Co-regulation of the Smooth Muscle Cell Alternative Splicing Programme by the RNA-Binding Proteins RBPMS, RBFOX2, MBNL1, and QKI
The phenotypic switching of vascular smooth muscle cells (VSMCs) from a contractile /differentiated state to a synthetic/ proliferative state is a hallmark of many cardiovascular diseases, driven in part by extensive alternative splicing (AS) regulation. RBPMS, a master splicing regulator in differentiated VSMCs, is responsible for approximately 20% of AS changes observed during dedifferentiation in PAC1 VSMCs (Nakagaki-Silva et al., 2019). However, the cooperative and antagonistic relationships between RBPMS and other RNAbinding proteins (RBPs) in this regulatory network remain unclear. In this study, I investigated RBFOX2, MBNL1, and QKI as potential co-regulators of RBPMS in VSMC AS regulation. RBFOX2 was identified as a key candidate due to enrichment of its GCAUG binding motifs around RBPMS-regulated exons in positions suggesting cooperative regulation. MBNL1 was selected for its physical interaction with RBPMS and overlapping regulation of AS events, while QKI was selected as a potential antagonist of the RBPMSpromoted splicing programme, promoting a more proliferative phenotype. Using RNA-seq following RBP knockdowns, I identified distinct contributions of these RBPs to the VSMC splicing programme. RBPMS, RBFOX2, and MBNL1 predominantly cooperated to promote AS patterns associated with VSMC differentiation. In contrast, QKI exhibited context-dependent regulatory effects, aligning with RBPMS in some cases while acting antagonistically in others. Gene Ontology (GO) analysis confirmed that ASEs regulated by RBPMS and its coregulatory RBPs are linked to key cellular processes involved in phenotype switching. Knockdown RBPs also demonstrated distinct effects on cell morphology and motility. Co-immunoprecipitation (Co-IP) assays demonstrated physical interactions between RBPMS and RBFOX2, MBNL1, and QKI. In the case of RBPMS and RBFOX2 aromatic residues in their intrinsically disordered domains were shown to be essential for both their physical interactions and for their splicing regulatory activities, suggesting that these two proteins interact directly to regulate target splicing events. These findings provide insights into the intricate networks of RBPs involved in AS regulation during VSMC phenotypic plasticity, advancing our understanding of vascular remodelling and offering new avenues for targeted therapeutic strategies in cardiovascular diseases.
Vascular smooth muscle cells (VSMCs), Phenotypic switching, Alternative splicing (AS), RNA-binding proteins (RBPs), RBPMS, RBFOX2, MBNL1, QKI, Cooperative regulation
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