Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Jason Hubbard
PROVIDER: E-GEOD-23522 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:To gain global insights into the role of the well-known repressive splicing regulator PTB we analyzed the consequences of PTB knockdown in HeLa cells using high-density oliogonucleotide splice-sensitive microarrays. The major class of identified PTB-regulated splicing event was PTB-repressed cassette exons, but there was also a substantial number of PTB-activated splicing events. PTB repressed and activated exons showed a distinct arrangement of motifs with pyrimidine-rich motif enrichment within and upstream of repressed exons, but downstream of activated exons. The N-terminal half of PTB was sufficient to activate splicing when recruited downstream of a PTB-activated exon. Moreover, insertion of an upstream pyrimidine tract was sufficient to convert a PTBactivated to a PTB-repressed exon. Our results demonstrate that PTB, an archetypal splicing repressor, has variable splicing activity that predictably depends upon its binding location with respect to target exons. Target was prepared from 3 biological replicates of PTB/nPTB knockdown and 3 control mock knockdowns from HeLa S3 cell line and hybridized to a custom Affymetrix array containing exon and exon-junction probes for more than 30,000 human genes
2010-09-01 | E-GEOD-23513 | biostudies-arrayexpress
Project description:To gain global insights into the role of the well-known repressive splicing regulator PTB we analyzed the consequences of PTB knockdown in HeLa cells using high-density oliogonucleotide splice-sensitive microarrays. The major class of identified PTB-regulated splicing event was PTB-repressed cassette exons, but there was also a substantial number of PTB-activated splicing events. PTB repressed and activated exons showed a distinct arrangement of motifs with pyrimidine-rich motif enrichment within and upstream of repressed exons, but downstream of activated exons. The N-terminal half of PTB was sufficient to activate splicing when recruited downstream of a PTB-activated exon. Moreover, insertion of an upstream pyrimidine tract was sufficient to convert a PTBactivated to a PTB-repressed exon. Our results demonstrate that PTB, an archetypal splicing repressor, has variable splicing activity that predictably depends upon its binding location with respect to target exons. Target was prepared from 6 biological replicates of PTB/nPTB knockdown and 6 control mock knockdowns from HeLa S3 cell line and hybridized to the Affymetrix Human Exon 1.0 ST Array. Two groups of three replicates each were collected at two different times.
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Project description:This work provides the first evidence that Qk is a global regulator of splicing in vertebrates, defines a new splicing regulatory network in muscle, and suggests that overlapping splicing networks contribute to the complexity of changes in alternative splicing during differentiation. Alternative splicing contributes to muscle development and differentiation, but the complete set of muscle splicing factors and their combinatorial interactions are not known. Previously work identifies ACUAA (STAR motif) as an enriched sequence near muscle-specific alternative exons such as Capzb exon 9. We did mass spectrometry of proteins selected by wild type and mutant Capzb intron 9 RNA affinity chromatography, and identified Quaking (Qk), a protein known to regulate mRNA function through ACUAA motifs in 3' UTRs. We show that in myoblasts, Qk promotes inclusion of Capzb exon 9 in opposition to repression by PTB. Qk knockdown in myoblasts has little effect on transcript levels, but alters inclusion of 824 cassette exons whose adjacent intron sequences are enriched in ACUAA motifs. During differentiation to myotubes, Qk levels increase 2-3 fold, suggesting a mechanism for Qk-responsive exon regulation. We captured the PTB splicing regulatory network and intersected it with the Qk network, identifying overlap between the functions of Qk and PTB. Approximately 60% of exons whose inclusion is altered during myogenesis appear to be under control of one or both of these splicing factors in myoblasts. This series is the C2C12 Qk and PTB siRNA data. It is 12 arrays: 3 PTB siRNA arrays , 3 Qk siRNA arrays, and 6 mock siRNA arrays.
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Project description:Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, cross-linking and immunoprecipitation coupled with high-throughput sequencing, and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins, and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and auto-regulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells. In triplicate, polyA-selected RNA was extracted from control, hnRNP A1, hnRNP A2/B1, hnRNP H1, hnRNP F, hnRNP M, and hnRNP U siRNA treated human 293T cells, and hybridized to custom splice-junction arrays
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Project description:This work provides the first evidence that Qk is a global regulator of splicing in vertebrates, defines a new splicing regulatory network in muscle, and suggests that overlapping splicing networks contribute to the complexity of changes in alternative splicing during differentiation. Alternative splicing contributes to muscle development and differentiation, but the complete set of muscle splicing factors and their combinatorial interactions are not known. Previously work identifies ACUAA (STAR motif) as an enriched sequence near muscle-specific alternative exons such as Capzb exon 9. We did mass spectrometry of proteins selected by wild type and mutant Capzb intron 9 RNA affinity chromatography, and identified Quaking (Qk), a protein known to regulate mRNA function through ACUAA motifs in 3' UTRs. We show that in myoblasts, Qk promotes inclusion of Capzb exon 9 in opposition to repression by PTB. Qk knockdown in myoblasts has little effect on transcript levels, but alters inclusion of 824 cassette exons whose adjacent intron sequences are enriched in ACUAA motifs. During differentiation to myotubes, Qk levels increase 2-3 fold, suggesting a mechanism for Qk-responsive exon regulation. We captured the PTB splicing regulatory network and intersected it with the Qk network, identifying overlap between the functions of Qk and PTB. Approximately 60% of exons whose inclusion is altered during myogenesis appear to be under control of one or both of these splicing factors in myoblasts. This series is the C2C12 differentiation data. It is 9 arrays, 3 timepoints, with 3 replicates. The time points are 0 hrs, 24 hrs, and 72hrs.
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Project description:Alternative splicing of pre-mRNA is a prominent mechanism to generate protein diversity, yet its regulation is poorly understood. Here, we demonstrate a direct role for histone modifications in alternative splicing. We find distinctive histone modification signatures which correlate with splicing outcome in a set of human genes. Modulation of histone modifications causes splice site switching. The mechanism for histone-mediated splice site selection involves a histone mark which is read by a chromatin protein, which in turn recruits a splicing regulator. These results outline an adaptor system for reading of histone marks by the pre-mRNA splicing machinery. To obtain an estimate of how many PTB-dependent alternative splicing events are regulated by SET2/MRG15-mediated recruitment of PTB, we carried out a genomewide comparative analysis of alternative splicing in hMSC cells depleted of either SETD2, MRG15 or PTB using specific siRNAs, or mock-depleted using a control siRNA.
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