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

项目总结 肌肉特异性剪接因子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。我们小组最近发现了一家核“拼接工厂”，涉及 RBM20热点，使来自不同染色体的多个共调控基因座接近。形成 这种三维(3D)染色质结构依赖于其主要剪接靶对RBM20焦点的成核， 编码巨蛋白titin(TTN)的前信使核糖核酸。消融TTN转录扰乱RBM20剪接 工厂和失调参与钙处理的基因的选择性剪接，包括L类型 钙通道(CACNA1C)和钙/钙调蛋白依赖的蛋白激酶II增量(CAMK2D)。因此， 在这项提议中检验的中心假设是RBM20组装了无膜大分子 控制选择性剪接以集中调节心脏发育和疾病的缩合物。我们的 具体目标是：(1)确定RBM20剪接工厂失控的功能后果；(2) 定义驱动RBM20拼接工厂组装的生物物理属性；(3)定义关键 RBM20拼接工厂的组件。在目标1和目标2中，我们将进行细胞实验来阐明 RBM20病灶形成的机制和疾病相关性，以及利用人类进行的功能研究 多能干细胞来源心肌细胞(hPSC-CMS)。补充hPSC-CM单层培养，我们 将表征3D工程心脏组织(3D-EHTS)和人体心肌移植物。我们将利用 CRISPR/Cas9基因组编辑以研究RBM20 DCM突变的影响以及产生荧光 记者专线研究生理相关模型中的焦点动力学。我们将描述疾病的特征-- RBM20拼接工厂内使用组合的3D染色质拓扑的相关变化 建立了基于测序和成像的方法。然后我们将合成这些生物物理的、拓扑的、 和生化变化与功能基因组范围的选择性剪接的变化，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，我们将探讨特定的RBM20调节的CACNA1C和CACNA1C剪接异构体的致病作用。 CAMK2D。在目标3中，我们将应用谢切纳实验室开发的一种新的交互发现方法， 寡核苷酸引导的生物素化(ODB)，对其组成进行无偏见的“多组学”分析 拼接厂。然后，我们将确定新发现的剪接靶在RBM20 DCM中的作用，以及 可能的RBM20辅助因子对RBM20斑点结构和心脏基因替代的调节 拼接。总而言之，这些实验将阐明LLP和亚核 架构合作，以推动DCM的替代剪接，潜在地揭示新的治疗靶点。

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.