Background:GATA4 (GATA-binding protein 4), a zinc finger–containing, DNA-binding transcription factor, is essential for normal cardiac development and homeostasis in mice and humans, and mutations in this gene have been reported in human heart defects. Defects in alternative splicing are associated with many heart diseases, yet relatively little is known about how cell type– or cell state–specific alternative splicing is achieved in the heart. Here, we show that GATA4 regulates cell type–specific splicing through direct interaction with RNA and the spliceosome in human induced pluripotent stem cell–derived cardiac progenitors.Methods:We leveraged a combination of unbiased approaches including affinity purification of GATA4 and mass spectrometry, enhanced cross-linking with immunoprecipitation, electrophoretic mobility shift assays, in vitro splicing assays, and unbiased transcriptomic analysis to uncover GATA4’s novel function as a splicing regulator in human induced pluripotent stem cell–derived cardiac progenitors.Results:We found that GATA4 interacts with many members of the spliceosome complex in human induced pluripotent stem cell–derived cardiac progenitors. Enhanced cross-linking with immunoprecipitation demonstrated that GATA4 also directly binds to a large number of mRNAs through defined RNA motifs in a sequence-specific manner. In vitro splicing assays indicated that GATA4 regulates alternative splicing through direct RNA binding, resulting in functionally distinct protein products. Correspondingly, knockdown of GATA4 in human induced pluripotent stem cell–derived cardiac progenitors resulted in differential alternative splicing of genes involved in cytoskeleton organization and calcium ion import, with functional consequences associated with the protein isoforms.Conclusions:This study shows that in addition to its well described transcriptional function, GATA4 interacts with members of the spliceosome complex and regulates cell type–specific alternative splicing via sequence-specific interactions with RNA. Several genes that have splicing regulated by GATA4 have functional consequences and many are associated with dilated cardiomyopathy, suggesting a novel role for GATA4 in achieving the necessary cardiac proteome in normal and stress-responsive conditions.

中文翻译：

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转录因子 GATA4 通过与人诱导多能干细胞衍生的心脏祖细胞中的 RNA 直接相互作用来调节细胞类型特异性剪接

背景：GATA4（GATA 结合蛋白 4）是一种含锌指的 DNA 结合转录因子，对于小鼠和人类的正常心脏发育和体内平衡至关重要，并且已报道该基因的突变导致人类心脏缺陷。选择性剪接的缺陷与许多心脏疾病有关，但对于细胞类型或细胞状态特异性的选择性剪接如何在心脏中实现的了解相对较少。在这里，我们证明 GATA4 通过与人类诱导多能干细胞衍生的心脏祖细胞中的 RNA 和剪接体直接相互作用来调节细胞类型特异性剪接。方法：我们利用了公正方法的组合，包括 GATA4 的亲和纯化和质谱分析，增强了通过免疫沉淀交联、电泳迁移率变动测定、体外剪接测定和无偏转录组分析来揭示 GATA4 作为人诱导多能干细胞来源的心脏祖细胞中剪接调节因子的新功能。结果：我们发现 GATA4 与许多成员相互作用人类诱导多能干细胞来源的心脏祖细胞中剪接体复合物的研究。通过免疫沉淀增强交联表明，GATA4 还可以通过特定的 RNA 基序以序列特异性方式直接结合大量 mRNA。体外剪接测定表明，GATA4 通过直接 RNA 结合调节选择性剪接，从而产生功能不同的蛋白质产物。相应地，在人诱导多能干细胞衍生的心脏祖细胞中敲低 GATA4 会导致参与细胞骨架组织和钙离子输入的基因发生差异选择性剪接，并产生与蛋白质亚型相关的功能后果。结论：这项研究表明，除了其GATA4 的转录功能已得到充分描述，它与剪接体复合体的成员相互作用，并通过与 RNA 的序列特异性相互作用来调节细胞类型特异性的选择性剪接。受 GATA4 调节剪接的几个基因具有功能性后果，并且许多基因与扩张型心肌病相关，这表明 GATA4 在正常和应激反应条件下获得必要的心脏蛋白质组方面具有新的作用。