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MRNA profiling of wildtype, germline depleted, NMD mutant C. elegans whole worms and wildtype dissected gonads

ABSTRACT: Adjacent alternative 3’ splice sites, those separated by ≤18nt, provide a unique problem in the study of alternative splicing regulation; there is overlap of the cis-elements that define the adjacent sites. Identification of the intron's 3' end depends upon sequence elements that define the branchpoint, polypyrimidine tract and terminal AG dinucleotide. Starting with RNA-seq data from germline-enriched and somatic cell-enriched C. elegans samples, we identify hundreds of introns with adjacent alternative 3’ splice sites. We identify 203 events that undergo tissue-specific alternative splicing. For these, the regulation is mono-directional, with somatic cells preferring to splice at the distal 3' splice site and germline cells showing a distinct shift towards usage of the adjacent proximal 3' splice site. Splicing patterns in somatic cells follow consensus rules of 3’ splice site definition, using sites with a short stretch of pyrimidines and an AG dinucleotide. Splicing in germline cells occurs at proximal 3' splice sites that frequently lack a polypyrimidine tract or, occasionally, the AG dinucleotide. We provide evidence that use of germline-specific proximal 3' splice sites is conserved across Caenorhabditis species. We propose that divergent mechanisms exist between germline and somatic cells in determining an intron terminus at adjacent alternative 3’ splice sites. Examination of alternative splicing changes between germline- and somatic-cell enriched samples as well as nonsense-mediated decay mutants.

ORGANISM(S): Caenorhabditis elegans

SUBMITTER: James Ragle

PROVIDER: E-GEOD-64672 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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mRNA profiling of wildtype, germline depleted, NMD mutant C. elegans whole worms and wildtype dissected gonads

Project description:Adjacent alternative 3’ splice sites, those separated by ≤18nt, provide a unique problem in the study of alternative splicing regulation; there is overlap of the cis-elements that define the adjacent sites. Identification of the intron's 3' end depends upon sequence elements that define the branchpoint, polypyrimidine tract and terminal AG dinucleotide. Starting with RNA-seq data from germline-enriched and somatic cell-enriched C. elegans samples, we identify hundreds of introns with adjacent alternative 3’ splice sites. We identify 203 events that undergo tissue-specific alternative splicing. For these, the regulation is mono-directional, with somatic cells preferring to splice at the distal 3' splice site and germline cells showing a distinct shift towards usage of the adjacent proximal 3' splice site. Splicing patterns in somatic cells follow consensus rules of 3’ splice site definition, using sites with a short stretch of pyrimidines and an AG dinucleotide. Splicing in germline cells occurs at proximal 3' splice sites that frequently lack a polypyrimidine tract or, occasionally, the AG dinucleotide. We provide evidence that use of germline-specific proximal 3' splice sites is conserved across Caenorhabditis species. We propose that divergent mechanisms exist between germline and somatic cells in determining an intron terminus at adjacent alternative 3’ splice sites.

2015-04-22 | GSE64672 | GEO

A conserved germline-specific Dsn1 alternative splice isoform supports oocyte and embryo development

Project description:Alternative RNA splicing can generate distinct protein isoforms to allow for the differential control of cell processes across cell types. The chromosome segregation and cell division programs associated with somatic mitosis and germline meiosis display dramatic differences such as kinetochore orientation, cohesin removal, or the presence of a gap phase. These changes in chromosome segregation require alterations to the established cell division machinery. However, it remains unclear what aspects of kinetochore function and its regulatory control differ between the mitotic and meiotic cell divisions to rewire these core processes. Additionally, the alternative splice isoforms that differentially modulate distinct cell division programs have remained elusive. Here, we demonstrate that mammalian germ cells express an alternative mRNA splice isoform for the kinetochore component, DSN1, a subunit of the MIS12 complex that links the centromeres to spindle microtubules during chromosome segregation. This germline DSN1 isoform bypasses the requirement for Aurora kinase phosphorylation for its centromere localization due to the absence of a key regulatory region allowing DSN1 to display persistent centromere localization. Expression of the germline DSN1 isoform in somatic cells results in constitutive kinetochore localization, chromosome segregation errors, and growth defects, providing an explanation for its tight cell type-specific expression. Reciprocally, precisely eliminating expression of the germline-specific DSN1 splice isoform in mouse models disrupts oocyte maturation and early embryonic divisions coupled with a reduction in fertility. Together, this work identifies a germline-specific splice isoform for a chromosome segregation component and implicates its role in mammalian fertility.

