GSE98836
EWSR1 influences alternative splicing through direct and indirect mechanisms
FET family RNA binding proteins are implicated in cancer and neurological disorders and yet their biological function is incompletely understood. We used cross-linking and immunoprecipitation followed by high-throughput sequencing (HITS-CLIP) as well as gene expression and alternative splicing analysis by RNAseq to characterize the molecular function of the FET family RNA binding protein EWSR1. In HeLA cells, EWSR1 binding is enriched at 3'UTRs and at both 5' exon/intron and 3' intron/exon boundaries.  RNAs bound by EWSR1 are functionally enriched for RNA binding and RNA processing genes, suggesting a key role for EWSR1 in RNA regulation. While EWSR1 knock-down altered expression of hundreds of genes, only a subset of the transcripts for these genes were directly bound by EWSR1. By overlapping the HITS-CLIP data with the RNAseq splicing analysis we identified genes where EWSR1 binding is more likely to directly affect splicing.  EWSR1 regulated splicing events were varied and included exon inclusion, alternative 3 exon usage and intron retention. These  results indicate that EWSR1 has pleiotropic effects on splicing and that these effects are caused both by direct regulation of splicing and through effects on a large number of RNAs that themselves code for splicing effectors. Further understanding of this complex splicing regulation network may be important to disentangle the role of EWSR1 in multiple human diseases. Overall design: CLIP: 2 replicates; RNAseq: 3 Controls (GFP siRNA), 4 Experimental (EWSR1 siRNA)