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.

哺乳动物环状RNA（circRNA）庞大家族可通过与蛋白质及核酸相互作用，被认为可调控诸多生物学过程。本研究通过对肌发生过程中差异表达的环状RNA开展RNA测序分析，发现小鼠C2C12成肌细胞在向肌管分化的过程中，circSamd4的表达量显著上调。此外，人和小鼠间保守的circSamd4经沉默后，会延缓两种物种来源的体外培养成肌细胞的肌发生进程，并降低肌源性标志物的表达水平。后续通过亲和纯化下拉结合定量质谱分析，发现circSamd4可与PURA和PURB结合——这两种蛋白均为肌发生的阻遏因子，通过抑制肌球蛋白重链（MYH）蛋白家族的转录发挥功能。该结果支持如下假说：circSamd4可与PUR蛋白形成复合物，从而阻断其与DNA的结合；沉默circSamd4会增强PUR蛋白与Myh启动子的结合，而过表达circSamd4则会干扰PUR蛋白与MYH启动子的结合。当使用缺失PUR结合位点的突变型circSamd4时，上述效应均被消除。本研究结果表明，PUR蛋白与circSamd4的结合可通过解除MYH转录的阻遏，直接促进肌发生进程。实验整体设计：先进行RNA测序，再开展环状RNA检测分析。