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 mass spectrometry revealed that circSamd4 associated with PURA and PURB, two repressors of myogenesis that inhibit transcription of the myosin heavy chain (MHC) protein family. Supporting the hypothesis that circSamd4 might complex with PUR proteins and thereby prevent their interaction with DNA, silencing circSamd4 enhanced the association of PUR proteins with the Mhc promoter, while overexpressing circSamd4 interfered with the binding of PUR proteins to the Mhc 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 enhances myogenesis by contributing to the derepression of MHC transcription.

Recent developments in high-throughput RNA sequencing methods coupled with innovative bioinformatic tools have uncovered thousands of circular (circ)RNAs. CircRNAs have emerged as a vast and novel class of regulatory RNAs with potential to modulate gene expression by acting as sponges for microRNAs (miRNAs) and RNA-binding proteins (RBPs). The biochemical enrichment of circRNAs by exoribonuclease treatment or by depletion of polyadenylated RNAs coupled with deep-sequencing is widely used for the systematic identification of circRNAs. Although these methods enrich circRNAs substantially, they do not eliminate efficiently nonpolyadenylated and highly-structured RNAs. Here, we describe a method we termed RPAD, based on initial RNase R treatment followed by Polyadenylation and poly(A) + RNA Depletion. These joint interventions drastically depleted linear RNAs leading to isolation of highly pure circRNAs from total RNA pools. By facilitating the isolation of highly pure circRNAs, RPAD enables the elucidation of circRNA biogenesis, sequence, and function.