Myogenesis is driven by specific changes in the transcriptome that occur during the different stages of muscle differentiation. In addition to controlled transcriptional transitions, several other post-transcriptional mechanisms direct muscle differentiation. Both alternative splicing and miRNA activity regulate gene expression and production of specialized protein isoforms. Importantly, disruption of either process often results in severe phenotypes as reported for several muscle diseases. Thus, broadening our understanding of the post-transcriptional pathways that operate in muscles will lay the foundation for future therapeutic interventions. We employed bioinformatics analysis in concert with the well-established C2C12 cell system for predicting and validating novel miR-1 and miR-206 targets engaged in muscle differentiation. We used reporter gene assays to test direct miRNA targeting and studied C2C12 cells stably expressing one of the cDNA candidates fused to a heterologous, miRNA-resistant 3′ UTR. We monitored effects on differentiation by measuring fusion index, myotube area, and myogenic gene expression during time course differentiation experiments. Gene ontology analysis revealed a strongly enriched set of putative miR-1 and miR-206 targets associated with RNA metabolism. Notably, the expression levels of several candidates decreased during C2C12 differentiation. We discovered that the splicing factor Srsf9 is a direct target of both miRNAs during myogenesis. Persistent Srsf9 expression during differentiation impaired myotube formation and blunted induction of the early pro-differentiation factor myogenin as well as the late differentiation marker sarcomeric myosin, Myh8. Our data uncover novel miR-1 and miR-206 cellular targets and establish a functional link between the splicing factor Srsf9 and myoblast differentiation. The finding that miRNA-mediated clearance of Srsf9 is a key myogenic event illustrates the coordinated and sophisticated interplay between the diverse components of the gene regulatory network.

中文翻译：

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miR-1 / 206下调剪接因子Srsf9以促进C2C12分化。

肌发生是由在肌肉分化的不同阶段发生的转录组的特定变化驱动的。除受控的转录过渡外，其他几种转录后机制还指导肌肉分化。可变剪接和miRNA活性均调节基因表达和特殊蛋白质同工型的产生。重要的是，任一过程的中断通常会导致严重的表型，如几种肌肉疾病所报道的那样。因此，拓宽我们对在肌肉中起作用的转录后途径的理解，将为未来的治疗性干预奠定基础。我们将生物信息学分析与完善的C2C12细胞系统配合使用，以预测和验证参与肌肉分化的新型miR-1和miR-206目标。我们使用报告基因分析法测试了直接对miRNA的靶向作用，并研究了C2C12细胞稳定表达融合了异源，耐miRNA的3'UTR的cDNA候选物之一。我们通过在时程分化实验中测量融合指数，肌管面积和成肌基因表达来监测对分化的影响。基因本体分析显示与RNA代谢相关的推定的miR-1和miR-206靶标高度丰富。值得注意的是，在C2C12分化过程中，几种候选基因的表达水平下降。我们发现剪接因子Srsf9是成肌过程中两个miRNA的直接目标。分化过程中持续的Srsf9表达削弱了肌管的形成，并削弱了早期促分化因子肌生成素以及晚期分化标记肌节肌球蛋白Myh8的诱导。我们的数据揭示了新型的miR-1和miR-206细胞靶标，并在剪接因子Srsf9和成肌细胞分化之间建立了功能联系。miRNA介导的Srsf9清除是一个关键的成肌事件的发现说明了基因调节网络的各个组成部分之间协调而复杂的相互作用。