METTL3 Promotes Proliferation and Myogenic Differentiation Through m <sup>6</sup>A RNA Methylation/YTHDF1/2 Signaling Axis in Myoblasts

Skeletal muscle development has an important impact on muscle-related diseases and domestic animal meat production. The m6A RNA methylation is a common post-transcriptional modification, affecting the development and metabolism of various organs. However, the effect and regulatory mechanism of methyltransferase like 3 (METTL3) on myogenesis are still unclear. Here, we showed that the mRNA levels of METTL3 was greater in skeletal muscles including extensor digitorum longus (EDL), soleus (SOL), tibialis anterior (TA) and gastrocnemius (GAS). Moreover, METTL3 highly expressed in the early stage of myoblast proliferation at hour 0 and the late stage of myoblast differentiation at day 8, indicating it was involved in myogenesis. Interestingly, METTL3 knockdown inhibited myoblast proliferation and myogenic differentiation, whereas METTL3 overexpression promoted these processes. Mechanically, METTL3 overexpression increased the ratio of mRNA m6A/A and shortened the time of P21 and P27 mRNA half level, causing the mRNAs downregulation via reducing their stability. Meanwhile, the promotion of cell proliferation by METTL3 overexpression was attenuated by YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) knockdown. Furthermore, the promotion of myogenic differentiation by METTL3 overexpression was weakened by YTHDF1 knockdown through reducing the mRNA translation of MRFs including MyHC, MyoD and MyoG. Therefore, METTL3 facilitates myoblast proliferation and myogenic differentiation. Overall, these findings suggest that METTL3/m6A RNA methylation/YTHDF1/2 signaling axis is a novel strategy for the regulation of skeletal muscle development.