Abstract
Introduction/Background: A dysregulation in vascular smooth muscle cell (VSMC) cytoskeletal components may contribute to impaired contraction, leading to vascular disease. Post-transcriptional mechanisms of mRNA stability is an understudied, yet key mechanism in the regulation of VSMC contractility. m6A mRNA methylation is the most prevalent post-transcriptional modification and recognized to regulate mRNA stability. We recently showed that inducible expression of RNA binding protein FXR1 in injured arteries and stimulated hVSMCs regulates VSMC cytoskeletal gene mRNA stability, and FXR1 smc/smc mice have decreased VSMC contractility and blood pressure. Research Questions/Hypothesis: FXR1 functions as an m6A mRNA reader and regulates the stability of m6A methylated transcripts, altering VSMC contractility. Goals/Aims: To determine if the m6A methylation of mRNA transcripts by m6A methylases METTL3 and METTL14 play an important role in regulating cytoskeletal mRNA abundance and VSMC contractility and if transcript recognition by FXR1 is modified by transcript methylation state. Methods/Approach: m6A profiling was performed to investigate methylation status of hVSMC mRNA transcripts. Contraction assay following knockdown of METTL3 was performed to assess VSMC contraction. RNA immunoprecipitation experiments were performed to identify FXR1- interacting cytoskeletal transcripts following METTL3 knockdown. Biotinylated pulldown experiments were performed to assess FXR1’s interaction with methylated RNA probes. Results/Data: Cytoskeletal-related mRNA are the most highly methylated category of methylated transcripts. FXR1 interacts with methylated rather than unmodified target probes. Knockdown of the m6A methylase, METTL3 resulted in significantly decreased contraction of hVSMC. METTL3 knockdown significantly reduced FXR1-cytoskeletal mRNA interactions. Conclusions: These data support our hypothesis that FXR1 is an m6A methylated mRNA reader which regulates cytoskeletal mRNA stability and VSMC contractility.
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