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

RBM20液-液相分离驱动心肌细胞生理学和扩张型心肌病的机制研究

基本信息

批准号：
10570894
负责人：
Aidan Mandy Fenix
金额：
$ 1.12万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-16 至 2023-03-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10570894
关键词：
3-Dimensional Affect Alternative Splicing Arginine Arrhythmia Automobile Driving Behavior Binding Sites Biochemical Biological Models Biological Phenomena Biology Biophysics Calcium Cardiac Cardiac Myocytes Cells Characteristics Chromatin Chromatin Structure Chromosomes DNA DNA Repair DNA Sequence Alteration Data Dilated Cardiomyopathy Disease Disease model Electrophysiology (science)Engineering Fluorescence Recovery After Photobleaching Fluorescent in Situ Hybridization Genes Goals Health Heart Heart Diseases Heart failure Homeostasis Human Image Imaging Techniques In Vitro Inherited Knock-out Laboratory Research Lead Liquid substance Magnetism Measures Mediating Membrane Mentorship Microscopy Modeling Molecular Biology Muscle Mutation Myocardial dysfunction N-terminal Nuclear Nuclear RNA Nuclear Structure Organelles Organoids Pathogenicity Performance Phase Phenotype Photobleaching Physical condensation Physiology Point Mutation Protein Binding Domain Proteins RNA RNA Binding RNA Recognition Motif RNA Splicing RNA purification Reaction Recovery Regenerative Medicine Regulation Reporter Repression Role Scheme Serine Structure Testing Training Universities Washington Work biophysical properties cardiac tissue engineering career collaborative environment connectin disease-causing mutation early onset experimental study force sensor genomic locus heart function human disease human pluripotent stem cell human stem cells lens live cell microscopy mRNA Precursor membrane assembly monomer mutant new therapeutic target novel novel therapeutics overexpression segregation stem cell model stem cells sudden cardiac death superresolution microscopy supportive environment tool

项目摘要

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.

项目摘要/摘要肌肉特异性RNA剪接因子RBM20中的突变是最近确定的侵略性扩张的原因心肌病（DCM）的特征是严重的心律不齐。但是，基本机制仍然是不清楚，因此没有疗法可用。我们最近发现了存在核RBM20剪接的存在从不同染色体带来邻近多个共同调节的DNA基因座的工厂。形成三维染色质结构依赖于RBM20灶，其主要剪接靶标成核的焦点编码巨型蛋白滴定（TTN）的MRNA。我的初步数据表明RBM20 FOCI经历了液态液相分离（LLP），一种被认为有助于隔离和调节的机制亚核室。我的初步数据还表明RBM20 LLP在RBM20 DCM中受到干扰突变体。因此，该提案中检验的中央假设是RBM20组装了膜无膜心脏稳态功能所需的大分子冷凝物。我的具体目的是：（1）确定RBM20剪接工厂的生物物理特性和DCM突变的影响；（2）确定RBM20 LLP是否需要RBM20功能，以及RBM20突变驱动DCM 表型。我将使用纯化的人RBM20和使用细胞实验进行体外实验人多能干细胞衍生的心肌细胞（HPSC-CMS），以确定RBM20的机制 LLP和DCM突变的影响。我将利用超分辨率显微镜，照片的组合漂白和光转换显微镜和分子生物学，以研究LLP。关联生物物理和由于具有心脏功能的RBM20 LLP扰动引起的拓扑变化，我将表征三个尺寸工程性心脏组织（3D-EHTS），一种心脏器官模型。测试是否需要LLP 对于RBM20功能，我将执行靶基因的原位原位杂交和RT-QPCR，以测量剪接工厂组装和替代剪接。揭示RBM20突变如何驱动DCM 表型，我将测量WT和突变体3D-EHT的电生理和收缩性能。总的来说，这些实验将详尽地表征RBM20突变的方式 DCM，影响RBM20的生物物理特性，细胞动力学和功能。另外，这些实验将揭示RBM20 DCM突变体如何影响心脏生理和驱动疾病，并有可能揭示 RBM20 DCM的新治疗选择。从更广泛的角度来看，我们的工作将增强我们对LLP的理解 - 介导的分室化驱动了人类健康以及LLP的扰动如何对疾病有效。这项目将在干细胞研究所的高度支持和协作环境中进行华盛顿大学再生医学。在我的赞助商和共同赞助商的指导下（Charles E. Murry博士和Nathan J Sniadecki博士），该项目将提供所需的培训我建立独立研究实验室的职业目标。