RBPMS promotes contractile smooth muscle splicing and alters phenotypic behaviour of human embryonic stem cell derived vascular smooth muscle cells

Abstract

Differentiated Vascular Smooth Muscle Cells (VSMCs) express a unique network of splice isoforms (smooth muscle specific alternative splicing - SM-AS) in functionally critical genes including those comprising the contractile machinery. We previously described RNA Binding Protein Multiple Splicing (RBPMS) as a potent driver of contractile, aortic tissue like SM-AS in VSMCs using rodent models. What is unknown is how RBPMS affects VSMC phenotype and behaviour. Here, we use human embryonic stem cell-derived VSMCs (hES-VSMCs) to dissect the role of RBPMS in SM-AS in human cells and determine the impact on VSMC phenotypic properties. hES-VSMCs are inherently immature and display only partially differentiated SM-AS patterns while RBPMS levels are undetectable endogenously. Hence, we used an over-expression system and found that RBPMS induces SM-AS patterns in hES-VSMCs akin to the contractile tissue VSMC splicing patterns in multiple events. We present in silico and experimental findings that support RBPMS splicing activity as mediated through direct binding and via functional cooperativity with splicing factor RBFOX2 on a significant subset of targets. Finally, we demonstrate that RBPMS is capable of altering the motility and the proliferative properties of hES-VSMCs to mimic a more differentiated state. Overall, this study emphasizes a critical splicing regulatory role for RBPMS in human VSMCs and provides evidence of phenotypic modulation by RBPMS.

Mechanism of an alternative splicing switch mediated by cell-specific and general splicing regulators

“…However, in human breast cancer cells RBFOX1 promoted skipping of the same exon, as part of epithelial-mesenchymal transition (Li et al, 2018). Other lines of evidence also suggest widespread functional cooperation of RBPMS and RBFOX proteins including: enrichment around RBPMS-regulated exons not only of RBPMS dual CAC binding motifs but also of RBFOX binding GCAUG motifs (Jacob et al, 2022); the identification of a GCAUG containing motif as the top intronic binding site for RBPMS in ES cells (Bartsch et al, 2021); and the association of both RBFOX and RBPMS binding motifs with ERG (E26 related gene protein)-repressed exons in HeLa cells (Saulnier et al, 2021). Furthermore, the SMC transcription factor MYOCD was recently found to indirectly drive a set of SMC AS changes via changes in the expression levels of RBPMS, RBFOX2 and MBNL1 (Liu et al, 2022), and both Rbpms and Mbnl1 were found by scRNA-Seq to be part of a contractile VSMC gene signature (Dobnikar et al, 2018).…”