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

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肌病。这些以肌肉无力和运动障碍为特征的疾病，形成了一组影响儿童和成人的遗传性疾病。由于遗传、表型和临床异质性的复杂性，对确定肌肉疾病的致病基因提出了巨大的挑战。此外，即使在已经识别出缺陷基因的情况下，这些知识通常也无法转化为针对相关神经肌肉疾病的特定有效疗法的开发。需要合适的动物模型来更好地了解病理生理学，并提供高通量分析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有关的新基因，申请人在脊椎动物模型斑马鱼（Danio rerio）中进行了前向遗传学筛选，并确定了13种具有缺陷骨骼肌的独特突变体。这项K01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osoi鱼的遗传图谱中发现了一种新的DEAD-box RNA解旋酶（一种RNA结合蛋白）中20个碱基对的缺失，导致移码突变。古普塔博士的初步工作确定了这种RNA解旋酶的突变是骨骼肌萎缩的原因。病理结果发现较小尺寸的肌纤维，伴有与中央核相关的肌肉组织紊乱。中心成核使人联想到核中性肌病，一种人类先天性肌病和肌强直性营养不良症，而肌纤维萎缩通常是先天性肌病和几种形式的营养不良症的主要特征。在骨骼肌疾病中，目前正在大力开发旨在增加肌纤维大小的治疗方法。然而，缺乏合适的靶点一直是发展成功治疗的主要障碍。DEAD-box RNA解旋酶参与所有涉及RNA的细胞过程，包括转录、mRNA剪接、翻译、RNA修饰、转运、核糖体生物发生、miRNA生物合成、RNA/蛋白复合物组装和RNA降解。Gupta博士的初步数据确定了osoi突变体中的核糖体缺陷，并提出了研究肌肉生长和疾病中核糖体调节的新途径。通过更好地了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。特异性目的1将在体内和体外研究与osoi位点相关的表型和病理变化。这一发现将应用于研究骨骼肌疾病的遗传基础，患者有类似的肌肉缺陷。专项目的2将探讨核糖体生物发生缺陷观察到在osoi鱼采用生化方法。本研究将进一步探讨核糖体生物发生和骨骼肌萎缩中不同保守解旋酶运动的结构-功能关系。Specific Aim 3将利用分子生物学和系统生物学方法，通过鉴定与该RNA解旋酶结合的RNA靶点，探索osoi位点的分子功能。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morpholino技术进行敲除来测试。作为一名独立研究者，Gupta博士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，以设计使用模式生物的治疗策略。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理想的机会，该实验室在研究肌肉疾病的遗传原因和治疗方面长期存在的兴趣和经验，优秀的导师，儿童医院和哈佛医学院社区内的可用资源和合作机会。K01奖提供的支持将帮助她完成培训，并为她提供一个跳板，为成功申请R01提供数据，为她未来的工作提供资金。

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.