Regulation of RBM20 expression and activity in cardiac development and disease

选择性剪接在基因表达调控和蛋白质组多样性的产生中起着重要作用。最近，人们越来越认识到剪接因子及其靶位的缺陷可引起人类疾病。因此，选择性剪接有可能作为中枢神经系统、心脏和骨骼肌的各种遗传性疾病的治疗靶点。我们最近建立了RNA结合基序蛋白20 （RBM20）作为心脏剪接的调节剂，具有广泛的底物谱，包括titin。RBM20的突变导致严重形式的人类扩张型心肌病（DCM），并伴有导致疾病状态的几种选择性剪接转录物的失调。由于RBM20调节调节心电和机械功能的选择底物的剪接，因此RBM20定向治疗有望改善心脏病理的各个方面。在过去的五年里，RBM20缺乏及其底物谱的临床相关性已经被描述，我们才刚刚开始了解其在RNA和蛋白质水平上的调控。因此，关于RBM20表达的调控、RBM20与基本剪接机制的相互作用、其剪接活性的调节以及RBM20突变导致心脏病理的潜在机制，我们知之甚少。为了解剖RBM20的分子功能，了解其调控机制，开发RBM20作为治疗靶点，我们提出以下目标：目的1:RBM20的表达调控假设：RBM20的表达在发育和患病的横纹肌中受到时空调控，以适应titin异构体的表达。试验1.2:RBM20在心脏重构和疾病中的水平。试验1.3：人RBM20启动子的表征。检验1.4:RBM20转录物水平的药理调节。目的2:RBM20介导剪接的调控假设：RBM20通过与RNA和剪接机制组分的特异性相互作用调控选择性剪接测试2.1:RBM20依赖性剪接的分子和机制基础。测试2.2:RBM20蛋白结构域参与选择性剪接的调控。目的3:RBM20突变的功能后果假设：与DCM相关的RBM20突变改变了蛋白质的功能、稳定性或定位测试3.1：突变RBM20的蛋白质稳态。试验3.2：突变体RBM20的核定位、RNA结合和剪接调控。

Alternative splicing plays a major role in the regulation of gene expression and in the generation of proteome diversity. Lately it has been increasingly recognized that defects in splice factors as well as their target sites can cause human disease. Accordingly, alternative splicing has the potential to serve as a therapeutic target for various inherited diseases of the central nervous system, heart, and skeletal muscle. We have recently established RNA binding motif protein 20 (RBM20) as a regulator of cardiac splicing with a broad substrate spectrum that includes titin. Mutations in RBM20 result in severe forms of human dilated cardiomyopathy (DCM) with deregulation of several alternatively spliced transcripts that contribute to the disease state. As RBM20 modulates splicing of select substrates that orchestrate both electrical and mechanical cardiac function, an RBM20 directed therapy is expected to improve diverse aspects of cardiac pathology.The clinical relevance of RBM20 deficiency and its substrate spectrum have been described in the past five years and we are only beginning to understand its regulation on the RNA and protein level. Thus, little is known about the regulation of RBM20 expression, about the interactions of RBM20 with the basic splicing machinery, about the modulation of its splicing activity, and about the underlying mechanisms by which mutations in RBM20 contribute to cardiac pathology. To dissect the molecular functions of RBM20, to understand its regulation and to develop RBM20 as a therapeutic target, we propose the following aims:Aim 1: Regulation of RBM20 expressionHypothesis: The expression of RBM20 is spatially and temporally regulated to adapt titin isoform expression in developing and diseased striated muscleTest 1.1: Physiological RBM20 RNA and protein expression.Test 1.2: RBM20 levels in cardiac remodeling and disease.Test 1.3: Characterization of the human RBM20 promoter.Test 1.4: Pharmacological modulation of RBM20 transcript levels.Aim 2: Regulation of RBM20-mediated splicingHypothesis: RBM20 regulates alternative splicing through specific interactions with RNA and components of the splicing machineryTest 2.1: The molecular and mechanistic basis of RBM20-dependent splicing.Test 2.2: RBM20 protein domains involved in the regulation of alternative splicing.Aim 3: Functional consequences of RBM20 mutationsHypothesis: Mutations of RBM20 that are linked to DCM alter function, stability or localization of the proteinTest 3.1: Protein homeostasis of mutant RBM20.Test 3.2: Nuclear localization, RNA binding and splicing regulation of mutant RBM20.