m(6)A Methylases Regulate Myoblast Proliferation, Apoptosis and Differentiation

SIMPLE SUMMARY: N(6)-methyladenosine (m(6)A) is the most prevalent methylation modification in eukaryotic mRNA, and it plays an important role in regulating gene expression. Previous studies found that m(6)A methylation plays a role in mammalian skeletal muscle development. Skeletal muscle is an important factor that regulates livestock muscle quality and maintains metabolic homeostasis, and skeletal myogenesis is regulated by a series of transcription factors. However, the role of m(6)A in bovine skeletal myogenesis is unclear. In this study, we examined the expression patterns of the m(6)A methylase genes METTL3, METTL14, WTAP, FTO and ALKBH5 in bovine skeletal muscle tissue and during myogenesis in myoblasts. Furthermore, we used bovine skeletal muscle myoblasts as the object of study to discover the regulatory role of these genes in the process of skeletal myogenesis in vitro. Our findings indicate that these five m(6)A methylases have pronounced and diverse functions in regulating bovine skeletal myoblast proliferation, apoptosis and myogenic differentiation, which can contribute to further understanding the roles of m(6)A in skeletal muscle development. ABSTRACT: N(6)-methyladenosine (m(6)A) plays an important role in regulating gene expression. Previous studies found that m(6)A methylation affects skeletal muscle development. However, the effect of m(6)A methylases on bovine skeletal myogenesis is still unclear. Here, we found that the expression of m(6)A demethylases (FTO and ALKBH5) was significantly higher in the longissimus dorsi muscle of adult cattle than in newborn cattle. In contrast, the expression of m(6)A methyltransferases (METTL3, METTL14 and WTAP) was reduced. The mRNA expression of all five genes was found to be increased during the myogenesis of myoblasts in vitro. Knockdown of FTO or METTL3 promoted myoblast proliferation, inhibited myoblast apoptosis and suppressed myogenic differentiation, whereas ALKBH5 knockdown had the opposite effect. METTL14 knockdown enhanced myoblast proliferation and impaired myogenic differentiation. WTAP knockdown attenuated proliferation and contributed to apoptosis but did not affect differentiation. Furthermore, the functional domains of these five m(6)A methylases are conserved across species. Our results suggest that m(6)A methylases are involved in regulating skeletal muscle development and that there may be a complex network of m(6)A methylation regulating skeletal myogenesis.