Identification of RBPMS as a smooth muscle master splicing regulator via association of its gene with super-enhancers

Abstract

Alternative splicing (AS) is primarily regulated by regulatory RNA-binding proteins (RBPs). It has been suggested that a small number of master splicing regulators might control cell-specific programs and these regulators could be identified via the association of their genes with transcriptional super-enhancers. Using this approach, RNA Binding Protein with Multiple Splicing (RBPMS) was identified as a critical splicing regulator in vascular smooth-muscle cells (SMCs). RBPMS is strongly downregulated during SMC dedifferentiation and is responsible for nearly 20% of the AS changes during this transition as indicated by mRNA-Seq of rat PAC1 cells with RBPMS-manipulated levels. RBPMS overexpression also promoted splicing events that are usually only observed in tissue SMCs. RBPMS targeted a network of proteins involved in the cytoskeleton and cell-adhesions, machineries remodelled during the transition from contractile to motile-dedifferentiated SMCs. RBPMS directly regulated target exons with a positional bias depending upon whether acting as an activator or repressor, as indicated by RBPMS-maps, in vivo transfections with minigene reporters, RBPMS RNA binding mutant, MS2 artificial tethering and lastly in vitro binding assays. RBPMS controlled splicing and activity of other splicing and post-transcriptional regulators (MBNL1, MBNL2 and LSM14B) as well as the key SMC transcription factor Myocardin. Structure-function analyses revealed that the two major RBPMS isoforms (RBPMS-A and B) have differential activity, and that dimerization and RBPMS C- terminus are essential to RBPMS splicing activity. Yet, RBPMS RRM was insufficient for splicing. In fact, a core section of the C-terminus of RBPMS-B antagonized its repressor-splicing activity. Additionally, two threonine residues of RBPMS could be phosphorylated differentially modulating RBPMS isoforms activity. Therefore, this study provides the strongest evidence to date for a molecular function of RBPMS as a splicing-regulator, matching many of the expected criteria of a master regulator of AS in differentiated VSMCs.