mRNA mC inhibits adipogenesis and promotes myogenesis by respectively facilitating YBX2 and SMO mRNA export in ALYREF-mC manner.

Although 5-methylcytosine (mC) has been identified as a novel and abundant mRNA modification and associated with energy metabolism, its regulation function in adipose tissue and skeletal muscle is still limited. This study aimed at investigating the effect of mRNA mC on adipogenesis and myogenesis using Jinhua pigs (J), Yorkshire pigs (Y) and their hybrids Yorkshire-Jinhua pigs (YJ). We found that Y grow faster than J and YJ, while fatness-related characteristics observed in Y were lower than those of J and YJ. Besides, total mRNA mC levels and expression rates of NSUN2 were higher both in backfat layer (BL) and longissimus dorsi muscle (LDM) of Y compared to J and YJ, suggesting that higher mRNA mC levels positively correlate with lower fat and higher muscle mass. RNA bisulfite sequencing profiling of mC revealed tissue-specific and dynamic features in pigs. Functionally, hyper-methylated mC-containing genes were enriched in pathways linked to impaired adipogenesis and enhanced myogenesis. In in vitro, mC inhibited lipid accumulation and promoted myogenic differentiation. Furthermore, YBX2 and SMO were identified as mC targets. Mechanistically, YBX2 and SMO mRNAs with mC modification were recognized and exported into the cytoplasm from the nucleus by ALYREF, thus leading to increased YBX2 and SMO protein expression and thereby inhibiting adipogenesis and promoting myogenesis, respectively. Our work uncovered the critical role of mRNA mC in regulating adipogenesis and myogenesis via ALYREF-mC-YBX2 and ALYREF-mC-SMO manners, providing a potential therapeutic target in the prevention and treatment of obesity, skeletal muscle dysfunction and metabolic disorder diseases.