulated. Recent findings have uncovered a post-transcriptional mechanism that maintains the integrity of the Microprocessor. These studies revealed that the Microprocessor is involved in the processing of the messenger RNA (mRNA) that encodes DGCR8. This regulatory feedback loop, along with the reported role played by DGCR8 in the stabilization of Drosha protein, is part of a newly identified regulatory mechanism controlling Microprocessor activity.

76 FR 28060 - Regional Habitat Conservation Plan, Hays County, TX

Federal Register 2010, 2011, 2012, 2013, 2014

2011-05-13

... alternative features the acquisition of a modestly sized, pre-determined preserve system and limits the amount... under Alternative B, this alternative involves a conservation program that utilizes a pre-determined..., adaptive management, and other strategies designed to avoid and minimize harm to the covered species and...

RNA Splicing: Regulation and Dysregulation in the Heart.

PubMed

van den Hoogenhof, Maarten M G; Pinto, Yigal M; Creemers, Esther E

2016-02-05

RNA splicing represents a post-transcriptional mechanism to generate multiple functional RNAs or proteins from a single transcript. The evolution of RNA splicing is a prime example of the Darwinian function follows form concept. A mutation that leads to a new mRNA (form) that encodes for a new functional protein (function) is likely to be retained, and this way, the genome has gradually evolved to encode for genes with multiple isoforms, thereby creating an enormously diverse transcriptome. Advances in technologies to characterize RNA populations have led to a better understanding of RNA processing in health and disease. In the heart, alternative splicing is increasingly being recognized as an important layer of post-transcriptional gene regulation. Moreover, the recent identification of several cardiac splice factors, such as RNA-binding motif protein 20 and SF3B1, not only provided important insight into the mechanisms underlying alternative splicing but also revealed how these splicing factors impact functional properties of the heart. Here, we review our current knowledge of alternative splicing in the heart, with a particular focus on the major and minor spliceosome, the factors controlling RNA splicing, and the role of alternative splicing in cardiac development and disease. © 2016 American Heart Association, Inc.

Mucin gene mRNA levels in broilers challenged with eimeria and/or Clostridium perfringens.

PubMed

Kitessa, Soressa M; Nattrass, Gregory S; Forder, Rebecca E A; McGrice, Hayley A; Wu, Shu-Biao; Hughes, Robert J

2014-09-01

The effects of Eimeria (EM) and Clostridium perfringens (CP) challenges on the mRNA levels of genes involved in mucin (Muc) synthesis (Muc2, Muc5ac, Muc13, and trefoil family factor-2 [TFF2]), inflammation (tumor necrosis factor alpha [TNF-alpha] and interleukin-18 [IL-18]), and metabolic processes (cluster of differentiation [CD]36) in the jejunum of broilers were investigated. Two parallel experiments involving 1) EM challenge and 2) EM and CP challenges were conducted. The first experiment was a 2 X 2 study with 12 birds per treatment (N = 48) involving fishmeal substitution (25%) in the diet (FM) and EM challenge. The treatments were: Control (FM-, EM-), Fishmeal (FM+, EM-), EM challenge (FM-, EM+), and fishmeal substitution and EM challenge (FM+, EM+). The second experiment was a 2 X 2 X 2 experiment with six birds per treatment (N = 48) involving fishmeal (FM-, FM+), Eimeria (EM-, EM+), and C perfringens (CP-, CP+). In both arms of the study, male broilers were given a starter diet for the whole period of 16 days, except those assigned to FM+, where 25% of the starter ration was replaced with fishmeal from days 8 to 14. EM inoculation was performed on day 9 and CP inoculation on days 14 and 15. The EM challenge birds were euthanatized for sampling on day 13; postmortem examination and sampling for the Eimeria plus C perfringens challenge arm of the study were on day 16. In the Eimeria challenge arm of the study, fishmeal supplementation significantly suppressed the mRNA levels of TNF-alpha, TFF2, and IL-18 pre-CP inoculation but simultaneously increased the levels of Muc13 and CD36 mRNAs. Birds challenged with Eimeria exhibited increased mRNA levels of Muc13, Muc5ac, TNF-alpha, and IL-18. In the Eimeria and C. perfringens challenge arm, birds exposed to EM challenge exhibited significantly lower mRNA levels of Muc2 and CD36. The mRNA levels of CD36 were also significantly suppressed by CP challenge. Our results showed that the transcription of mucin synthesis genes in the jejunum of broilers is modulated by fishmeal inclusion in the diet. Furthermore, we show for the first time suppression of CD36 mRNA levels in the intestine of broilers challenged with Eimeria or C. perfringens.

The gene and the genon concept: a functional and information-theoretic analysis

PubMed Central

Scherrer, Klaus; Jost, Jürgen

2007-01-01

‘Gene' has become a vague and ill-defined concept. To set the stage for mathematical analysis of gene storage and expression, we return to the original concept of the gene as a function encoded in the genome, basis of genetic analysis, that is a polypeptide or other functional product. The additional information needed to express a gene is contained within each mRNA as an ensemble of signals, added to or superimposed onto the coding sequence. To designate this programme, we introduce the term ‘genon'. Individual genons are contained in the pre-mRNA forming a pre-genon. A genomic domain contains a proto-genon, with the signals of transcription activation in addition to the pre-genon in the transcripts. Some contain several mRNAs and hence genons, to be singled out by RNA processing and differential splicing. The programme in the genon in cis is implemented by corresponding factors of protein or RNA nature contained in the transgenon of the cell or organism. The gene, the cis programme contained in the individual domain and transcript, and the trans programme of factors, can be analysed by information theory. PMID:17353929

Perception or Fact: Measuring the Effectiveness of the Terrorism Early Warning (TEW) Group

DTIC Science & Technology

2005-09-01

alternatives ” (Campbell 2005). The logic model process is a tool that has been used by evaluators for many years to identify performance measures and...pertinent information is obtained, this cell is responsible for the development (pre-event) and use (trans- and post-event) of playbooks and...

Transcriptome analysis reveals the complexity of alternative splicing regulation in the fungus Verticillium dahliae.

PubMed

Jin, Lirong; Li, Guanglin; Yu, Dazhao; Huang, Wei; Cheng, Chao; Liao, Shengjie; Wu, Qijia; Zhang, Yi

2017-02-06

Alternative splicing (AS) regulation is extensive and shapes the functional complexity of higher organisms. However, the contribution of alternative splicing to fungal biology is not well studied. This study provides sequences of the transcriptomes of the plant wilt pathogen Verticillium dahliae, using two different strains and multiple methods for cDNA library preparations. We identified alternatively spliced mRNA isoforms in over a half of the multi-exonic fungal genes. Over one-thousand isoforms involve TopHat novel splice junction; multiple types of combinatory alternative splicing patterns were identified. We showed that one Verticillium gene could use four different 5' splice sites and two different 3' donor sites to produce up to five mature mRNAs, representing one of the most sophisticated alternative splicing model in eukaryotes other than animals. Hundreds of novel intron types involving a pair of new splice sites were identified in the V. dahliae genome. All the types of AS events were validated by using RT-PCR. Functional enrichment analysis showed that AS genes are involved in most known biological functions and enriched in ATP biosynthesis, sexual/asexual reproduction, morphogenesis, signal transduction etc., predicting that the AS regulation modulates mRNA isoform output and shapes the V. dahliae proteome plasticity of the pathogen in response to the environmental and developmental changes. These findings demonstrate the comprehensive alternative splicing mechanisms in a fungal plant pathogen, which argues the importance of this fungus in developing complicate genome regulation strategies in eukaryotes.

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

Time-Dependent Changes in Increased Levels of Plasma Irisin and Muscle PGC-1α and FNDC5 after Exercise in Mice.

PubMed

Pang, Minhui; Yang, Jianwei; Rao, Jiaming; Wang, Haiqing; Zhang, Jiayi; Wang, Shengyong; Chen, Xiongfei; Dong, Xiaomei

2018-02-01

Exercise induces the expression of peroxisome proliferator-activated receptor gamma co-activator 1-α (PGC-1α) in skeletal muscle, which promotes the cleavage of fibronectin type III domain-containing protein 5 (FNDC5) to irisin. To explore the relationship between irisin and its regulators, we analyzed the plasma irisin levels and the muscle levels of FNDC5 and PGC-1α after exercise. Male C57BL/6J mice underwent a treadmill exercise (60% of VO 2max ) for 30 min or one hour (h), and blood and gastrocnemius samples were collected before exercise (pre-exercise), immediately after exercise, and during 24-h recovery after 1-h exercise. We found that plasma irisin levels were significantly increased during exercise (P < 0.05), while FNDC5 protein levels were not significantly increased. Moreover, PGC-1α mRNA and protein levels were significantly increased during 30-min exercise, but were decreased during 1-h exercise. After 1-h exercise, the irisin levels peaked at 6 h (20.71 ± 0.25 ng/ml) and decreased to pre-exercise levels by 24 h (15.45 ± 0.27 ng/ml). Likewise, PGC-1α mRNA and protein levels were increased at 1 h and maintained at elevated levels for 6 h; thereafter, the expression levels of PGC1-α protein were decreased to pre-exercise levels at 12 h. Thus, the restoration of PGC-1α expression to the pre-exercise levels was followed by the decrease in plasma irisin levels. By contrast, during 24-h recovery, the expression levels of FNDC5 mRNA and protein were maintained at elevated levels. These results suggest that the coordinated expression of FNDC5 and PGC-1α may contribute to the increased levels of plasma irisin after exercise.

The subthalamic nucleus during decision-making with multiple alternatives.

PubMed

Keuken, Max C; Van Maanen, Leendert; Bogacz, Rafal; Schäfer, Andreas; Neumann, Jane; Turner, Robert; Forstmann, Birte U

2015-10-01

Several prominent neurocomputational models predict that an increase of choice alternatives is modulated by increased activity in the subthalamic nucleus (STN). In turn, increased STN activity allows prolonged accumulation of information. At the same time, areas in the medial frontal cortex such as the anterior cingulate cortex (ACC) and the pre-SMA are hypothesized to influence the information processing in the STN. This study set out to test concrete predictions of STN activity in multiple-alternative decision-making using a multimodal combination of 7 Tesla structural and functional Magnetic Resonance Imaging, and ancestral graph (AG) modeling. The results are in line with the predictions in that increased STN activity was found with an increasing amount of choice alternatives. In addition, our study shows that activity in the ACC is correlated with activity in the STN without directly modulating it. This result sheds new light on the information processing streams between medial frontal cortex and the basal ganglia. © 2015 Wiley Periodicals, Inc.

Effect of hyperthyroidism on circulating prolactin and hypothalamic expression of tyrosine hydroxylase, prolactin signaling cascade members and estrogen and progesterone receptors during late pregnancy and lactation in the rat.

PubMed

Pennacchio, Gisela E; Neira, Flavia J; Soaje, Marta; Jahn, Graciela A; Valdez, Susana R

2017-02-15

Hyperthyroidism (HyperT) compromises pregnancy and lactation, hindering suckling-induced PRL release. We studied the effect of HyperT on hypothalamic mRNA (RT-qPCR) and protein (Western blot) expression of tyrosine hydroxylase (TH), PRL receptor (PRLR) and signaling pathway members, estrogen-α (ERα) and progesterone (PR) receptors on late pregnancy (days G19, 20 and 21) and early lactation (L2) in rats. HyperT advanced pre-partum PRL release, reduced circulating PRL on L2 and increased TH mRNA (G21 and L2), p-TH, PRLR mRNA, STAT5 protein (G19 and L2), PRLR protein (G21) and CIS protein (G19). PRs mRNAs and protein decreased on G19 but afterwards PRA mRNA (G20), PRB mRNA (G21) and PRA mRNA and protein (L2) increased. ERα protein increased on G19 and decreased on G20. Thus, the altered hypothalamic PRLR, STAT5, PR and ERα expression in hyperthyroid rats may induce elevated TH expression and activation, that consequently, elevate dopaminergic tone during lactation, blunting suckling-induced PRL release and litter growth. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Viperin mRNA is a novel target for the human RNase MRP/RNase P endoribonuclease.

PubMed

Mattijssen, Sandy; Hinson, Ella R; Onnekink, Carla; Hermanns, Pia; Zabel, Bernhard; Cresswell, Peter; Pruijn, Ger J M

2011-07-01

RNase MRP is a conserved endoribonuclease, in humans consisting of a 267-nucleotide RNA associated with 7-10 proteins. Mutations in its RNA component lead to several autosomal recessive skeletal dysplasias, including cartilage-hair hypoplasia (CHH). Because the known substrates of mammalian RNase MRP, pre-ribosomal RNA, and RNA involved in mitochondrial DNA replication are not likely involved in CHH, we analyzed the effects of RNase MRP (and the structurally related RNase P) depletion on mRNAs using DNA microarrays. We confirmed the upregulation of the interferon-inducible viperin mRNA by RNAi experiments and this appeared to be independent of the interferon response. We detected two cleavage sites for RNase MRP/RNase P in the coding sequence of viperin mRNA. This is the first study providing direct evidence for the cleavage of a mRNA by RNase MRP/RNase P in human cells. Implications for the involvement in the pathophysiology of CHH are discussed.

Effect of resistance exercise intensity on the expression of PGC-1α isoforms and the anabolic and catabolic signaling mediators, IGF-1 and myostatin, in human skeletal muscle.

PubMed

Schwarz, Neil A; McKinley-Barnard, Sarah K; Spillane, Mike B; Andre, Thomas L; Gann, Joshua J; Willoughby, Darryn S

2016-08-01

The purpose of this study was to investigate the acute messenger (mRNA) expression of the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) isoforms, insulin-like growth factor-1Ea (IGF-1Ea), and myostatin in response to 2 resistance exercise intensities. In a uniform-balanced, crossover design, 10 participants performed 2 separate testing sessions involving a lower body resistance exercise component consisting of a lower intensity (50% of 1-repetition maximum; 1RM) protocol and a higher intensity (80% of 1RM) protocol of equal volumes. Muscle samples were obtained at before exercise, 45 min, 3 h, 24 h, and 48 h postexercise. Resistance exercise did not alter total PGC-1α mRNA expression; however, distinct responses of each PGC-1α isoform were observed. The response of each isoform was consistent between sessions, suggesting no effect of resistance exercise intensity on the complex transcriptional expression of the PGC-1α gene. IGF-1Ea mRNA expression significantly increased following the higher intensity session compared with pre-exercise and the lower intensity session. Myostatin mRNA expression was significantly reduced compared with pre-exercise values at all time points with no difference between exercise intensity. Further research is needed to determine the effects of the various isoforms of PGC-1α in human skeletal muscle on the translational level as well as their relation to the expression of IGF-1 and myostatin.

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.

Histone and RNA-binding protein interaction creates crosstalk network for regulation of alternative splicing.

PubMed

Kim, Yong-Eun; Park, Chungoo; Kim, Kyoon Eon; Kim, Kee K

2018-04-30

Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by in vivo crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing. Copyright © 2018 Elsevier Inc. All rights reserved.

Promoter-Terminator Gene Loops Affect Alternative 3'-End Processing in Yeast.

PubMed

Lamas-Maceiras, Mónica; Singh, Badri Nath; Hampsey, Michael; Freire-Picos, María A

2016-04-22

Many eukaryotic genes undergo alternative 3'-end poly(A)-site selection producing transcript isoforms with 3'-UTRs of different lengths and post-transcriptional fates. Gene loops are dynamic structures that juxtapose the 3'-ends of genes with their promoters. Several functions have been attributed to looping, including memory of recent transcriptional activity and polarity of transcription initiation. In this study, we investigated the relationship between gene loops and alternative poly(A)-site. Using the KlCYC1 gene of the yeast Kluyveromyces lactis, which includes a single promoter and two poly(A) sites separated by 394 nucleotides, we demonstrate in two yeast species the formation of alternative gene loops (L1 and L2) that juxtapose the KlCYC1 promoter with either proximal or distal 3'-end processing sites, resulting in the synthesis of short and long forms of KlCYC1 mRNA. Furthermore, synthesis of short and long mRNAs and formation of the L1 and L2 loops are growth phase-dependent. Chromatin immunoprecipitation experiments revealed that the Ssu72 RNA polymerase II carboxyl-terminal domain phosphatase, a critical determinant of looping, peaks in early log phase at the proximal poly(A) site, but as growth phase advances, it extends to the distal site. These results define a cause-and-effect relationship between gene loops and alternative poly(A) site selection that responds to different physiological signals manifested by RNA polymerase II carboxyl-terminal domain phosphorylation status. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Alternating anoxic feast/aerobic famine condition for improving granular sludge formation in sequencing batch airlift reactor at reduced aeration rate.

PubMed

Wan, Junfeng; Bessière, Yolaine; Spérandio, Mathieu

2009-12-01

In this study the influence of a pre-anoxic feast period on granular sludge formation in a sequencing batch airlift reactor is evaluated. Whereas a purely aerobic SBR was operated as a reference (reactor R2), another reactor (R1) was run with a reduced aeration rate and an alternating anoxic-aerobic cycle reinforced by nitrate feeding. The presence of pre-anoxic phase clearly improved the densification of aggregates and allowed granular sludge formation at reduced air flow rate (superficial air velocity (SAV)=0.63cms(-1)). A low sludge volume index (SVI(30)=45mLg(-1)) and a high MLSS concentration (9-10gL(-1)) were obtained in the anoxic/aerobic system compared to more conventional results for the aerobic reactor. A granular sludge was observed in the anoxic/aerobic system whilst only flocs were observed in the aerobic reference even when operated at a high aeration rate (SAV=2.83cms(-1)). Nitrification was maintained efficiently in the anoxic/aerobic system even when organic loading rate (OLR) was increased up to 2.8kgCODm(-3)d(-1). In the contrary nitrification was unstable in the aerobic system and dropped at high OLR due to competition between autotrophic and heterotrophic growth. The presence of a pre-anoxic period positively affected granulation process via different mechanisms: enhancing heterotrophic growth/storage deeper in the internal anoxic layer of granule, reducing the competition between autotrophic and heterotrophic growth. These processes help to develop dense granular sludge at a moderate aeration rate. This tends to confirm that oxygen transfer is the most limiting factor for granulation at reduced aeration. Hence the use of an alternative electron acceptor (nitrate or nitrite) should be encouraged during feast period for reducing energy demand of the granular sludge process.

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.

The “Tail” of Connexin43: An Unexpected Journey from Alternative Translation to Trafficking

PubMed Central

Basheer, Wassim; Shaw, Robin

2015-01-01

With each heartbeat, Connexin43 (Cx43) cell-cell communication gap junctions are needed to rapidly spread and coordinate excitation signals for an effective heart contraction. The correct formation and delivery of channels to their respective membrane subdomain is referred to as protein trafficking. Altered Cx43 trafficking is a dangerous complication of diseased myocardium which contributes to the arrhythmias of sudden cardiac death. Cx43 has also been found to regulate many other cellular processes that cannot be explained by cell-cell communication. We recently identified the existence of up to six endogenous internally translated Cx43 N-terminal truncated isoforms from the same full-length mRNA molecule. This is the first evidence that alternative translation is possible for human ion channels and in human heart. Interestingly, we found that these internally translated isoforms, more specifically the 20 kDa isoform (GJA1-20k), is important for delivery of Cx43 to its respective membrane subdomain. This review covers recent advances in Cx43 trafficking and potential importance of alternatively translated Cx43 truncated isoforms. PMID:26526689

Various dietary fats differentially change the gene expression of neuropeptides involved in body weight regulation in rats.

