Differentially expressed genes in myotubes derived from MBNL knockouts hiPSCs generated by CRISPR/Cas9

Alternative splicing has emerged as a fundamental mechanism not only for the diversification of protein isoforms but also for the spatiotemporal control of development. Therefore, a better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of MBNL proteins, a family of tissue-specific splicing regulators whose loss of function is associated with Myotonic Dystrophy type 1, an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the molecular and functional consequences of this loss on the generation of skeletal muscle cells. Our results indicated that MBNL proteins are specifically required for the late myogenic maturation but not for early myogenic commitment. By a transcriptomic analysis, we further demonstrated that MBNL proteins are not only important for the regulation of alternative splicing but also for the regulation of gene expression. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope the loss of function of these MBNL proteins. 9 samples were analyzed with 3 replicates per condition: 3 WT hiPSC-derived myotubes (control), 3 MBNL1(-/-) hiPSC-derived myotubes and 3 MBNL1(-/-); MBNL2(-/-) DKO hiPSC-derived myotubes.

可变剪接（Alternative splicing）已成为一种核心生物学机制，不仅可推动蛋白质异构体的多样化生成，还能实现发育过程的时空精准调控。因此，深入解析该机制的调控规律，不仅有助于阐明基础生物学原理，还能揭示正常剪接网络失调相关疾病的病理机制。本研究借助人多能干细胞（human pluripotent stem cells），在人类肌发生过程中解析了MBNL蛋白家族的功能。该家族属于组织特异性剪接调控因子，其功能丧失与1型强直性肌营养不良（Myotonic Dystrophy type 1）——一种遗传性神经肌肉疾病——密切相关。借助CRISPR/Cas9基因编辑技术，我们构建了MBNL蛋白缺失的诱导性多能干细胞（human-induced pluripotent stem cells, hiPSCs），并评估了该蛋白缺失对骨骼肌细胞生成的分子与功能影响。研究结果显示，MBNL蛋白仅对肌发生的晚期成熟阶段具有特异性必需作用，而不参与早期肌源定型过程。通过转录组学分析，我们进一步证实，MBNL蛋白不仅参与可变剪接调控，还可调控基因表达。综上，本研究阐明了MBNL蛋白在人类肌发生过程中的时序依赖性需求，有望为针对该类蛋白功能丧失的新型治疗策略开发提供指导。本研究共分析9个样本，每组设置3次生物学重复：3组野生型（WT）hiPSC来源的肌管（对照组）、3组MBNL1基因敲除（MBNL1(-/-)）hiPSC来源的肌管，以及3组MBNL1(-/-); MBNL2(-/-)双敲除（DKO）hiPSC来源的肌管。