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.

中文翻译：

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circSamd4 抑制 PUR 蛋白的肌源性转录活性。

通过与蛋白质和核酸相互作用，哺乳动物 circRNA 的庞大家族被认为可以影响许多生物过程。在这里，对肌发生过程中差异表达的 circRNA 进行 RNA 测序分析表明，在分化为肌管的小鼠 C2C12 成肌细胞中，circSamd4 表达急剧增加。此外，沉默在人类和小鼠之间保守的circSamd4会延迟两个物种培养的成肌细胞中的肌生成并降低肌源标记物的表达。亲和力下拉和质谱分析显示 circSamd4 与 PURA 和 PURB 相关，PURA 和 PURB 是两种肌生成抑制因子，可抑制肌球蛋白重链 (MHC) 蛋白家族的转录。沉默 circSamd4 增强了 PUR 蛋白与 Mhc 启动子的关联，而过表达 circSamd4 则干扰 PUR 蛋白与 Mhc 启动子的结合，这支持了 circSamd4 可能与 PUR 蛋白复合从而阻止其与 DNA 相互作用的假设。当使用缺乏 PUR 结合位点的突变 circSamd4 时，这些效应被消除。我们的结果表明，PUR 蛋白与 circSamd4 的关联通过促进 MHC 转录的去抑制来增强肌生成。