PubMed

Dziedzic, B; Szemraj, J; Bartkowiak, J; Walczewska, A

2007-05-01

Various high-fat diets are obesogenic but not to the same extent. The aim of the present study was to investigate the effects of saturated fat n-6 and n-3 polyunsaturated fatty acids (PUFAs) on the central neuropeptidergic system in adult rats. Using reverse transcriptase-polymerase chain reaction and in situ hybridisation, we evaluated the net effect of feeding in these fats, comparing the effects of a high- to low-fat diet, and the diversity of the effects of these fats in the same amount within the diet. We also determined plasma lipids, glucose, insulin and leptin concentrations. Six-week feeding with high-saturated fat evoked hyperpahagia and the largest weight gain compared to both high-PUFA diets. Rats fed high-saturated fat were found to have decreased neuropeptide Y (NPY) mRNA expression in the arcuate nucleus (ARC) and the compact zone of the dorsomedial nucleus (DMHc), unchanged pro-opiomelanocortin (POMC), galanin-like peptide (GALP) mRNA expression in the ARC, as well as melanin-concentrating hormone (MCH) and prepro-orexin (preORX) mRNA expression in the lateral hypothalamus, compared to low-saturated fed rats. By contrast, feeding with both high-PUFA diets increased POMC and GALP mRNA expression in the ARC compared to the corresponding low-fat diet and the high-saturated fat diet. Furthermore, feeding with both low-PUFA diets reduced NPY mRNA expression compared to the low-saturated fat diet exclusively in the DMHc. Uniquely, the high n-3 PUFA feeding halved MCH and preORX mRNA expression in the lateral hypothalamus compared to the other high-fat and low n-3 PUFA diets. In rats fed three high-fat diets, plasma insulin and leptin concentrations were significantly increased and the type of fat had no effect on these hormone levels. Rats fed high-saturated fat had both hyperglycaemia and hypertriacylglycerolemia and rats fed high n-3 PUFA only had hyperglycaemia. The present study demonstrates that various forms of dietary fat differentially change the expression of neuropeptide genes involved in energy homeostasis.

Performance of biofuel processes utilising separate lignin and carbohydrate processing.

PubMed

Melin, Kristian; Kohl, Thomas; Koskinen, Jukka; Hurme, Markku

2015-09-01

Novel biofuel pathways with increased product yields are evaluated against conventional lignocellulosic biofuel production processes: methanol or methane production via gasification and ethanol production via steam-explosion pre-treatment. The novel processes studied are ethanol production combined with methanol production by gasification, hydrocarbon fuel production with additional hydrogen produced from lignin residue gasification, methanol or methane synthesis using synthesis gas from lignin residue gasification and additional hydrogen obtained by aqueous phase reforming in synthesis gas production. The material and energy balances of the processes were calculated by Aspen flow sheet models and add on excel calculations applicable at the conceptual design stage to evaluate the pre-feasibility of the alternatives. The processes were compared using the following criteria: energy efficiency from biomass to products, primary energy efficiency, GHG reduction potential and economy (expressed as net present value: NPV). Several novel biorefinery concepts gave higher energy yields, GHG reduction potential and NPV. Copyright © 2015 Elsevier Ltd. All rights reserved.

Distributed Attention Is Implemented through Theta-Rhythmic Gamma Modulation.

PubMed

Landau, Ayelet Nina; Schreyer, Helene Marianne; van Pelt, Stan; Fries, Pascal

2015-08-31

When subjects monitor a single location, visual target detection depends on the pre-target phase of an ∼8 Hz brain rhythm. When multiple locations are monitored, performance decrements suggest a division of the 8 Hz rhythm over the number of locations, indicating that different locations are sequentially sampled. Indeed, when subjects monitor two locations, performance benefits alternate at a 4 Hz rhythm. These performance alternations were revealed after a reset of attention to one location. Although resets are common and important events for attention, it is unknown whether, in the absence of resets, ongoing attention samples stimuli in alternation. Here, we examined whether spatially specific attentional sampling can be revealed by ongoing pre-target brain rhythms. Visually induced gamma-band activity plays a role in spatial attention. Therefore, we hypothesized that performance on two simultaneously monitored stimuli can be predicted by a 4 Hz modulation of gamma-band activity. Brain rhythms were assessed with magnetoencephalography (MEG) while subjects monitored bilateral grating stimuli for a unilateral target event. The corresponding contralateral gamma-band responses were subtracted from each other to isolate spatially selective, target-related fluctuations. The resulting lateralized gamma-band activity (LGA) showed opposite pre-target 4 Hz phases for detected versus missed targets. The 4 Hz phase of pre-target LGA accounted for a 14.5% modulation in performance. These findings suggest that spatial attention is a theta-rhythmic sampling process that is continuously ongoing, with each sampling cycle being implemented through gamma-band synchrony. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification by high-throughput imaging of the histone methyltransferase EHMT2 as an epigenetic regulator of VEGFA alternative splicing.

PubMed

Salton, Maayan; Voss, Ty C; Misteli, Tom

2014-12-16

Recent evidence points to a role of chromatin in regulation of alternative pre-mRNA splicing (AS). In order to identify novel chromatin regulators of AS, we screened an RNAi library of chromatin proteins using a cell-based high-throughput in vivo assay. We identified a set of chromatin proteins that regulate AS. Using simultaneous genome-wide expression and AS analysis, we demonstrate distinct and non-overlapping functions of these chromatin modifiers on transcription and AS. Detailed mechanistic characterization of one dual function chromatin modifier, the H3K9 methyltransferase EHMT2 (G9a), identified VEGFA as a major chromatin-mediated AS target. Silencing of EHMT2, or its heterodimer partner EHMT1, affects AS by promoting exclusion of VEGFA exon 6a, but does not alter total VEGFA mRNA levels. The epigenetic regulatory mechanism of AS by EHMT2 involves an adaptor system consisting of the chromatin modulator HP1γ, which binds methylated H3K9 and recruits splicing regulator SRSF1. The epigenetic regulation of VEGFA is physiologically relevant since EHMT2 is transcriptionally induced in response to hypoxia and triggers concomitant changes in AS of VEGFA. These results characterize a novel epigenetic regulatory mechanism of AS and they demonstrate separate roles of epigenetic modifiers in transcription and alternative splicing. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by US Government employees and is in the public domain in the US.

A comprehensive evaluation of the accuracy of cervical pre-cancer detection methods in a high-risk area in East Congo

PubMed Central

Hovland, S; Arbyn, M; Lie, A K; Ryd, W; Borge, B; Berle, E J; Skomedal, H; Kadima, T M; Kyembwa, L; Billay, E M; Mukwege, D; Chirimwami, R B; Mvula, T M; Snijders, P J; Meijer, C J L M; Karlsen, F

2010-01-01

Background: Given the high burden of cervical cancer in low-income settings, there is a need for a convenient and affordable method for detecting and treating pre-cancerous lesions. Methods: Samples for comparing the accuracy of cytology, virology and histology were collected. Identification of HPV E6/E7 mRNA was performed using PreTect HPV-Proofer. HPV DNA detection was performed by GP5+/6+ PCR, followed by reverse line blot (RLB) for typing. Results: A total of 343 women, aged 25–60 years, attending gynaecological polyclinics in DR Congo were included for sample enrolment. The test positivity rate was conventional and liquid-based cytology (LBC) at cutoff ASCUS+ of 6.9 and 6.6%, respectively; PreTect HPV-Proofer of 7.3% and consensus DNA PCR for 14 HR types of 18.5%. Sixteen cases of CIN2+ lesions were identified. Of these, conventional cytology identified 66.7% with a specificity of 96.2%, LBC identified 73.3% with a specificity of 96.9%, all at cutoff ASCUS+. HR-HPV DNA detected all CIN2+ cases with a specificity of 85.9%, whereas PreTect HPV-Proofer gave a sensitivity of 81.3% and a specificity of 96.6%. Conclusion: Both HPV detection assays showed a higher sensitivity for CIN2+ than did cytological methods. Detecting E6/E7 mRNA from only a subset of HR HPVs, as is the case with PreTect HPV-Proofer, resulted in a similar specificity to cytology and a significantly higher specificity than consensus HR HPV DNA (P<0.0001). PMID:20197765

Differential expression of alternatively spliced transcripts related to energy metabolism in colorectal cancer.

PubMed

Snezhkina, Anastasiya Vladimirovna; Krasnov, George Sergeevich; Zaretsky, Andrew Rostislavovich; Zhavoronkov, Alex; Nyushko, Kirill Mikhailovich; Moskalev, Alexey Alexandrovich; Karpova, Irina Yurievna; Afremova, Anastasiya Isaevna; Lipatova, Anastasiya Valerievna; Kochetkov, Dmitriy Vladimitovich; Fedorova, Maria Sergeena; Volchenko, Nadezhda Nikolaevna; Sadritdinova, Asiya Fayazovna; Melnikova, Nataliya Vladimirovna; Sidorov, Dmitry Vladimirovich; Popov, Anatoly Yurievich; Kalinin, Dmitry Valerievich; Kaprin, Andrey Dmitrievich; Alekseev, Boris Yakovlevich; Dmitriev, Alexey Alexandrovich; Kudryavtseva, Anna Viktorovna

2016-12-28

Colorectal cancer (CRC) is one of the most common malignant tumors worldwide. CRC molecular pathogenesis is heterogeneous and may be followed by mutations in oncogenes and tumor suppressor genes, chromosomal and microsatellite instability, alternative splicing alterations, hypermethylation of CpG islands, oxidative stress, impairment of different signaling pathways and energy metabolism. In the present work, we have studied the alterations of alternative splicing patterns of genes related to energy metabolism in CRC. Using CrossHub software, we analyzed The Cancer Genome Atlas (TCGA) RNA-Seq datasets derived from colon tumor and matched normal tissues. The expression of 1014 alternative mRNA isoforms involved in cell energy metabolism was examined. We found 7 genes with differentially expressed alternative transcripts whereas overall expression of these genes was not significantly altered in CRC. A set of 8 differentially expressed transcripts of interest has been validated by qPCR. These eight isoforms encoded by OGDH, COL6A3, ICAM1, PHPT1, PPP2R5D, SLC29A1, and TRIB3 genes were up-regulated in colorectal tumors, and this is in concordance with the bioinformatics data. The alternative transcript NM_057167 of COL6A3 was also strongly up-regulated in breast, lung, prostate, and kidney tumors. Alternative transcript of SLC29A1 (NM_001078177) was up-regulated only in CRC samples, but not in the other tested tumor types. We identified tumor-specific expression of alternative spliced transcripts of seven genes involved in energy metabolism in CRC. Our results bring new knowledge on alternative splicing in colorectal cancer and suggest a set of mRNA isoforms that could be used for cancer diagnosis and development of treatment methods.

Maternal programming of offspring hypothalamic-pituitary-interrenal axis in wild sockeye salmon (Oncorhynchus nerka).

PubMed

Sopinka, N M; Jeffrey, J D; Burnett, N J; Patterson, D A; Gilmour, K M; Hinch, S G

2017-02-01

In fishes, maternal exposure to a stressor can influence offspring size and behavior. However, less is known about how maternal stress influences physiological processes in offspring, such as function of the hypothalamic-pituitary-interrenal (HPI) axis. We examined the impact of chronic maternal exposure to an acute chase stressor on the stress response/HPI activity of progeny in wild sockeye salmon (Oncorhynchus nerka). Resting plasma cortisol and brain preoptic area (POA) corticotropin-releasing factor (CRF) mRNA levels did not vary between offspring reared from undisturbed, control females and offspring reared from females exposed to the stressor. However, resting levels of POA glucocorticoid receptors (GR1 and GR2), and head kidney melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR), and cytochrome P450 side chain cleavage enzyme (P450scc) were elevated in offspring reared from stressor-exposed females. Offspring reared from stressor-exposed females had lower plasma cortisol levels 1-h after an acute chase stressor compared to cortisol levels in offspring reared from control females. In offspring reared from chased females, mRNA levels of genes associated with cortisol biosynthesis were reduced in the head kidney post-chase. In offspring reared from control females, mRNA levels in the head kidney did not vary pre- to post-chase. Together, the results of the present study suggest maternal programming of progeny with respect to baseline and stressor-induced mediators of HPI axis activity. Copyright © 2015 Elsevier Inc. All rights reserved.

Deliberation before determination: the definition and evaluation of good decision making.

PubMed

Elwyn, Glyn; Miron-Shatz, Talya

2010-06-01

In this article, we examine definitions of suggested approaches to measure the concept of good decisions, highlight the ways in which they converge, and explain why we have concerns about their emphasis on post-hoc estimations and post-decisional outcomes, their prescriptive concept of knowledge, and their lack of distinction between the process of deliberation, and the act of decision determination. There has been a steady trend to involve patients in decision making tasks in clinical practice, part of a shift away from paternalism towards the concept of informed choice. An increased understanding of the uncertainties that exist in medicine, arising from a weak evidence base and, in addition, the stochastic nature of outcomes at the individual level, have contributed to shifting the responsibility for decision making from physicians to patients. This led to increasing use of decision support and communication methods, with the ultimate aim of improving decision making by patients. Interest has therefore developed in attempting to define good decision making and in the development of measurement approaches. We pose and reflect whether decisions can be judged good or not, and, if so, how this goodness might be evaluated. We hypothesize that decisions cannot be measured by reference to their outcomes and offer an alternative means of assessment, which emphasizes the deliberation process rather than the decision's end results. We propose decision making comprises a pre-decisional process and an act of decision determination and consider how this model of decision making serves to develop a new approach to evaluating what constitutes a good decision making process. We proceed to offer an alternative, which parses decisions into the pre-decisional deliberation process, the act of determination and post-decisional outcomes. Evaluating the deliberation process, we propose, should comprise of a subjective sufficiency of knowledge, as well as emotional processing and affective forecasting of the alternatives. This should form the basis for a good act of determination.

Perturbation of Chromatin Structure Globally Affects Localization and Recruitment of Splicing Factors

PubMed Central

Risso, Guillermo J.; Pawellek, Andrea; Ule, Jernej; Lamond, Angus I.; Kornblihtt, Alberto R.

2012-01-01

Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3′ splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ∼20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin structure is essential for efficient co-transcriptional recruitment of general and regulatory splicing factors to pre-mRNA. PMID:23152763

Analysis of alterative cleavage and polyadenylation by 3′ region extraction and deep sequencing

PubMed Central

Hoque, Mainul; Ji, Zhe; Zheng, Dinghai; Luo, Wenting; Li, Wencheng; You, Bei; Park, Ji Yeon; Yehia, Ghassan; Tian, Bin

2012-01-01

Alternative cleavage and polyadenylation (APA) leads to mRNA isoforms with different coding sequences (CDS) and/or 3′ untranslated regions (3′UTRs). Using 3′ Region Extraction And Deep Sequencing (3′READS), a method which addresses the internal priming and oligo(A) tail issues that commonly plague polyA site (pA) identification, we comprehensively mapped pAs in the mouse genome, thoroughly annotating 3′ ends of genes and revealing over five thousand pAs (~8% of total) flanked by A-rich sequences, which have hitherto been overlooked. About 79% of mRNA genes and 66% of long non-coding RNA (lncRNA) genes have APA; but these two gene types have distinct usage patterns for pAs in introns and upstream exons. Promoter-distal pAs become relatively more abundant during embryonic development and cell differentiation, a trend affecting pAs in both 3′-most exons and upstream regions. Upregulated isoforms generally have stronger pAs, suggesting global modulation of the 3′ end processing activity in development and differentiation. PMID:23241633

Human TREX2 components PCID2 and centrin 2, but not ENY2, have distinct functions in protein export and co-localize to the centrosome.

PubMed

Cunningham, Corey N; Schmidt, Casey A; Schramm, Nathaniel J; Gaylord, Michelle R; Resendes, Karen K

2014-01-15

TREX-2 is a five protein complex, conserved from yeast to humans, involved in linking mRNA transcription and export. The centrin 2 subunit of TREX-2 is also a component of the centrosome and is additionally involved in a distinctly different process of nuclear protein export. While centrin 2 is a known multifunctional protein, the roles of other human TREX-2 complex proteins other than mRNA export are not known. In this study, we found that human TREX-2 member PCID2 but not ENY2 is involved in some of the same cellular processes as those of centrin 2 apart from the classical TREX-2 function. PCID2 is present at the centrosome in a subset of HeLa cells and this localization is centrin 2 dependent. Furthermore, the presence of PCID2 at the centrosome is prevalent throughout the cell cycle as determined by co-staining with cyclins E, A and B. PCID2 but not ENY2 is also involved in protein export. Surprisingly, siRNA knockdown of PCID2 delayed the rate of nuclear protein export, a mechanism distinct from the effects of centrin 2, which when knocked down inhibits export. Finally we showed that co-depletion of centrin 2 and PCID2 leads to blocking rather than delaying nuclear protein export, indicating the dominance of the centrin 2 phenotype. Together these results represent the first discovery of specific novel functions for PCID2 other than mRNA export and suggest that components of the TREX-2 complex serve alternative shared roles in the regulation of nuclear transport and cell cycle progression. © 2013 Published by Elsevier Inc.

Identification of a reference gene for the quantification of mRNA and miRNA expression during skin wound healing.

PubMed

Etich, Julia; Bergmeier, Vera; Pitzler, Lena; Brachvogel, Bent

2017-03-01

Wound healing is a coordinated process to restore tissue homeostasis and reestablish the protective barrier of the skin. miRNAs may modulate the expression of target genes to contribute to repair processes, but due to the complexity of the tissue it is challenging to quantify gene expression during the distinct phases of wound repair. Here, we aimed to identify a common reference gene to quantify changes in miRNA and mRNA expression during skin wound healing. Quantitative real-time PCR and bioinformatic analysis tools were used to identify suitable reference genes during skin repair and their reliability was tested by studying the expression of mRNAs and miRNAs. Morphological assessment of wounds showed that the injury model recapitulates the distinct phases of skin repair. Non-degraded RNA could be isolated from skin and wounds and used to study the expression of non-coding small nuclear RNAs during wound healing. Among those, RNU6B was most constantly expressed during skin repair. Using this reference gene we could confirm the transient upregulation of IL-1β and PTPRC/CD45 during the early phase as well as the increased expression of collagen type I at later stages of repair and validate the differential expression of miR-204, miR-205, and miR-31 in skin wounds. In contrast to Gapdh the normalization to multiple reference genes gave a similar outcome. RNU6B is an accurate alternative normalizer to quantify mRNA and miRNA expression during the distinct phases of skin wound healing when analysis of multiple reference genes is not feasible.

Alternative Fuels Data Center: Natural Gas Vehicle Availability

Science.gov Websites

options for converting conventional vehicles to run on natural gas. Pre-Owned Vehicles Learn about buying and selling pre-owned alternative fuel and advanced technology vehicles. More Information For more

Differential regulation of the lung endothelin system by urban particulate matter and ozone.

