The differentiation and regeneration of skeletal muscle from myoblasts to myotubes involves myogenic transcription factors, such as myocardin-related transcription factor A (MRTF-A) and serum response factor (SRF). In addition, post-transcriptional regulation by miRNAs is required during myogenesis. Here, we provide evidence for novel mechanisms regulating MRTF-A during myogenic differentiation. Endogenous MRTF-A protein abundance and activity decreased during C2C12 differentiation, which was attributable to miRNA-directed inhibition. Conversely, overexpression of MRTF-A impaired differentiation and myosin expression. Applying miRNA trapping by RNA affinity purification (miTRAP), we identified miRNAs which directly regulate MRTF-A via its 3′UTR, including miR-1a-3p, miR-206-3p, miR-24-3p and miR-486-5p. These miRNAs were upregulated during differentiation and specifically recruited to the 3′UTR of MRTF-A. Concomitantly, Ago2 recruitment to the MRTF-A 3′UTR was considerably increased, whereas Dicer1 depletion or 3′UTR deletion elevated MRTF-A and inhibited differentiation. MRTF-A protein expression was inhibited by ectopic miRNA expression in murine C2C12 and primary human myoblasts. 3′UTR reporter activity diminished upon differentiation or miRNA expression, whereas deletion of the predicted binding sites reversed these effects. Furthermore, TGF-β abolished MRTF-A reduction and decreased miR-486-5p expression. Our findings implicate miR-24-3p and miR-486-5p in the repression of MRTF-A and suggest a complex network of transcriptional and post-transcriptional mechanisms regulating myogenesis.

中文翻译：

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在成肌细胞成肌分化过程中，miRNA对MRTF-A进行转录后调控。

骨骼肌从成肌细胞到肌管的分化和再生涉及成肌转录因子，例如心肌相关转录因子A（MRTF-A）和血清反应因子（SRF）。此外，在成肌过程中需要通过miRNA进行转录后调控。在这里，我们为成肌分化过程中调节MRTF-A的新机制提供证据。内源性MRTF-A蛋白的丰度和活性在C2C12分化过程中降低，这归因于miRNA定向抑制。相反，MRTF-A的过表达损害了分化和肌球蛋白的表达。应用通过RNA亲和纯化（miTRAP）捕获miRNA，我们鉴定了通过其3'UTR直接调节MRTF-A的miRNA，包括miR-1a-3p，miR-206-3p，miR-24-3p和miR-486-5p 。这些miRNA在分化过程中被上调，并特异性募集到MRTF-A的3'UTR中。同时，Ago2募集至MRTF-A 3'UTR显着增加，而Dicer1耗竭或3'UTR缺失则增加MRTF-A并抑制分化。MRTF-A蛋白表达在小鼠C2C12和原代人成肌细胞中被异位miRNA表达抑制。3'UTR报告基因活性在分化或miRNA表达后减弱，而预测的结合位点的缺失则逆转了这些作用。此外，TGF-β消除了MRTF-A的减少，并减少了miR-486-5p的表达。我们的发现暗示了miR-24-3p和miR-486-5p抑制MRTF-A，并暗示了调控肌发生的转录和转录后机制的复杂网络。