Role of mRNA-interacting circRNA in C2C12 muscle cell differentiation

Muscle cell differentiation myogenesis is tightly regulated through precise changes in gene expression via transcriptional and post-transcriptional processes by various transcription factors, RNA-binding proteins, microRNAs, long noncoding RNAs lncRNAs, and recently discovered circular RNAs circRNAs. The past few years have witnessed an exciting increase in studies emphasizing the functional relevance of circRNA by interacting with microRNAs and proteins. However, circRNA interactions with protein-coding mRNAs remain largely unknown. We employed the CliPP-Seq method, wherein AMT-mediated crosslinking of RNA-RNA duplexes was performed followed by poly-A RNA pulldown and high-throughput sequencing to identify mRNA-interacting circRNAs in C2C12 myoblast and myotube cells. Using this method, we identified hundreds of mRNA-interacting circRNAs enriched in the mRNA samples. In addition, custom BLAST analysis of the circRNAs with mRNAs identified their potential interacting partners. Interestingly, the silencing of C2C12 myotube-specific and mRNA-interacting circPde4dip altered the expression of target mRNA Zfp143 and inhibited the differentiation of C2C12 myoblasts into myotubes. In summary, circRNAs may regulate muscle cell differentiation by directly interacting with mRNAs.