PubMed

Thomson, Errol; Kumarathasan, Prem; Goegan, Patrick; Aubin, Rémy A; Vincent, Renaud

2005-11-01

Periodic elevation of ambient particulate matter and ozone levels is linked to acute cardiac morbidity and mortality. Increased plasma levels of the potent vasoconstrictor endothelin (ET)-1, a prognostic indicator of cardiac mortality, have been detected in both animal models and humans after exposure to air pollutants. The lungs are the primary source of circulating ET-1, but the direct effects of individual air pollutants and their interaction in modulating the pulmonary endothelin system are unknown. Fischer-344 rats were exposed to particles (0, 5, 50 mg/m3 EHC-93), ozone (0, 0.4, 0.8 ppm), or combinations of particles and ozone for 4 h. Changes in gene expression were measured using real-time reverse transcription polymerase chain reaction immediately after exposure and following 24 h recovery in clean air. Both pollutants individually increased preproET-1, endothelin converting enzyme-1, and endothelial nitric oxide synthase mRNA levels in the lungs shortly after exposure, consistent with the concomitant increase in plasma of the 21 amino acid ET-1[1-21] peptide measured by HPLC-fluorescence. PreproET-1 mRNA remained elevated 24 h after exposure to particles but not after ozone, in line with previously documented changes of the peptide in plasma. Both pollutants transiently increased endothelin-B receptor mRNA expression, while ozone decreased endothelin-A receptor mRNA levels. Coexposure to particles plus ozone increased lung preproET-1 mRNA but not plasma ET-1[1-21], suggesting alternative processing or degradation of endothelins. This coincided with an increase in the lungs of matrix metalloproteinase-2 (MMP-2), an enzyme that cleaves bigET-1 to ET-1[1-32]. Taken together, our data indicate that ozone and particulate matter independently regulate the expression of lung endothelin system genes, but show complex toxicological interaction with respect to plasma ET-1.

Similar Properties of Chondrocytes from Osteoarthritis Joints and Mesenchymal Stem Cells from Healthy Donors for Tissue Engineering of Articular Cartilage

PubMed Central

Fernandes, Amilton M.; Herlofsen, Sarah R.; Karlsen, Tommy A.; Küchler, Axel M.; Fløisand, Yngvar; Brinchmann, Jan E.

2013-01-01

Lesions of hyaline cartilage do not heal spontaneously, and represent a therapeutic challenge. In vitro engineering of articular cartilage using cells and biomaterials may prove to be the best solution. Patients with osteoarthritis (OA) may require tissue engineered cartilage therapy. Chondrocytes obtained from OA joints are thought to be involved in the disease process, and thus to be of insufficient quality to be used for repair strategies. Bone marrow (BM) derived mesenchymal stem cells (MSCs) from healthy donors may represent an alternative cell source. We have isolated chondrocytes from OA joints, performed cell culture expansion and tissue engineering of cartilage using a disc-shaped alginate scaffold and chondrogenic differentiation medium. We performed real-time reverse transcriptase quantitative PCR and fluorescence immunohistochemistry to evaluate mRNA and protein expression for a range of molecules involved in chondrogenesis and OA pathogenesis. Results were compared with those obtained by using BM-MSCs in an identical tissue engineering strategy. Finally the two populations were compared using genome-wide mRNA arrays. At three weeks of chondrogenic differentiation we found high and similar levels of hyaline cartilage-specific type II collagen and fibrocartilage-specific type I collagen mRNA and protein in discs containing OA and BM-MSC derived chondrocytes. Aggrecan, the dominant proteoglycan in hyaline cartilage, was more abundantly distributed in the OA chondrocyte extracellular matrix. OA chondrocytes expressed higher mRNA levels also of other hyaline extracellular matrix components. Surprisingly BM-MSC derived chondrocytes expressed higher mRNA levels of OA markers such as COL10A1, SSP1 (osteopontin), ALPL, BMP2, VEGFA, PTGES, IHH, and WNT genes, but lower levels of MMP3 and S100A4. Based on the results presented here, OA chondrocytes may be suitable for tissue engineering of articular cartilage. PMID:23671648

On the Presentation of Pre-Calculus and Calculus Topics: An Alternate View

ERIC Educational Resources Information Center

Davydov, Aleksandr; Sturm-Beiss, Rachel

2008-01-01

The orders of presentation of pre-calculus and calculus topics, and the notation used, deserve careful study as they affect clarity and ultimately students' level of understanding. We introduce an alternate approach to some of the topics included in this sequence. The suggested alternative is based on years of teaching in colleges within and…

MUC1-ARF-A Novel MUC1 Protein That Resides in the Nucleus and Is Expressed by Alternate Reading Frame Translation of MUC1 mRNA.

PubMed

Chalick, Michael; Jacobi, Oded; Pichinuk, Edward; Garbar, Christian; Bensussan, Armand; Meeker, Alan; Ziv, Ravit; Zehavi, Tania; Smorodinsky, Nechama I; Hilkens, John; Hanisch, Franz-Georg; Rubinstein, Daniel B; Wreschner, Daniel H

2016-01-01

Translation of mRNA in alternate reading frames (ARF) is a naturally occurring process heretofore underappreciated as a generator of protein diversity. The MUC1 gene encodes MUC1-TM, a signal-transducing trans-membrane protein highly expressed in human malignancies. Here we show that an AUG codon downstream to the MUC1-TM initiation codon initiates an alternate reading frame thereby generating a novel protein, MUC1-ARF. MUC1-ARF, like its MUC1-TM 'parent' protein, contains a tandem repeat (VNTR) domain. However, the amino acid sequence of the MUC1-ARF tandem repeat as well as N- and C- sequences flanking it differ entirely from those of MUC1-TM. In vitro protein synthesis assays and extensive immunohistochemical as well as western blot analyses with MUC1-ARF specific monoclonal antibodies confirmed MUC1-ARF expression. Rather than being expressed at the cell membrane like MUC1-TM, immunostaining showed that MUC1-ARF protein localizes mainly in the nucleus: Immunohistochemical analyses of MUC1-expressing tissues demonstrated MUC1-ARF expression in the nuclei of secretory luminal epithelial cells. MUC1-ARF expression varies in different malignancies. While the malignant epithelial cells of pancreatic cancer show limited expression, in breast cancer tissue MUC1-ARF demonstrates strong nuclear expression. Proinflammatory cytokines upregulate expression of MUC1-ARF protein and co-immunoprecipitation analyses demonstrate association of MUC1-ARF with SH3 domain-containing proteins. Mass spectrometry performed on proteins coprecipitating with MUC1-ARF demonstrated Glucose-6-phosphate 1-dehydrogenase (G6PD) and Dynamin 2 (DNM2). These studies not only reveal that the MUC1 gene generates a previously unidentified MUC1-ARF protein, they also show that just like its 'parent' MUC1-TM protein, MUC1-ARF is apparently linked to signaling and malignancy, yet a definitive link to these processes and the roles it plays awaits a precise identification of its molecular functions. Comprising at least 524 amino acids, MUC1-ARF is, furthermore, the longest ARF protein heretofore described.

MUC1-ARF—A Novel MUC1 Protein That Resides in the Nucleus and Is Expressed by Alternate Reading Frame Translation of MUC1 mRNA

PubMed Central

Pichinuk, Edward; Garbar, Christian; Bensussan, Armand; Meeker, Alan; Ziv, Ravit; Zehavi, Tania; Smorodinsky, Nechama I.; Hilkens, John; Hanisch, Franz-Georg; Rubinstein, Daniel B.; Wreschner, Daniel H.

2016-01-01

Translation of mRNA in alternate reading frames (ARF) is a naturally occurring process heretofore underappreciated as a generator of protein diversity. The MUC1 gene encodes MUC1-TM, a signal-transducing trans-membrane protein highly expressed in human malignancies. Here we show that an AUG codon downstream to the MUC1-TM initiation codon initiates an alternate reading frame thereby generating a novel protein, MUC1-ARF. MUC1-ARF, like its MUC1-TM 'parent’ protein, contains a tandem repeat (VNTR) domain. However, the amino acid sequence of the MUC1-ARF tandem repeat as well as N- and C- sequences flanking it differ entirely from those of MUC1-TM. In vitro protein synthesis assays and extensive immunohistochemical as well as western blot analyses with MUC1-ARF specific monoclonal antibodies confirmed MUC1-ARF expression. Rather than being expressed at the cell membrane like MUC1-TM, immunostaining showed that MUC1-ARF protein localizes mainly in the nucleus: Immunohistochemical analyses of MUC1-expressing tissues demonstrated MUC1-ARF expression in the nuclei of secretory luminal epithelial cells. MUC1-ARF expression varies in different malignancies. While the malignant epithelial cells of pancreatic cancer show limited expression, in breast cancer tissue MUC1-ARF demonstrates strong nuclear expression. Proinflammatory cytokines upregulate expression of MUC1-ARF protein and co-immunoprecipitation analyses demonstrate association of MUC1-ARF with SH3 domain-containing proteins. Mass spectrometry performed on proteins coprecipitating with MUC1-ARF demonstrated Glucose-6-phosphate 1-dehydrogenase (G6PD) and Dynamin 2 (DNM2). These studies not only reveal that the MUC1 gene generates a previously unidentified MUC1-ARF protein, they also show that just like its ‘parent’ MUC1-TM protein, MUC1-ARF is apparently linked to signaling and malignancy, yet a definitive link to these processes and the roles it plays awaits a precise identification of its molecular functions. Comprising at least 524 amino acids, MUC1-ARF is, furthermore, the longest ARF protein heretofore described. PMID:27768738

Induction of IgA B cell differentiation of bone marrow-derived B cells by Peyer's patch autoreactive helper T cells.

PubMed

Kihira, T; Kawanishi, H

1995-08-01

The objective of this study was to demonstrate in vitro that bone marrow-derived pro/pre-B cells bearing mu mRNA can switch their Ig heavy-chain isotype to that of alpha mRNA-expressing B cells after contact with Peyer's patches-derived activated autoreactive CD4+ T cells. Bone marrow-derived pro/pre-B cells and activated autoreactive Peyer's patch, mesenteric lymph node, or spleen CD4+ T cells were co-cultured in the presence of recombinant (r) IL-2, rIL-7, and Con A for 3 days. The mixed cultured cells were isolated for preparation of total RNA. Dot/slot hybridization, using murine C mu (pu3741) and C alpha (P alpha J558) Ig heavy-chain cDNA probes, detected C mu and C alpha Ig heavy-chain mRNA transcripts. The magnitude of each mRNA expression was measured demsitometrically. In addition, the secreted class-specific Ig contents from the co-cultured supernatants were measured. The results indicate that activated autoreactive Peyer's patch and mesenteric lymph node CD4+ T cells provide a specific Ig heavy-chain switch from mu to alpha (Peyer's patch CD4+ T cells > mesenteric lymph node CD4+ T cells) in bone marrow-derived pro/pre-B cells and also assist to develop IgA-secreting plasma cells. The alpha heavy-chain switch and IgA production do not occur in the presence of activated autoreactive spleen CD4+ T cells. These results support the view that autoreactive gut Peyer's patch CD4+ T cells, at least, regulate IgA B cell heavy-chain switching and terminal differentiation during gut mucosal B cell development.

Oriental Medicine Kyung-Ok-Ko Prevents and Alleviates Dehydroepiandrosterone-Induced Polycystic Ovarian Syndrome in Rats

PubMed Central

Lee, Jin Moo; Bae, Chun-Sik; Kim, Sung-Hoon; Ryu, Jong Hoon; Cho, Ik-Hyun

2014-01-01

Kyung-Ok-Ko (KOK), a traditional herbal prescription composed of Rehmannia glutinosa Liboschitz var. purpurae, Lycium chinense, Aquillaria agallocha, Poria cocos, Panax ginseng, and honey, has been widely used in traditional Oriental medicine as a vitalizing medicine or as the prescription for patients with age-associated disorders such as amnesia and stroke. However, the potential protective value of KOK for the treatment of polycystic ovarian syndrome (PCOS) is largely unknown. We investigated whether pre-administration (daily from 2 hours before PCOS induction) and post-administration (daily after induction of PCOS) of KOK (0.5, 1.0, and 2.0 g/kg/day, p.o.) could have a protective effect in a dehydroepiandrosterone (DHEA, s.c.)-induced PCOS rat model. Pre-administration of KOK significantly decreased the elevated body weight and ovary weight, elevated size and number of follicular cysts, elevated level of serum glucose, and estradiol after DHEA injection. KOK reduced the elevated percentage of CD8 (+) T lymphocytes in lymph nodes, the elevated mRNA expression of CD11b and CD3 in ovaries, and infiltration of macrophages in ovarian tissue with PCOS. KOK diminished the increased mRNA expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), chemokines (IL-8, MCP-1), and iNOS in the ovaries, and increased the reduced mRNA expression of growth factors (EGF, TGF-β) by DHEA injection. Post-administration of KOK also improved the DHEA-induced PCOS-like symptoms, generally similar to those evident from pre-administration of KOK. KOK may effectively prevent and improve DHEA-induced PCOS via anti-inflammatory action, indicating its preventive and therapeutic potential for suppressing PCOS. PMID:24520334

Residual social, memory and oxytocin-related changes in rats following repeated exposure to γ-hydroxybutyrate (GHB), 3,4-methylenedioxymethamphetamine (MDMA) or their combination.

PubMed

van Nieuwenhuijzen, Petra S; Long, Leonora E; Hunt, Glenn E; Arnold, Jonathon C; McGregor, Iain S

2010-12-01

There has been little investigation of the possible lasting adverse effects of γ-hydroxybutyrate (GHB). This study aims to study whether GHB produces residual adverse effects on memory and social behaviour in rats and lasting changes in brain monoamines and oxytocin-related gene expression. Rats received daily intraperitoneal injections of GHB (500 mg/kg), methylenedioxymethamphetamine (MDMA; 5 mg/kg) or their combination (GHB/MDMA) over ten consecutive days. Locomotor activity and body weight were assessed during the dosing period and withdrawal-related anxiety was assessed 24 h after drug cessation. After a washout of 4 weeks, rats were tested on the emergence, social interaction, and object recognition tasks over a 2-week period. Monoamine levels in cortex and striatum, and hypothalamic oxytocin and oxytocin receptor mRNA, were then assessed. MDMA and GHB/MDMA caused modest sensitization of locomotor activity over time, while sedative effects of GHB diminished with repeated exposure. GHB-treated rats showed reduced social interaction 24 h after the final dose, indicating GHB withdrawal-induced anxiety. All drug-treated groups displayed residual deficits in social interaction and object recognition. No changes in monoamine levels were detected 8 weeks post-drug. However, MDMA pre-exposure increased hypothalamic oxytocin mRNA while GHB pre-exposure upregulated oxytocin receptor mRNA. GHB/MDMA pre-exposure caused intermediate changes in both of these measures. GHB treatment caused residual impairments in memory and social behaviour and increases in anxiety, paralleling the lasting adverse effects of MDMA. Both drugs caused lasting neuroadaptations in brain oxytocin systems and this may be related to the long-term social interaction deficiencies caused by both drugs.

Serum from pregnant women with gestational diabetes mellitus increases the expression of FABP4 mRNA in primary subcutaneous human pre-adipocytes

PubMed Central

Li, Lan; Lee, Se Jin; Kook, Song Yi; Ahn, Tae Gyu; Lee, Ji Yeon

2017-01-01

Objective Gestational diabetes mellitus (GDM) is defined as glucose intolerance first detected during pregnancy. It can result in pregnancy complications such as birth injury, stillbirth. Fatty acid-binding protein 4 (FABP4), found in adipose tissue, is associated with insulin resistance, and type 2 diabetes. The aim of this study was to investigate whether FABP4 in the placenta and decidua of pregnant women with GDM is higher than that in normal pregnant women, and whether serum from pregnant women with GDM may cause adipocytes to secrete more FABP4 than does serum from a normal pregnant group. Methods We obtained placentas, deciduas, and serum from 12 pregnant women with GDM and 12 normal pregnant women and performed enzyme-linked immunosorbent assay, real time quantitative-polymerase chain reaction. We cultured human pre-adipocytes for 17 days with GDM and non-GDM serum and performed western blot, real time quantitative-polymerase chain reaction, and oil red O staining. Results Expression of FABP4 in serum, placenta and decidua of pregnant women with GDM was significantly higher than that in normal pregnant women. Serum from pregnant women with GDM increased the expression of FABP4 mRNA and decreased the expression of adiponectin mRNA in human pre-adipocytes significantly. Adipocyte cultured in GDM serum showed significantly greater lipid accumulation than those cultured in normal serum. Conclusion Our results suggest that FABP4 is higher in placenta and decidua from pregnant women with GDM. Increased circulating FABP4 in maternal serum from pregnant women with GDM may originate from adipocytes and the placenta. Circulating FABP4 can induce increased insulin resistance and decreased insulin sensitivity. PMID:28534013

Serum from pregnant women with gestational diabetes mellitus increases the expression of FABP4 mRNA in primary subcutaneous human pre-adipocytes.

PubMed

Li, Lan; Lee, Se Jin; Kook, Song Yi; Ahn, Tae Gyu; Lee, Ji Yeon; Hwang, Jong Yun

2017-05-01

Gestational diabetes mellitus (GDM) is defined as glucose intolerance first detected during pregnancy. It can result in pregnancy complications such as birth injury, stillbirth. Fatty acid-binding protein 4 (FABP4), found in adipose tissue, is associated with insulin resistance, and type 2 diabetes. The aim of this study was to investigate whether FABP4 in the placenta and decidua of pregnant women with GDM is higher than that in normal pregnant women, and whether serum from pregnant women with GDM may cause adipocytes to secrete more FABP4 than does serum from a normal pregnant group. We obtained placentas, deciduas, and serum from 12 pregnant women with GDM and 12 normal pregnant women and performed enzyme-linked immunosorbent assay, real time quantitative-polymerase chain reaction. We cultured human pre-adipocytes for 17 days with GDM and non-GDM serum and performed western blot, real time quantitative-polymerase chain reaction, and oil red O staining. Expression of FABP4 in serum, placenta and decidua of pregnant women with GDM was significantly higher than that in normal pregnant women. Serum from pregnant women with GDM increased the expression of FABP4 mRNA and decreased the expression of adiponectin mRNA in human pre-adipocytes significantly. Adipocyte cultured in GDM serum showed significantly greater lipid accumulation than those cultured in normal serum. Our results suggest that FABP4 is higher in placenta and decidua from pregnant women with GDM. Increased circulating FABP4 in maternal serum from pregnant women with GDM may originate from adipocytes and the placenta. Circulating FABP4 can induce increased insulin resistance and decreased insulin sensitivity.

Oriental medicine Kyung-Ok-Ko prevents and alleviates dehydroepiandrosterone-induced polycystic ovarian syndrome in rats.

