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 3'-end selection through complementary mechanisms with varying degrees of independence from U1 snRNP. Within 3' UTRs, largely independent of U1 snRNP, SRSF1 binds RNA near proximal PASs and promotes their usage. Congruent with this observation, breast cancer tumors with altered SRSF1 levels display shifted 3'-end selection. At PASs modulated by both SRSF1 and U1 snRNP, SRSF1 acts on sites through U1 snRNP-mediated Pol II interactions. Consistent with co-transcriptional regulation, SRSF1 reduces the Pol II elongation index and 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 co-transcriptional coordination.