Deciphering the role of the RNA-binding protein, FXR1, in cardiac muscle assembly

描述（由申请人提供）：心肌的正常功能需要成千上万的细胞骨架和调节蛋白高度协调地组装成单个肌节（收缩单位）和膜相关的连接复合物。在这个高度调控的过程中，一个关键但研究非常不足的方面是mRNA在蛋白质功能位点的定位和翻译要求。本研究的长期目标是确定在心脏发育和肥厚过程中调控mRNA调控的分子机制。在本研究中，我们重点研究了脆性X蛋白家族横纹肌特异性成员RNA结合蛋白FXR1在控制心肌特异性mrna表达中的作用。通过结合细胞、分子和遗传方法，我们获得了广泛的初步数据，确定了心脏中FXR1的第一个mRNA靶点。其中包括编码与特殊细胞骨架组装相关的蛋白质的mrna，如costameres （talin）和嵌入盘（desmoplakin）。值得注意的是，电子和共聚焦显微镜显示，连接复合物中存在严重的结构扰动，这与我们发现的mRNA靶点失调一致，并为FXR1基因敲除（KO）胚胎致死的原因提供了合理的解释。初步结果还表明，FXR1蛋白（以及我们确定的其直接靶点）在小鼠和人类心脏肥厚中被相同的错误调节，并且FXR1 KO心脏表现出许多心脏病的分子特征。我们的研究结果与目前的脆性X家族成员功能模型构成了我们假设的基础，即FXR1通过控制在正常和病理状态下编码必需细胞骨架蛋白的特定mRNA靶点的定位、稳定性和/或翻译来调节心肌的发育和功能。使用小鼠心脏组织和原代心肌细胞，我们的目标是：1)使用广泛的高分辨率免疫荧光和电子显微镜确定KO心脏中FXR1缺失的表型后果。这些实验将通过siRNA敲除与原代心肌细胞的活细胞成像相结合来补充；2)利用候选基因和全基因组方法鉴定FXR1的mRNA靶点，并确定它们受FXR1调控的方式。最先进的分子方法将用于确定FXR1是否直接结合其mRNA靶标，并建立调控模式（mRNA的定位、稳定和/或翻译）；3)破译FXR1在心脏肥大/应激中的作用，并确定其新的肥大特异性靶点。Aim 2中描述的几种方法将与小鼠心肌肥厚模型和心肌生理学功能评估结合使用。通过这种综合方法，我们能够很好地破译FXR1在新生心肌组装和肌病/应激过程中利用的分子机制。据我们所知，我们是唯一一个研究肌肉发育这一关键方面的小组。此外，在正常发育和患病状态下，FXR1调控的mRNA靶点的发现可能为心脏病的治疗找到新的方法。

DESCRIPTION (provided by applicant): The proper function of cardiac muscle requires the highly orchestrated assembly of thousands of cytoskeletal and regulatory proteins into individual sarcomeres (contractile units) and membrane-associated junctional complexes. A critical, yet extremely understudied, aspect of this highly regulated process is the requirement for mRNA localization and translation at the site of protein function. The long-term goal of this research is to identify the molecular mechanisms governing mRNA regulation during cardiac development and hypertrophy. In this proposal, we focus on the role of the RNA binding protein FXR1, the striated muscle- specific member of the Fragile X protein family, in controlling the expression of specific mRNAs in cardiac muscle. Using a combined cellular, molecular and genetic approach we obtained extensive preliminary data that identified the first mRNA targets of FXR1 in the heart. These include mRNAs that encode proteins associated with specialized cytoskeletal assemblies such as costameres (talin) and intercalated discs (desmoplakin). Remarkably, electron and confocal microscopy revealed severe structural perturbations in junctional complexes that are consistent with dysregulation of the mRNA targets we identified, and provide a plausible explanation for the cause of the FXR1 knockout (KO) embryonic lethality. Preliminary results also showed that FXR1 protein (and its direct targets that we identified) is misregulated identically in mouse and human cardiac hypertrophy, and that FXR1 KO hearts exhibits numerous molecular signatures of heart disease. Our results together with current models for the function of Fragile X family members form the basis for our hypothesis that FXR1 regulates cardiac muscle development and function by controlling the localization, stability and/or translation of specific mRNA targets encoding essential cytoskeletal proteins during normal and pathological conditions. Using mouse heart tissue and primary cardiomyocytes, we aim to: 1) determine the phenotypic consequences of FXR1 loss in KO hearts using extensive high-resolution immunofluorescence and electron microscopy. These experiments will be complemented by siRNA knock-down coupled with live-cell imaging in primary cardiac myocytes; 2) identify mRNA targets of, and determine the manner in which they are regulated by FXR1 using both candidate and genome-wide approaches. State-of-the-art molecular methods will be utilized to determine whether FXR1 directly binds its mRNA targets and to establish the mode of regulation (localization, stability and/or translation of the mRNA); and 3) decipher the role of FXR1 in cardiac hypertrophy/stress and identify its novel hypertrophy-specific targets. Several methods described in Aim 2 will be used in conjunction with mouse models of cardiac hypertrophy and functional assessment of cardiac muscle physiology. With this integrative approach, we are well positioned to decipher the molecular mechanisms utilized by FXR1 during de novo cardiac muscle assembly and myopathy/stress. To our knowledge we are the only group investigating this critical aspect of muscle development. Furthermore, the discovery of mRNA targets regulated by FXR1 during normal development and in diseased states may identify novel therapeutic approaches for heart disease.