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Illumina mRNA-Seq of control and hnRNP H knockdown in 293T cells

ABSTRACT: Pre-mRNA splicing is regulated through combinatorial activity of RNA motifs including splice sites and splicing regulatory elements (SREs). Here, we show that the activity of a major class of mammalian SREs is highly sensitive to the strength of the adjacent 5' splice site (5'ss) sequence, and that this has important functional and evolutionary implications. Activity of G-run SREs was higher for intermediate strength 5'ss by ~4-fold relative to weak 5'ss, and by ~1.3-fold relative to strong 5'ss. The dependence on 5'ss strength was supported both by comparative genomics and by microarray and Illumina mRNA-Seq analyses of splicing changes following RNAi against the splicing factor heterogeneous nuclear ribonucleoprotein (hnRNP) H, which binds G-runs. This dependence implies that the responsiveness of exons to changes in hnRNP H levels is a bivariate function of both SRE abundance and 5'ss strength; this relationship may hold also for other splicing factors. This pattern of activity enables G-runs and hnRNP H to buffer the effects of 5'ss mutations, augmenting both the frequency of 5'ss polymorphism and the evolution of new splicing patterns.

ORGANISM(S): Homo sapiens

PROVIDER: GSE16642 | GEO | 2009/09/13

SECONDARY ACCESSION(S): PRJNA116223

REPOSITORIES: GEO

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Illumina mRNA-Seq of control and hnRNP H knockdown in 293T cells

Project description:Pre-mRNA splicing is regulated through combinatorial activity of RNA motifs including splice sites and splicing regulatory elements (SREs). Here, we show that the activity of a major class of mammalian SREs is highly sensitive to the strength of the adjacent 5' splice site (5'ss) sequence, and that this has important functional and evolutionary implications. Activity of G-run SREs was higher for intermediate strength 5'ss by ~4-fold relative to weak 5'ss, and by ~1.3-fold relative to strong 5'ss. The dependence on 5'ss strength was supported both by comparative genomics and by microarray and Illumina mRNA-Seq analyses of splicing changes following RNAi against the splicing factor heterogeneous nuclear ribonucleoprotein (hnRNP) H, which binds G-runs. This dependence implies that the responsiveness of exons to changes in hnRNP H levels is a bivariate function of both SRE abundance and 5'ss strength; this relationship may hold also for other splicing factors. This pattern of activity enables G-runs and hnRNP H to buffer the effects of 5'ss mutations, augmenting both the frequency of 5'ss polymorphism and the evolution of new splicing patterns. Examine mRNA expression in 293T cells following hnRNP H or control siRNA knockdown

2009-09-12 | E-GEOD-16642 | biostudies-arrayexpress

Affymetrix exon array analysis of control and hnRNP H knockdown in 293T cells

Project description:Splice site strength and the presence of splicing regulatory elements (SREs) play central roles in the control of pre-mRNA splicing1-3 and in exon evolution4. However, the degree to which SRE function is dependent or independent of the strength of nearby splice sites is not well understood. Here, we show that the activity of a major class of mammalian SREs is highly sensitive to the strength of the adjacent 5' splice site (5'ss) sequence, with important functional and evolutionary implications. Using extensive splicing reporter assays, we found that activity of identical 'G-run' intronic splicing enhancers (ISEs)5, 6 was higher by ~4-fold for 5'ss of intermediate strength relative to weak 5'ss, and higher by ~1.3-fold relative to strong 5'ss, based on established 5'ss scoring criteria7. This dependence on 5'ss strength was supported both by comparative genomic analyses and by high-throughput transcriptome sequencing analyses of splicing changes following RNAi against the G-run-binding factor heterogeneous nuclear ribonucleoprotein (hnRNP) H. This pattern and an inverse pattern of 5'ss-dependent activity observed for G-run exonic splicing silencers (ESSs) enables G-runs and hnRNP H to buffer 5'ss mutations and to function as effective evolutionary capacitors8 of splicing change. Human exons flanked by G-runs exhibited increased 5'ss polymorphism and a ~30% increased frequency of evolutionary change between constitutive and alternative splicing, supporting a role in facilitating splicing-level evolution. Evolutionary conservation indicated a substantially greater role for intronic elements generally in splicing of exons with weak and intermediate 5'ss, and supported 5'ss strength-dependent activity for several other intronic motifs, suggesting widespread functional and evolutionary differences between exons of differing 5'ss strength. Keywords: gene expression array-based, exon and protein coding gene analysis

2009-09-13 | GSE12386 | GEO

Octamer sequences with HEXplorer score amplitude of -10.35, –0.15 or +10.32

Project description:Correct pre-mRNA processing in higher eukaryotes vastly depends on splice site recognition. Beyond conserved 5’ss and 3’ss motifs, splicing regulatory elements (SREs) play a pivotal role in this recognition process. Here, we present in silico designed sequences with arbitrary a priori prescribed splicing regulatory HEXplorer properties that can be concatenated to arbitrary length without changing their regulatory properties. We performed pulldown analysis to identify RNA-binding proteins, that bind either sequence segments with a HEXplorer score amplitude of +10.32, –0.15 or -10.35.

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Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins

Project description:This SuperSeries is composed of the following subset Series: GSE34992: Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins (splice array) GSE34993: Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins (CLIP-Seq) GSE34995: Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins (RNA-Seq) Refer to individual Series

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Project description:Spliced messages constitute one-fourth of expressed mRNAs in the yeast Saccharomyces cerevisiae, and most mRNAs in metazoans. Splicing requires 5' splice site (5'SS), branch point (BP), and 3' splice site (3'SS) elements, but the role of the BP in splicing control is poorly understood because BP identification remains difficult. We developed a high-throughput method, Branch-seq, to map BP and 5'SS of isolated RNA lariats. Applied to S. cerevisiae, Branch-seq detected 76% of expressed, annotated BPs and identified a comparable number of novel BPs. We used RNA-seq to confirm associated 3'SS locations, identifying some 200 novel splice junctions, including an AT-AC intron. We show that several yeast introns use two or even three different BPs, with effects on 3'SS choice, protein coding potential, or RNA stability and identify novel introns whose splicing changes during meiosis or in response to stress. Together, these findings reveal BP-based regulation and demonstrate unanticipated complexity of splicing in yeast.

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Nuclear Matrix Factor hnRNP U/SAF-A Exerts a Global Control of Alternative Splicing by Regulating U2 snRNP Maturation

Project description:The nuclear matrix associated hnRNP U/SAF-A protein has been implicated in diverse pathways from transcriptional regulation to telomere length control to X inactivation, but the precise mechanism underlying each of these processes has remained elusive. Here, we report hnRNP U as a regulator of SMN2 splicing from a custom RNAi screen. Genome-wide analysis by CLIP-seq reveals that hnRNP U binds virtually to all classes of regulatory non-coding RNAs, including all snRNAs required for splicing of both major and minor classes of introns, leading to the discovery that hnRNP U regulates U2 snRNP maturation and Cajal body morphology in the nucleus. Global analysis of hnRNP U-dependent splicing by RNA-seq coupled with bioinformatic analysis of associated splicing signals suggests a general rule for splice site selection through modulating the core splicing machinery. These findings exemplify hnRNP U/SAF-A as a potent regulator of nuclear ribonucleoprotein particles in diverse gene expression pathways. Examination of hnRNP U regulated splicing in Hela cells with CLIP-seq (two biological replicates) and paired-end RNA-seq (control and hnRNP U knockdown)

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Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins (splicing arrays)

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: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.

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