SRSF1 shapes 3′-end site selection with differential dependence on U1 snRNP

Proper polyadenylation site (PAS) selection is critical for RNA isoform determination. Core spliceosomal components, including U1 snRNP, regulate PAS choice, but whether they work with other splicing factors in this role remains unclear. Here, we establish that the splicing factor SRSF1 regulates PAS selection independently of and through interactions with U1 snRNP. Independent of U1 snRNP, SRSF1 binds RNA near proximal PASs within 3' UTRs to promote their usage, and, in line with this observation, breast cancer tumors with altered SRSF1 levels display shifted 3'-end selection. In conjunction with U1 snRNP, SRSF1 acts on PASs through U1 snRNP-mediated SRSF1-Pol II interactions. Consistent with co-transcriptional regulation, SRSF1 reduces the Pol II elongation index and limits transcription readthrough. Together, our results reveal that SRSF1 shapes RNA isoform determination beyond its canonical role in splicing, through a combination of direct RNA binding and U1 snRNP-dependent coordination with Pol II.