Alternative Splicing Modulates the Activity of CaV3.1, an Ion Channel Gene Involved in Spinocerebellar Ataxia, Epilepsy, and Autism Spectrum Disorders.

项目概要/摘要 中枢神经系统由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着重要的功能作用。为了更好地理解贡献 RNA剪接与神经细胞生物学的关系，并帮助阐明剪接在神经元生理学中的功能 和神经系统疾病，有必要描述特定外显子的包含或跳过如何 调节分子的生理特性（例如离子通道），这对神经元至关重要 功能，并表征这些剪接事件如何在细胞和分子水平上受到调节。我们的 长期目标是了解调节蛋白质-RNA 网络的分子机制，这些网络控制着 大脑中的选择性剪接，以及它们与神经元生物学和神经疾病的关系 系统。该提案的目的是研究 CaV3.1（一种电压门控钙）的选择性剪接如何 通道对细胞膜兴奋性的调节有显着贡献——特别是在肌肉中 和神经元——脊髓小脑共济失调 42 (SCA42) 患者中发生突变，在不同的环境中受到调节 神经元细胞类型，以及它如何有助于通道活动的调节。中心假设为 该提议认为 CaV3.1 在 C 末端的神经元细胞类型特异性选择性剪接塑造了 该电压门控离子通道的生理特性。 在目标 1 中，我们将检验 CaV3.1 选择性剪接外显子在 大脑中不同的神经元细胞类型。为了解决这个问题，我们开发了基于 RNAseq 的 生物信息学管道将使我们能够询问定义的神经元亚类之间的差异剪接 处于不同的层级。该方法不仅提供选择性剪接的快照 CaV3.1 在大脑不同神经元亚类中的景观，但它也将使我们能够生成 关于如何调控这些选择性剪接事件的预测。在目标 2 中，我们将检验以下假设： C 末端的选择性剪接显着有助于调节生理活性 这个离子通道。由于 CaV3.1 中的几个与疾病相关的突变映射到可变剪接的外显子， 了解选择性剪接如何调节通道活动至关重要。 由于患有 CaV3.1 相关疾病的患者表现出钙电流特性缺陷， 了解选择性剪接如何调节 CaV3.1 的生物学功能以及这如何 调节受到调节，可能对脊髓小脑共济失调、癫痫、 和自闭症谱系障碍，它可以为新型治疗策略的设计提供信息。而且，这 该项目将为本科生和研究生提供独特的学习机会 分子生物学和生物医学研究的基础知识，并帮助他们追求职业生涯 生物医学领域。

PROJECT SUMMARY/ABSTRACT 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 splicing to nerve cell biology, and to help elucidate the function that splicing plays in neuron physiology and neurologic disorders it is necessary to characterize how the inclusion or skipping of specific exons modulates the physiological properties of molecules — such as ion channels — that are critical for neuronal function, and to characterize how these splicing events are regulated at the cellular and molecular level. Our long-term goal is to understand the molecular mechanisms regulating protein-RNA networks that control alternative splicing in the brain, and how they relate to the biology of neurons and to disorders of the nervous system. The objective of this proposal is to study how alternative splicing of CaV3.1, a voltage-gated Calcium channel that significantly contributes to the regulation of cell membrane excitability — particularly in muscle and neurons — and that is mutated in patients with spinocerebellar ataxia-42 (SCA42) is regulated in different neuron cell types, and how it may contributes to the modulation of channel activity. The central hypothesis of this proposal is that neuronal cell type-specific alternative splicing of CaV3.1 at the C-terminus shapes the physiological properties of this voltage-gated ion channel. In Aim 1 we will test the hypothesis that CaV3.1 alternatively spliced exons are differentially expressed in different neuronal cell types in the brain. To tackle this question, we have developed an RNAseq-based bioinformatics pipeline that will allow us to interrogate differential splicing between neuronal subclasses defined at different hierarchical levels. This methodology will not only provide a snapshot of the alternative splicing landscape of CaV3.1 in different neuronal subclasses in the brain, but it will also allow us to generate predictions on how these alternative splicing events are regulated. In Aim 2 we will test the hypothesis that alternative splicing at the C-terminus significantly contributes to the regulation of the physiological activity of this ion channel. Since several disease-associated mutations in CaV3.1 map to alternatively spliced exons, understanding how alternative splicing modulates channel activity is critical. Since patients with CaV3.1-associated pathologies display defects in Calcium current properties, understanding how alternative splicing may modulate the biological functions of CaV3.1 and how this modulation is regulated, may have broad and significant clinical implications in spinocerebellar ataxia, epilepsy, and autism spectrum disorders, and it may inform the design of novel therapeutic strategies. Moreover, 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.