RNA 结合蛋白 RBM24 通过可变剪接促进心肌成肌发生具有分化阶段特异性。

Alternative Splicing Mediated by RNA-Binding Protein RBM24 Facilitates Cardiac Myofibrillogenesis in a Differentiation Stage-Specific Manner.

机构信息

Department of Life Science (S.H.L., K.-Z.L., L.-Y.S., Y.-C.Y., C.-Y.P., S.-Y.T.), National Taiwan University, Taiwan.

Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli County, Taiwan (P.W.H.).

出版信息

Circ Res. 2022 Jan 7;130(1):112-129. doi: 10.1161/CIRCRESAHA.121.320080. Epub 2021 Nov 24.

DOI:10.1161/CIRCRESAHA.121.320080

PMID:34816743

Abstract

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 from human embryonic stem cells (hESCs) using CRISPR/Cas9 techniques.

RESULTS

Although 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 . 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 in hESCs, as well as forced expression of full-length ACTN2 in 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 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.

摘要

背景

编码肌节蛋白的基因突变导致肌节组装失败，肌节是收缩肌肉的结构基础，从而导致心肌病成为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，构成肌节的结构完整性。RBM24（RNA 结合基序蛋白 24）是一种组织特异性剪接调节剂，在心脏发生中发挥重要作用。然而，尚不清楚 RBM24 促进的发育阶段特异性选择性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

我们使用 CRISPR/Cas9 技术从人胚胎干细胞（hESC）中敲除。

结果

尽管 hESC 仍然分化为含有肌节的心肌细胞，但由于早期肌原纤维发生过程中肌球蛋白替换受损，它们表现出肌节结构破坏，有斑点状 Z 线。转录组学揭示了 >4000 个受调节的基因。其中，核心肌节发生蛋白（例如 ACTN2[α-辅肌动蛋白 2]、TTN[伴肌球蛋白重链]和 MYH10[非肌肉肌球蛋白 IIB]）发生错剪接。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预肌纤维阶段停滞，并导致肌节破坏。有趣的是，我们发现 Z 线锚定蛋白 ACTN2 的 ABD（肌动蛋白结合结构域；由外显子 6 编码）在早期心脏分化中主要被排除在外，而在人类成年心脏中始终包含。CRISPR/Cas9 介导的 hESC 中外显子 6 的缺失以及全长 ACTN2 在 hESC 中的强制表达进一步证实了外显子 6 的包含对于肌节组装至关重要。总的来说，我们已经证明 RBM24 在心脏分化的不同阶段促进中外显子 6 的包含是进化保守的，对于肌节组装和完整性至关重要。