PubMed

Jang, Minhee; Lee, Min Jung; Lee, Jin Moo; Bae, Chun-Sik; Kim, Sung-Hoon; Ryu, Jong Hoon; Cho, Ik-Hyun

2014-01-01

Kyung-Ok-Ko (KOK), a traditional herbal prescription composed of Rehmannia glutinosa Liboschitz var. purpurae, Lycium chinense, Aquillaria agallocha, Poria cocos, Panax ginseng, and honey, has been widely used in traditional Oriental medicine as a vitalizing medicine or as the prescription for patients with age-associated disorders such as amnesia and stroke. However, the potential protective value of KOK for the treatment of polycystic ovarian syndrome (PCOS) is largely unknown. We investigated whether pre-administration (daily from 2 hours before PCOS induction) and post-administration (daily after induction of PCOS) of KOK (0.5, 1.0, and 2.0 g/kg/day, p.o.) could have a protective effect in a dehydroepiandrosterone (DHEA, s.c.)-induced PCOS rat model. Pre-administration of KOK significantly decreased the elevated body weight and ovary weight, elevated size and number of follicular cysts, elevated level of serum glucose, and estradiol after DHEA injection. KOK reduced the elevated percentage of CD8 (+) T lymphocytes in lymph nodes, the elevated mRNA expression of CD11b and CD3 in ovaries, and infiltration of macrophages in ovarian tissue with PCOS. KOK diminished the increased mRNA expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), chemokines (IL-8, MCP-1), and iNOS in the ovaries, and increased the reduced mRNA expression of growth factors (EGF, TGF-β) by DHEA injection. Post-administration of KOK also improved the DHEA-induced PCOS-like symptoms, generally similar to those evident from pre-administration of KOK. KOK may effectively prevent and improve DHEA-induced PCOS via anti-inflammatory action, indicating its preventive and therapeutic potential for suppressing PCOS.

Investigation of Mental Models of Turkish Pre-Service Physics Students for the Concept of "Spin"

ERIC Educational Resources Information Center

Özcan, Özgür

2013-01-01

Problem Statement: Difficulties in the learning process usually emerge from the problem of mental representations constructed by students in their interactions with the world. This previous knowledge and these ideas are in contradiction with scientific facts, and are known as misconceptions or alternative ideas. Thus, an analysis of the mental…

Comparing the effects of tetrabromobisphenol-A, bisphenol A, and their potential replacement alternatives, TBBPA-bis(2,3-dibromopropyl ether) and bisphenol S, on cell viability and messenger ribonucleic acid expression in chicken embryonic hepatocytes.

PubMed

Ma, Melissa; Crump, Doug; Farmahin, Reza; Kennedy, Sean W

2015-02-01

A market for alternative brominated flame retardants (BFRs) has emerged recently due to the phase out of persistent and inherently toxic BFRs. Several of these replacement compounds have been detected in environmental matrices, including wild birds. A chicken embryonic hepatocyte (CEH) assay was utilized to assess the effects of the BFR, tetrabromobisphenol-A (TBBPA), and its replacement alternative, tetrabromobisphenol A bis(2,3-dibromopropyl ether [TBBPA-DBPE]) on cell viability and messenger ribonucleic acid (mRNA) expression. Bisphenol A (BPA) and 1 of its replacement alternatives, bisphenol S (BPS), were also screened for effects. Both TBBPA and BPA decreased CEH viability with calculated median lethal concentration (LC50) values of 40.6 μM and 61.7 μM, respectively. However, the replacement alternatives, TBBPA-DBPE and BPS, did not affect cell viability (up to 300 μM). Effects on mRNA expression were determined using an Avian ToxChip polymerse chain reaction (PCR) array and a real-time (RT)-PCR assay for the estrogen-responsive genes, apolipoproteinII (ApoII) and vitellogenin (Vtg). A luciferase reporter gene assay was used to assess dioxin-like effects. Tetrabromobisphenol-A altered mRNA levels of 4 genes from multiple toxicity pathways and increased luciferase activity in the luciferase reporter gene assay, whereas its alternative, TBBPA-DBPE, only altered 1 gene on the array, Cyp1a4, and increased luciferase activity. At 300 μM, a concentration that decreased cell viability for TBBPA and BPA, the BPA replacement, BPS, altered the greatest number of transcripts, including both ApoII and Vtg. Bisphenol A exposure did not alter any genes on the array but did up-regulate Vtg at 10 μM. Characterization of the potential toxicological and molecular-level effects of these compounds will ideally be useful to chemical regulators tasked with assessing the risk of new and existing chemicals. © 2014 SETAC.

Alternative Fuels Data Center: Flexible Fuel Vehicle Availability

Science.gov Websites

options for converting vehicles to run on E85 and other ethanol-gasoline blends. Pre-Owned Vehicles Learn about buying and selling pre-owned alternative fuel and advanced vehicles. Learn More Interested in

Universal Poisson Statistics of mRNAs with Complex Decay Pathways.

PubMed

Thattai, Mukund

2016-01-19

Messenger RNA (mRNA) dynamics in single cells are often modeled as a memoryless birth-death process with a constant probability per unit time that an mRNA molecule is synthesized or degraded. This predicts a Poisson steady-state distribution of mRNA number, in close agreement with experiments. This is surprising, since mRNA decay is known to be a complex process. The paradox is resolved by realizing that the Poisson steady state generalizes to arbitrary mRNA lifetime distributions. A mapping between mRNA dynamics and queueing theory highlights an identifiability problem: a measured Poisson steady state is consistent with a large variety of microscopic models. Here, I provide a rigorous and intuitive explanation for the universality of the Poisson steady state. I show that the mRNA birth-death process and its complex decay variants all take the form of the familiar Poisson law of rare events, under a nonlinear rescaling of time. As a corollary, not only steady-states but also transients are Poisson distributed. Deviations from the Poisson form occur only under two conditions, promoter fluctuations leading to transcriptional bursts or nonindependent degradation of mRNA molecules. These results place severe limits on the power of single-cell experiments to probe microscopic mechanisms, and they highlight the need for single-molecule measurements. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

SRSF3 represses the expression of PDCD4 protein by coordinated regulation of alternative splicing, export and translation

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

Park, Seung Kuk; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

2016-02-05

Gene expression is regulated at multiple steps, such as transcription, splicing, export, degradation and translation. Considering diverse roles of SR proteins, we determined whether the tumor-related splicing factor SRSF3 regulates the expression of the tumor-suppressor protein, PDCD4, at multiple steps. As we have reported previously, knockdown of SRSF3 increased the PDCD4 protein level in SW480 colon cancer cells. More interestingly, here we showed that the alternative splicing and the nuclear export of minor isoforms of pdcd4 mRNA were repressed by SRSF3, but the translation step was unaffected. In contrast, only the translation step of the major isoform of pdcd4 mRNAmore » was repressed by SRSF3. Therefore, overexpression of SRSF3 might be relevant to the repression of all isoforms of PDCD4 protein levels in most types of cancer cell. We propose that SRSF3 could act as a coordinator of the expression of PDCD4 protein via two mechanisms on two alternatively spliced mRNA isoforms.« less

An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

PubMed

Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

2012-07-01

cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

Rrp6 is recruited to transcribed genes and accompanies the spliced mRNA to the nuclear pore

PubMed Central

Hessle, Viktoria; von Euler, Anne; González de Valdivia, Ernesto; Visa, Neus

2012-01-01

Rrp6 is an exoribonuclease involved in the quality control of mRNA biogenesis. We have analyzed the association of Rrp6 with the Balbiani ring pre-mRNPs of Chironomus tentans to obtain insight into the role of Rrp6 in splicing surveillance. Rrp6 is recruited to transcribed genes and its distribution along the genes does not correlate with the positions of exons and introns. In the nucleoplasm, Rrp6 is bound to both unspliced and spliced transcripts. Rrp6 is released from the mRNPs in the vicinity of the nuclear pore before nucleo-cytoplasmic translocation. We show that Rrp6 is associated with newly synthesized transcripts during all the nuclear steps of gene expression and is associated with the transcripts independently of their splicing status. These observations suggest that the quality control of pre-mRNA splicing is not based on the selective recruitment of the exoribonuclease Rrp6 to unprocessed mRNAs. PMID:22745224

The Effects of Low-Dose Bisphenol A and Bisphenol F on Neural Differentiation of a Fetal Brain-Derived Neural Progenitor Cell Line.

PubMed

Fujiwara, Yuki; Miyazaki, Wataru; Koibuchi, Noriyuki; Katoh, Takahiko

2018-01-01

Environmental chemicals are known to disrupt the endocrine system in humans and to have adverse effects on several organs including the developing brain. Recent studies indicate that exposure to environmental chemicals during gestation can interfere with neuronal differentiation, subsequently affecting normal brain development in newborns. Xenoestrogen, bisphenol A (BPA), which is widely used in plastic products, is one such chemical. Adverse effects of exposure to BPA during pre- and postnatal periods include the disruption of brain function. However, the effect of BPA on neural differentiation remains unclear. In this study, we explored the effects of BPA or bisphenol F (BPF), an alternative compound for BPA, on neural differentiation using ReNcell, a human fetus-derived neural progenitor cell line. Maintenance in growth factor-free medium initiated the differentiation of ReNcell to neuronal cells including neurons, astrocytes, and oligodendrocytes. We exposed the cells to BPA or BPF for 3 days from the period of initiation and performed real-time PCR for neural markers such as β III-tubulin and glial fibrillary acidic protein (GFAP), and Olig2. The β III-tubulin mRNA level decreased in response to BPA, but not BPF, exposure. We also observed that the number of β III-tubulin-positive cells in the BPA-exposed group was less than that of the control group. On the other hand, there were no changes in the MAP2 mRNA level. These results indicate that BPA disrupts neural differentiation in human-derived neural progenitor cells, potentially disrupting brain development.

Acetylcholine-induced seizure-like activity and modified cholinergic gene expression in chronically epileptic rats.

PubMed

Zimmerman, Gabriel; Njunting, Marleisje; Ivens, Sebastian; Tolner, Else A; Tolner, Elsa; Behrens, Christoph J; Gross, Miriam; Soreq, Hermona; Heinemann, Uwe; Friedman, Alon

2008-02-01

The entorhinal cortex (EC) plays an important role in temporal lobe epilepsy. Under normal conditions, the enriched cholinergic innervation of the EC modulates local synchronized oscillatory activity; however, its role in epilepsy is unknown. Enhanced neuronal activation has been shown to induce transcriptional changes of key cholinergic genes and thus alter cholinergic responses. To examine cholinergic modulations in epileptic tissue we studied molecular and electrophysiological cholinergic responses in the EC of chronically epileptic rats following exposure to pilocarpine or kainic acid. We confirmed that while the total activity of the acetylcholine (ACh)-hydrolysing enzyme, acetylcholinesterase (AChE) was not altered, epileptic rats showed alternative splicing of AChE pre-mRNA transcripts, accompanied by a shift from membrane-bound AChE tetramers to soluble monomers. This was associated with increased sensitivity to ACh application: thus, in control rats, ACh (10-100 microm) induced slow (< 1Hz), periodic events confined to the EC; however, in epileptic rats, ACh evoked seconds-long seizure-like events with initial appearance in the EC, and frequent propagation to neighbouring cortical regions. ACh-induced seizure-like events could be completely blocked by the non-specific muscarinic antagonist, atropine, and were partially blocked by the muscarinic-1 receptor antagonist, pirenzepine; but were not affected by the non-specific nicotinic antagonist, mecamylamine. Epileptic rats presented reduced transcript levels of muscarinic receptors with no evidence of mRNA editing or altered mRNA levels for nicotinic ACh receptors. Our findings suggest that altered cholinergic modulation may initiate seizure events in the epileptic temporal cortex.

Alternative pre-approved and novel therapies for the treatment of anthrax.

PubMed

Head, Breanne M; Rubinstein, Ethan; Meyers, Adrienne F A

2016-11-03

Bacillus anthracis, the causative agent of anthrax, is a spore forming and toxin producing rod-shaped bacterium that is classified as a category A bioterror agent. This pathogenic microbe can be transmitted to both animals and humans. Clinical presentation depends on the route of entry (direct contact, ingestion, injection or aerosolization) with symptoms ranging from isolated skin infections to more severe manifestations such as cardiac or pulmonary shock, meningitis, and death. To date, anthrax is treatable if antibiotics are administered promptly and continued for 60 days. However, if treatment is delayed or administered improperly, the patient's chances of survival are decreased drastically. In addition, antibiotics are ineffective against the harmful anthrax toxins and spores. Therefore, alternative therapeutics are essential. In this review article, we explore and discuss advances that have been made in anthrax therapy with a primary focus on alternative pre-approved and novel antibiotics as well as anti-toxin therapies. A literature search was conducted using the University of Manitoba search engine. Using this search engine allowed access to a greater variety of journals/articles that would have otherwise been restricted for general use. In order to be considered for discussion for this review, all articles must have been published later than 2009. The alternative pre-approved antibiotics demonstrated high efficacy against B. anthracis both in vitro and in vivo. In addition, the safety profile and clinical pharmacology of these drugs were already known. Compounds that targeted underexploited bacterial processes (DNA replication, RNA synthesis, and cell division) were also very effective in combatting B. anthracis. In addition, these novel compounds prevented bacterial resistance. Targeting B. anthracis virulence, more specifically the anthrax toxins, increased the length of which treatment could be administered. Several novel and pre-existing antibiotics, as well as toxin inhibitors, have shown increasing promise. A combination treatment that targets both bacterial growth and toxin production would be ideal and probably necessary for effectively combatting this armed bacterium.

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

Retrotransposons as regulators of gene expression

PubMed Central

Elbarbary, Reyad A.; Lucas, Bronwyn A.; Maquat, Lynne E.

2016-01-01

Transposable elements (TEs) are both a boon and a bane to eukaryotic organisms, depending on where they integrate into the genome and how their sequences function once integrated. We focus on two types of TEs: long interspersed elements (LINEs) and short interspersed elements (SINEs). LINEs and SINEs are retrotransposons; that is, they transpose via an RNA intermediate. We discuss how LINEs and SINEs have expanded in eukaryotic genomes and contribute to genome evolution. An emerging body of evidence indicates that LINEs and SINEs function to regulate gene expression by affecting chromatin structure, gene transcription, pre-mRNA processing, or aspects of mRNA metabolism. We also describe how adenosine-to-inosine editing influences SINE function and how ongoing retrotransposition is countered by the body’s defense mechanisms. PMID:26912865

In situ synthesis of protein arrays.

PubMed

He, Mingyue; Stoevesandt, Oda; Taussig, Michael J

2008-02-01

In situ or on-chip protein array methods use cell free expression systems to produce proteins directly onto an immobilising surface from co-distributed or pre-arrayed DNA or RNA, enabling protein arrays to be created on demand. These methods address three issues in protein array technology: (i) efficient protein expression and availability, (ii) functional protein immobilisation and purification in a single step and (iii) protein on-chip stability over time. By simultaneously expressing and immobilising many proteins in parallel on the chip surface, the laborious and often costly processes of DNA cloning, expression and separate protein purification are avoided. Recently employed methods reviewed are PISA (protein in situ array) and NAPPA (nucleic acid programmable protein array) from DNA and puromycin-mediated immobilisation from mRNA.

Quantitative identification of proteins that influence miRNA biogenesis by RNA pull-down-SILAC mass spectrometry (RP-SMS).

PubMed

Choudhury, Nila Roy; Michlewski, Gracjan

2018-06-08

RNA-binding proteins mediate and control gene expression. As some examples, they regulate pre-mRNA synthesis and processing; mRNA localisation, translation and decay; and microRNA (miRNA) biogenesis and function. Here, we present a detailed protocol for RNA pull-down coupled to stable isotope labelling by amino acids in cell culture (SILAC) mass spectrometry (RP-SMS) that enables quantitative, fast and specific detection of RNA-binding proteins that regulate miRNA biogenesis. In general, this method allows for the identification of RNA-protein complexes formed using in vitro or chemically synthesized RNAs and protein extracts derived from cultured cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Late emerging effects of prenatal and early postnatal nicotine exposure on the cholinergic system and anxiety-like behavior.

PubMed

Eppolito, Amy K; Bachus, Susan E; McDonald, Craig G; Meador-Woodruff, James H; Smith, Robert F

2010-01-01

Animal models of prenatal nicotine exposure clearly indicate that nicotine is a neuroteratogen. Some of the persisting effects of prenatal nicotine exposure include low birth weight, behavioral changes and deficits in cognitive function, although few studies have looked for neurobehavioral and neurochemical effects that might persist throughout the lifespan. Pregnant rats were given continuous infusions of nicotine (0.96mg/kg/day or 2.0mg/kg/day, freebase) continuing through the third trimester equivalent, a period of rapid brain development. Because the third trimester equivalent occurs postnatally in the rat (roughly the first week of life) nicotine administration to neonate pups continued via maternal milk until postnatal day (P) 10. Exposure to nicotine during pre- and early postnatal development had an anxiogenic effect on adult rats (P75) in the elevated plus maze (EPM), and blocked extinction learning in a fear conditioning paradigm, suggesting that pre- and postnatal nicotine exposure affect anxiety-like behavior and cognitive function well into adulthood. In contrast, nicotine exposure had no effect on anxiety-like behaviors in the EPM in adolescent animals (P30). Analysis of mRNA for the alpha4, alpha7, and beta2 subunits of nicotinic acetylcholine receptors revealed lower expression of these subunits in the adult hippocampus and medial prefrontal cortex following pre- and postnatal nicotine exposure, suggesting that nicotine altered the developmental trajectory of the brain. These long-term behavioral and neurochemical changes strengthen the case for discouraging cigarette smoking during pregnancy and clearly indicate that the use of the patch as a smoking cessation aid during pregnancy is not a safe alternative.

Pyrvinium pamoate changes alternative splicing of the serotonin receptor 2C by influencing its RNA structure

PubMed Central

Shen, Manli; Bellaousov, Stanislav; Hiller, Michael; de La Grange, Pierre; Creamer, Trevor P.; Malina, Orit; Sperling, Ruth; Mathews, David H.; Stoilov, Peter; Stamm, Stefan

2013-01-01

The serotonin receptor 2C plays a central role in mood and appetite control. It undergoes pre-mRNA editing as well as alternative splicing. The RNA editing suggests that the pre-mRNA forms a stable secondary structure in vivo. To identify substances that promote alternative exons inclusion, we set up a high-throughput screen and identified pyrvinium pamoate as a drug-promoting exon inclusion without editing. Circular dichroism spectroscopy indicates that pyrvinium pamoate binds directly to the pre-mRNA and changes its structure. SHAPE (selective 2′-hydroxyl acylation analysed by primer extension) assays show that part of the regulated 5′-splice site forms intramolecular base pairs that are removed by this structural change, which likely allows splice site recognition and exon inclusion. Genome-wide analyses show that pyrvinium pamoate regulates >300 alternative exons that form secondary structures enriched in A–U base pairs. Our data demonstrate that alternative splicing of structured pre-mRNAs can be regulated by small molecules that directly bind to the RNA, which is reminiscent to an RNA riboswitch. PMID:23393189

Genetic and environmental effects on the muscle structure response post-mortem.

