Abstract Body: Hypertension affects about half of the US population. Vascular changes that occur in hypertension may be in part due to changes in vascular smooth muscle cell (VSMC) cytoskeletal dynamics and actomyosin interactions. RhoA is a small GTPase that has been shown to modulate VSMC cytoskeletal dynamics by activating Rho kinase (ROCK1/2), which phosphorylates various downstream effectors. Changes in mRNA stability is mediated by RNA binding proteins (RBPs) that bind mRNA transcripts, leading to their stabilization or degradation. This greatly influences the VSMC transcriptome but is an understudied yet key mechanism in the regulation of vascular disease. It has recently been appreciated that m6A modification of mRNA can greatly impact mRNA stability and subsequent protein abundance. Fragile X-related protein 1 (FXR1) is an RBP that our lab has shown to regulate the stability of inflammatory and cytoskeletal RNA. Our hypothesis is that FXR1 regulates RhoA by multiple mechanisms, contributing to changes to cytoskeletal dynamics and VSMC physiology.
Aortas isolated from tamoxifen-injected, VSMC-specific conditional knockout (FXR1smc/smc) mice had significantly decreased RhoA mRNA and protein compared with oil-injected FXR1smc/smc mice as well as FXR1fl/fl and Cre driver control mice. In hVSMCs, when FXR1 is knocked down with siRNA, RhoA mRNA abundance and stability, protein abundance, as well as VSMC contraction are significantly reduced. RNA immunoprecipitation demonstrates that FXR1 binds RhoA mRNA. FXR1 preferentially binds to m6A methylated probes compared with naïve biotinylated probes. When the major m6A methylase METTL3 is knocked down with siRNA, FXR1’s ability to pull down RhoA mRNA is reduced, suggesting that the FXR1-RhoA mRNA interaction is modulated by the methylation state of the RhoA transcript. Mass spectrometry and co-immunoprecipitation demonstrated an interaction between FXR1 and ROCK1. FXR1 deletion reduced phosphorylation of MYPT1, an effector of RhoA/ROCK1 involved in VSMC contraction. Using pull-down assays, when FXR1 is knocked down, ROCK1/RhoA interaction is decreased, implying that FXR1 acts as a scaffold to keep ROCK1 in reactive proximity with RhoA.
We conclude that FXR1 may be an m6A mRNA binding protein and regulates cytoskeletal dynamics by at least 1 of 2 mechanisms: 1- regulation of RhoA mRNA stability, and 2- regulation RhoA activity by reducing its proximity with its effector protein ROCK1.