Unknown,Transcriptomics,Genomics,Proteomics

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Functional splicing network reveals extensive regulatory plasticity of the core Spliceosomal machinery

ABSTRACT: Pre-mRNA splicing relies on the still poorly understood dynamic interplay between more than 150 protein components of the Spliceosome, and the steps at which splicing can be regulated remain largely unknown. Here we systematically analyze the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and use this information to reconstruct a network of functional interactions. The network accurately captures well-established physical and functional associations and identifies new, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during Spliceosome assembly. In contrast with standard models of regulation at early events of splice site recognition, factors involved in catalytic activation of the Spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF and on targets of anti-tumor drugs, and can be widely used to identify mechanisms of splicing regulation. RNA from 3 biological replicates of 72 hours knockdowns of human IK or SMU1 and a control set were used. Changes between the control and knockdowns were measured based on using a splice-junction array (Affymetrix HJAY).

ORGANISM(S): Homo sapiens

SUBMITTER: Juan Valcarcel

PROVIDER: E-GEOD-56605 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Alternative RNA splicing analysis in Hep3B cell cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1? (HIF1?) and 2? (HIF2?) to activate gene transcription, which promotes tumor cell survival. 95% of human genes are alternatively spliced, producing RNA isoforms that code functionally distinct proteins. Thus, effective hypoxia response requires increased HIF target gene transcription as well as proper RNA splicing of these HIF target genes. However, it is unclear if and how hypoxia regulates RNA splicing of HIF target genes. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in Hep3B cells and characterized the role of HIF in regulating AS of HIF induced genes. The results indicated that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF target genes including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirmed that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of the PDK1 minigene by other transcription factor in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing, demonstrating a novel role of HIF target gene(s) in regulating RNA splicing of HIF target genes. Implications:This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. We analyzed total RNA from Hep3B cells cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Altanalyze software version 2.0.7. Techinical replicates were performed for Nx and Hx treated Hep3B cells

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Functional splicing network reveals extensive regulatory plasticity of the core Spliceosomal machinery

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