PubMed

Thompson, J M; Perry, D; Daly, B; Gardner, G E; Johnston, D J; Pethick, D W

2006-09-01

This paper reviewed the mechanisms by which glycolytic rate and pre-rigor stretching of muscle impact on meat quality. If muscle is free to shorten during the rigor process extremes in glycolytic rate can impact negatively on meat quality by inducing either cold or rigor shortening. Factors that contribute to variation in glycolytic rate include the glycogen concentration at slaughter and fibre type of the muscle. Glycolysis is highly sensitive to temperature, which is an important factor in heavy grain fed carcasses. An alternative solution to controlling glycolysis is to stretch the muscle pre-rigor so that it cannot shorten, thus providing an insurance against extremes in processing conditions. Results are presented which show a large reduction in variance (both additive and phenotypic) in tenderness caused by pre-rigor stretching. Whilst this did not impact on the heritability of shear force, it did reduce genotype differences. The implications of these results on the magnitude of genotype effects on tenderness is discussed.

Transcriptional, post-transcriptional and chromatin-associated regulation of pri-miRNAs, pre-miRNAs and moRNAs

PubMed Central

Nepal, Chirag; Coolen, Marion; Hadzhiev, Yavor; Cussigh, Delphine; Mydel, Piotr; Steen, Vidar M.; Carninci, Piero; Andersen, Jesper B.; Bally-Cuif, Laure; Müller, Ferenc; Lenhard, Boris

2016-01-01

MicroRNAs (miRNAs) play a major role in the post-transcriptional regulation of target genes, especially in development and differentiation. Our understanding about the transcriptional regulation of miRNA genes is limited by inadequate annotation of primary miRNA (pri-miRNA) transcripts. Here, we used CAGE-seq and RNA-seq to provide genome-wide identification of the pri-miRNA core promoter repertoire and its dynamic usage during zebrafish embryogenesis. We assigned pri-miRNA promoters to 152 precursor-miRNAs (pre-miRNAs), the majority of which were supported by promoter associated post-translational histone modifications (H3K4me3, H2A.Z) and RNA polymerase II (RNAPII) occupancy. We validated seven miR-9 pri-miRNAs by in situ hybridization and showed similar expression patterns as mature miR-9. In addition, processing of an alternative intronic promoter of miR-9–5 was validated by 5′ RACE PCR. Developmental profiling revealed a subset of pri-miRNAs that are maternally inherited. Moreover, we show that promoter-associated H3K4me3, H2A.Z and RNAPII marks are not only present at pri-miRNA promoters but are also specifically enriched at pre-miRNAs, suggesting chromatin level regulation of pre-miRNAs. Furthermore, we demonstrated that CAGE-seq also detects 3′-end processing of pre-miRNAs on Drosha cleavage site that correlates with miRNA-offset RNAs (moRNAs) production and provides a new tool for detecting Drosha processing events and predicting pre-miRNA processing by a genome-wide assay. PMID:26673698

Synaptic Plasticity and Translation Initiation

ERIC Educational Resources Information Center

Klann, Eric; Antion, Marcia D.; Banko, Jessica L.; Hou, Lingfei

2004-01-01

It is widely accepted that protein synthesis, including local protein synthesis at synapses, is required for several forms of synaptic plasticity. Local protein synthesis enables synapses to control synaptic strength independent of the cell body via rapid protein production from pre-existing mRNA. Therefore, regulation of translation initiation is…

Increase of prolactin mRNA in the rat hypothalamus after intracerebroventricular injection of VIP or PACAP.

PubMed

Bredow, S; Kacsóh, B; Obál, F; Fang, J; Krueger, J M

1994-10-17

Vasoactive intestinal peptide (VIP), the structurally homologous pituitary adenylate cyclase-activating peptide (PACAP) and the pituitary hormone, prolactin (PRL) enhance rapid eye movement sleep (REMS). VIP and PACAP are both inducers of PRL gene expression and release in the pituitary gland. Little is known about PRL regulation in the brain although it is hypothesized that the REMS-promoting activity of i.c.v. administered VIP may be mediated via the activation of cerebral PRL. To test whether VIP or PACAP in fact increase intracerebral mRNA, the peptides (VIP: 30 or 300 pmol; PACAP: 220 pmol) were injected i.c.v. into rats at dark onset. 1 h later, cDNA was synthesized from purified hypothalamic mRNA. Standardized amounts were analysed for PRL using the polymerase chain reaction followed by Southern blotting and hybridization. Compared with beta-actin mRNA levels, both VIP and PACAP increased PRL mRNA levels in a dose-dependent fashion though VIP was more effective on a molar basis. The previously reported alternatively spliced PRL mRNA (lacking exon 4) was not detected. The data support the hypothesis that the REMS-promoting activity of central VIP and PACAP might be mediated by cerebral PRL.

Ribosomal proteins L7 and L8 function in concert with six A3 assembly factors to propagate assembly of domains I and II of 25S rRNA in yeast 60S ribosomal subunits

PubMed Central

Jakovljevic, Jelena; Ohmayer, Uli; Gamalinda, Michael; Talkish, Jason; Alexander, Lisa; Linnemann, Jan; Milkereit, Philipp; Woolford, John L.

2012-01-01

Ribosome biogenesis is a complex multistep process that involves alternating steps of folding and processing of pre-rRNAs in concert with assembly of ribosomal proteins. Recently, there has been increased interest in the roles of ribosomal proteins in eukaryotic ribosome biogenesis in vivo, focusing primarily on their function in pre-rRNA processing. However, much less is known about participation of ribosomal proteins in the formation and rearrangement of preribosomal particles as they mature to functional subunits. We have studied ribosomal proteins L7 and L8, which are required for the same early steps in pre-rRNA processing during assembly of 60S subunits but are located in different domains within ribosomes. Depletion of either leads to defects in processing of 27SA3 to 27SB pre-rRNA and turnover of pre-rRNAs destined for large ribosomal subunits. A specific subset of proteins is diminished from these residual assembly intermediates: six assembly factors required for processing of 27SA3 pre-rRNA and four ribosomal proteins bound to domain I of 25S and 5.8S rRNAs surrounding the polypeptide exit tunnel. In addition, specific sets of ribosomal proteins are affected in each mutant: In the absence of L7, proteins bound to domain II, L6, L14, L20, and L33 are greatly diminished, while proteins L13, L15, and L36 that bind to domain I are affected in the absence of L8. Thus, L7 and L8 might establish RNP structures within assembling ribosomes necessary for the stable association and function of the A3 assembly factors and for proper assembly of the neighborhoods containing domains I and II. PMID:22893726

Identification and characterization of yak (Bos grunniens) b-Boule gene and its alternative splice variants.

PubMed

Li, Bojiang; Ngo, Sherry; Wu, Wangjun; Xu, Hongtao; Xie, Zhuang; Li, Qifa; Pan, Zengxiang

2014-10-25

Boule is responsible for meiotic arrest of sperms and male sterility during mammalian spermatogenesis. In the present study, we first identified yak b-Boule gene and its two alternative splice variants. The full length coding region of yak b-Boule is 888bp and encodes a 295-amino acid protein with a typical RNA-recognition motif (RRM) and a Deleted in Azoospermia (DAZ) repetitive sequence motif. Two alternative splice variants of yak b-Boule were generated following the consensus "GT-AG" rule and named b-Boule1 (36bp deletion in exon 3) and b-Boule2 (deletion of integral exon 7), respectively. In male yak, b-Boule, b-Boule1 and b-Boule2 were found to be exclusively expressed in the testes at a ratio of 81:0.1:1. Intriguingly, the mRNA expression levels of b-Boule and b-Boule1 in yak testis were significantly higher than those in cattle-yak, although no significant difference was observed for b-Boule2 expression between the yak and cattle-yak. These results suggest that b-Boule gene, which is partially regulated by alternative splicing, may be involved in the process of yak spermatogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

Intervention of rosiglitazone on myocardium Glut-4 mRNA expression during ischemia-reperfusion injury in cardio-pulmonary bypass in dogs.

PubMed

Liu, Bin; Liang, Guiyou; Xu, Gang; Liu, Daxin; Cai, Qingyong; Gao, Zhenyu

2013-01-01

During cardiac pulmonary bypass (CPB), myocardial ischemia-reperfusion (I/R) induces heart glucose metabolism impairment. Our previous research showed that the decreased glucose utilization is due to decreased glucose transporter-4 (Glut-4) expression and translocation to myocyte surface membranes. This study further examined whether rosiglitazone, a synthetic agonist of peroxisome proliferator-activated receptor γ, could intervene glucose metabolism by regulating Glut-4 mRNA during I/R in dogs. Cardiac ischemia was induced by cardiopulmonary bypass for 30 or 120 min. Plasma insulin and glucose concentrations were measured at pre-bypass (control), aortic cross-clamp off (I/R) at 15, 45, and 75 min. The left ventricle biopsies were taken for the expression of Glut-4 mRNA by real-time RT-PCR. In dogs receiving 120 min ischemia, coronary arterial, venous glucose concentrations, plasma insulin levels, and insulin resistant index (IRI) were increased, but the expression of Glut-4 mRNA was decreased obviously at 15 min of reperfusion, and recovered gradually. On the other hand, these changes were relatively mild in dogs treated with rosiglitazone in cardioplegic solution and expression of Glut-4 mRNA was increased remarkably. It is concluded that the decrease in total amount of Glut-4 mRNA expression could be one of the important molecular mechanisms, which causes the myocardium insulin resistance. The longer the ischemia period, the decrease in amount of Glut-4 mRNA was more dramatic. Adding rosiglitazone into the cardioplegic solution during I/R can increase the amount of Glut-4 mRNA expression, mitigate the myocardium insulin resistance and improve the myocardium I/R injury during CPB.

Update on soil fumigation: MBr alternatives and reregistration decisions

Treesearch

Scott A. Enebak

2011-01-01

This article gives a brief history of the importance of methyl bromide in the production of forest seedlings in the southern United States and the timeline for the Montreal Protocol and Clean Air Act to phase out ozone-depleting compounds. In addition, the process, steps, and potential for continued MBr use under the Critical Use Exemption and Quarantine Pre-shipment...

Violent aggression predicted by multiple pre-adult environmental hits.

PubMed

Mitjans, Marina; Seidel, Jan; Begemann, Martin; Bockhop, Fabian; Moya-Higueras, Jorge; Bansal, Vikas; Wesolowski, Janina; Seelbach, Anna; Ibáñez, Manuel Ignacio; Kovacevic, Fatka; Duvar, Oguzhan; Fañanás, Lourdes; Wolf, Hannah-Ulrike; Ortet, Generós; Zwanzger, Peter; Klein, Verena; Lange, Ina; Tänzer, Andreas; Dudeck, Manuela; Penke, Lars; van Elst, Ludger Tebartz; Bittner, Robert A; Schmidmeier, Richard; Freese, Roland; Müller-Isberner, Rüdiger; Wiltfang, Jens; Bliesener, Thomas; Bonn, Stefan; Poustka, Luise; Müller, Jürgen L; Arias, Bárbara; Ehrenreich, Hannelore

2018-05-24

Early exposure to negative environmental impact shapes individual behavior and potentially contributes to any mental disease. We reported previously that accumulated environmental risk markedly decreases age at schizophrenia onset. Follow-up of matched extreme group individuals (≤1 vs. ≥3 risks) unexpectedly revealed that high-risk subjects had >5 times greater probability of forensic hospitalization. In line with longstanding sociological theories, we hypothesized that risk accumulation before adulthood induces violent aggression and criminal conduct, independent of mental illness. We determined in 6 independent cohorts (4 schizophrenia and 2 general population samples) pre-adult risk exposure, comprising urbanicity, migration, physical and sexual abuse as primary, and cannabis or alcohol as secondary hits. All single hits by themselves were marginally associated with higher violent aggression. Most strikingly, however, their accumulation strongly predicted violent aggression (odds ratio 10.5). An epigenome-wide association scan to detect differential methylation of blood-derived DNA of selected extreme group individuals yielded overall negative results. Conversely, determination in peripheral blood mononuclear cells of histone-deacetylase1 mRNA as 'umbrella mediator' of epigenetic processes revealed an increase in the high-risk group, suggesting lasting epigenetic alterations. Together, we provide sound evidence of a disease-independent unfortunate relationship between well-defined pre-adult environmental hits and violent aggression, calling for more efficient prevention.

BPAG1a and b associate with EB1 and EB3 and modulate vesicular transport, Golgi apparatus structure, and cell migration in C2.7 myoblasts.

PubMed

Poliakova, Kseniia; Adebola, Adijat; Leung, Conrad L; Favre, Bertrand; Liem, Ronald K H; Schepens, Isabelle; Borradori, Luca

2014-01-01

BPAG1a and BPAG1b (BPAG1a/b) constitute two major isoforms encoded by the dystonin (Dst) gene and show homology with MACF1a and MACF1b. These proteins are members of the plakin family, giant multi-modular proteins able to connect the intermediate filament, microtubule and microfilament cytoskeletal networks with each other and to distinct cell membrane sites. They also serve as scaffolds for signaling proteins that modulate cytoskeletal dynamics. To gain better insights into the functions of BPAG1a/b, we further characterized their C-terminal region important for their interaction with microtubules and assessed the role of these isoforms in the cytoskeletal organization of C2.7 myoblast cells. Our results show that alternative splicing does not only occur at the 5' end of Dst and Macf1 pre-mRNAs, as previously reported, but also at their 3' end, resulting in expression of additional four mRNA variants of BPAG1 and MACF1. These isoform-specific C-tails were able to bundle microtubules and bound to both EB1 and EB3, two microtubule plus end proteins. In the C2.7 cell line, knockdown of BPAG1a/b had no major effect on the organization of the microtubule and microfilament networks, but negatively affected endocytosis and maintenance of the Golgi apparatus structure, which became dispersed. Finally, knockdown of BPAG1a/b caused a specific decrease in the directness of cell migration, but did not impair initial cell adhesion. These data provide novel insights into the complexity of alternative splicing of Dst pre-mRNAs and into the role of BPAG1a/b in vesicular transport, Golgi apparatus structure as well as in migration in C2.7 myoblasts.

BPAG1a and b Associate with EB1 and EB3 and Modulate Vesicular Transport, Golgi Apparatus Structure, and Cell Migration in C2.7 Myoblasts

PubMed Central

Poliakova, Kseniia; Adebola, Adijat; Leung, Conrad L.; Favre, Bertrand; Liem, Ronald K. H.; Schepens, Isabelle; Borradori, Luca

2014-01-01

BPAG1a and BPAG1b (BPAG1a/b) constitute two major isoforms encoded by the dystonin (Dst) gene and show homology with MACF1a and MACF1b. These proteins are members of the plakin family, giant multi-modular proteins able to connect the intermediate filament, microtubule and microfilament cytoskeletal networks with each other and to distinct cell membrane sites. They also serve as scaffolds for signaling proteins that modulate cytoskeletal dynamics. To gain better insights into the functions of BPAG1a/b, we further characterized their C-terminal region important for their interaction with microtubules and assessed the role of these isoforms in the cytoskeletal organization of C2.7 myoblast cells. Our results show that alternative splicing does not only occur at the 5′ end of Dst and Macf1 pre-mRNAs, as previously reported, but also at their 3′ end, resulting in expression of additional four mRNA variants of BPAG1 and MACF1. These isoform-specific C-tails were able to bundle microtubules and bound to both EB1 and EB3, two microtubule plus end proteins. In the C2.7 cell line, knockdown of BPAG1a/b had no major effect on the organization of the microtubule and microfilament networks, but negatively affected endocytosis and maintenance of the Golgi apparatus structure, which became dispersed. Finally, knockdown of BPAG1a/b caused a specific decrease in the directness of cell migration, but did not impair initial cell adhesion. These data provide novel insights into the complexity of alternative splicing of Dst pre-mRNAs and into the role of BPAG1a/b in vesicular transport, Golgi apparatus structure as well as in migration in C2.7 myoblasts. PMID:25244344

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

PubMed

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

2015-03-31

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

Differential utilization of decapping enzymes in mammalian mRNA decay pathways

PubMed Central

Li, You; Song, Mangen; Kiledjian, Megerditch

2011-01-01

mRNA decapping is a crucial step in the regulation of mRNA stability and gene expression. Dcp2 is an mRNA decapping enzyme that has been widely studied. We recently reported the presence of a second mammalian cytoplasmic decapping enzyme, Nudt16. Here we address the differential utilization of the two decapping enzymes in specified mRNA decay processes. Using mouse embryonic fibroblast (MEF) cell lines derived from a hypomorphic knockout of the Dcp2 gene with undetectable levels of Dcp2 or MEF cell lines harboring a Nudt16-directed shRNA to generate reduced levels of Nudt16, we demonstrate the distinct roles for Dcp2 and Nudt16 in nonsense-mediated mRNA decay (NMD), decay of ARE-containing mRNA and miRNA-mediated silencing. Our results indicated that NMD preferentially utilizes Dcp2 rather than Nudt16; Dcp2 and Nudt16 are redundant in miRNA-mediated silencing; and Dcp2 and Nudt16 are differentially utilized for ARE-mRNA decay. These data demonstrate that the two distinct decapping enzymes can uniquely function in specific mRNA decay processes in mammalian cells. PMID:21224379

Molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets

PubMed Central

Arango, Daniel; Morohashi, Kengo; Yilmaz, Alper; Kuramochi, Kouji; Parihar, Arti; Brahimaj, Bledi; Grotewold, Erich; Doseff, Andrea I.

2013-01-01

Flavonoids constitute the largest class of dietary phytochemicals, adding essential health value to our diet, and are emerging as key nutraceuticals. Cellular targets for dietary phytochemicals remain largely unknown, posing significant challenges for the regulation of dietary supplements and the understanding of how nutraceuticals provide health value. Here, we describe the identification of human cellular targets of apigenin, a flavonoid abundantly present in fruits and vegetables, using an innovative high-throughput approach that combines phage display with second generation sequencing. The 160 identified high-confidence candidate apigenin targets are significantly enriched in three main functional categories: GTPase activation, membrane transport, and mRNA metabolism/alternative splicing. This last category includes the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2), a factor involved in splicing regulation, mRNA stability, and mRNA transport. Apigenin binds to the C-terminal glycine-rich domain of hnRNPA2, preventing hnRNPA2 from forming homodimers, and therefore, it perturbs the alternative splicing of several human hnRNPA2 targets. Our results provide a framework to understand how dietary phytochemicals exert their actions by binding to many functionally diverse cellular targets. In turn, some of them may modulate the activity of a large number of downstream genes, which is exemplified here by the effects of apigenin on the alternative splicing activity of hnRNPA2. Hence, in contrast to small-molecule pharmaceuticals designed for defined target specificity, dietary phytochemicals affect a large number of cellular targets with varied affinities that, combined, result in their recognized health benefits. PMID:23697369

Purification and Characterization of Mouse Soluble Receptor for Advanced Glycation End Products (sRAGE)*

PubMed Central

Hanford, Lana E.; Enghild, Jan J.; Valnickova, Zuzana; Petersen, Steen V.; Schaefer, Lisa M.; Schaefer, Todd M.; Reinhart, Todd A.; Oury, Tim D.

