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Determining the impact of alternative splicing events on transcriptome dynamics

ABSTRACT: Background The complete sequencing of the human genome and its subsequent analysis revealed a predominant role for alternative splicing in the generation of proteome diversity. Splice switching oligonucleotides (SSOs) are a powerful and specific tool to experimentally control alternative splicing of endogenous messenger RNAs in living cells. SSOs also have therapeutic potential to treat diseases that are caused by aberrant splicing. The assignment of biological roles to alternative splicing events of currently unknown function promises to provide a largely untapped source of potential new therapeutic targets. Here we have developed a protocol that combines high sensitivity microarrays with the transfection of SSOs to monitor global changes in gene expression downstream of alternate, endogenous splice events. Findings When applied to a well-characterized splicing event in the Bcl-x gene, the application of high sensitivity microarrays revealed a link between the induction of the Bcl-xS isoform and the repression of genes involved in protein synthesis. Conclusions The strategy introduced herein provides a useful approach to define the biological impact of any given alternative splicing event on global gene expression patterns. Furthermore, our data provide the first link between Bcl-xS expression and the repression of ribosomal protein gene expression.

ORGANISM(S): Homo sapiens

PROVIDER: GSE9851 | GEO | 2008/10/02

SECONDARY ACCESSION(S): PRJNA103827

REPOSITORIES: GEO

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Determining the impact of alternative splicing events on transcriptome dynamics

Project description:Background The complete sequencing of the human genome and its subsequent analysis revealed a predominant role for alternative splicing in the generation of proteome diversity. Splice switching oligonucleotides (SSOs) are a powerful and specific tool to experimentally control alternative splicing of endogenous messenger RNAs in living cells. SSOs also have therapeutic potential to treat diseases that are caused by aberrant splicing. The assignment of biological roles to alternative splicing events of currently unknown function promises to provide a largely untapped source of potential new therapeutic targets. Here we have developed a protocol that combines high sensitivity microarrays with the transfection of SSOs to monitor global changes in gene expression downstream of alternate, endogenous splice events. Findings When applied to a well-characterized splicing event in the Bcl-x gene, the application of high sensitivity microarrays revealed a link between the induction of the Bcl-xS isoform and the repression of genes involved in protein synthesis. Conclusions The strategy introduced herein provides a useful approach to define the biological impact of any given alternative splicing event on global gene expression patterns. Furthermore, our data provide the first link between Bcl-xS expression and the repression of ribosomal protein gene expression. Biological triplicates, 2 conditions: control oligonucleotide, Bcl-x oligonucleotide

2008-10-02 | E-GEOD-9851 | biostudies-arrayexpress

TCERG1 regulates alternative splicing of Bcl-x gene by modulating the rate of RNAPII transcription

Project description:Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative splicing. Pausing sites and changes in the rate of transcription by RNAPII may therefore have a fundamental impact in the regulation of alternative splicing. Here, we show that the elongation and splicing-related factor TCERG1 regulates alternative splicing of the apoptosis gene Bcl-x in a promoter-dependent manner. TCERG1 promotes the splicing of the short isoform of Bcl-x (Bcl-xs) through the SB1 regulatory element located in the first half of exon 2. Consistent with these results, we show evidence for in vitro and in vivo interaction of TCERG1 with the Bcl-x pre-mRNA. Transcription profile analysis reveals that the RNA sequences required for the effect of TCERG1 on Bcl-x alternative splicing coincide with a putative polymerase pause site. Furthermore, TCERG1 modifies the impact of a slow polymerase on Bcl-x alternative splicing. In support of a role for an elongation mechanism in the transcriptional control of Bcl-x alternative splicing, we found that TCERG1 modifies the amount of pre-mRNAs generated at distal regions of the endogenous Bcl-x. Most importantly, TCERG1 affects the rate of RNAPII transcription of endogenous human Bcl-x. We propose that TCERG1 modulates the elongation rate of RNAPII to relieve pausing, thereby activating the pro-apoptotic Bcl-xS 5’ splice site. ChIP-Seq

2012-01-12 | E-GEOD-33716 | biostudies-arrayexpress

TCERG1 regulates alternative splicing of Bcl-x gene by modulating the rate of RNAPII transcription

2012-01-12 | GSE33716 | GEO

Inactivation of the splicing factor RBM10 limits EGFR targeted therapy response in EGFR mutant lung cancer

Project description:Oncogene-driven lung cancers such as those with activating mutations in the epidermal growth factor receptor (EGFR) often harbor additional co-occurring genetic alterations. The significance of most alterations co-occurring with mutant EGFR remains unclear. We report the impact of loss of the mRNA splicing factor RBM10 in human EGFR mutant lung cancer. RBM10 loss decreased EGFR inhibitor efficacy in patient-derived EGFR mutant tumor models. RBM10 regulated mRNA splicing of the mitochondrial apoptotic regulator Bcl-x. Genetic inactivation of RBM10 diminished EGFR inhibitor-mediated apoptosis by altering Bcl-x splicing, decreasing Bcl-xS (pro-apoptotic) and increasing Bcl-xL (anti-apoptotic) levels. Co-inhibition of Bcl-xL and mutant EGFR overcomes resistance induced by RBM10 loss. RBM10 loss was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Inactivation of the splicing factor RBM10 is a key co-occurring genetic alteration in EGFR mutant tumors that limits EGFR inhibitor efficacy and a potential biomarker of Bcl-xL inhibitor response.

