Identifying Therapeutic Targets for RNA Splicing-Related Cardiomyopathy

 描述（由申请人提供） 扩张型心肌病（DCM）是西方国家心脏移植最常见的适应症。RBM20（RNA结合基序蛋白20）是最近描述的心脏特异性RNA剪接蛋白，其在2 - 3%的DCM中突变。RBM20直接与许多心肌病相关基因的RNA转录物结合，并确保心脏特异性蛋白质亚型的产生。人体研究揭示了一个惊人的，无法解释的，重复的模式，7个紧密聚集的单一氨基酸DCM相关的取代附近的RS域的RBM20。DCM患者中杂合突变的紧密模式和RBM20结合蛋白的生化研究表明，人DCM突变可能对RBM20剪接复合物具有显性负干扰作用。我们的中心假设是RBM20 RS结构域突变体引起显性负干扰，导致不同于功能丧失突变的病理生理RNA剪接。我们计划开发一系列同基因的 人诱导多能干细胞（iPSC）-心肌细胞（iPS-CM）。我们已经在iPSC中进行了多个单碱基RBM20突变，而没有抗生素选择（"无瘢痕"）。我们可以有效地产生RBM20 RS结构域突变体（R636S）iPS-CM，并且细胞显示出与DCM一致的细胞病理学。R636S突变体iPS-CM的RNA-Seq研究揭示了> 360个可变剪接事件，包括许多先前报道的事件。我们的目标是：目标1。通过RNA-Seq分析剪接中的病理变化，验证RBM20 DCM引起的点突变具有显性负效应的假设。我们将把七种重复的人类DCM突变改造成同基因iPSC，以及RBM20无效突变。RNA-Seq研究将集中于确定可以解释DCM病理的最病理性的选择性剪接事件。 目标二。为了验证RBM20 RS结构域突变（R636S）具有不同的RNA和蛋白质结合的假设，我们将分别使用FLAG和APEX表位标记来选择性地鉴定结合伴侣。我们将使用FLAG-RBM20-RNA结合测定（CLIP-Seq）来确定确切的RBM20 RNA结合位点。FLAG-和APEX-APMS将确定RBM20的推定结合伴侣和调节剂。 目标3。确定假定的结合伴侣是否是RBM20剪接调控所必需的。我们将有条件地沉默推定的结合伴侣的表达。我们将使用CRISPRi来调节每个基因的表达，以确定每个蛋白质是否是潜在的治疗靶点。 随着这些目标的完成，我们将直接检验我们的假设， 人RBM20突变的模式，并提供病理性剪接的分子机制。我们将确定RBM20的分子靶标和蛋白质伴侣。改变RBM20活性的药物可以通过一种新的机制增强心脏健康和修复。这些研究将为开发基于人类iPS-CM的平台以开发新的治疗方法提供基础。

 DESCRIPTION (provided by applicant) Dilated cardiomyopathy (DCM) is the most common indication for heart transplantation in the western world. RBM20 (RNA binding motif protein 20) is a recently described cardiac-specific, RNA splicing protein that is mutated in 2-3% of DCM. RBM20 directly binds to the RNA transcripts of many cardiomyopathy-associated genes and ensures the production of cardiac-specific protein isoforms. Human studies revealed a striking, unexplained, recurring pattern of seven tightly clustered single amino acid DCM-associated substitutions near the RS domain of RBM20. The tight pattern of heterozygous mutations in DCM patients and biochemical studies of RBM20 binding proteins suggest that human DCM mutations could have dominant negative interfering effects on the RBM20 splicing complex. Our central hypothesis is that the RBM20 RS domain mutants cause dominant negative interference resulting in pathophysiological RNA splicing that are distinct from loss-of-function mutations. We plan to develop a series of isogenic human induced pluripotent stem cell (iPSC)-cardiomyocytes (iPS-CMs). We have made multiple single-base RBM20 mutations without antibiotic selection ("scarless") in iPSCs. We can efficiently produce RBM20 RS domain mutant (R636S) iPS-CMs, and the cells display cellular pathology consistent with DCM. RNA-Seq studies of R636S mutant iPS-CMs reveal >360 alternative splicing events, including many that have been previously reported. Our aims are: Aim 1. To test the hypothesis that RBM20 DCM-causing point mutations have a dominant negative effect, by analyzing pathological changes in splicing by RNA-Seq. We will engineer the seven recurring human DCM mutations into isogenic iPSCs, as well as RBM20 null mutations. The RNA-Seq studies will focus on identifying the most pathological alternative splicing events that could explain the DCM pathology. Aim 2. To test the hypothesis that RBM20 RS domain mutation (R636S) has different RNA- and protein- binding, we will use FLAG and APEX epitope tagging respectively, to selectively identify binding partners. We will use FLAG-RBM20-RNA binding assays (CLIP-Seq) to determine the exact RBM20 RNA binding sites. FLAG- and APEX-APMS will identify putative binding partners and regulators of RBM20. Aim 3. To determine if the putative binding partners are essential for RBM20 splice regulation. We will conditionally silence the expression of putative binding partners. We will use CRISPRi to modulate the expression of each gene, to determine if each protein is a potential therapeutic target. With the completion of these aims, we will directly test our hypothesis that would explain the pattern of human RBM20 mutations, and provide a molecular mechanism for pathological splicing. We will have defined the molecular targets and protein partners of RBM20. Drugs that alter RBM20 activity could enhance cardiac health and repair, via a novel mechanism. These studies will provide a foundation for developing a human iPS-CM-based platform to develop new therapeutics.