Roles of RNA helicase and ribosomal biogenesis in normal and diseased muscle

RNA解旋酶和核糖体生物发生在正常和患病肌肉中的作用

• 批准号: 8720697
• 负责人: Vandana Gupta
• 金额: $ 12.93万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8720697
• 关键词: Adult; Affect; Amino Acids; Animal Model; Base Pairing; Behavior; Binding; Biochemical; Biogenesis; Boston; Boxing; Cell Nucleus; Cell physiology; Centronuclear myopathy; Child; Chromosome Mapping; Clinical; Communities; Conserved Sequence; Data; Defect; Development; Diagnosis; Disease; Dissection; Exhibits; Family; Fishes; Frameshift Mutation; Functional disorder; Funding; Future; Genes; Genetic; Genetic Transcription; Genome; Goals; Growth; Heredity; Heterogeneity; Human; Immunofluorescence Immunologic; Immunoprecipitation; In Situ Hybridization; In Vitro; Inborn Genetic Diseases; Knowledge; Laboratories; Locomotion; Mentored Research Scientist Development Award; Mentorship; Messenger RNA; Methods; MicroRNAs; Modeling; Modification; Molecular; Molecular Biology; Muscle; Muscle Cells; Muscle Development; Muscle Weakness; Mutation; Myoblasts; Myopathy; Myotonic Dystrophy; Neuromuscular Diseases; Pathogenesis; Pathway interactions; Patients; Pediatric Hospitals; Penetrance; Pharmaceutical Preparations; Phenotype; Physiologic pulse; Positioning Attribute; Protein Subunits; RNA; RNA Binding; RNA Degradation; RNA Helicase; RNA Splicing; RNA biosynthesis; RNA-Binding Proteins; Regulation; Research; Research Personnel; Resources; Ribosomal RNA; Ribosomes; Role; Sequence Homology; Skeletal Muscle; Structure; Synteny; System; Systems Biology; Technology; Testing; Therapeutic; Training; Translating; Translations; Vertebrates; Work; Yeasts; Zebrafish; base; career development; cell motility; congenital myopathy; developmental genetics; effective therapy; experience; forward genetics; helicase; high throughput screening; improved; in vivo; interest; knock-down; loss of function mutation; medical schools; member; mortality; muscle hypertrophy; mutant; novel; protein complex; public health relevance; skeletal muscle growth; therapy design; therapy development; tool; translation assay; treatment strategy; zebrafish development

DESCRIPTION (provided by applicant): Primary inherited disorders of muscle include both dystrophies and non-dystrophic myopathies. These conditions, characterized by muscle weakness and impaired locomotion, form a group of heredity diseases affecting both children and adults. Complexity due to genetic, phenotypic and clinical heterogeneity poses a great challenge in identifying causative genes underlying muscle disorders. Furthermore, even in instances where a defective gene has been identified, that knowledge has often not translated into development of specific and effective therapies for the relevant neuromuscular disorders. Suitable animal models are needed to better understand the pathophysiology, and to provide high throughput assay systems to identify potentially therapeutic drugs. Therefore, to identify novel genes involved in neuromuscular disorders, the applicant performed a forward genetics screen in a vertebrate animal model zebrafish (Danio rerio) and identified 13 unique mutants with defective skeletal muscle. This K01 proposal is aimed at supporting the career development of Dr. Vandana Gupta as she explores the cellular processes and molecular mechanisms associated with skeletal muscle growth and diseases, by studying a zebrafish mutant, osoi. Genetic mapping in osoi fish identified a 20 base pair deletion in a novel DEAD-box RNA helicase, a RNA binding protein, resulting in a frameshift mutation. Dr. Gupta's preliminary work firmly establishes mutation in this RNA helicase as a cause of skeletal muscle hypotrophy. The pathological findings identified smaller size myofibers with sarcomeric disorganization associated with central nuclei. The central nucleation is reminiscent of centronuclear myopathy, a form of human congenital myopathy and myotonic dystrophies, and myofiber hypotrophy in general is a central feature of congenital myopathies and several forms of dystrophies. In skeletal muscle diseases, strong efforts are currently being devoted to develop therapies aimed at increasing myofiber size. However, lack of suitable targets has been a major hindrance in development of successful treatments. DEAD-box RNA helicases are involved in all cellular processes involving RNA, from transcription, mRNA splicing, translation, RNA modification, transport, ribosome biogenesis, miRNA biosynthesis, RNA/protein complex assembly and RNA degradation. Dr. Gupta's preliminary data identifies a ribosomal defect in osoi mutant and puts forward a novel path to investigate ribosomal regulation in muscle growth and diseases. Through better understanding of the molecular pathways in skeletal muscle hypotrophy, the applicant hopes to begin development of corrective therapies for such muscle defects. Specific Aim 1 will investigate the phenotypic and pathological changes associated with osoi loci in vivo and in vitro. Findings from this will be applied to study the genetic basis of skeletal muscle diseases in patients with similar muscle defects. Specific Aim 2 will explore the ribosomal biogenesis defects observed in osoi fish employing biochemical approaches. This aim will further investigate the structure-functional relationship of different conserved helicase motis in ribosomal biogenesis and skeletal muscle hypotrophy. Specific Aim 3 will explore the molecular functions of osoi loci by identifying RNA targets bound to this RNA helicase in vivo using molecular and system biology methods. The in vivo functional significance of targets identified in this aim will be tested by knock-downs using morpholino technology in zebrafish. Dr. Gupta's long term aim as an independent investigator is to study the genetic causes of human neuromuscular disorders and their molecular basis to devise treatment strategies using model organisms. Her research position in Beggs Laboratory at Children's Hospital Boston provides an ideal opportunity for her training, with the lab's long standing interest and experience in studying genetic causes and treatments of muscle disorders, excellent mentorship, available resources and collaborative opportunities within Children's Hospital and the Harvard Medical School community. The support provided with K01 award will help her to complete her training and with provide her with a launch pad to obtain data for a successful R01 application to fund her future work.

