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: 8519309
• 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 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8519309
• 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 Screening; 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; 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(申请人提供)：肌肉的原发遗传性疾病包括营养不良和非营养不良肌病。这些疾病以肌肉无力和运动障碍为特征，形成了一组影响儿童和成人的遗传性疾病。由于遗传、表型和临床的异质性导致的复杂性给确定肌肉疾病的致病基因带来了巨大的挑战。此外，即使在识别出缺陷基因的情况下，这种知识往往也没有转化为针对相关神经肌肉疾病的具体和有效的治疗方法。需要合适的动物模型来更好地了解病理生理学，并提供高通量的检测系统来识别潜在的治疗药物。因此，为了确定与神经肌肉疾病相关的新基因，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查，并确定了13个骨骼肌缺陷的独特突变。这项K01提案旨在支持Vandana Gupta博士的职业发展，她通过研究斑马鱼突变体osoi来探索与骨骼肌生长和疾病相关的细胞过程和分子机制。在乌贼鱼的遗传图谱中发现了一种新的死盒RNA解旋酶，一种RNA结合蛋白，它有一个20碱基对的缺失，导致了移码突变。古普塔博士的初步工作确定这种RNA解旋酶的突变是造成骨骼肌营养不良的原因之一。病理发现发现较小的肌纤维与中央核相关的肌节组织紊乱。中央核使人联想到中央核肌病，这是人类先天性肌病和强直性肌营养不良的一种形式，一般情况下，肌纤维减少是先天性肌病和几种形式的肌营养不良的中心特征。在骨骼肌疾病方面，目前正致力于开发旨在增加肌纤维尺寸的治疗方法。然而，缺乏合适的靶点一直是开发成功的治疗方法的主要障碍。DEAD-box RNA解旋酶参与了从转录、mRNA剪接、翻译、RNA修饰、转运、核糖体生物发生、miRNA生物合成、RNA/蛋白质复合体组装到RNA降解的所有细胞过程。古普塔博士的初步数据确定了osoi突变中的一个核糖体缺陷，并提出了一条研究核糖体在肌肉生长和疾病中的调控的新途径。通过更好地了解骨骼肌营养不良的分子途径，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osoI基因座相关的表型和病理变化。这一发现将被应用于研究患有类似肌肉缺陷的患者骨骼肌疾病的遗传基础。具体目标2将利用生化方法探索在鱼体中观察到的核糖体生物发生缺陷。这将进一步研究不同保守的解旋酶MOTIS在核糖体生物发生和骨骼肌营养不良中的结构与功能关系。具体目标3将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靶标来探索osoi基因座的分子功能。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啡技术进行击倒来测试。古普塔博士作为一名独立研究员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。她在波士顿儿童医院贝格斯实验室的研究职位为她的培训提供了一个理想的机会，因为该实验室在研究肌肉疾病的遗传原因和治疗方面具有长期的兴趣和经验、出色的指导、可用的资源以及儿童医院和哈佛医学院社区的合作机会。K01奖项提供的支持将帮助她完成培训，并为她提供一个发射台，为她成功申请R01获得数据，为她未来的工作提供资金。