Alternative splicing factor hnRNPA1 induces U2AF2 association with Alu-derived RNA

Alternative splicing drives transcriptome and proteome diversification. While splicing regulatory proteins govern this process, their global mechanisms remain enigmatic. We generated high resolution transcriptome-wide protein-RNA interaction maps to determine how the splicing repressor hnRNPA1 influences the global association of spliceosome assembly factor U2AF2 and SRSF1 with pre-mRNA. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3' splice sites of cassette exons but not constitutive exons upon hnRNPA1 overexpression. By contrast, SRSF1 crosslinking patterns relative to splice sites are independent of hnRNPA1 expression levels. We also observed an hnRNPA1-dependent increase in U2AF2 but not SRSF1 crosslinking to exon proximal antisense Alu elements. Thus Alu elements can serve as splicing factor-responsive sinks for U2AF2. These results not only demonstrate a novel mechanism for alternative splicing regulation but also implicate retrotransposon-derived sequences in the evolution of species-specific alternative splicing. Overall design: Perform iCLIP and RNA-seq from cells expressing endogenous or overexpressed levels of hnRNP A1. iCLIP was performed on hnRNP A1 and two splicing factors seen to be influenced by modulation of hnRNP A1 levels in vitro, SRSF1 and U2AF2. RNA-seq was performed to determine splicing changes between two conditions for comparison to changes seen between iCLIP datasets under same conditions. These changes are summarized globally, as well as at observed splicing changes, and on representative examples.