2007-01-01

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface proteins that has been implicated as a progression factor in a number of pathologic conditions from chronic inflammation to cancer to Alzheimer’s disease. In such conditions, RAGE acts to facilitate pathogenic processes. Its secreted isoform, soluble RAGE or sRAGE, has the ability to prevent RAGE signaling by acting as a decoy. sRAGE has been used successfully in animal models of a range of diseases to antagonize RAGE-mediated pathologic processes. In humans, sRAGE results from alternative splicing of RAGE mRNA. This study was aimed to determine whether the same holds true for mouse sRAGE and, in addition, to biochemically characterize mouse sRAGE. The biochemical characteristics examined include glycosylation and disulfide patterns. In addition, sRAGE was found to bind heparin, which may mediate its distribution in the extracellular matrix and cell surfaces of tissues. Finally, our data indicated that sRAGE in the mouse is likely produced by carboxyl-terminal truncation, in contrast to the alternative splicing mechanism reported in humans. PMID:15381690

Changes in cholecystokinin and peptide Y gene expression with feeding in yellowtail (Seriola quinqueradiata): relation to pancreatic exocrine regulation.

PubMed

Murashita, Koji; Fukada, Haruhisa; Hosokawa, Hidetsuyo; Masumoto, Toshiro

2007-03-01

In fish, the regulation of digestive enzyme secretion by hormonal control such as cholecystokinin (CCK) and neuropeptide Y (NPY)-related peptide is not well understood. To investigate the roles of fish CCK and peptide Y (PY) in digestive enzyme secretion, mRNA levels of CCK and PY, pyloric caeca enzyme activities and mRNA levels of pancreatic digestive enzymes (lipase, trypsin and amylase) were measured at pre- and post-prandial stages in yellowtail. Pyloric caeca were sampled at 0, 0.5, 1.5, 3, 6, 12, 24 and 48 h after feeding. The mRNA levels of trypsin and amylase increased after feeding, suggesting that transcription was induced by feed ingestion. Digestive enzyme activities decreased in exocrine pancreas after feeding, suggesting the stored enzyme was secreted from pancreas post-prandially. mRNA levels for CCK displayed a time-dependent increase, peaking between 1.5 and 3 h after-feeding followed by a rapid decrease 3 to 6 h after feeding. The mRNA expression pattern of PY was inverse to the pattern of CCK, decreasing until 1.5 h after feeding and then rising to initial levels by 12 h after feeding. These results suggest that CCK and PY work antagonistically in the exocrine pancreas of yellowtail.

Intracellular calcium: a prerequisite for aldosterone action.

PubMed

Schäfer, C; Shahin, V; Albermann, L; Schillers, H; Hug, M J; Oberleithner, H

2003-12-01

Transport of salt and water in various tissues is under control of the mineralocorticoid hormone aldosterone. As a liphophilic hormone, aldosterone diffuses through the plasma membrane and, then, binds to cytosolic mineralocorticoid receptors in the target cells. After binding to nuclear pore complexes, the activated receptor is translocated to the nucleus where transcription processes are initiated. After a lag period of about 20 minutes hormone-specific early mRNA transcripts leave the nucleus through nuclear pores. Some of the steps in this cascade can be followed by electrophysiology in Xenopus laevis oocyte nuclei. In addition to the genomic pathway, aldosterone exerts a rapid pre-genomic response that involves an increase in intracellular calcium. In this study, we tested for the potential role of Ca(2+) in the genomic response of the hormone. We measured the electrical resistance across the nuclear envelope in response to aldosterone, in presence and absence of intracellular Ca(2+). Nuclear envelope electrical resistance reflects receptor binding to the nuclear pore complexes ("early" resistance peak, 2 minutes after aldosterone), ongoing transcription ("transient" resistance drop, 5-15 minutes after aldosterone) and mRNA export ("late" resistance peak, 20 minutes after aldosterone). Pre-injection of the Ca(2+) chelator EGTA eliminated all electrical responses evoked by aldosterone. The transient resistance drop and the late resistance peak, induced by the hormone, were prevented by the transcription inhibitor actinomycin D, coinjected with aldosterone, while the early resistance peak remained unaffected. We conclude that (i). the presence of intracellular Ca(2+) is a prerequisite for the genomic action of aldosterone. (ii). Intracellular calcium plays a role early in the signaling cascade, either in agonist-receptor interaction, or receptor transport/docking to the nuclear pore complexes.

Implementing Effective Mission Systems Engineering Practices During Early Project Formulation Phases

NASA Technical Reports Server (NTRS)

Moton, Tryshanda

2016-01-01

Developing and implementing a plan for a NASA space mission can be a complicated process. The needs, goals, and objectives of any proposed mission or technology must be assessed early in the Project Life Cycle. The key to successful development of a space mission or flight project is the inclusion of systems engineering in early project formulation, namely during Pre-phase A, Phase A, and Phase B of the NASA Project Life Cycle. When a space mission or new technology is in pre-development, or "pre-Formulation", feasibility must be determined based on cost, schedule, and risk. Inclusion of system engineering during project formulation is key because in addition to assessing feasibility, design concepts are developed and alternatives to design concepts are evaluated. Lack of systems engineering involvement early in the project formulation can result in increased risks later in the implementation and operations phases of the project. One proven method for effective systems engineering practice during the pre-Formulation Phase is the use of a mission conceptual design or technology development laboratory, such as the Mission Design Lab (MDL) at NASA's Goddard Space Flight Center (GSFC). This paper will review the engineering process practiced routinely in the MDL for successful mission or project development during the pre-Formulation Phase.

Galectin-3 is a non-classic RNA binding protein that stabilizes the mucin MUC4 mRNA in the cytoplasm of cancer cells.

PubMed

Coppin, Lucie; Vincent, Audrey; Frénois, Frédéric; Duchêne, Belinda; Lahdaoui, Fatima; Stechly, Laurence; Renaud, Florence; Villenet, Céline; Van Seuningen, Isabelle; Leteurtre, Emmanuelle; Dion, Johann; Grandjean, Cyrille; Poirier, Françoise; Figeac, Martin; Delacour, Delphine; Porchet, Nicole; Pigny, Pascal

2017-03-06

Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing. However a broader role of Gal-3 in mRNA fate is unexplored. We report herein that Gal-3 increases MUC4 mRNA stability through an intermediate, hnRNP-L which binds to a conserved CA repeat element in the 3'UTR in a Gal-3 dependent manner and also controls Muc4 mRNA levels in epithelial tissues of Gal3 -/- mice. Gal-3 interacts with hnRNP-L in the cytoplasm, especially during cell mitosis, but only partly associates with protein markers of P-Bodies or Stress Granules. By RNA-IP plus RNA-seq analysis and imaging, we demonstrate that Gal-3 binds to mature spliced MUC4 mRNA in the perinuclear region, probably in hnRNP-L-containing RNA granules. Our findings highlight a new role for Gal-3 as a non-classic RNA-binding protein that regulates MUC4 mRNA post-transcriptionally.

Galectin-3 is a non-classic RNA binding protein that stabilizes the mucin MUC4 mRNA in the cytoplasm of cancer cells

PubMed Central

Coppin, Lucie; Vincent, Audrey; Frénois, Frédéric; Duchêne, Belinda; Lahdaoui, Fatima; Stechly, Laurence; Renaud, Florence; Villenet, Céline; Seuningen, Isabelle Van; Leteurtre, Emmanuelle; Dion, Johann; Grandjean, Cyrille; Poirier, Françoise; Figeac, Martin; Delacour, Delphine; Porchet, Nicole; Pigny, Pascal

2017-01-01

Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing. However a broader role of Gal-3 in mRNA fate is unexplored. We report herein that Gal-3 increases MUC4 mRNA stability through an intermediate, hnRNP-L which binds to a conserved CA repeat element in the 3′UTR in a Gal-3 dependent manner and also controls Muc4 mRNA levels in epithelial tissues of Gal3−/− mice. Gal-3 interacts with hnRNP-L in the cytoplasm, especially during cell mitosis, but only partly associates with protein markers of P-Bodies or Stress Granules. By RNA-IP plus RNA-seq analysis and imaging, we demonstrate that Gal-3 binds to mature spliced MUC4 mRNA in the perinuclear region, probably in hnRNP-L-containing RNA granules. Our findings highlight a new role for Gal-3 as a non-classic RNA-binding protein that regulates MUC4 mRNA post-transcriptionally. PMID:28262838

Zinc oxide nanoflowers make new blood vessels

NASA Astrophysics Data System (ADS)

Barui, Ayan Kumar; Veeriah, Vimal; Mukherjee, Sudip; Manna, Joydeb; Patel, Ajay Kumar; Patra, Sujata; Pal, Krishnendu; Murali, Shruthi; Rana, Rohit K.; Chatterjee, Suvro; Patra, Chitta Ranjan

2012-11-01

It is well established that angiogenesis is the process of formation of new capillaries from pre-existing blood vessels. It is a complex process, involving both pro- and anti-angiogenic factors, and plays a significant role in physiological and pathophysiological processes such as embryonic development, atherosclerosis, post-ischemic vascularization of the myocardium, tumor growth and metastasis, rheumatoid arthritis etc. This is the first report of zinc oxide (ZnO) nanoflowers that show significant pro-angiogenic properties (formation of new capillaries from pre-existing blood vessels), observed by in vitro and in vivo angiogenesis assays. The egg yolk angiogenesis assay using ZnO nanoflowers indicates the presence of matured blood vessels formation. Additionally, it helps to promote endothelial cell (EA.hy926 cells) migration in wound healing assays. Formation of reactive oxygen species (ROS), especially hydrogen peroxide (H2O2)--a redox signaling molecule, might be the plausible mechanism for nanoflower-based angiogenesis. Angiogenesis by nanoflowers may provide the basis for the future development of new alternative therapeutic treatment strategies for cardiovascular and ischemic diseases, where angiogenesis plays a significant role.It is well established that angiogenesis is the process of formation of new capillaries from pre-existing blood vessels. It is a complex process, involving both pro- and anti-angiogenic factors, and plays a significant role in physiological and pathophysiological processes such as embryonic development, atherosclerosis, post-ischemic vascularization of the myocardium, tumor growth and metastasis, rheumatoid arthritis etc. This is the first report of zinc oxide (ZnO) nanoflowers that show significant pro-angiogenic properties (formation of new capillaries from pre-existing blood vessels), observed by in vitro and in vivo angiogenesis assays. The egg yolk angiogenesis assay using ZnO nanoflowers indicates the presence of matured blood vessels formation. Additionally, it helps to promote endothelial cell (EA.hy926 cells) migration in wound healing assays. Formation of reactive oxygen species (ROS), especially hydrogen peroxide (H2O2)--a redox signaling molecule, might be the plausible mechanism for nanoflower-based angiogenesis. Angiogenesis by nanoflowers may provide the basis for the future development of new alternative therapeutic treatment strategies for cardiovascular and ischemic diseases, where angiogenesis plays a significant role. Electronic supplementary information (ESI) available: See DOI: 10.1039/c2nr32369a

Hypoxic Air Inhalation and Ischemia Interventions Both Elicit Preconditioning Which Attenuate Subsequent Cellular Stress In vivo Following Blood Flow Occlusion and Reperfusion.

PubMed

Barrington, James H; Chrismas, Bryna C R; Gibson, Oliver R; Tuttle, James; Pegrum, J; Govilkar, S; Kabir, Chindu; Giannakakis, N; Rayan, F; Okasheh, Z; Sanaullah, A; Ng Man Sun, S; Pearce, Oliver; Taylor, Lee

2017-01-01

Ischemic preconditioning (IPC) is valid technique which elicits reductions in femoral blood flow occlusion mediated reperfusion stress (oxidative stress, Hsp gene transcripts) within the systemic blood circulation and/or skeletal muscle. It is unknown whether systemic hypoxia, evoked by hypoxic preconditioning (HPC) has efficacy in priming the heat shock protein (Hsp) system thus reducing reperfusion stress following blood flow occlusion, in the same manner as IPC. The comparison between IPC and HPC being relevant as a preconditioning strategy prior to orthopedic surgery. In an independent group design, 18 healthy men were exposed to 40 min of (1) passive whole-body HPC (FiO 2 = 0.143; no ischemia. N = 6), (2) IPC (FiO 2 = 0.209; four bouts of 5 min ischemia and 5 min reperfusion. n = 6), or (3) rest (FiO 2 = 0.209; no ischemia. n = 6). The interventions were administered 1 h prior to 30 min of tourniquet derived femoral blood flow occlusion and were followed by 2 h subsequent reperfusion. Systemic blood samples were taken pre- and post-intervention. Systemic blood and gastrocnemius skeletal muscle samples were obtained pre-, 15 min post- (15PoT) and 120 min (120PoT) post-tourniquet deflation. To determine the cellular stress response gastrocnemius and leukocyte Hsp72 mRNA and Hsp32 mRNA gene transcripts were determined by RT-qPCR. The plasma oxidative stress response (protein carbonyl, reduced glutathione/oxidized glutathione ratio) was measured utilizing commercially available kits. In comparison to control, at 15PoT a significant difference in gastrocnemius Hsp72 mRNA was seen in HPC (-1.93-fold; p = 0.007) and IPC (-1.97-fold; p = 0.006). No significant differences were observed in gastrocnemius Hsp32 and Hsp72 mRNA, leukocyte Hsp72 and Hsp32 mRNA, or oxidative stress markers ( p > 0.05) between HPC and IPC. HPC provided near identical amelioration of blood flow occlusion mediated gastrocnemius stress response (Hsp72 mRNA), compared to an established IPC protocol. This was seen independent of changes in systemic oxidative stress, which likely explains the absence of change in Hsp32 mRNA transcripts within leukocytes and the gastrocnemius. Both the established IPC and novel HPC interventions facilitate a priming of the skeletal muscle, but not leukocyte, Hsp system prior to femoral blood flow occlusion. This response demonstrates a localized tissue specific adaptation which may ameliorate reperfusion stress.

Hypoxic Air Inhalation and Ischemia Interventions Both Elicit Preconditioning Which Attenuate Subsequent Cellular Stress In vivo Following Blood Flow Occlusion and Reperfusion

PubMed Central

Barrington, James H.; Chrismas, Bryna C. R.; Gibson, Oliver R.; Tuttle, James; Pegrum, J.; Govilkar, S.; Kabir, Chindu; Giannakakis, N.; Rayan, F.; Okasheh, Z.; Sanaullah, A.; Ng Man Sun, S; Pearce, Oliver; Taylor, Lee

2017-01-01

Ischemic preconditioning (IPC) is valid technique which elicits reductions in femoral blood flow occlusion mediated reperfusion stress (oxidative stress, Hsp gene transcripts) within the systemic blood circulation and/or skeletal muscle. It is unknown whether systemic hypoxia, evoked by hypoxic preconditioning (HPC) has efficacy in priming the heat shock protein (Hsp) system thus reducing reperfusion stress following blood flow occlusion, in the same manner as IPC. The comparison between IPC and HPC being relevant as a preconditioning strategy prior to orthopedic surgery. In an independent group design, 18 healthy men were exposed to 40 min of (1) passive whole-body HPC (FiO2 = 0.143; no ischemia. N = 6), (2) IPC (FiO2 = 0.209; four bouts of 5 min ischemia and 5 min reperfusion. n = 6), or (3) rest (FiO2 = 0.209; no ischemia. n = 6). The interventions were administered 1 h prior to 30 min of tourniquet derived femoral blood flow occlusion and were followed by 2 h subsequent reperfusion. Systemic blood samples were taken pre- and post-intervention. Systemic blood and gastrocnemius skeletal muscle samples were obtained pre-, 15 min post- (15PoT) and 120 min (120PoT) post-tourniquet deflation. To determine the cellular stress response gastrocnemius and leukocyte Hsp72 mRNA and Hsp32 mRNA gene transcripts were determined by RT-qPCR. The plasma oxidative stress response (protein carbonyl, reduced glutathione/oxidized glutathione ratio) was measured utilizing commercially available kits. In comparison to control, at 15PoT a significant difference in gastrocnemius Hsp72 mRNA was seen in HPC (−1.93-fold; p = 0.007) and IPC (−1.97-fold; p = 0.006). No significant differences were observed in gastrocnemius Hsp32 and Hsp72 mRNA, leukocyte Hsp72 and Hsp32 mRNA, or oxidative stress markers (p > 0.05) between HPC and IPC. HPC provided near identical amelioration of blood flow occlusion mediated gastrocnemius stress response (Hsp72 mRNA), compared to an established IPC protocol. This was seen independent of changes in systemic oxidative stress, which likely explains the absence of change in Hsp32 mRNA transcripts within leukocytes and the gastrocnemius. Both the established IPC and novel HPC interventions facilitate a priming of the skeletal muscle, but not leukocyte, Hsp system prior to femoral blood flow occlusion. This response demonstrates a localized tissue specific adaptation which may ameliorate reperfusion stress. PMID:28824456

Comparison of the QuantiGene 2.0 Assay and Real-Time RT-PCR in the Detection of p53 Isoform mRNA Expression in Formalin-Fixed Paraffin-Embedded Tissues- A Preliminary Study

PubMed Central

Morten, Brianna C.; Scott, Rodney J.; Avery-Kiejda, Kelly A.

2016-01-01

p53 is expressed as multiple smaller isoforms whose functions in cancer are not well understood. The p53 isoforms demonstrate abnormal expression in different cancers, suggesting they are important in modulating the function of full-length p53 (FLp53). The quantification of relative mRNA expression has routinely been performed using real-time PCR (qPCR). However, there are serious limitations when detecting p53 isoforms using this method, particularly for formalin-fixed paraffin-embedded (FFPE) tissues. The use of FFPE tumours would be advantageous to correlate expression of p53 isoforms with important clinical features of cancer. One alternative method of RNA detection is the hybridization-based QuantiGene 2.0 Assay, which has been shown to be advantageous for the detection of RNA from FFPE tissues. In this pilot study, we compared the QuantiGene 2.0 Assay to qPCR for the detection of FLp53 and its isoform Δ40p53 in matched fresh frozen (FF) and FFPE breast tumours. FLp53 mRNA expression was detected using qPCR in FF and FFPE tissues, but Δ40p53 mRNA was only detectable in FF tissues. Similar results were obtained for the QuantiGene 2.0 Assay. FLp53 relative mRNA expression was shown to be strongly correlated between the two methods (R2 = 0.9927, p = 0.0031) in FF tissues, however Δ40p53 was not (R2 = 0.4429, p = 0.3345). When comparing the different methods for the detection of FLp53 mRNA from FFPE and FF samples, no correlation (R2 = 0.0002, p = 0.9863) was shown using the QuantiGene 2.0 Assay, and in contrast, the level of expression was highly correlated between the two tissues using qPCR (R2 = 0.8753, p = 0.0644). These results suggest that both the QuantiGene 2.0 Assay and qPCR methods are inadequate for the quantification of Δ40p53 mRNA in FFPE tissues. Therefore, alternative methods of RNA detection and quantification are required to study the relative expression of Δ40p53 in FFPE samples. PMID:27832134

From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.

PubMed

Marinov, Georgi K; Williams, Brian A; McCue, Ken; Schroth, Gary P; Gertz, Jason; Myers, Richard M; Wold, Barbara J

2014-03-01

Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30-100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states.

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.

