GSE136004
circSamd4 represses myogenic transcriptional activity of PUR proteins
By interacting with proteins and nucleic acids, the vast family of mammalian circRNAs is proposed to influence many biological processes. Here, RNA sequencing analysis of circRNAs differentially expressed during myogenesis revealed that circSamd4 expression increased robustly in mouse C2C12 myoblasts differentiating into myotubes. Moreover, silencing circSamd4, which is conserved between human and mouse, delayed myogenesis and lowered the expression of myogenic markers in cultured myoblasts from both species. Affinity pulldown followed by quantitative mass spectrometry revealed that circSamd4 associated with PURA and PURB, two repressors of myogenesis that function by impairing transcription of the myosin heavy chain (MYH) protein family. Supporting the hypothesis that circSamd4 might complex with PUR proteins thereby preventing their interaction with DNA, silencing circSamd4 enhanced the association of PUR proteins with the Myh promoter, while overexpressing circSamd4 interfered with the binding of PUR proteins to the MYH promoter. These effects were abrogated when using a mutant circSamd4 lacking the PUR binding site. Our results indicate that the association of PUR proteins with circSamd4 directly contributes to myogenesis by derepressing MYH transcription. Overall design: RNA sequencing followed by circular RNA detection.