Myocardin regulates exon usage in smooth muscle cells through induction of splicing regulatory factors

Abstract

Differentiation of smooth muscle cells (SMCs) depends on serum response factor (SRF) and its co-activator myocardin (MYOCD). The role of MYOCD for the SMC program of gene transcription is well established. In contrast, the role of MYOCD in control of SMC-specific alternative exon usage, including exon splicing, has not been explored. In the current work we identified four splicing factors (MBNL1, RBPMS, RBPMS2, and RBFOX2) that correlate with MYOCD across human SMC tissues. Forced expression of MYOCD family members in human coronary artery SMCs in vitro upregulated expression of these splicing factors. For global profiling of transcript diversity, we performed RNA-sequencing after MYOCD transduction. We analyzed alternative transcripts with three different methods. Exon-based analysis identified 1637 features with differential exon usage. For example, usage of 3´ exons in MYLK that encode telokin increased relative to 5´ exons, as did the 17 kDa telokin to 130 kDa MYLK protein ratio. Dedicated event-based analysis identified 239 MYOCD-driven splicing events. Events involving MBNL1, MCAM, and ACTN1 were among the most prominent, and this was confirmed using variant-specific PCR analyses. In support of a role for RBPMS and RBFOX2 in MYOCD-driven splicing we found enrichment of their binding motifs around differentially spliced exons. Moreover, knockdown of either RBPMS or RBFOX2 antagonized splicing events stimulated by MYOCD, including those involving ACTN1, VCL, and MBNL1. Supporting an in vivo role of MYOCD-SRF-driven splicing, we demonstrate altered Rbpms expression and splicing in inducible and SMC-specific Srf knockout mice. We conclude that MYOCD-SRF, in part via RBPMS and RBFOX2, induce a program of differential exon usage and alternative splicing as part of the broader program of SMC differentiation.

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

“…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). Indeed, it has been suggested that gastrointestinal dysfunction in myotonic dystrophy is associated with dysregulation of an MBNL1 regulated splicing program in visceral smooth muscle cells (Peterson & Cooper, 2022).…”

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

“…A critical link between RBPMS-regulated SM-AS and the mature VSMC transcriptional network is MYOCD. A recent study indicated that MYOCD transcriptionally upregulates RBPMS in VSMCs leading to SM-AS induction upon over-expression [65]. We have shown that RBPMS, in turn, activates splicing of the VSMC-specific exon 2a in MYOCD [7], which allows for the generation of an N-terminal truncated protein that has been shown, in vitro , to act as the optimal co-factor with SRF to drive expression of the CArG box genes when compared to the non-VSMC isoform (exon 2a skipped) [19].…”

Emerging role of YAP/TAZ in vascular mechanotransduction and disease

“…6,7,26 Since myocardin is suggested to promote its own splicing, reduced myocardin expression may also affect the activity of the expressed myocardin. 22 Dysregulation of the Hippo pathway has been associated with various diseases, including vascular disease. 43,47 Understanding the molecular mechanisms and cellular processes involved in disease progression due to YAP/TAZ dysregulation can lead us to identify novel therapeutic targets.…”