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N   6  -methyladenosine (m 6 A) is the most prevalent methylation modification of eukaryotic mRNA, and it plays an important role in regulating gene expression. Previous studies have found that m6A methylation plays a role in mammalian skeletal muscle development. However, the effect of m 6 A on bovine skeletal myogenesis are still unclear. Here, we selected proliferating myoblasts (GM) and differentiated myotubes (on the 4th day of differentiation, DM) for m 6 A-seq and RNA-seq to explore the m 6 A methylation modification pattern during bovine skeletal myogenesis. m 6 A-seq analysis revealed that m 6 A methylation was an abundant modification of the mRNA in bovine myoblasts and myotubes. We scanned 5,691–8,094 m 6 A-modified transcripts, including 1,437 differentially methylated genes (DMGs). GO and KEGG analyses revealed that DMGs were primarily involved in transcriptional regulation and RNA metabolism, as well as insulin resistance and metabolic pathways related to muscle development. The combined analysis further identified 268 genes that had significant changes at both m 6 A and mRNA levels, suggesting that m 6 A modification may regulate myoblast differentiation by mediating the expression of these genes. Furthermore, we experimentally confirmed four genes related to myogenesis, including MYOZ2, TWIST1, KLF5 and MYOD1, with differential changes in both m 6 A and mRNA levels during bovine myoblast differentiation, indicating that they can be potential candidate targets for m 6 A regulation of skeletal myogenesis. Our results may provide new insight into molecular genetics and breeding of beef cattle, and provide a reference for investigating the mechanism of m 6 A regulating skeletal muscle development.

Transcriptome-wide N6-Methyladenosine Methylome Profiling Reveals m6A Regulation of Skeletal Myoblast Differentiation in Cattle (Bos taurus)