2024-08-28 | PXD054119 | Pride

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

Project description:Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many Saccharomyces cerevisiae intron-containing genes exhibit usage of alternative splice sites, but most transcripts generated by splicing from these sites are non-functional because they introduce premature termination codons leading to transcript 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 3M-bM-^@M-^Y splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3M-bM-^@M-^Y-splice site. The use of non-productive alternative splice sites can limit the expression of some transcripts and can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results reveal that alternative splicing is frequent in S.cerevisiae but masked by RNA degradation and that the use of alternative splice sites is mostly aimed at controlling transcript levels rather than increasing proteome diversity. mRNA-Seq profiling of 3 mutants in the nonsense-mediated mRNA decay pathway and wildtype yeast

2014-02-21 | E-GEOD-55213 | biostudies-arrayexpress

Genentech Lung Cancer Sequencing

Project description:Version 1 Whole genome sequencing was applied to tumor and adjacent normal lung tissue in an individual non-small-cell lung cancer patient. We present an analysis of somatic changes identified throughout the tumor genome, including single-nucleotide variants, copy number variants, and large-scale chromosomal rearrangements. Over 50,000 high-confidence single-nucleotide variants were identified, revealing evidence of substantial smoking-related DNA damage as well as distinct mutational pressures within the tumor resulting in uneven distribution of somatic mutations across the genome. Version 2 Lung cancer is a highly heterogeneous disease in terms of both underlying genetic lesions and response to therapeutic treatments. We performed deep whole genome sequencing and transcriptome sequencing on 19 lung cancer cell lines and 3 lung tumor/normal pairs. Overall, our data show that cell line models exhibit similar mutation spectra to human tumor samples. Smoker and never-smoker cancer samples exhibit distinguishable patterns of mutations. A number of epigenetic regulators are frequently altered by mutations or copy number changes. A systematic survey of splice-site mutations identified over 100 splice site mutations associated with cancer specific aberrant splicing, including mutations in several known cancer-related genes. Differential usages of splice isoforms were also studied. Taken together, these data present a comprehensive genomic landscape of a large number of lung cancer samples and further demonstrate that cancer specific alternative splicing is a widespread phenomenon that has potential utility as therapeutic biomarkers.

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RBFOX2 alter splicing outcome in distinct binding modes with multiple protein partners

Project description:RBFOX2 controls the splicing of a large number of transcripts implicated in cell differentiation and development. Parsing RNA-binding protein datasets, we uncover that RBFOX2 can interact with hnRNPC, hnRNPM and SRSF1 to regulate splicing of a broad range of splicing events using different sequence motifs and binding modes. Using immunoprecipitation, specific RBP knockdown, RNA-seq and splice-sensitive PCR, we show that RBFOX2 can target splice sites using three binding configurations: single, multiple or secondary modes. In the single binding mode RBFOX2 is recruited to its target splice sites through a single canonical binding motif, while in the multiple binding mode RBFOX2 binding sites include the adjacent binding of at least one other RNA binding protein partner. Finally, in the secondary binding mode RBFOX2 likely does not bind the RNA directly but is recruited to splice sites lacking its canonical binding motif through the binding of one of its protein partners. These dynamic modes bind distinct sets of transcripts at different positions and distances relative to alternative splice sites explaining the heterogeneity of RBFOX2 targets and splicing outcomes.

