The ubiquitin proteasomal system is a critical regulator of muscle physiology, and impaired UPS is key in many muscle pathologies. Yet, little is known about the function of deubiquitinating enzymes (DUBs) in the muscle cell context. We performed a genetic screen to identify DUBs as potential regulators of muscle cell differentiation. Surprisingly, we observed that the depletion of ubiquitin-specific protease 18 (USP18) affected the differentiation of muscle cells. USP18 depletion first stimulated differentiation initiation. Later, during differentiation, the absence of USP18 expression abrogated myotube maintenance. USP18 enzymatic function typically attenuates the immune response by removing interferon-stimulated gene 15 (ISG15) from protein substrates. However, in muscle cells, we found that USP18, predominantly nuclear, regulates differentiation independent of ISG15 and the ISG response. Exploring the pattern of RNA expression profiles and protein networks whose levels depend on USP18 expression, we found that differentiation initiation was concomitant with reduced expression of the cell-cycle gene network and altered expression of myogenic transcription (co) factors. We show that USP18 depletion altered the calcium channel gene network, resulting in reduced calcium flux in myotubes. Additionally, we show that reduced expression of sarcomeric proteins in the USP18 proteome was consistent with reduced contractile force in an engineered muscle model. Our results revealed nuclear USP18 as a critical regulator of differentiation initiation and maintenance, independent of ISG15 and its role in the ISG response.

泛素蛋白酶体系统是肌肉生理学的关键调节因子，受损的 UPS 是许多肌肉病理的关键。然而，人们对去泛素化酶 (DUB) 在肌肉细胞中的功能知之甚少。我们进行了基因筛选，以确定 DUB 作为肌肉细胞分化的潜在调节剂。令人惊讶的是，我们观察到泛素特异性蛋白酶 18 (USP18) 的消耗影响了肌肉细胞的分化。 USP18 耗尽首先刺激分化起始。后来，在分化过程中，USP18 表达的缺失导致肌管维持失效。 USP18 酶功能通常通过从蛋白质底物中去除干扰素刺激基因 15 (ISG15) 来减弱免疫反应。然而，在肌肉细胞中，我们发现 USP18（主要是细胞核）独立于 ISG15 和 ISG 反应调节分化。通过探索 RNA 表达谱和其水平取决于 USP18 表达的蛋白质网络的模式，我们发现分化起始伴随着细胞周期基因网络表达的减少和肌源性转录 (co) 因子表达的改变。我们发现 USP18 耗竭改变了钙通道基因网络，导致肌管中的钙通量减少。此外，我们发现 USP18 蛋白质组中肌节蛋白表达的减少与工程肌肉模型中收缩力的减少是一致的。我们的结果表明核 USP18 是分化起始和维持的关键调节因子，独立于 ISG15 及其在 ISG 反应中的作用。