Phosphorylation of the smooth muscle master splicing regulator, RBPMS-A, regulates its splicing activity

We have previously identified RBPMS as a master regulator of alternative splicing in differentiated smooth muscle cells (SMCs), responsible for controlling numerous components critical to differentiated SMC function (Nakagaki-Silva et al., 2019). Therefore, the precise control of this splicing regulator is vital in determining SMC fate. Although RBPMS is transcriptionally downregulated during SMC dedifferentiation, protein activity is also expected to be directly regulated. Post-translational modifications, specifically phosphorylation, provide the cell with a highly dynamic and reversible mechanism to regulate the activity of proteins. Examining publicly available phosphoproteomic databases, I found several RBPMS residues reported to be phosphorylated. In particular, Thr113 and Thr118 are immediately adjacent to the RRM domain, and their phosphorylation could potentially affect RNA-binding, nuclear localization, dimerization, higher-order oligomerization, and splicing regulatory activity. I, therefore, created a series of phosphomimetic (T/E) and non-phosphomimetic (T/A) mutants to explore what effect phosphorylation of these residues might have on splicing. Transfection of these mutant constructs showed that Thr113 and Thr118 are critical in regulating the splicing activity of RBPMS-A, with the T/E mutant showing reduced activity. Furthermore, the RBPMS T/E mutant showed a decrease in nuclear localization which potentially could explain reduced splicing regulation. However, in vitro experiments with the recombinant mutated protein also showed reduced splicing regulation, independent of localization. Recombinant RBPMS-A proteins harboring these phosphomimetic mutations showed a significant reduction in splicing in vitro as well as a reduced RNA-binding and reduced higher-order oligomerization. However, NMR analysis also revealed that the T113/118E peptide acts as an RNA mimic which can loop back and antagonize RNA-binding by the RRM domain. Finally, I identified ERK2 as the most likely kinase responsible for phosphorylation at Thr113 and Thr118. Collectively, these data identify a potential mechanism for rapid modulation of the SMC splicing program in response to external signals during the vascular injury response and atherogenesis.