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Function, composition, and mechanism of RNA splicing factories in cardiomyopathy

RNA剪接工厂在心肌病中的功能、组成和机制

基本信息

批准号：
10583011
负责人：
Charles E Murry
金额：
$ 58.66万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-10 至 2026-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10583011
关键词：
3-Dimensional Ablation Action Potentials Adherent Culture Adult Affect Alternative Splicing Amino Acids Architecture Area Arrhythmia Binding Sites Biochemical Biogenesis Biophysical Process Biophysics Biotinylation CRISPR/Cas technology Ca(2+)-Calmodulin Dependent Protein Kinase Calcium Cardiac Cardiac Myocytes Cardiomyopathies Cells Chromatin Chromatin Structure Chromosomes Collaborations Complement Data Deoxyribonucleases Development Dilated Cardiomyopathy Disease Dissection Electrophysiology (science)Engineering Functional disorder Gene Expression Genes Genetic Transcription Goals Hand Heart Heart Diseases Heart failure Hi-C Human Image L-Type Calcium Channels Laboratories Left Link Liquid substance Membrane Messenger RNA Methods Modeling Molecular Muscle Muscle Proteins Mutation Myocardial Nuclear Oligonucleotides Onset of illness Pathogenesis Pathogenicity Patients Phase Phenotype Physical condensation Physiological Play Property Protein Isoforms Proteins RNA RNA Binding RNA Splicing Regulation Reporter Role Sampling Spliced Genes Structure Testing Therapeutic Intervention Work biophysical properties calmodulin-dependent protein kinase II cardiac tissue engineering cardiogenesis cofactor connectin experimental study genome editing genome-wide genomic locus heart function human pluripotent stem cell imaging modality in vivo insight loss of function mRNA Precursor membrane assembly molecular dynamics molecular targeted therapies multiple omics mutant negative affect new therapeutic target novel release of sequestered calcium ion into cytoplasm scaffold superresolution microscopy therapeutic target tool

项目摘要

PROJECT SUMMARY Mutations in the muscle-specific 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. Our group recently discovered a nuclear “splicing factory” involving RBM20 hotspots, which brings into proximity multiple co-regulated loci from different chromosomes. Formation of this three-dimensional (3D) chromatin structure relies the nucleation of RBM20 foci by its main splicing target, the pre-mRNA encoding for the giant protein titin (TTN). Ablating TTN transcription disrupts the RBM20 splicing factory and dysregulates the alternative splicing of genes involved in calcium handling, including the L-type calcium channel (CACNA1C) and calcium/calmodulin-dependent protein kinase II delta (CAMK2D). Thus, the central hypothesis tested in this proposal is that RBM20 assembles membraneless macromolecular condensates that control alternative splicing to centrally regulate cardiac development and disease. Our specific aims are: (1) Identify the functional consequences of dysregulating the RBM20 splicing factory; (2) Define the biophysical properties that drive assembly of the RBM20 splicing factory; (3) Define the key components of the RBM20 splicing factory. In Aims 1 and 2, we will perform cellular experiments to clarify the mechanisms and disease relevance of RBM20 focus formation, as well as functional studies using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). Complementing hPSC-CM monolayer cultures, we will characterize 3D engineered heart tissues (3D-EHTs) and human myocardial grafts. We will utilize CRISPR/Cas9 genome editing to study the effect of RBM20 DCM mutations, as well as to generate fluorescent reporter lines to study focus dynamics in physiologically relevant models. We will characterize disease- associated changes in 3D chromatin topology within the RBM20 splicing factory using a combination of established sequencing- and imaging-based methods. We will then synthesize these biophysical, topological, and biochemical changes with functional genome-wide alterations in alternative splicing, to understand the dysregulation of cardiac electrophysiological and contractile properties. Aiming to elucidate the disease mechanism, we will probe the pathogenic role of specific RBM20-regulated splicing isoforms for CACNA1C and CAMK2D. In Aim 3, we will apply a novel interaction-discovery method developed in the Shechner laboratory, oligonucleotide-directed biotinylation (ODB), to perform an unbiased “multi-omic” analysis of the composition of the splicing factory. We will then determine the role of the newly identified splicing targets in RBM20 DCM, and of the putative RBM20 cofactors in the regulation of RBM20 puncta architecture and of cardiac genes' alternative splicing. Collectively, these experiments will elucidate the mechanisms by which LLPS and subnuclear architecture collaborate to drive alternative splicing in DCM, potentially revealing novel therapeutic targets.

项目摘要肌肉特异性剪接因子RBM 20的突变是最近发现的侵袭性扩张性肌萎缩症的原因。心肌病（DCM），其特征在于严重的心律失常。然而，潜在的机制仍然是不清楚，因此没有可用的治疗方法。我们小组最近发现了一个核“拼接工厂”， RBM 20热点，其使来自不同染色体的多个共调节基因座接近。形成这种三维（3D）染色质结构的形成依赖于RBM 20焦点通过其主要剪接靶点的成核，编码巨蛋白肌联蛋白（TTN）的前mRNA。破坏TTN转录破坏RBM 20剪接与钙处理有关的基因的选择性剪接，包括L型钙通道（CACNA 1C）和钙/钙调蛋白依赖性蛋白激酶II δ（CAMK 2D）。因此在该提议中测试的中心假设是RBM 20组装无膜大分子，控制选择性剪接以集中调节心脏发育和疾病的缩合物。我们具体目标是：（1）确定RBM 20剪接工厂失调的功能后果;（2）定义驱动RBM 20剪接工厂组装的生物物理特性;（3）定义关键 RBM 20拼接工厂的组件。在目标1和2中，我们将进行细胞实验以阐明 RBM 20焦点形成的机制和疾病相关性，以及使用人类的功能研究多能干细胞衍生的心肌细胞（hPSC-CM）。补充hPSC-CM单层培养，我们将表征3D工程心脏组织（3D-EHTs）和人类心肌移植物。我们将利用 CRISPR/Cas9基因组编辑研究RBM 20 DCM突变的影响，以及产生荧光报告线，以研究生理相关模型中的焦点动态。我们将描述疾病的特征- RBM 20剪接工厂内3D染色质拓扑结构的相关变化，建立了测序和成像方法。然后我们将综合这些生物物理，拓扑，和生物化学变化与功能性基因组改变的选择性剪接，以了解心脏电生理和收缩特性的失调。旨在阐明这种疾病机制，我们将探讨特异性RBM 20调节的CACNA 1C剪接异构体的致病作用， CAMK 2D。在目标3中，我们将应用Shechner实验室开发的一种新的相互作用发现方法，通过寡核苷酸指导的生物素化（ODB），对重组蛋白的组成进行无偏的“多组学”分析。拼接工厂然后，我们将确定新鉴定的剪接靶点在RBM 20 DCM中的作用，推测RBM 20辅因子在调节RBM 20点结构和心脏基因的选择性拼接总的来说，这些实验将阐明LLPS和亚核这一研究表明，在DCM中，DNA结构协同驱动选择性剪接，可能揭示新的治疗靶点。