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.

项目概要肌肉特异性剪接因子 RBM20 的突变是最近发现的积极扩张的原因以严重心律失常为特征的心肌病（DCM）然而，其潜在机制仍然存在。尚不清楚，因此我们的小组最近发现了一个涉及核“剪接工厂”的方法。 RBM20 热点，使来自不同染色体形成的多个共同调控基因座接近。这种三维 (3D) 染色质结构的依赖于 RBM20 焦点通过其主要剪接目标的成核，编码巨蛋白肌联蛋白 (TTN) 的前 mRNA 会破坏 RBM20 剪接。工厂并失调参与钙处理的基因的选择性剪接，包括 L 型钙通道 (CACNA1C) 和钙/钙调蛋白依赖性蛋白激酶 II δ (CAMK2D)。该提案测试的中心假设是 RBM20 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。具体目标是：(1) 确定 RBM20 剪接工厂失调的功能后果；(2) (3)定义密钥在目标 1 和 2 中，我们将进行细胞实验来阐明 RBM20 剪接工厂的组成部分。 RBM20 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) 是对 hPSC-CM 单层培养物的补充。我们将利用 3D 工程心脏组织 (3D-EHT) 和人体心肌移植物来表征。 CRISPR/Cas9 基因组编辑研究 RBM20 DCM 突变的影响，并产生荧光记者线研究生理相关模型中的焦点动态我们将描述疾病的特征。 RBM20 剪接工厂内 3D 染色质拓扑的相关变化，使用以下组合然后我们将综合这些生物物理、拓扑、以及选择性剪接中功能性全基因组改变的生化变化，以了解心脏电生理和收缩特性的失调旨在阐明该疾病。机制，我们将探讨特定 RBM20 调节的剪接亚型对 CACNA1C 和在 CAMK2D 中，我们将应用 Shechner 实验室开发的一种新颖的交互发现方法，寡核苷酸定向生物素化（ODB），对以下物质的组成进行公正的“多组学”分析然后我们将确定新识别的拼接目标在 RBM20 DCM 中的作用，以及假定的 RBM20 辅因子在 RBM20 斑点结构和心脏基因替代调节中的作用总的来说，这些实验将阐明 LLPS 和亚核的机制。结构协作驱动 DCM 中的选择性剪接，有可能揭示新的治疗靶点。