Background:Mutations in genes encoding sarcomeric proteins lead to failures in sarcomere assembly, the building blocks of contracting muscles, resulting in cardiomyopathies that are a leading cause of morbidity and mortality worldwide. Splicing variants of sarcomeric proteins are crucial at different stages of myofibrillogenesis, accounting for sarcomeric structural integrity. RBM24 (RNA-binding motif protein 24) is known as a tissue-specific splicing regulator that plays an essential role in cardiogenesis. However, it had been unclear if the developmental stage-specific alternative splicing facilitated by RBM24 contributes to sarcomere assembly and cardiogenesis. Our aim is to study the molecular mechanism by which RBM24 regulates cardiogenesis and sarcomere assembly in a temporal-dependent manner.Methods:We ablated RBM24 from human embryonic stem cells (hESCs) using CRISPR/Cas9 techniques.Results:Although RBM24^−/− hESCs still differentiated into sarcomere-hosting cardiomyocytes, they exhibited disrupted sarcomeric structures with punctate Z-lines due to impaired myosin replacement during early myofibrillogenesis. Transcriptomics revealed >4000 genes regulated by RBM24. Among them, core myofibrillogenesis proteins (eg, ACTN2 [α-actinin 2], TTN [titin], and MYH10 [non-muscle myosin IIB]) were misspliced. Consequently, MYH6 (muscle myosin II) cannot replace nonmuscle myosin MYH10, leading to myofibrillogenesis arrest at the early premyofibril stage and causing disrupted sarcomeres. Intriguingly, we found that the ABD (actin-binding domain; encoded by exon 6) of the Z-line anchor protein ACTN2 is predominantly excluded from early cardiac differentiation, whereas it is consistently included in human adult heart. CRISPR/Cas9-mediated deletion of exon 6 from ACTN2 in hESCs, as well as forced expression of full-length ACTN2 in RBM24^−/− hESCs, further corroborated that inclusion of exon 6 is critical for sarcomere assembly. Overall, we have demonstrated that RBM24-facilitated inclusion of exon 6 in ACTN2 at distinct stages of cardiac differentiation is evolutionarily conserved and crucial to sarcomere assembly and integrity.Conclusions:RBM24 acts as a master regulator to modulate the temporal dynamics of core myofibrillogenesis genes and thereby orchestrates sarcomere organization.

中文翻译：

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由 RNA 结合蛋白 RBM24 介导的可变剪接以特定于分化阶段的方式促进心脏肌原纤维生成

背景：编码肌节蛋白的基因突变导致肌节装配失败，肌节是肌肉收缩的组成部分，导致心肌病，这是全球发病率和死亡率的主要原因。肌节蛋白的剪接变体在肌原纤维发生的不同阶段至关重要，这说明了肌节结构的完整性。RBM24（RNA 结合基序蛋白 24）被称为组织特异性剪接调节剂，在心脏发生中起重要作用。然而，尚不清楚 RBM24 促进的发育阶段特异性可变剪接是否有助于肌节组装和心脏发生。我们的目的是研究RBM24以时间依赖性方式调节心脏发生和肌节组装的分子机制。方法：我们消融RBM24使用 CRISPR/Cas9 技术从人类胚胎干细胞 (hESCs) 中提取。结果：尽管RBM24 ^-/- hESCs 仍然分化为承载肌节的心肌细胞，但由于早期肌原纤维生成过程中肌球蛋白置换受损，它们表现出带有点状 Z 线的肌节结构破坏。转录组学揭示了超过 4000 个受RBM24调控的基因. 其中，核心肌原纤维发生蛋白（例如，ACTN2 [α-肌动蛋白 2]、TTN [titin] 和 MYH10 [非肌肉肌球蛋白 IIB]）被错误拼接。因此，MYH6（肌肉肌球蛋白 II）不能替代非肌肉肌球蛋白 MYH10，导致肌原纤维发生在早期肌原纤维阶段停滞并导致肌节破裂。有趣的是，我们发现 Z 线锚蛋白 ACTN2 的 ABD（肌动蛋白结合域；由外显子 6 编码）主要被排除在早期心脏分化之外，而它始终包含在人类成人心脏中。CRISPR/Cas9 介导的hESC 中ACTN2外显子 6 的缺失，以及 RBM24 中全长 ACTN2 的强制表达- ^/-hESCs 进一步证实了外显子 6 的包含对于肌节组装至关重要。总体而言，我们已经证明 RBM24 促进的ACTN2外显子 6 在心脏分化的不同阶段在进化上是保守的，对肌节组装和完整性至关重要。结论：RBM24 作为调节核心肌原纤维发生基因的时间动态的主要调节因子和从而协调肌节组织。