The role of RNA m⁶A methylation modification in muscle development has been continuously discovered in recent years. The aim of this study was to explore the mRNA expression of m⁶A methylases, including METTL3, METTL14, WTAP, FTO, and ALKBH5, in bovine muscle tissue and in the proliferation and differentiation of skeletal muscle satellite cells (SMSCs). Meanwhile, the changes of m⁶A level during SMSCs myogenic differentiation in vitro were analyzed. This study could provide a reference for clarifying the role and mechanism of m⁶A modification in skeletal muscle development.

The expression of m⁶A methylases in skeletal muscle of newborn and adult cattle was detected by real-time quantitative PCR (RT-qPCR). Then, SMSCs were isolated from the Longissimus dorsi muscle of Qinchuan beef cattle. The proliferation and myogenic differentiation of SMSCs were verified by cell growth curve, immunofluorescence and RT-qPCR, and the temporal expression profiles of m⁶A methylases in proliferation (24 h, 36 h, 48 h, 60 h, and 72 h) and differentiation (Day 0, 2, 4, 6, and 8) of SMSCs were detected by RT-qPCR. Finally, the m⁶A levels during SMSCs differentiation were detected using LC-MS/MS and dot blot assays.

The mRNA expression levels of m⁶A methyltransferases, including METTL3, METTL14, and WTAP, in the Longissimus dorsi muscle, forelegs muscle and hind legs muscle of adult cattle were significantly lower than those of newborn cattle (P<0.01). The mRNA expression of demethylases such as FTO and ALKBH5 in the hind leg muscle of adult cattle was higher (P<0.01), and ALKBH5 was higher in the Longissimus dorsi muscle of adult cattle (P<0.01). The isolated SMSCs had the functions of normal growth, proliferation and myogenic differentiation. The expression of METTL3 decreased gradually in SMSCs proliferation, but increased significantly at 72 h. The expression of METTL14 did not change significantly, while WTAP reached the highest level at 48 h, and then decreased gradually. The temporal expression profiles of FTO and ALKBH5 were similar in the proliferative phase. They did not change obviously before 60 h and increased significantly at 72 h. The expression patterns of METTL3, METTL14 and WTAP were consistent during SMSCs differentiation, with an increase in the early stage of differentiation, followed by a decrease, and an increase in the late stage of differentiation. The expression of FTO increased gradually with differentiation. The expression of ALKBH5 increased during the first 4 days of differentiation and then continuously decreased. Furthermore, the overall m6A level of mRNA declined during the myogenic differentiation in SMSCs (P<0.01).

The expression changes of m⁶A methyltransferases and demethylases in skeletal muscle of newborn and adult cattle were significantly different, suggesting that m⁶A modification might have an important role in the development of skeletal muscle in Qinchuan cattle. Meanwhile, these m⁶A methylases might regulate the proliferation and differentiation of bovine SMSCs. These results provide a theoretical basis for the study of the role and molecular mechanism of m⁶A methylation modifications in regulating skeletal myogenesis.