Evolutionary conserved circular MEF2A RNAs regulate myogenic differentiation and skeletal muscle development

Circular RNAs (circRNAs) have been recognized as critical regulators of skeletal muscle development. Myocyte enhancer factor 2A (MEF2A) is an evolutionarily conserved transcriptional factor that regulates myogenesis. However, it remains unclear whether MEF2A produces functional circRNAs. In this study, we identified two evolutionarily conserved circular MEF2A RNAs (circMEF2As), namely circMEF2A1 and circMEF2A2, in chicken and mouse muscle stem cells. Our findings revealed that circMEF2A1 promotes myogenesis by regulating the miR-30a-3p/PPP3CA/NFATC1 axis, whereas circMEF2A2 facilitates myogenic differentiation by targeting the miR-148a-5p/SLIT3/ROBO2/β-catenin signaling pathway. Furthermore, in vivo experiments demonstrated that circMEF2As both promote skeletal muscle growth. We also discovered that the linear MEF2A mRNA-derived MEF2A protein binds to its own promoter region, accelerating the transcription of MEF2A and upregulating the expression of both linear MEF2A and circMEF2As, forming a MEF2A autoregulated positive feedback loop. Moreover, circMEF2As positively regulate the expression of linear MEF2A by adsorbing miR-30a-3p and miR-148a-5p, which directly contribute to the MEF2A autoregulated feedback loop. Importantly, we found that mouse circMEF2As are essential for the myogenic differentiation of C2C12 cells. Collectively, our results demonstrated the evolution, function, and underlying mechanisms of circMEF2As in animal myogenesis, which may provide novel insight for both the farm animal meat industry and human medicine.Author summary: Skeletal muscles play important motor and metabolic functions in animals. The development of skeletal muscles is a complex biological process regulated by numerous genes, including MEF2A, a well-known muscle growth regulator. MEF2A messenger RNA-derived protein is a transcription factor that typically controls the expression of myogenic genes. In our study, we uncovered a non-canonical role of the MEF2A gene in skeletal muscle development, wherein it generates two non-coding circRNAs. These circRNAs regulate skeletal muscle myogenesis independently of the MEF2A protein. Circular RNAs have a different structure from linear messenger RNAs and were previously considered an erroneous product of genome transcription. However, our study not only highlights the potential role of MEF2A-derived circular RNAs in skeletal muscle development, but also reveals their evolutionary conservation and expression in multiple species, which proves that circRNAs are not errors to the organisms. In summary, our study demonstrated the multiple roles of the MEF2A gene in skeletal muscle development and provides strong evidence that circRNAs are important for animal physiological regulation.