The requirement of Mettl3-promoted <i>MyoD</i> mRNA maintenance in proliferative myoblasts for skeletal muscle differentiation

Abstract

Myogenic progenitor/stem cells retain their skeletal muscle differentiation potential by maintaining myogenic transcription factors such as MyoD. However, the mechanism of how MyoD expression is maintained in proliferative progenitor cells has not been elucidated. Here, we found that <i>MyoD</i> expression was reduced at the mRNA level by cell cycle arrest in S and G2 phases, which in turn led to the absence of skeletal muscle differentiation. The reduction of <i>MyoD</i> mRNA was correlated with the reduced expression of factors regulating RNA metabolism, including methyltransferase like 3 (Mettl3), which induces N⁶-methyladenosine (m⁶A) modifications of RNA. Knockdown of Mettl3 revealed that <i>MyoD</i> RNA was specifically downregulated and that this was caused by a decrease in processed, but not unprocessed, mRNA. Potential m⁶A modification sites were profiled by m⁶A sequencing and identified within the 5' untranslated region (UTR) of <i>MyoD</i> mRNA. Deletion of the 5' UTR revealed that it has a role in <i>MyoD</i> mRNA processing. These data showed that Mettl3 is required for <i>MyoD</i> mRNA expression in proliferative myoblasts.