An RBPMS-driven splicing regulatory axis including MBNL1, RBFOX2, and QK promotes smooth muscle cell contractile identity.

Phenotype switching of vascular smooth muscle cells (SMCs) between contractile and more proliferative and motile states is associated with cardiovascular disease and is underpinned by transcriptional and alternative splicing (AS) programs. We previously showed the RNA-binding protein (RBP) RNA Binding Protein with Multiple Splicing (RBPMS) to be a master regulator of AS in differentiated SMCs. Although changes in master regulator activities can drive AS programs, such proteins rarely act alone. Here we investigated how MBNL1, RBFOX2, and QK act as coregulators with RBPMS to promote contractile smooth muscle AS and phenotypic properties. All four RBPs largely promoted contractile phenotype splicing, with RBPMS showing the highest degree of alignment with the program. Coregulated splicing events were enriched for functions associated with actin filaments and focal adhesions indicating RBPMS-coordinated remodelling of the cellular contractile and motility machinery. Strikingly, while knockdown of each RBP affected various cell morphological and functional properties, knockdown of RBPMS alone induced all aspects of phenotype switching, including lower contraction, higher proliferation, and motility. Our results highlight how a master regulatory RBP can guide an axis of more widely expressed regulators to drive key cellular phenotype changes independently of a transcriptional program.