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Alternative Splicing in Erythropoiesis

ABSTRACT: Differentiating erythroid cells execute a unique gene expression program that insures synthesis of the appropriate proteome at each stage of maturation. Standard expression microarrays provide important insight into erythroid gene expression, but cannot detect qualitative changes in transcript structure, mediated by RNA processing, that alter structure and function of encoded proteins. We analyzed stage-specific changes in the late erythroid transcriptome via use of high resolution microarrays that detect altered expression of individual exons. Ten differentiation-associated changes in erythroblast splicing patterns were identified, including the previously known activation of protein 4.1R exon 16 splicing. Six new alternative splicing switches involving enhanced inclusion of internal cassette exons were discovered, as well as three changes in use of alternative first exons. All of these erythroid stage-specific splicing events represent activated inclusion of authentic annotated exons, suggesting they represent an active regulatory process rather than a general loss of splicing fidelity. The observation that three of the regulated transcripts encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may indicate significant changes in the RNA processing machinery of late erythroblasts. Together these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for late erythroid differentiation. Keywords: Time course; Splicing-sensitive microarray

ORGANISM(S): Homo sapiens

PROVIDER: GSE14588 | GEO | 2009/07/01

SECONDARY ACCESSION(S): PRJNA111707

REPOSITORIES: GEO

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Alternative Splicing in Erythropoiesis

Project description:Differentiating erythroid cells execute a unique gene expression program that insures synthesis of the appropriate proteome at each stage of maturation. Standard expression microarrays provide important insight into erythroid gene expression, but cannot detect qualitative changes in transcript structure, mediated by RNA processing, that alter structure and function of encoded proteins. We analyzed stage-specific changes in the late erythroid transcriptome via use of high resolution microarrays that detect altered expression of individual exons. Ten differentiation-associated changes in erythroblast splicing patterns were identified, including the previously known activation of protein 4.1R exon 16 splicing. Six new alternative splicing switches involving enhanced inclusion of internal cassette exons were discovered, as well as three changes in use of alternative first exons. All of these erythroid stage-specific splicing events represent activated inclusion of authentic annotated exons, suggesting they represent an active regulatory process rather than a general loss of splicing fidelity. The observation that three of the regulated transcripts encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may indicate significant changes in the RNA processing machinery of late erythroblasts. Together these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for late erythroid differentiation. Keywords: Time course; Splicing-sensitive microarray For Exon array hybridizations: 3 biological replicates of day 7, 2 biological replicates of day 10, 3 biological replicates of day 14; For HJAY array hybridizations: 5 biological replicates each were analyzed from day 7 and day 14. One day 14 replicate was deemed an outlier and removed from subsequent analyses.

2010-05-16 | E-GEOD-14588 | biostudies-arrayexpress

RNAi profiling by array of human HeLa cells with hnRNP C siRNA knockdown using a splice-junction microarray to detect changes in alternative splicing.

Project description:poly(A)+ RNA samples from hnRNP C siRNA knockdown and control HeLa cells were compared on a splice-junction microarray to detect changes in alternative splicing. Using the ASPIRE3 algorithm, we detected changes in splicing at 1,340 alternative exons. We observed a similar incidence of increased or decreased exon inclusion in hnRNP C knockdown cells, indicating that hnRNP C can either silence or enhance exon inclusion, respectively. We validated changes at 26 exons by RT-PCR with a 92% success rate.

2010-10-01 | E-MTAB-367 | biostudies-arrayexpress

Regulation of alternative splicing by the core spliceosomal machinery

Project description:Abstract: Alternative splicing (AS) plays a major role in the generation of proteomic diversity and in gene regulation. However, the role of the basal splicing machinery in regulating AS remains poorly understood. Here we show that the core snRNP protein SmB/B’ self-regulates its expression by promoting the inclusion of a highly-conserved alternative exon in its own pre-mRNA that targets the spliced transcript for nonsense-mediated mRNA decay (NMD). Depletion of SmB/B’ in human cells results in reduced levels of snRNPs and in a striking reduction in the inclusion levels of hundreds of alternative exons, with comparatively few effects on constitutive exon splicing levels. The affected alternative exons are enriched in genes encoding RNA processing and other RNA binding factors, and a subset of these exons also regulate gene expression by activating NMD. Our results thus demonstrate a role for the core spliceosomal machinery in controlling an exon network that appears to modulate the levels of many RNA processing factors.

2011-02-01 | GSE26463 | GEO

Mouse Alt-Splice Tissue Panel (Brain vs Non-Brain)

Project description:22 Normal adult mouse tissues on custom alternative transcript sensitive Affymetrix microarray used to address differeneces in tissue specific alternative splicing. Abstract: Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe expression of RNA containing at least two alternative splice junctions for about a third of the 3200 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues, and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif strikingly similar to the branchpoint consensus, but which is located downstream of the 5' splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase (MAGUK) genes reveals that each contains a paralogous tissue regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families. Keywords: Alternative splicing isoform specific, adult mouse tissues

2006-01-19 | GSE3063 | GEO

Regulation of alternative splicing by the core spliceosomal machinery

2011-02-01 | E-GEOD-26463 | biostudies-arrayexpress

Quaking is a global regulator of muscle-specific alternative splicing in vertebrates [differentiation data]

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.

2013-06-05 | E-GEOD-40956 | biostudies-arrayexpress

Quaking is a global regulator of muscle-specific alternative splicing in vertebrates [siRNA data]

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.

2013-06-05 | E-GEOD-40961 | biostudies-arrayexpress

Quaking is a global regulator of muscle-specific alternative splicing in vertebrates [differentiation data]

2013-06-05 | GSE40956 | GEO

Quaking is a global regulator of muscle-specific alternative splicing in vertebrates [siRNA data]

2013-06-05 | GSE40961 | GEO

Global impact of RNA polymerase II elongation inhibition on alternative splicing regulation (expression)

Project description:The rate of RNA polymerase II (pol II) elongation can influence splice site selection in nascent transcripts, yet the extent and physiological relevance of this kinetic coupling between transcription and alternative splicing is not well understood. We performed experiments to perturb pol II elongation and then globally compared alternative splicing patterns with genome-wide pol II occupancy. RNA binding and RNA processing functions were significantly enriched among the genes with pol II elongation inhibition-dependent changes in alternative splicing. Under conditions that interfere with pol II elongation, including cell stress, increased pol II occupancy was detected in the intronic regions flanking the alternative exons in these genes, and these exons generally became more included. A disproportionately high fraction of these exons introduced premature termination codons that elicited nonsense-mediated mRNA decay (NMD), thereby further reducing transcript levels. Our results provide evidence that kinetic coupling between transcription, alternative splicing and NMD affords a rapid mechanism by which cells can respond to changes in growth conditions, including cell stress, to coordinate the levels of RNA processing factors with mRNA levels. In order to identify alternative splicing events influenced by changes in pol II elongation, we performed quantitative alternative splicing microarray profiling (Pan et al., 2004 (PMID 15610736); Shai et al., 2006 (PMID 16403798)) of RNA isolated from stimulated Jurkat T lymphoma cells, cultured separately in the presence or absence of two different drugs that can inhibit pol II elongation: 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) and camptothecin.

2010-12-09 | E-GEOD-25287 | biostudies-arrayexpress

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