Alternative Polyadenylation Regulates CELF1/CUGBP1 Target Transcripts Following T Cell Activation

PubMed Central

Beisang, Daniel; Reilly, Cavan; Bohjanen, Paul R.

2014-01-01

Alternative polyadenylation (APA) is an evolutionarily conserved mechanism for regulating gene expression. Transcript 3′ end shortening through changes in polyadenylation site usage occurs following T cell activation, but the consequences of APA on gene expression are poorly understood. We previously showed that GU-rich elements (GREs) found in the 3′ untranslated regions of select transcripts mediate rapid mRNA decay by recruiting the protein CELF1/CUGBP1. Using a global RNA sequencing approach, we found that a network of CELF1 target transcripts involved in cell division underwent preferential 3′ end shortening via APA following T cell activation, resulting in decreased inclusion of CELF1 binding sites and increased transcript expression. We present a model whereby CELF1 regulates APA site selection following T cell activation through reversible binding to nearby GRE sequences. These findings provide insight into the role of APA in controlling cellular proliferation during biological processes such as development, oncogenesis and T cell activation PMID:25123787

The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis.

PubMed

Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

2014-04-01

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival.

The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis

PubMed Central

Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

2014-01-01

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5′ splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival. PMID:24514149

Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain

PubMed Central

Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; Mapelli, Marina; Walters, Kylie J.; Polo, Simona

2016-01-01

Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VIshort and myosin VIlong, which differ in the C-terminal region. Their physiological and pathological role remains unknown. Here we identified an isoform-specific regulatory helix, named α2-linker that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a novel clathrin-binding domain that is unique to myosin VIlong and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, where alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VIshort for tumor cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VIshort. Thus the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VIlong) or migratory (myosin VIshort) functional roles. PMID:26950368

Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain.

PubMed

Wollscheid, Hans-Peter; Biancospino, Matteo; He, Fahu; Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; Mapelli, Marina; Walters, Kylie J; Polo, Simona

2016-04-01

Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VI(short) and myosin VI(long), which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VI(long) and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VI(short) in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VI(short). Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VI(long)) or migratory (myosin VI(short)) functional roles.

Viability assessment of regional biomass pre-processing center based bioethanol value chains

NASA Astrophysics Data System (ADS)

Carolan, Joseph E.

Petroleum accounts for 94% of all liquid fuels and 36% of the total of all energy consumed in the United States. Petroleum dependence is problematic because global petroleum reserves are estimated to last only for 40 to 60 years at current consumption rates; global supplies are often located in politically unstable or unfriendly regions; and fossil fuels have negative environmental footprints. Domestic policies have aimed at promoting alternative, renewable liquid fuels, specifically bio-fuels derived from organic matter. Cellulosic bio-ethanol is one promising alternative fuel that has featured prominently in federal bio-fuel mandates under the Energy Independence and Security Act, 2007. However, the cellulosic bio-ethanol industry faces several technical, physical and industrial organization challenges. This dissertation examines the concept of a network of regional biomass pre-treatment centers (RBPC) that form an extended biomass supply chain feeding into a simplified biorefinery as a way to overcome these challenges. The analyses conducted address the structural and transactional issues facing bio-ethanol value chain establishment; the technical and financial feasibility of a stand alone pre-treatment center (RBPC); the impact of distributed pre-treatment on biomass transport costs; a comparative systems cost evaluation of the performance of the RBPC chain versus a fully integrated biorefinery (gIBRh), followed by application of the analytical framework to three case study regions.

CLP1 founder mutation links tRNA splicing and maturation to cerebellar development and neurodegeneration.

PubMed

Schaffer, Ashleigh E; Eggens, Veerle R C; Caglayan, Ahmet Okay; Reuter, Miriam S; Scott, Eric; Coufal, Nicole G; Silhavy, Jennifer L; Xue, Yuanchao; Kayserili, Hulya; Yasuno, Katsuhito; Rosti, Rasim Ozgur; Abdellateef, Mostafa; Caglar, Caner; Kasher, Paul R; Cazemier, J Leonie; Weterman, Marian A; Cantagrel, Vincent; Cai, Na; Zweier, Christiane; Altunoglu, Umut; Satkin, N Bilge; Aktar, Fesih; Tuysuz, Beyhan; Yalcinkaya, Cengiz; Caksen, Huseyin; Bilguvar, Kaya; Fu, Xiang-Dong; Trotta, Christopher R; Gabriel, Stacey; Reis, André; Gunel, Murat; Baas, Frank; Gleeson, Joseph G

2014-04-24

Neurodegenerative diseases can occur so early as to affect neurodevelopment. From a cohort of more than 2,000 consanguineous families with childhood neurological disease, we identified a founder mutation in four independent pedigrees in cleavage and polyadenylation factor I subunit 1 (CLP1). CLP1 is a multifunctional kinase implicated in tRNA, mRNA, and siRNA maturation. Kinase activity of the CLP1 mutant protein was defective, and the tRNA endonuclease complex (TSEN) was destabilized, resulting in impaired pre-tRNA cleavage. Germline clp1 null zebrafish showed cerebellar neurodegeneration that was rescued by wild-type, but not mutant, human CLP1 expression. Patient-derived induced neurons displayed both depletion of mature tRNAs and accumulation of unspliced pre-tRNAs. Transfection of partially processed tRNA fragments into patient cells exacerbated an oxidative stress-induced reduction in cell survival. Our data link tRNA maturation to neuronal development and neurodegeneration through defective CLP1 function in humans. Copyright © 2014 Elsevier Inc. All rights reserved.

Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene

PubMed Central

Nakajima, Yoko; Meijer, Judith; Zhang, Chunhua; Wang, Xu; Kondo, Tomomi; Ito, Tetsuya; Dobritzsch, Doreen; Van Kuilenburg, André B. P.

2016-01-01

Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients. PMID:26771602

Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene.

PubMed

Nakajima, Yoko; Meijer, Judith; Zhang, Chunhua; Wang, Xu; Kondo, Tomomi; Ito, Tetsuya; Dobritzsch, Doreen; Van Kuilenburg, André B P

2016-01-12

Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.

Alternate promoter selection within a human cytomegalovirus immediate-early and early transcription unit (UL119-115) defines true late transcripts containing open reading frames for putative viral glycoproteins.

PubMed Central

Leatham, M P; Witte, P R; Stinski, M F

1991-01-01

The human cytomegalovirus open reading frames (ORFs) UL119 through UL115 (UL119-115) are located downstream of the immediate-early 1 and 2 transcription units. The promoter upstream of UL119 is active at all times after infection and drives the synthesis of a spliced 3.1-kb mRNA. The viral mRNA initiates in UL119, contains UL119-117 and UL116, and terminates just downstream of UL115. True late transcripts that are detected only after viral DNA synthesis originate from this transcription unit. True late mRNAs of 2.1 kb, containing ORFs UL116 and UL115, and 1.2 kb, containing ORF UL115 only, are synthesized. The true late viral mRNAs are 3' coterminal with the 3.1-kb mRNA. This transcription unit is an example of late promoters nested within an immediate-early-early transcription unit. The gene products of UL119-117, UL116, and UL115 are predicted to be glycoproteins. Efficient expression of the downstream ORFs at late times after infection may be related to alternate promoter usage and downstream cap site selection. Images PMID:1717716

Tissue- and case-specific retention of intron 40 in mature dystrophin mRNA.

PubMed

Nishida, Atsushi; Minegishi, Maki; Takeuchi, Atsuko; Niba, Emma Tabe Eko; Awano, Hiroyuki; Lee, Tomoko; Iijima, Kazumoto; Takeshima, Yasuhiro; Matsuo, Masafumi

2015-06-01

The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), comprises 79 exons that show multiple alternative splicing events. Intron retention, a type of alternative splicing, may control gene expression. We examined intron retention in dystrophin introns by reverse-transcription PCR from skeletal muscle, focusing on the nine shortest (all <1000 bp), because these are more likely to be retained. Only one, intron 40, was retained in mRNA; sequencing revealed insertion of a complete intron 40 (851 nt) between exons 40 and 41. The intron 40 retention product accounted for 1.2% of the total product but had a premature stop codon at the fifth intronic codon. Intron 40 retention was most strongly observed in the kidney (36.6%) and was not obtained from the fetal liver, lung, spleen or placenta. This indicated that intron retention is a tissue-specific event whose level varies among tissues. In two DMD patients, intron 40 retention was observed in one patient but not in the other. Examination of splicing regulatory factors revealed that intron 40 had the highest guanine-cytosine content of all examined introns in a 30-nt segment at its 3' end. Further studies are needed to clarify the biological role of intron 40-retained dystrophin mRNA.

An Alternative Nested Reading Frame May Participate in the Stress-Dependent Expression of a Plant Gene

PubMed Central

Sheshukova, Ekaterina V.; Komarova, Tatiana V.; Ershova, Natalia M.; Shindyapina, Anastasia V.; Dorokhov, Yuri L.

2017-01-01

Although plants as sessile organisms are affected by a variety of stressors in the field, the stress factors for the above-ground and underground parts of the plant and their gene expression profiles are not the same. Here, we investigated NbKPILP, a gene encoding a new member of the ubiquitous, pathogenesis-related Kunitz peptidase inhibitor (KPI)-like protein family, that we discovered in the genome of Nicotiana benthamiana and other representatives of the Solanaceae family. The NbKPILP gene encodes a protein that has all the structural elements characteristic of KPI but in contrast to the proven A. thaliana KPI (AtKPI), it does not inhibit serine peptidases. Unlike roots, NbKPILP mRNA and its corresponding protein were not detected in intact leaves, but abiotic and biotic stressors drastically affected NbKPILP mRNA accumulation. In search of the causes of suppressed NbKPILP mRNA accumulation in leaves, we found that the NbKPILP gene is “matryoshka,” containing an alternative nested reading frame (ANRF) encoding a 53-amino acid (aa) polypeptide (53aa-ANRF) which has an amphipathic helix (AH). We confirmed ANRF expression experimentally. A vector containing a GFP-encoding sequence was inserted into the NbKPILP gene in frame with 53aa-ANRF, resulting in a 53aa-GFP fused protein that localized in the membrane fraction of cells. Using the 5′-RACE approach, we have shown that the expression of ANRF was not explained by the existence of a cryptic promoter within the NbKPILP gene but was controlled by the maternal NbKPILP mRNA. We found that insertion of mutations destroying the 53aa-ANRF AH resulted in more than a two-fold increase of the NbKPILP mRNA level. The NbKPILP gene represents the first example of ANRF functioning as a repressor of a maternal gene in an intact plant. We proposed a model where the stress influencing the translation initiation promotes the accumulation of NbKPILP and its mRNA in leaves. PMID:29312392

An Alternative Nested Reading Frame May Participate in the Stress-Dependent Expression of a Plant Gene.

PubMed

Sheshukova, Ekaterina V; Komarova, Tatiana V; Ershova, Natalia M; Shindyapina, Anastasia V; Dorokhov, Yuri L

2017-01-01

Although plants as sessile organisms are affected by a variety of stressors in the field, the stress factors for the above-ground and underground parts of the plant and their gene expression profiles are not the same. Here, we investigated NbKPILP , a gene encoding a new member of the ubiquitous, pathogenesis-related Kunitz peptidase inhibitor (KPI)-like protein family, that we discovered in the genome of Nicotiana benthamiana and other representatives of the Solanaceae family. The NbKPILP gene encodes a protein that has all the structural elements characteristic of KPI but in contrast to the proven A. thaliana KPI (AtKPI), it does not inhibit serine peptidases. Unlike roots, NbKPILP mRNA and its corresponding protein were not detected in intact leaves, but abiotic and biotic stressors drastically affected NbKPILP mRNA accumulation. In search of the causes of suppressed NbKPILP mRNA accumulation in leaves, we found that the NbKPILP gene is "matryoshka," containing an alternative nested reading frame (ANRF) encoding a 53-amino acid (aa) polypeptide (53aa-ANRF) which has an amphipathic helix (AH). We confirmed ANRF expression experimentally. A vector containing a GFP-encoding sequence was inserted into the NbKPILP gene in frame with 53aa-ANRF, resulting in a 53aa-GFP fused protein that localized in the membrane fraction of cells. Using the 5'-RACE approach, we have shown that the expression of ANRF was not explained by the existence of a cryptic promoter within the NbKPILP gene but was controlled by the maternal NbKPILP mRNA. We found that insertion of mutations destroying the 53aa-ANRF AH resulted in more than a two-fold increase of the NbKPILP mRNA level. The NbKPILP gene represents the first example of ANRF functioning as a repressor of a maternal gene in an intact plant. We proposed a model where the stress influencing the translation initiation promotes the accumulation of NbKPILP and its mRNA in leaves.

Boric acid reversibly inhibits the second step of pre-mRNA splicing.

PubMed

Shomron, Noam; Ast, Gil

2003-09-25

Several approaches have been used to identify the factors involved in mRNA splicing. None of them, however, comprises a straightforward reversible method for inhibiting the second step of splicing using an external reagent other than a chelator. This investigation demonstrates that the addition of boric acid to an in vitro pre-mRNA splicing reaction causes a dose-dependent reversible inhibition effect on the second step of splicing. The mechanism of action does not involve chelation of several metal ions; hindrance of 3' splice-site; or binding to hSlu7. This study presents a novel method for specific reversible inhibition of the second step of pre-mRNA splicing.

Expression of insulin-like growth factor I receptors at mRNA and protein levels during metamorphosis of Japanese flounder (Paralichthys olivaceus).

PubMed

Zhang, Junling; Shi, Zhiyi; Cheng, Qi; Chen, Xiaowu

2011-08-01

Insulin-like growth factor I (IGF-I) is an important regulator of fish growth and development, and its biological actions are initiated by binding to IGF-I receptor (IGF-IR). Our previous study has revealed that IGF-I could play an important role during metamorphosis of Japanese flounder, Paralichthys olivaceus. The analysis of IGF-IR expression thus helps further elucidate the IGF-I regulation of metamorphic processes. In this study, the spatial-temporal expression of two distinct IGF-IR mRNAs was investigated by real-time RT-PCR. The spatial distribution of two IGF-IR mRNAs in adult tissues is largely overlapped, but they exhibit distinct temporal expression patterns during larval development. A remarkable decrease in IGF-IR-2 mRNA was detected during metamorphosis. In contrast, a significant increase in IGF-IR-1 mRNA was determined from pre-metamorphosis to metamorphic completion. These indicate that they may play different function roles during the flounder metamorphosis. The levels and localization of IGF-IR proteins during larval development were further studied by Western blotting and immunohistochemistry. Immunoreactive IGF-IRs were detected throughout larval development, and the IGF-IR proteins displayed a relatively abundant expression during metamorphosis. Moreover, the IGF-IR proteins appeared in key tissues, such as thickened skin beneath the migrating eye, developing intestine, gills and kidney during metamorphosis. These results further suggest that the IGF-I system may be involved in metamorphic development of Japanese flounder. Copyright © 2011 Elsevier Inc. All rights reserved.

Pre- and Perinatal Risk for Attention-Deficit Hyperactivity Disorder: Does Neuropsychological Weakness Explain the Link?

PubMed

Wiggs, Kelsey; Elmore, Alexis L; Nigg, Joel T; Nikolas, Molly A

2016-11-01

Etiological investigations of attention-deficit hyperactivity disorder (ADHD) and disruptive behavior problems support multiple causal pathways, including involvement of pre- and perinatal risk factors. Because these risks occur early in life, well before observable ADHD and externalizing symptoms emerge, the relation between risk and symptoms may be mediated by neurodevelopmental effects that manifest later in neuropsychological functioning. However, potential dissociable effects of pre/perinatal risk elements on ADHD and familial confounds must also be considered to test alternative hypotheses. 498 youth aged 6-17 years (55.0 % male) completed a multi-stage, multi-informant assessment including parent and teacher symptom reports of symptoms and parent ratings of pre/perinatal health risk indicators. Youth completed a neuropsychological testing battery. Multiple mediation models examined direct effects of pre- and perinatal health risk on ADHD and other disruptive behavior disorder symptoms and indirect effects via neuropsychological functioning. Parental ADHD symptoms and externalizing status was covaried to control for potential familial effects. Effects of prenatal substance exposure on inattention were mediated by memory span and temporal processing deficits. Further, effects of perinatal health risk on inattention, hyperactivity-impulsivity, and ODD were mediated by deficits in response variability and temporal processing. Further, maternal health risks during pregnancy appeared to exert direct rather than indirect effects on outcomes. Results suggest that after controlling for familial relatedness of ADHD between parent and child, early developmental health risks may influence ADHD via effects on neuropsychological processes underpinning the disorder.

A Function for the hnRNP A1/A2 Proteins in Transcription Elongation.

PubMed

Lemieux, Bruno; Blanchette, Marco; Monette, Anne; Mouland, Andrew J; Wellinger, Raymund J; Chabot, Benoit

2015-01-01

The hnRNP A1 and A2 proteins regulate processes such as alternative pre-mRNA splicing and mRNA stability. Here, we report that a reduction in the levels of hnRNP A1 and A2 by RNA interference or their cytoplasmic retention by osmotic stress drastically increases the transcription of a reporter gene. Based on previous work, we propose that this effect may be linked to a decrease in the activity of the transcription elongation factor P-TEFb. Consistent with this hypothesis, the transcription of the reporter gene was stimulated when the catalytic component of P-TEFb, CDK9, was inhibited with DRB. While low levels of A1/A2 stimulated the association of RNA polymerase II with the reporter gene, they also increased the association of CDK9 with the repressor 7SK RNA, and compromised the recovery of promoter-distal transcription on the Kitlg gene after the release of pausing. Transcriptome analysis revealed that more than 50% of the genes whose expression was affected by the siRNA-mediated depletion of A1/A2 were also affected by DRB. RNA polymerase II-chromatin immunoprecipitation assays on DRB-treated and A1/A2-depleted cells identified a common set of repressed genes displaying increased occupancy of polymerases at promoter-proximal locations, consistent with pausing. Overall, our results suggest that lowering the levels of hnRNP A1/A2 elicits defective transcription elongation on a fraction of P-TEFb-dependent genes, hence favoring the transcription of P-TEFb-independent genes.

