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

选择性剪接调节 CaV3.1 的活性，CaV3.1 是一种与脊髓小脑共济失调、癫痫和自闭症谱系障碍有关的离子通道基因。

• 批准号: 10579415
• 负责人: Matteo Ruggiu
• 金额: $ 49.2万
• 依托单位: ST. JOHN'S UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-12 至 2025-09-11
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10579415
• 关键词: Absence Epilepsy; Action Potentials; Affect; Alternative Splicing; Biological Process; Biology; Biomedical Research; Biophysics; Bipolar Disorder; Brain; C-terminal; CACNA1G gene; Calcium; Calcium Channel; Calmodulin; Cardiac; Cell membrane; Cells; Cellular biology; Clinical; Data; Defect; Disease; Epilepsy; Event; Exons; Functional disorder; Gene Expression; Genes; Goals; Hormonal; Human; Ion Channel; Ion Channel Gating; Kinetics; Knockout Mice; Learning; Location; Malignant hyperpyrexia due to anesthesia; Maps; Membrane; Membrane Potentials; Messenger RNA; Methodology; Migraine; Molecular; Molecular Biology; Muscle; Mutate; Mutation; Neuraxis; Neurons; Pathology; Patients; Permeability; Physiological; Physiological Processes; Physiology; Play; Potassium Channel; Property; Proteins; RNA; RNA Splicing; RNA-Binding Proteins; Regulation; Role; Shapes; Signal Transduction Pathway; Spinocerebellar Ataxias; Structure; Testing; Thalamic structure; Timothy syndrome; Tissues; Variant; autism spectrum disorder; base; bioinformatics pipeline; biophysical properties; career; cell type; design; differential expression; experimental study; graduate student; nervous system disorder; neurotransmitter release; novel therapeutic intervention; sensor; success; transcriptome sequencing; undergraduate student; voltage

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.

项目总结/摘要 中枢神经系统包括在神经系统中具有最高比率的选择性剪接的组织和细胞。 RNA结合蛋白在神经元中起着重要的功能作用。为了更好地理解 RNA剪接的神经细胞生物学，并帮助阐明剪接在神经元生理学中发挥的作用 和神经系统疾病，有必要描述特定外显子的包含或跳跃如何影响 调节分子的生理特性--如离子通道--这对神经元的功能至关重要。 功能，并表征这些剪接事件如何在细胞和分子水平上调节。我们 长期目标是了解调节蛋白质-RNA网络的分子机制， 大脑中的选择性剪接，以及它们如何与神经元的生物学和神经系统疾病联系在一起。 系统本研究的目的是研究电压门控性钙通道CaV3.1的选择性剪接， 一种对调节细胞膜兴奋性（尤其是肌肉）有重要作用的通道 在脊髓小脑共济失调患者中发生突变的SCA 42（SCA 42）在不同的 神经元细胞类型，以及它如何有助于调节通道活动。的中心假设 这一建议是，在C末端的CaV3.1的神经元细胞类型特异性选择性剪接形成了 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CaV3.1可变剪接外显子在人乳腺癌中差异表达的假设。 大脑中不同的神经细胞类型。为了解决这个问题，我们开发了一种基于RNAseq的 生物信息学管道，这将使我们能够询问定义的神经元亚类之间的差异剪接 在不同的层次上。这种方法不仅提供了选择性剪接的快照， CaV3.1在大脑中不同神经元亚类中的分布，但它也将使我们能够产生 预测这些选择性剪接事件是如何调节的。在目标2中，我们将检验以下假设： 在C-末端的选择性剪接显著有助于调节 这个离子通道由于CaV3.1中的几种疾病相关突变映射到选择性剪接的外显子， 理解选择性剪接如何调节通道活性是至关重要的。 由于患有CaV3.1相关病理的患者显示出钙电流特性的缺陷， 了解选择性剪接如何调节CaV3.1的生物学功能以及这如何 调节被调节，可能在脊髓小脑共济失调，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。而且这 项目将为本科生和研究生提供一个独特的机会，学习 分子生物学和生物医学研究的基础知识，并帮助他们在他们的职业生涯的追求， 生物医学领域