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

 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起着主要的功能作用。为了更好地了解RNA加工对神经细胞生物学的贡献，并帮助阐明RNA加工调控因子在神经元生理学和神经系统疾病中所起的作用，有必要确定哪些RNA结合蛋白参与了这些生物途径，并在分子水平上表征它们的工作原理。我们的长期目标是了解控制选择性剪接的蛋白质-RNA网络的调节分子机制，以及它们与神经元生物学和神经系统疾病的关系。这项建议的目的是研究NOVA，一种参与自身免疫性运动疾病的神经元特异性剪接因子，如何调节无处不在的蛋白质集聚蛋白的神经细胞特异性选择性剪接的分子基础。集聚蛋白是神经肌肉接头处神经-肌肉突触的主要设计师，与人类先天性肌无力综合征(CMS)有关。我们的初步数据表明，两个Nova1和Nova2基因缺失的小鼠无法产生神经源性集聚蛋白亚型--称为Z+集聚蛋白--这对神经肌肉连接的形成、发育和维持至关重要。然而，Nova调节这一重要发育开关的具体机制仍不清楚。这一建议的中心假设是，Nova直接调节集聚蛋白在Z位点的选择性剪接来形成神经肌肉突触，并且这种剪接开关构成了治疗特定神经系统疾病的新切入点。在目标1中，我们将检验这样的假设，即一种新的内含子剪接增强剂直接介导依赖于NovA的集聚蛋白Z外显子的包含。为了解决这个问题，我们开发了一种基于细胞的剪接试验，这将使我们能够测试Nova蛋白与agrin miniges结合的功能。在目标2中，我们将利用Nova缺乏症的诱导性神经细胞模型系统，分析Nova缺乏症在核糖核蛋白复合体形成中的后果及其与神经系统病理学的关系。该系统解决了从小鼠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的调制是否是治疗干预的可行切入点。了解Nova-agrin调节开关是如何调控的，可能对RNA介导的神经退行性疾病、CMS病理、阿尔茨海默病和癫痫具有临床意义。此外，该项目将为本科生和研究生提供一个学习分子生物学和生物医学研究基础的独特机会，并帮助他们在生物医学领域追求职业生涯。

 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.