描述（由申请人提供）：原发性遗传性肌肉疾病包括营养不良和非营养不良性肌病。这些疾病的特征是肌肉无力和运动障碍，形成了一组影响儿童和成人的遗传疾病。由于遗传、表型和临床异质性的复杂性，在确定肌肉疾病的致病基因方面提出了巨大的挑战。此外，即使在已经鉴定出缺陷基因的情况下，这些知识通常也没有转化为针对相关神经肌肉疾病的特异性和有效疗法的开发。需要合适的动物模型来更好地理解病理生理学，并提供高通量测定系统来鉴定潜在的治疗药物。因此，为了鉴定涉及神经肌肉疾病的新基因，申请人在脊椎动物模型斑马鱼（Danio rerio）中进行了正向遗传学筛选，并鉴定了13种具有缺陷性骨骼肌的独特突变体。这个K 01提案旨在支持Vandana Gupta博士的职业发展，因为她通过研究斑马鱼突变体oepa来探索与骨骼肌生长和疾病相关的细胞过程和分子机制。在斑马鱼的遗传作图中发现了一种新型的DEAD盒RNA解旋酶（一种RNA结合蛋白）中的20个碱基对缺失，导致移码突变。古普塔博士的初步工作坚定地确立了这种RNA解旋酶的突变是骨骼肌萎缩的一个原因。病理结果显示，较小尺寸的肌纤维与中央核相关的肌节紊乱。中央成核使人联想到中央性肌病，一种人类先天性肌病和肌强直性营养不良的形式，并且肌纤维萎缩通常是先天性肌病和几种形式的营养不良的中心特征。在骨骼肌疾病中，目前正在努力开发旨在增加肌纤维尺寸的疗法。然而，缺乏合适的靶点一直是开发成功治疗的主要障碍。DEAD盒RNA解旋酶参与所有涉及RNA的细胞过程，从转录、mRNA剪接、翻译、RNA修饰、转运、核糖体生物合成、miRNA生物合成、RNA/蛋白质复合物组装和RNA降解。Gupta博士的初步数据确定了一个核糖体缺陷的突变体，并提出了一个新的途径来研究核糖体调控肌肉生长和疾病。通过更好地理解骨骼肌萎缩的分子途径，申请人希望开始开发针对此类肌肉缺陷的矫正疗法。具体目标1将在体内和体外研究与卵巢基因座相关的表型和病理变化。这一发现将用于研究具有类似肌肉缺陷的患者的骨骼肌疾病的遗传基础。具体目标2将探讨核糖体生物合成缺陷观察到的斑马鱼采用生化方法。本研究旨在进一步探讨不同保守解旋酶motis在核糖体生物合成和骨骼肌萎缩中的结构与功能关系。具体目标3将探索通过使用分子和系统生物学方法在体内鉴定与该RNA解旋酶结合的RNA靶标的卵巢癌基因座的分子功能。在这一目标中确定的目标的体内功能意义将通过在斑马鱼中使用吗啉代技术的敲除来测试。 古普塔博士作为一名独立研究者的长期目标是研究人类神经肌肉疾病的遗传原因及其分子基础，以利用模式生物设计治疗策略。她在贝格斯实验室在儿童医院波士顿的研究职位为她的培训提供了一个理想的机会，与实验室的长期兴趣和研究遗传原因和肌肉疾病的治疗经验，优秀的导师，可用的资源和合作机会内儿童医院和哈佛医学院社区。K 01奖提供的支持将帮助她完成培训，并为她提供一个发射台，以获得成功申请R 01的数据，为她未来的工作提供资金。