2021-06-23 | GSE140679 | GEO

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

Project description:Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many Saccharomyces cerevisiae intron-containing genes exhibit usage of alternative splice sites, but most transcripts generated by splicing from these sites are non-functional because they introduce premature termination codons leading to transcript 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. The use of non-productive alternative splice sites can limit the expression of some transcripts and can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results reveal that alternative splicing is frequent in S.cerevisiae but masked by RNA degradation and that the use of alternative splice sites is mostly aimed at controlling transcript levels rather than increasing proteome diversity.

2014-02-21 | GSE55213 | GEO

Development of a novel splice array platform and its application in the identification of alternative splice variants

Project description:Changes in alternative splicing are associated with several pathological conditions, including cancer. Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array platform was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events. The array consists of exon probes and thermodynamically balanced junction probes. Suboptimal probes are tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was first validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms for 8000 genes in lung cancer samples compared to matched normal lung tissue. The expression of lung cancer-associated splice isoforms was validated by RT-PCR. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer. In conclusion, this highly accurate methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies. 20 normal/tumor paired specimens. Tumor samples are from non-small cell lung cancer (NSCLC) whereas normals are from adjacent normal lung tissue.

2010-06-21 | E-GEOD-18346 | biostudies-arrayexpress

Microarray validation of an alternative splicing database

Project description:Alternative splicing generates functional diversity in higher organisms through alternative first and last exons, skipped and included exons, intron retentions and alternative donor and acceptor sites. In large-scale microarray studies in human and mouse, emphasis so far has been placed on exon-skip events, leaving the prevalence and importance of other splice types largely unexplored. Using a new human splice variant database and a genome-wide microarray to probes thousands of splice events of each type, we measured differential expression of splice types across 6 pairs of diverse cell lines and validated the database annotation process. Results suggest that splicing in human is more complex than simple exon skip events, which account for a minority of splicing differences. The relative frequency of differential expression of the splice types correlates with what is found by our annotation efforts. In conclusion, alternative splicing in human cells is considerably more complex than the canonical example of the exon-skip. The complementary approaches of genome-wide annotation of alternative splicing in human and design of genome-wide splicing microarrays to measure differential splicing in biological samples provide a powerful high-throughput tool to study the role of alternative splicing in human biology. Keywords: alternative splicing

2007-12-08 | GSE9734 | GEO

Alternative splicing factor hnRNPA1 induces U2AF2 association with Alu-derived RNA

Project description:Alternative splicing drives transcriptome and proteome diversification. While splicing regulatory proteins govern this process, their global mechanisms remain enigmatic. We generated high resolution transcriptome-wide protein-RNA interaction maps to determine how the splicing repressor hnRNPA1 influences the global association of spliceosome assembly factor U2AF2 and SRSF1 with pre-mRNA. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3’ splice sites of cassette exons but not constitutive exons upon hnRNPA1 overexpression. By contrast, SRSF1 crosslinking patterns relative to splice sites are independent of hnRNPA1 expression levels. We also observed an hnRNPA1-dependent increase in U2AF2 but not SRSF1 crosslinking to exon proximal antisense Alu elements. Thus Alu elements can serve as splicing factor-responsive sinks for U2AF2. These results not only demonstrate a novel mechanism for alternative splicing regulation but also implicate retrotransposon-derived sequences in the evolution of species-specific alternative splicing.

2017-06-01 | GSE83923 | GEO

The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript

Project description:The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex which binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. Here, we describe a novel function for the EJC and its splicing subunit RnpS1 in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where in the absence of RnpS1 the fourth intron of piwi is retained. Within this intron the polypyrimidine tract is disrupted by a transposon-adjacent A/T-rich sequence that confers dependence on RnpS1. Finally, we demonstrate that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing. Small-RNA libraries from two control samples and four knockdowns in germline or somatic tissues of the Drosophila melanogaster ovary.

2014-08-14 | E-GEOD-59327 | biostudies-arrayexpress

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