Leaching of Au, Ag, and Pd from waste printed circuit boards of mobile phone by iodide lixiviant after supercritical water pre-treatment

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

Xiu, Fu-Rong, E-mail: xiu_chem@hotmail.com; Qi, Yingying; Zhang, Fu-Shen

Highlights: • We report a novel process for recovering Au, Ag, and Pd from waste PCBs. • The effect of SCWO on the leaching of Au, Ag, and Pd in waste PCBs was studied. • SCWO was highly efficient for enhancing the leaching of Au, Ag, and Pd. • The optimum leaching parameters for Au, Ag, and Pd in iodine–iodide were studied. - Abstract: Precious metals are the most attractive resources in waste printed circuit boards (PCBs) of mobile phones. In this work, an alternative process for recovering Au, Ag, and Pd from waste PCBs of mobile phones by supercriticalmore » water oxidation (SCWO) pre-treatment combined with iodine–iodide leaching process was developed. In the process, the waste PCBs of mobile phones were pre-treated in supercritical water, then a diluted hydrochloric acid leaching (HL) process was used to recovery the Cu, whose leaching efficiency was approximately 100%, finally the resulting residue was subjected to the iodine–iodide leaching process for recovering the Au, Ag, and Pd. Experimental results indicated that SCWO pre-treatment temperature, time, and pressure had significant influence on the Au, Ag, and Pd leaching from (SCWO + HL)-treated waste PCBs. The optimal SCWO pre-treatment conditions were 420 °C and 60 min for Au and Pd, and 410 °C and 30 min for Ag. The optimum dissolution parameters for Au, Pd, and Ag in (SCWO + HL)-treated PCBs with iodine–iodide system were leaching time of 120 min (90 min for Ag), iodine/iodide mole ratio of 1:5 (1:6 for Ag), solid-to-liquid ratio (S/L) of 1:10 g/mL (1:8 g/mL for Ag), and pH of 9, respectively. It is believed that the process developed in this study is environment friendly for the recovery of Au, Ag, and Pd from waste PCBs of mobile phones by SCWO pre-treatment combined with iodine–iodide leaching process.« less

Brahma regulates a specific trans-splicing event at the mod(mdg4) locus of Drosophila melanogaster

PubMed Central

Yu, Simei; Waldholm, Johan; Böhm, Stefanie; Visa, Neus

2014-01-01

The mod(mdg4) locus of Drosophila melanogaster contains several transcription units encoded on both DNA strands. The mod(mdg4) pre-mRNAs are alternatively spliced, and a very significant fraction of the mature mod(mdg4) mRNAs are formed by trans-splicing. We have studied the transcripts derived from one of the anti-sense regions within the mod(mdg4) locus in order to shed light on the expression of this complex locus. We have characterized the expression of anti-sense mod(mdg4) transcripts in S2 cells, mapped their transcription start sites and cleavage sites, identified and quantified alternatively spliced transcripts, and obtained insight into the regulation of the mod(mdg4) trans-splicing. In a previous study, we had shown that the alternative splicing of some mod(mdg4) transcripts was regulated by Brahma (BRM), the ATPase subunit of the SWI/SNF chromatin-remodeling complex. Here we show, using RNA interference and overexpression of recombinant BRM proteins, that the levels of BRM affect specifically the abundance of a trans-spliced mod(mdg4) mRNA isoform in both S2 cells and larvae. This specific effect on trans-splicing is accompanied by a local increase in the density of RNA polymerase II and by a change in the phosphorylation state of the C-terminal domain of the large subunit of RNA polymerase II. Interestingly, the regulation of the mod(mdg4) splicing by BRM is independent of the ATPase activity of BRM, which suggests that the mechanism by which BRM modulates trans-splicing is independent of its chromatin-remodeling activity. PMID:24526065

Mixing Metaphors in the Cerebral Hemispheres: What Happens When Careers Collide?

PubMed Central

Chettih, Selmaan; Durgin, Frank H.; Grodner, Daniel J.

2014-01-01

Are processes of figurative comparison and figurative categorization different? An experiment combining alternative-sense and matched-sense metaphor priming with a divided visual field assessment technique sought to isolate processes of comparison and categorization in the two cerebral hemispheres. For target metaphors presented in the RVF/LH, only matched-sense primes were facilitative. Literal primes and alternative-sense primes had no effect on comprehension time compared to the unprimed baseline. The effects of matched-sense primes were additive with the rated conventionality of the targets. For target metaphors presented to the LVF/RH, matched-sense primes were again additively facilitative. However, alternative-sense primes, though facilitative overall, seemed to eliminate the pre-existing advantages of conventional target metaphor senses in the LVF/RH in favor of metaphoric senses similar to those of the primes. These findings are consistent with tightly controlled categorical coding in the LH and coarse, and flexible, context dependent coding in the RH. PMID:22103787

Triage of women with low-grade cervical lesions--HPV mRNA testing versus repeat cytology.

PubMed

Sørbye, Sveinung Wergeland; Arbyn, Marc; Fismen, Silje; Gutteberg, Tore Jarl; Mortensen, Elin Synnøve

2011-01-01

In Norway, women with low-grade squamous intraepithelial lesions (LSIL) are followed up after six months in order to decide whether they should undergo further follow-up or be referred back to the screening interval of three years. A high specificity and positive predictive value (PPV) of the triage test is important to avoid unnecessary diagnostic and therapeutic procedures. At the University Hospital of North Norway, repeat cytology and the HPV mRNA test PreTect HPV-Proofer, detecting E6/E7 mRNA from HPV types 16, 18, 31, 33 and 45, are used in triage of women with ASC-US and LSIL. In this study, women with LSIL cytology in the period 2005-2008 were included (n = 522). Two triage methods were evaluated in two separate groups: repeat cytology only (n = 225) and HPV mRNA testing in addition to repeat cytology (n = 297). Histologically confirmed cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) was used as the study endpoint. Of 522 women with LSIL, 207 had biopsies and 125 of them had CIN2+. The sensitivity and specificity of repeat cytology (ASC-US or worse) were 85.7% (95% confidence interval (CI): 72.1, 92.2) and 54.4 % (95% CI: 46.9, 61.9), respectively. The sensitivity and specificity of the HPV mRNA test were 94.2% (95% CI: 88.7, 99.7) and 86.0% (95% CI: 81.5, 90.5), respectively. The PPV of repeat cytology was 38.4% (95% CI: 29.9, 46.9) compared to 67.0% (95% CI: 57.7, 76.4) of the HPV mRNA test. HPV mRNA testing was more sensitive and specific than repeat cytology in triage of women with LSIL cytology. In addition, the HPV mRNA test showed higher PPV. These data indicate that the HPV mRNA test is a better triage test for women with LSIL than repeat cytology.

Effect of environmental pollutants on oxytocin synthesis and secretion from corpus luteum and on contractions of uterus from pregnant cows

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

Mlynarczuk, Jaroslaw; Wrobel, Michal H.; Kotwica, Jan, E-mail: j.kotwica@pan.olsztyn.p

Chloro-organic compounds are persistent environmental pollutants and affect many reproductive processes. Oxytocin (OT) synthesized in luteal cells is a local regulator of ovarian activity and uterine contractions. Therefore the effect of xenobiotics on the OT prohormone synthesis, secretion of OT and progesterone (P4) from luteal cells and on myometrial contractions during early pregnancy in cows was investigated. Luteal cells and myometrial strips from a cow at early pregnancy were treated with polychlorinated biphenyl 77 (PCB 77), dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene (DDE) and hexachlorocyclohexane (HCH) (1 or 10 ng/ml). The mRNA expression of neurophysin-I/oxytocin (NP-I/OT) and peptidyl-glycine-{alpha}-amidating mono-oxygenase (PGA) and concentration ofmore » OT and P4 were determined by RT-PCR and EIA, respectively. Moreover, the effect of xenobiotics given with P4 (12 ng/ml) on the basal and OT (10{sup -7} M) stimulated contractions of myometrial strips was studied. Xenobiotics increased (P < 0.05) OT secretion but DDE only stimulated P4 secretion. The ratio of P4 to OT in culture medium was decreased by all xenobiotics during 9-12 weeks of pregnancy. All xenobiotics, except HCH, increased (P < 0.05) mRNA expression of NP-I/OT during all stages of pregnancy and all treatments decreased (P < 0.05) expression of mRNA for PGA during 9-12 weeks of pregnancy. Myometrial strips were relaxed (P < 0.01) after pre-incubation with P4, while each of the xenobiotics jointly with P4 increased (P < 0.01) myometrial contractions. In conclusion, the xenobiotics used increased both expression of mRNA for genes involved in OT synthesis and secretion of OT from luteal cells. This decreases the ratio of P4 to OT and presumably, in this manner, the chloro-organic compounds can influence uterine contractions and enhance risk of abortions in pregnant females.« less

Bone morphogenetic protein 15 and growth differentiation factor 9 expression in the ovary of European sea bass (Dicentrarchus labrax): cellular localization, developmental profiles, and response to unilateral ovariectomy.

PubMed

García-López, Ángel; Sánchez-Amaya, María Isabel; Halm, Silke; Astola, Antonio; Prat, Francisco

2011-12-01

Vertebrate oocytes actively contribute to follicle development by secreting a variety of growth factors, among which bone morphogenetic protein 15 (BMP15/Bmp15) and growth differentiation factor 9 (GDF9/Gdf9) have been paid particular attention. In the present study, we describe the cellular localization, the developmental profiles, and the response to unilateral ovariectomy (a procedure implying the surgical removal of one of the ovaries) of protein and mRNA steady-state levels of Bmp15 and Gdf9 in the ovary of European sea bass, an important fish species for marine aquaculture industry. In situ hybridization and immunohistochemistry demonstrated that the oocyte is the main production site of Bmp15 and Gdf9 in European sea bass ovary. During oocyte development, Bmp15 protein expression started to be detected only from the lipid vesicle stage onwards but not in primary pre-vitellogenic (i.e. perinucleolar) oocytes as the bmp15 mRNA already did. Gdf9 protein and gdf9 mRNA expression were both detected in primary perinucleolar oocytes and followed similar decreasing patterns thereafter. Unilateral ovariectomy induced a full compensatory growth of the remaining ovary in the 2-month period following surgery (Á. García-López, M.I. Sánchez-Amaya, C.R. Tyler, F. Prat 2011). The compensatory growth elicited different changes in the expression levels of mRNA and protein of both factors, although the involvement of Bmp15 and Gdf9 in the regulatory network orchestrating such process remains unclear at present. Altogether, our results establish a solid base for further studies focused on elucidating the specific functions of Bmp15 and Gdf9 during primary and secondary oocyte growth in European sea bass. Copyright © 2011 Elsevier Inc. All rights reserved.

The use of telephone in genetic counseling versus in-person counseling: a randomized study on counselees' outcome.

PubMed

Platten, Ulla; Rantala, Johanna; Lindblom, Annika; Brandberg, Yvonne; Lindgren, Gunilla; Arver, Brita

2012-09-01

Increased demand for genetic counseling services necessitates exploring alternatives to in-person counseling. Telephone counseling is a less time-consuming and more cost-effective alternative. So far there is insufficient evidence to support a pre-counseling telephone model. This randomized questionnaire study aims to evaluate the oncogenetic counseling process and to compare the impact of the initial part of the oncogenetic counseling, when conducted via telephone versus in-person. The aspects of evaluations were: patients' expectations, satisfaction and experiences of genetic counseling, worry for developing hereditary cancer and health related quality of life. A total of 215 participants representing several cancer syndromes were randomized to counseling via telephone or in-person. The questionnaires were completed before and after oncogenetic nurse counseling, and 1 year after the entire counseling process. Overall, a high satisfaction rate with the oncogenetic counseling process was found among the participants regardless of whether the oncogenetic nurse counseling was conducted by telephone or in-person. The results show that a considerable number of participants experienced difficulties with the process of creating a pedigree and dissatisfaction with information on surveillance and prevention. Affected participants reported lower levels in most SF-36 domains compared to non-affected and both groups reported lower levels as compared to a Swedish reference group. The results indicate that telephone pre-counseling works as well as in-person counseling. Emotional support during genetic counseling and information on recommended cancer prevention and surveillance should be improved.

Evolution of the Antisense Overlap between Genes for Thyroid Hormone Receptor and Rev-erbα and Characterization of an Exonic G-Rich Element That Regulates Splicing of TRα2 mRNA

PubMed Central

Munroe, Stephen H.; Morales, Christopher H.; Duyck, Tessa H.; Waters, Paul D.

2015-01-01

The α-thyroid hormone receptor gene (TRα) codes for two functionally distinct proteins: TRα1, the α-thyroid hormone receptor; and TRα2, a non-hormone-binding variant. The final exon of TRα2 mRNA overlaps the 3’ end of Rev-erbα mRNA, which encodes another nuclear receptor on the opposite strand of DNA. To understand the evolution of this antisense overlap, we sequenced these genes and mRNAs in the platypus Orthorhynchus anatinus. Despite its strong homology with other mammals, the platypus TRα/Rev-erbα locus lacks elements essential for expression of TRα2. Comparative analysis suggests that alternative splicing of TRα2 mRNA expression evolved in a stepwise fashion before the divergence of eutherian and marsupial mammals. A short G-rich element (G30) located downstream of the alternative 3’splice site of TRα2 mRNA and antisense to the 3’UTR of Rev-erbα plays an important role in regulating TRα2 splicing. G30 is tightly conserved in eutherian mammals, but is absent in marsupials and monotremes. Systematic deletions and substitutions within G30 have dramatically different effects on TRα2 splicing, leading to either its inhibition or its enhancement. Mutations that disrupt one or more clusters of G residues enhance splicing two- to three-fold. These results suggest the G30 sequence can adopt a highly structured conformation, possibly a G-quadruplex, and that it is part of a complex splicing regulatory element which exerts both positive and negative effects on TRα2 expression. Since mutations that strongly enhance splicing in vivo have no effect on splicing in vitro, it is likely that the regulatory role of G30 is mediated through linkage of transcription and splicing. PMID:26368571

40 CFR Appendix A to Subpart Dddd... - Alternative Procedure To Determine Capture Efficiency From Enclosures Around Hot Presses in the...

Code of Federal Regulations, 2012 CFR

2012-07-01

... of average velocity during the run and using these data, in conjunction with the pre- and post-test..., you may choose to follow the post-test calibration procedures of Method 320 in appendix A to 40 CFR... of hazardous air pollutants during the press process. This test method requires a minimum of three...

Learning about Bones at a Science Museum: Examining the Alternate Hypotheses of Ceiling Effect and Prior Knowledge

ERIC Educational Resources Information Center

Judson, Eugene

2012-01-01

Groups of children at a science museum were pre- and post-assessed with a type of concept map, known as personal meaning maps, to determine what new understandings, if any, they were gaining from participation in a series of structured hands-on activities about bones and the process of bones healing. Close examination was made regarding whether…

Adiabatic reduction of a model of stochastic gene expression with jump Markov process.

PubMed

Yvinec, Romain; Zhuge, Changjing; Lei, Jinzhi; Mackey, Michael C

2014-04-01

This paper considers adiabatic reduction in a model of stochastic gene expression with bursting transcription considered as a jump Markov process. In this model, the process of gene expression with auto-regulation is described by fast/slow dynamics. The production of mRNA is assumed to follow a compound Poisson process occurring at a rate depending on protein levels (the phenomena called bursting in molecular biology) and the production of protein is a linear function of mRNA numbers. When the dynamics of mRNA is assumed to be a fast process (due to faster mRNA degradation than that of protein) we prove that, with appropriate scalings in the burst rate, jump size or translational rate, the bursting phenomena can be transmitted to the slow variable. We show that, depending on the scaling, the reduced equation is either a stochastic differential equation with a jump Poisson process or a deterministic ordinary differential equation. These results are significant because adiabatic reduction techniques seem to have not been rigorously justified for a stochastic differential system containing a jump Markov process. We expect that the results can be generalized to adiabatic methods in more general stochastic hybrid systems.

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

PubMed

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

2014-01-01

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

Survey of the transcriptome of Aspergillus oryzae via massively parallel mRNA sequencing

PubMed Central

Wang, Bin; Guo, Guangwu; Wang, Chao; Lin, Ying; Wang, Xiaoning; Zhao, Mouming; Guo, Yong; He, Minghui; Zhang, Yong; Pan, Li

2010-01-01

Aspergillus oryzae, an important filamentous fungus used in food fermentation and the enzyme industry, has been shown through genome sequencing and various other tools to have prominent features in its genomic composition. However, the functional complexity of the A. oryzae transcriptome has not yet been fully elucidated. Here, we applied direct high-throughput paired-end RNA-sequencing (RNA-Seq) to the transcriptome of A. oryzae under four different culture conditions. With the high resolution and sensitivity afforded by RNA-Seq, we were able to identify a substantial number of novel transcripts, new exons, untranslated regions, alternative upstream initiation codons and upstream open reading frames, which provide remarkable insight into the A. oryzae transcriptome. We were also able to assess the alternative mRNA isoforms in A. oryzae and found a large number of genes undergoing alternative splicing. Many genes and pathways that might be involved in higher levels of protein production in solid-state culture than in liquid culture were identified by comparing gene expression levels between different cultures. Our analysis indicated that the transcriptome of A. oryzae is much more complex than previously anticipated, and these results may provide a blueprint for further study of the A. oryzae transcriptome. PMID:20392818

Survey of the transcriptome of Aspergillus oryzae via massively parallel mRNA sequencing.

PubMed

Wang, Bin; Guo, Guangwu; Wang, Chao; Lin, Ying; Wang, Xiaoning; Zhao, Mouming; Guo, Yong; He, Minghui; Zhang, Yong; Pan, Li

2010-08-01

Aspergillus oryzae, an important filamentous fungus used in food fermentation and the enzyme industry, has been shown through genome sequencing and various other tools to have prominent features in its genomic composition. However, the functional complexity of the A. oryzae transcriptome has not yet been fully elucidated. Here, we applied direct high-throughput paired-end RNA-sequencing (RNA-Seq) to the transcriptome of A. oryzae under four different culture conditions. With the high resolution and sensitivity afforded by RNA-Seq, we were able to identify a substantial number of novel transcripts, new exons, untranslated regions, alternative upstream initiation codons and upstream open reading frames, which provide remarkable insight into the A. oryzae transcriptome. We were also able to assess the alternative mRNA isoforms in A. oryzae and found a large number of genes undergoing alternative splicing. Many genes and pathways that might be involved in higher levels of protein production in solid-state culture than in liquid culture were identified by comparing gene expression levels between different cultures. Our analysis indicated that the transcriptome of A. oryzae is much more complex than previously anticipated, and these results may provide a blueprint for further study of the A. oryzae transcriptome.

Position-specific binding of FUS to nascent RNA regulates mRNA length

PubMed Central

Masuda, Akio; Takeda, Jun-ichi; Okuno, Tatsuya; Okamoto, Takaaki; Ohkawara, Bisei; Ito, Mikako; Ishigaki, Shinsuke; Sobue, Gen

2015-01-01

More than half of all human genes produce prematurely terminated polyadenylated short mRNAs. However, the underlying mechanisms remain largely elusive. CLIP-seq (cross-linking immunoprecipitation [CLIP] combined with deep sequencing) of FUS (fused in sarcoma) in neuronal cells showed that FUS is frequently clustered around an alternative polyadenylation (APA) site of nascent RNA. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) of RNA polymerase II (RNAP II) demonstrated that FUS stalls RNAP II and prematurely terminates transcription. When an APA site is located upstream of an FUS cluster, FUS enhances polyadenylation by recruiting CPSF160 and up-regulates the alternative short transcript. In contrast, when an APA site is located downstream from an FUS cluster, polyadenylation is not activated, and the RNAP II-suppressing effect of FUS leads to down-regulation of the alternative short transcript. CAGE-seq (cap analysis of gene expression [CAGE] combined with deep sequencing) and PolyA-seq (a strand-specific and quantitative method for high-throughput sequencing of 3' ends of polyadenylated transcripts) revealed that position-specific regulation of mRNA lengths by FUS is operational in two-thirds of transcripts in neuronal cells, with enrichment in genes involved in synaptic activities. PMID:25995189