m6A modified pre-miR-503-5p contributes to myogenic differentiation through the activation of mTOR pathway

The post-transcriptional regulation of epigenetic modification is a hot topic in skeletal muscle development research. Both m6A modifications and miRNAs have been well-established as crucial regulators in skeletal muscle development. However, the interacting regulatory mechanisms between m6A modifications and miRNAs in skeletal muscle development remain unclear. In this study, miRNA sequencing analysis of goat primary myoblasts (GPMs) pre- and post-differentiation revealed that miR-503-5p was upregulated during myogenic differentiation, and its precursor was identified to contain m6A modification sites. Combined analysis of RIP, qRT-PCR and mRNA stability assay showed that Ythdf2 could recognize and bind the m6A site on pre-miR-503-5p, thereby facilitating the maturation of pre-miR-503-5p in an m6A-dependent manner. Moreover, the overexpression of miR-503-5p significantly inhibits the proliferation of GPMs, promotes myogenic differentiation, and enhances mitochondrial biogenesis while activating the mTOR pathway. However, the suppression of mTOR activity can effectively counteract the accelerated myogenic differentiation induced by miR-503-5p overexpression. Collectively, our results indicate that Ythdf2-dependent m6A modification facilitates the maturation of pre-miR-503-5p, thereby promoting skeletal muscle differentiation through the activation of the mTOR pathway. These insights lay a valuable foundation for further investigation into the complexities of skeletal muscle development and the potential implications of epigenetic regulation in this process.