Investigating Mechanisms of RBM20 Liquid-liquid Phase Separation Driving Cardiomyocyte Physiology and Dilated Cardiomyopathy

项目摘要/摘要 肌肉特异性RNA剪接因子RBM20的突变是最近发现的侵袭性扩张的原因 以严重心律失常为特征的心肌病(DCM)。然而，潜在的机制仍然是 不清楚，因此没有可用的治疗方法。我们最近发现了核RBM20剪接的存在 使来自不同染色体的多个共同调节的DNA基因座接近的工厂。这一点的形成 染色质的三维结构依赖于RBM20焦点，这些焦点由其主要剪接靶点 编码巨型蛋白titin(TTN)的前信使核糖核酸。我的初步数据显示，RBM20病灶经历了 液-液相分离(LLP)，一种被认为有助于分离和调节 亚核舱。我的初步数据还表明，RBM20有限合伙人在RBM20 DCM中受到干扰 变种人。因此，在这项提议中检验的中心假设是RBM20组装无膜 心脏内平衡功能所需的大分子冷凝物。我的具体目标是：(1) 确定RBM20剪接工厂的生物物理性质和DCM突变的影响；和(2) 确定RBM20功能是否需要RBM20 LLP，以及RBM20突变如何驱动DCM 表型。我将使用纯化的人RBM20进行体外实验，并使用 人多能干细胞来源的心肌细胞(HPSC-CMS)研究RBM20的作用机制 LLP和DCM突变的影响。我将利用超分辨率显微镜，照片- 漂白和光转换显微镜，以及分子生物学，以研究LLP。将生物物理和 具有心功能的RBM20 LLP微扰引起的拓扑变化，我将描述三个特征： 三维工程心脏组织(3D-EHTS)，一种心脏器官模型。测试是否需要有限责任合伙人 对于RBM20的功能，我将对目的基因进行DNA荧光原位杂交和RT-qPCR，以检测 拼接工厂装配和替换拼接。揭示RBM20突变如何驱动DCM 表型方面，我将测量WT和突变型3D-EHTS的电生理和收缩性能。 总的来说，这些实验将详尽地描述RBM20突变是如何驱动攻击性的 DCM影响RBM20的生物物理性质、细胞动力学和功能。此外，这些实验 将揭示RBM20 DCM突变是如何影响心脏生理和推动疾病的，并有可能揭示 RBM20弥漫性心肌病的新治疗选择。更广泛地说，我们的工作将加强我们对有限责任合伙如何- 调节的区间化推动人类健康，以及LLP的扰动如何导致疾病。这 该项目将在干细胞研究所高度支持和合作的环境中进行 华盛顿大学的再生医学。在我的赞助商和共同赞助商的指导下 (分别是Charles E.Murry博士和Nathan J Sniadecki博士)，该项目将提供 我的职业目标是建立一个独立的研究实验室。

PROJECT SUMMARY/ABSTRACT Mutations in the muscle-specific RNA splicing factor RBM20 are a recently identified cause of aggressive dilated cardiomyopathy (DCM) characterized by severe arrhythmias. However, the underlying mechanisms are still unclear, and thus no therapies are available. We recently uncovered the existence of a nuclear RBM20 splicing factory that brings into proximity multiple co-regulated DNA loci from different chromosomes. Formation of this three-dimensional chromatin structure relies on RBM20 foci that are nucleated by its main splicing target, the pre-mRNA encoding for the giant protein titin (TTN). My preliminary data suggests that RBM20 foci undergo liquid-liquid phase separation (LLPS), a mechanism thought to contribute to the segregation and regulation of sub-nuclear compartments. My preliminary data also suggests RBM20 LLPS is perturbed in RBM20 DCM mutants. Thus, the central hypothesis tested in this proposal is that RBM20 assembles membrane-less macromolecular condensates required for homeostatic function in the heart. My specific aims are to: (1) determine the biophysical properties of the RBM20 splicing factory and impact of DCM mutations; and (2) determine whether RBM20 LLPS is required for RBM20 function, and how RBM20 mutations drive DCM phenotypes. I will perform in vitro experiments using purified human RBM20, and cellular experiments using human pluripotent stem cell-derived cardiomyocytes (HPSC-CMs), to determine the mechanisms of RBM20 LLPS and impact of DCM mutations. I will leverage a combination of super-resolution microscopy, photo- bleaching and photo-conversion microscopy, and molecular biology, to study LLPS. To correlate biophysical and topological changes due to perturbation of RBM20 LLPS with cardiac function, I will characterize three- dimensional engineered heart tissues (3D-EHTs), a cardiac organoid model. To test whether LLPS is required for RBM20 function, I will perform DNA fluorescent in situ hybridization and RT-qPCR of target genes, to measure splice factory assembly and alternative splicing, respectively. To reveal how RBM20 mutations drive DCM phenotypes, I will measure electrophysiological and contractile performance of WT and mutant 3D-EHTs. Collectively, these experiments will exhaustively characterize how RBM20 mutations, which drive aggressive DCM, affect the biophysical properties, cellular dynamics, and function of RBM20. In addition, these experiments will reveal how RBM20 DCM mutants affect cardiac physiology and drive disease, with the potential to reveal new therapeutic options for RBM20 DCM. More broadly, our work will enhance our understanding of how LLPS- mediated compartmentalization drives human health and how perturbation of LLPS contributes to disease. This project will take place in the highly supportive and collaborative environment of the Institute for Stem Cell and Regenerative Medicine at the University of Washington. With the mentorship of my Sponsor and Co-Sponsor (Dr. Charles E. Murry and Dr. Nathan J Sniadecki, respectively), this project will provide the training required for my career goal of establishing an independent research laboratory.