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（申请人提供）：中枢神经系统由体内选择性剪接率最高的组织和细胞组成，RNA结合蛋白在神经元中发挥主要功能作用，以更好地了解RNA加工对神经的贡献。细胞生物学，并帮助阐明 RNA 加工调节剂在神经元生理学和神经系统疾病中发挥的功能，有必要确定哪些 RNA 结合蛋白参与这些生物途径，并在分子水平上表征它们如何发挥作用。长期目标目的是了解控制选择性剪接的蛋白质-RNA 网络的分子机制，以及它们与神经元生物学和神经系统疾病的关系。本提案的目的是研究 NOVA（一种神经元）如何发挥作用的分子基础。参与自身免疫运动疾病的特定剪接因子，调节无处不在的集聚蛋白蛋白的神经细胞特异性选择性剪接，集聚蛋白是神经肌肉接头处神经肌肉突触的主要架构分子，并且参与其中我们的初步数据表明，Nova1 和 Nova2 两个基因缺失的小鼠无法产生 agrin 的神经源性剪接亚型（称为 Z+ agrin），这对于形成、发育至关重要。然而，Nova 调节这一重要发育开关的具体机制仍不清楚。该提议的中心假设是 NOVA 直接调节 agrin 的选择性剪接。 Z 位点塑造神经肌肉突触，并且这种剪接开关构成了神经系统特定疾病治疗干预的新切入点。在目标 1 中，我们将测试一种新的内含子剪接增强子介导 Nova 依赖性 Agrin Z 包含的假设。为了解决这个问题，我们开发了一种基于细胞的剪接测定，使我们能够测试 Nova 蛋白与集聚蛋白小基因结合的功能。在目标 2 中，我们将分析结果。通过使用 Nova 缺陷的诱导神经元细胞模型系统，研究 Nova 缺陷在核糖核蛋白复合物形成中的作用及其与神经系统病理学病因学的关系。该系统解决了从小鼠大脑中产生剪接活性提取物的固有技术困难。同时，它提供了一个灵活的平台来测试 Z 位点的集聚蛋白剪接调节是否是治疗干预的可行切入点，了解 Nova-集聚蛋白调节开关的调节方式可能对 RNA 介导的临床意义。此外，该项目将为本科生和研究生提供学习分子生物学和生物医学研究基础知识的独特机会，并帮助他们追求生物医学领域的职业生涯。场地。