Investigating the Mechanism Regulating Alternative Splicing of Neural Agin: A Novel Therapeutic Entry Point for Congenital Myasthenic Syndrome

研究调节神经Agin选择性剪接的机制：先天性肌无力综合征的新治疗切入点

基本信息

批准号：
9098986
负责人：
Matteo Ruggiu
金额：
$ 49.5万
依托单位：
ST. JOHN'S UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2021-08-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The central nervous system comprises the tissues and cells with the highest rate of alternative splicing in the body, and RNA-binding proteins play a major functional role in neurons. To better understand the contribution of RNA processing to nerve cell biology, and to help elucidate the function that RNA processing regulators play in neuron physiology and neurologic disorders it is necessary to identify which RNA-binding proteins are involved in these biological pathways, and to characterize how they work at the molecular level. Our long-term goal is to understand the molecular mechanisms regulating protein-RNA networks that control alternative splicing, and how they relate to neuron biology, and to disease of the nervous system. The objective of this proposal is to study the molecular basis of how NOVA, a neuron-specific splicing factor involved in an autoimmune motor disease, regulates nerve cell-specific alternative splicing of the ubiquitous protein agrin - a molecule that is the master architect of nerve-muscle synapses at the neuromuscular junction and that is involved in congenital myasthenic syndrome (CMS) in humans. Our preliminary data indicate that mice that are null for the two Nova1 and Nova2 genes fail to make a nerve-derived splice isoform of agrin - termed Z+ agrin - that is critical for the formation, development, and maintenance of the neuromuscular junction. However, the specific mechanism by which Nova regulates this essential developmental switch is still unknown. The central hypothesis of this proposal is that NOVA directly regulates alternative splicing of agrin at the Z site to shape neuromuscular synapses, and that this splicing switch constitutes a novel entry point for therapeutic intervention in specific disorders of the nervous system. In Aim 1 we will test the hypothesis that a novel intronic splicing enhancer mediates Nova-dependent inclusion of agrin Z exons directly. To tackle this question we have developed a cell-based splicing assay that will allow us to test the function of Nova proteins in combination with agrin minigenes. In Aim 2 we will analyze the consequences of Nova deficiency in the formation of ribonucleoprotein complexes and its relationship to the etiology of nervous system pathologies by using an inducible neuronal cell model system of Nova deficiency. This system addresses the inherent technical difficulties in generating splicing-active extracts from mouse brain, while at the same time it provides a flexible platform to test whether modulation of agrin splicing at the Z site is feasible entry point for therapeutic intervention. Understanding how the Nova-agrin regulatory switch is regulated may have clinical implications in RNA-mediated neurodegenerative disorders, CMS pathology, Alzheimer's disease, and epilepsy. Furthermore, this project will provide both undergraduate and graduate students with a unique opportunity to learn the fundamentals of molecular biology and biomedical research, and help them in their pursue of a career in the biomedical field.

描述（由适用提供）：中枢神经系统包括体内替代剪接速率最高的组织和细胞，RNA结合蛋白在神经元中起主要功能作用。为了更好地了解RNA处理对神经细胞生物学的贡献，并帮助阐明RNA加工调节剂在神经元生理和神经系统疾病中发挥作用的功能，有必要确定这些生物学途径中涉及哪些RNA结合蛋白，并表征它们在分子水平上的作用。我们的长期目标是了解分子机制调节控制替代剪接的蛋白质-RNA网络，以及它们与神经元生物学的关系以及与神经系统疾病的关系。 The objective of this proposal is to study the molecular basis of how NOVA, a neuron-specific splicing factor involved in an autoimmune motor disease, regulates nerveve Cell-specific alternative splicing of the ubiquitous protein agrin - a molecule that is the master architect of nerve-muscle synapses at the neuromuscular junction and that is involved in congenital myasthenic syndrome (CMS)在人类中。我们的初步数据表明，对两个Nova1和Nova2基因无效的小鼠无法使Agrin的神经衍生的剪接亚型 - 称为Z+ Agrin，这对于神经肌肉连接的形成，发育和维持至关重要。但是，NOVA调节这种基本发展开关的特定机制仍然未知。该提议的中心假设是，NOVA直接调节Z位置上的Agrin的替代剪接以塑造神经肌肉突触，并且该剪接开关构成了针对神经系统特定疾病的治疗干预的新型切入点。在AIM 1中，我们将检验以下假设：一种新型的内含子剪接增强子直接介导Nova依赖性的Agrin Z外显子。为了解决这个问题，我们开发了一种基于细胞的剪接测定法，这将使我们能够与Agrin Minigenes结合使用Nova蛋白的功能。在AIM 2中，我们将通过使用NOVA缺乏症的可诱导神经元细胞模型系统来分析NOVA缺乏症在形成色带核蛋白复合物中的后果及其与神经系统病理学病因的关系。该系统解决了从鼠标大脑中生成剪接活性提取物的固有技术困难，而与此同时，它提供了一个灵活的平台，以测试Z位点的Agrin剪接调制是否是可行的治疗干预切入点。了解如何调节Nova-Agrin调节转换可能对RNA介导的神经退行性疾病，CMS病理学，阿尔茨海默氏病和癫痫病具有临床意义。此外，该项目将为本科生和研究生提供一个独特的机会，以学习分子生物学和生物医学研究的基础知识，并帮助他们从事生物医学领域的职业。