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

项目摘要/摘要 中枢神经系统包括具有最高选择性剪接率的组织和细胞 而RNA结合蛋白在神经元中起着主要的功能作用。为了更好地理解这些贡献 RNA剪接对神经细胞生物学的影响，并帮助阐明剪接在神经元生理学中所起的作用 和神经系统疾病，有必要表征如何包含或跳过特定的外显子 调节对神经元至关重要的分子的生理特性，如离子通道 功能，并表征这些剪接事件是如何在细胞和分子水平上调节的。我们的 长期目标是了解调控蛋白质-RNA网络的分子机制 大脑中的选择性剪接，以及它们与神经元生物学和神经紊乱的关系 系统。这项建议的目的是研究电压门控钙离子CaV3.1的替代剪接 对调节细胞膜兴奋性有显著贡献的通道--特别是在肌肉中 和神经元-脊髓小脑性共济失调患者的突变-42(SCA42)在不同的 神经细胞类型，以及它如何可能对通道活动的调节起到作用。的中心假说 这一提议是，C端CaV3.1的神经细胞类型特异性选择性剪接塑造了 这种电压门控离子通道的生理特性。 在目标1中，我们将检验这样的假设，即CaV3.1选择性剪接外显子在 大脑中不同类型的神经细胞。为了解决这个问题，我们开发了一个基于RNAseq的 生物信息学管道，将允许我们询问定义的神经元亚类之间的差异剪接 在不同的层级。这种方法不仅提供了替代剪接的快照 CaV3.1的景观在大脑中不同的神经元亚类中，但它也会让我们产生 对这些选择性剪接事件如何调控的预测。在目标2中，我们将检验假设 C-末端的选择性剪接显著有助于调节细胞的生理活性 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选择性剪接的外显子， 了解选择性剪接如何调节通道活动是至关重要的。 由于患有CaV3.1相关病理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 调节，可能在脊髓小脑性共济失调，癫痫， 和自闭症谱系障碍，它可能会为新的治疗策略的设计提供信息。此外，这一点 该项目将为本科生和研究生提供一个独特的学习机会 分子生物学和生物医学研究的基础知识，并帮助他们在 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.