RNA-seq of P19 cells after knockdown of Srsf3, Srsf7 and Cpsf6 and after differentiation into neuronal cells
Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3' untranslated regions (3'UTRs). APA regulates stage- and tissue-specific gene expression by affecting the stability, subcellular localization and translation rate of transcripts. We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained, however, unknown. Here, we combined iCLIP, RNA-Seq and 3'-end sequencing to find that both proteins bind upstream of proximal PASs (pPASs), yet they exert opposite effects on 3'UTR length. We show that SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, thereby generating short 3'UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, and hypo-phosphorylation contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3'UTRs by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon reduced expression of SRSF3, CFIm levels strongly decrease and 3'UTRs are globally shortened. In SRSF3-regulated transcripts, CFIm and FIP1 bind upstream of dPASs and promote their usage. Surprisingly, both factors are also recruited to pPASs under conditions where their usage is blocked, suggesting the formation of inactive cleavage complexes. Thus, we identify SRSF3 as a novel regulator of CFIm activity, provide evidence that CFIm inhibits pPAS usage and show that small differences in the domain architecture of SR proteins confer opposite effects on PAS selection. Overall design: P19 cells were either depleted of SRSF3, SRSF7 and CPSF6 using esiRNAs or differentiated into neuronal cells using retinoic acid. 2-3 biological replicates each were subjected to RNA-seq.