circMEF2D Negatively Regulated by HNRNPA1 Inhibits Proliferation and Differentiation of Myoblasts via miR-486-PI3K/AKT Axis

Circular RNA (circRNA) is a form of endogenous RNA that can regulate gene expression and participate in the regulation of myogenesis. However, the molecular mechanisms and potential roles of circRNAs in bovine muscle development remain largely unknown. Nevertheless, the RNA splicing factors regulating the biogenesis of bovine circRNA have not yet been characterized. In this study, we identified a novel circRNA, circMEF2D, formed by back-splicing of constitutive exons (exons 5-7) of the bovine MEF2D gene. Functional assays showed that circMEF2D inhibited the proliferation and differentiation of bovine myoblasts. Importantly, we showed that circMEF2D regulated the PI3K-AKT signaling pathway through direct and competitive binding to miR-486. Furthermore, to explore the formation mechanism of circMEF2D, we explored the MEF2D gene alternative splicing progress. Four alternative linear variants of MEF2D were found. Due to its role in alternative splicing, the RNA-binding protein HNRNPA1 was selected for further study and the modulation of HNRNPA1 levels showed that it negatively regulated both back-splicing and linear splicing of MEF2D gene. Overall, in addition to the characterization of bovine circRNAs, these findings revealed the crucial role of HNRNPA1 in MEF2D gene alternative splicing and demonstrated a regulatory circMEF2D-miR-486-PI3K-AKT axis.