A unified activation mechanism for mRNA 3' end processing and splicing mediated by SR superfamily proteins

Alternative mRNA processing is a critical mechanism for proteome expansion and gene regulation in higher eukaryotes. The SR family proteins play important roles in splicing regulation. Intriguingly, mammalian genomes encode many poorly characterized SR-like proteins, including subunits of the mRNA 3' processing factor CFIm, CFIm68 and CFIm59. Here we demonstrate that CFIm functions as an enhancer-dependent activator of mRNA 3' processing. CFIm regulates global alternative polyadenylation (APA) by specifically binding and activating enhancer-containing poly(A) sites (PAS). Importantly, the CFIm activator functions are mediated by the arginine-serine repeat (RS) domains of CFIm68/59, which bind specifically to an RS-like region in the CPSF subunit Fip1, and this interaction is modulated by phosphorylation. The remarkable functional similarities between CFIm and SR proteins suggest that interactions between RS-like regions in regulatory and core factors may provide a common activation mechanism for mRNA 3' processing, splicing, and potentially other steps in RNA metabolism. Overall design: Total RNAs from control HEK293T cells, CFIm59 KO cell lines (2 different cell lines), and CFIm68 KO cell lines (2 different cell llines) were fragmented, and reverse transcribed with an oligo(dT) primer containing a linker sequence. cDNAs were circularized, re-linearized by restriction digestion, and PCR amplified to generate libraries. The libraries were sequenced on HiSeq 2000 (single end 150 nucleotides).