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

破译 RNA 结合蛋白 FXR1 在心肌组装中的作用

基本信息

批准号：
8816117
负责人：
Carol C Gregorio
金额：
$ 46.98万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-03-01 至 2017-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8816117
关键词：
Area Autistic Disorder Binding Biological Assay Biology Cardiac Cardiac Myocytes Cardiac development Cardiomyopathies Complement Complex Confocal Microscopy Coupled Cytoskeletal Proteins Data Development Disease Electron Microscopy Embryo Exhibits FMRP FXR1 gene FXR2 gene Family Family member Fluorescent in Situ Hybridization Fragile X Syndrome Gene Expression Genetic Translation Goals Heart Heart Diseases Heart Hypertrophy Heart failure Human Hypertrophy Immunofluorescence Immunologic Immunofluorescence Microscopy Immunoprecipitation Individual Inherited Intercalated disc Investigation Knock-out Knockout Mice Lead Life Luciferases Mediating Membrane Mental Retardation Messenger RNA Methods Modeling Molecular Molecular Genetics Molecular Profiling Mus Muscle Muscle Cells Muscle Development Muscle function Myocardium Myofibrils Myopathy Physiology Play Polyribosomes Positioning Attribute Process Protein Family Proteins RNA RNA Binding RNA-Binding Proteins Regulation Research Resolution Role Sarcomeres Site Small Interfering RNA Stress Striated Muscles Structural defect Structure Talin Testing Time Tissues Translational Repression Translations Western Blotting base cardiogenesis cellular imaging desmoplakin genetic approach genetic regulatory protein genome-wide in vivo insight knock-down mRNA Expression member mouse model muscle hypertrophy muscular structure neonate novel novel therapeutic intervention protein complex protein function research study small hairpin RNA therapeutic target

项目摘要

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.

描述（由申请人提供）：心肌的正常功能需要将数千个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。这一高度调控过程的一个关键但尚未得到充分研究的方面是蛋白质功能位点对 mRNA 定位和翻译的要求。这项研究的长期目标是确定心脏发育和肥大过程中控制 mRNA 调节的分子机制。在本提案中，我们重点关注 RNA 结合蛋白 FXR1（脆性 X 蛋白家族的横纹肌特异性成员）在控制心肌中特定 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 靶标。其中包括编码与特殊细胞骨架组件相关的蛋白质的 mRNA，例如肋节 (talin) 和闰盘 (desmoplakin)。值得注意的是，电子和共聚焦显微镜揭示了连接复合物中严重的结构扰动，这与我们确定的 mRNA 靶标的失调一致，并为 FXR1 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（及其我们确定的直接靶标）在小鼠和人类心脏肥大中同样受到错误调节，并且 FXR1 KO 心脏表现出许多心脏病的分子特征。我们的结果与目前脆弱 X 家族成员的功能模型一起构成了我们假设的基础，即 FXR1 在正常和病理条件下通过控制编码必需细胞骨架蛋白的特定 mRNA 靶标的定位、稳定性和/或翻译来调节心肌发育和功能。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 丢失的表型后果。这些实验将通过 siRNA 敲低以及原代心肌细胞活细胞成像得到补充； 2) 使用候选方法和全基因组方法识别 FXR1 的 mRNA 靶标，并确定它们受 FXR1 调节的方式。将利用最先进的分子方法来确定 FXR1 是否直接结合其 mRNA 靶点并建立调节模式（mRNA 的定位、稳定性和/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厚特异性靶点。目标 2 中描述的几种方法将与心脏肥大的小鼠模型和心肌生理学的功能评估结合使用。通过这种综合方法，我们能够很好地破译 FXR1 在心肌从头组装和肌病/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受 FXR1 调节的 mRNA 靶标的发现可能会确定心脏病的新治疗方法。