2022-06-04 | GSE199240 | GEO

Genome-wide analysis of alternative splicing in Caenorhabditis elegans

Project description:Alternative splicing (AS) plays a crucial role in the diversification of gene function and regulation. Consequently, the systematic identification and characterization of temporally regulated splice variants is of critical importance to understanding animal development. We have used high-throughput RNA sequencing and microarray profiling to analyze AS in C. elegans across various stages of development. This analysis identified thousands of novel splicing events, including hundreds of developmentally regulated AS events. To make these data easily accessible and informative, we constructed the C. elegans Splice Browser, a web resource in which researchers can mine AS events of interest and retrieve information about their relative levels and regulation across development. The data presented in this study, along with the Splice Browser, provides the most comprehensive set of annotated splice variants in C. elegans to date, and is therefore expected to faciliate focused, high resolution in vivo functional assays of AS function. Alternative splicing events were identified from alignments of C. elegans mRNA/EST sequences (UniGene Build #26) to C. elegans genomic sequence (NCBI timestamp: Sept. 25, 2006), essentially as previously described (Pan et al. 2005; Pan et al. 2004). In total, 499 cassette type AS events were identified. For each AS event, 3 exon probes and 3 exon junction probes were designed to profile the AS event on the microarray, essentially as previously described (Pan et al. 2004). This submission represents the expression microarray component of the study.

2010-12-14 | E-GEOD-25927 | biostudies-arrayexpress

Heritability of alternative splicing in the human genome

Project description:Alternative pre-mRNA splicing increases proteomic diversity and provides a potential mechanism underlying both phenotypic diversity and susceptibility to genetic disorders in human populations. To investigate the variation in splicing among humans on a genomewide scale, we use a comprehensive exon-targeted microarray to examine alternative splicing in lymphoblasts derived from the CEPH HapMap population. We show the identification of transcripts containing annotated and novel sequence verified exon skipping, intron retention, and cryptic splice site usage that are specific between individuals. Using family-based linkage analysis, we demonstrate Mendelian inheritance and segregation of specific splice isoforms with regulatory haplotypes for three genes. Allelic association was further used to identify individual SNPs or regulatory haplotype blocks linked to the alternative splicing event, taking advantage of the high-resolution genotype information from the CEPH HapMap population. Keywords: Comparative genomic hybridiation within a 3 generation pedigree We used 14 individuals from the 3 generation CEPH/UTAH 1444 pedigree for our analysis of looking at heritability of differentially spliced exons within the family. 3 biological growths of individuals NA12739, NA12740, NA12750, and NA12751 were used and a single biological growth for the remaining 10 individuals.

2007-08-07 | E-GEOD-7952 | biostudies-arrayexpress

MCF10A cells expressing empty vector, siRNA-resistant wild-type and siRNA-resistant catalytically dead TIP60 treated with control or TIP60 siRNA

Project description:Alternative splicing of RNA transcripts can result in a multitude of variations from the genome. Since splicing can occur co-transcriptionally, chromatin structure has been reported to affect the choice of a splice site. TIP60 is a haploinsufficient tumor suppressor gene that is frequently downregulated in cancers. Since TIP60 is a writer of histone and non-histone modifications, we investigated TIP60’s role in modulating alternative splicing. RNA sequencing was performed in TIP60-depleted MCF10A cells and with cells rescued with wild-type or catalytically inactive TIP60. The majority of the differential splicing events were skipped exon events, including exon 25 skipping event of ITGA6. To identify the splicing factor that regulates this event, a siRNA screen of 77 splicing factors was employed. TIP60 depletion results in an increase in the ITGA6 isoform that lacks exon 25 and the siRNA screen identified ESRP2 to regulate splicing of ITGA6 exon 25. This study suggests that alternative splicing of ITGA6 exon 25 is regulated by the TIP60-ESRP2 axis.

2023-06-19 | GSE152863 | GEO

Heritability of alternative splicing in the human genome

2007-08-08 | GSE7952 | GEO

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. Examination of alternative splicing changes between germline- and somatic-cell enriched samples as well as nonsense-mediated decay mutants.

2015-04-22 | E-GEOD-64672 | biostudies-arrayexpress

Gene expression and alternative splicing in pancreatic ductal adenocarcinoma (PDAC) [gene level]

Project description:Alternative splicing is a key event to human transcriptome and proteome diversity and complexity. Recent evidence suggests that pancreatic cancer might possess particular patterns of splice variation that influence the function of individual genes contributing to tumour progression in this disease. The identification of new pancreatic cancer-associated splice variants would offer opportunities for novel diagnostics and potentially also represent novel therapeutic targets. In this dataset, we investigated the alterations in the splicing machinery in pancreatic adenocarcinoma (PDAC) specimens with full clinicopathological details, in comparisons to adjacent pancreatic tissues and normal tissues from donors.

2014-11-10 | GSE56560 | GEO

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