转录因子 GATA4 通过与人诱导多能干细胞衍生的心肌祖细胞中的 RNA 直接相互作用调节细胞类型特异性剪接。
Transcription Factor GATA4 Regulates Cell Type-Specific Splicing Through Direct Interaction With RNA in Human Induced Pluripotent Stem Cell-Derived Cardiac Progenitors.
机构信息
Gladstone Institutes, San Francisco, CA (L.Z., K.C., B.G-T., Y-S.A., R.T., N.R.S., L.L., P.Z., Y.H., A.P., F.K., A.P., N.S., A.H., M.W.C., C.A.G., J.G.v.B., R.H., B.R.C., B.G.B., N.J.K., K.S.P., D.S.).
Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, CA (L.Z., B.G-T., Y-S.A., N.R.S., L.L., P.Z., Y.H., A.P., F.K., A.P., N.S., M.W.C., C.A.G., J.G.v.B., B.R.C., B.G.B., D.S.).
出版信息
Circulation. 2022 Sep 6;146(10):770-787. doi: 10.1161/CIRCULATIONAHA.121.057620. Epub 2022 Aug 8.
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.
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稳态至关重要，并且该基因的突变已在人类心脏缺陷中报道。选择性剪接的缺陷与许多心脏疾病有关，但对于心脏中如何实现细胞类型或细胞状态特异性的选择性剪接知之甚少。在这里，我们表明 GATA4 通过与人类诱导多能干细胞衍生的心脏祖细胞中的 RNA 和剪接体的直接相互作用来调节细胞类型特异性剪接。
方法
我们利用了包括 GATA4 的无偏亲和纯化和质谱、增强的免疫沉淀交联、电泳迁移率变动分析、体外剪接分析和无偏转录组分析在内的多种无偏方法的组合，以揭示 GATA4 在人类诱导多能干细胞衍生的心脏祖细胞中作为剪接调节剂的新功能。
结果
我们发现 GATA4 在人类诱导多能干细胞衍生的心脏祖细胞中与剪接体复合物的许多成员相互作用。增强的免疫沉淀交联表明，GATA4 还通过定义的 RNA 基序以序列特异性的方式直接结合大量 mRNA。体外剪接分析表明，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，在人类诱导多能干细胞衍生的心脏祖细胞中敲低 GATA4 导致参与细胞骨架组织和钙离子导入的基因的差异选择性剪接，与与蛋白质异构体相关的功能后果。
结论
这项研究表明，除了其描述良好的转录功能外，GATA4 还与剪接体复合物的成员相互作用，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选择性剪接。几个由 GATA4 调节剪接的基因具有功能后果，并且许多与扩张型心肌病相关，这表明 GATA4 在实现正常和应激反应条件下所需的心脏蛋白质组方面具有新的作用。
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