Insight Into The RNA Processing And Decay Pathways Critical For Proper Neuronal Development And Function Through Focus On Mutations That Cause Pontocerebellar Hypoplasia

通过关注导致脑桥小脑发育不全的突变，深入了解对神经元正常发育和功能至关重要的 RNA 加工和衰变途径

• 批准号: 10268003
• 负责人: ANITA H. CORBETT
• 金额: $ 3.8万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-11 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10268003
• 关键词: Affect; Amino Acid Substitution; Amino Acids; Animal Model; Atrophic; Biochemical; Biology; Brain region; Cells; Cerebellum; Child; Collaborations; Complex; Couples; Critical Pathways; Data; Defect; Disease; Drosophila genus; Enzymes; Etiology; Event; Functional disorder; Gene Expression; Genes; Genetic Transcription; Goals; Hoof; Human; Impairment; Life; Link; Longevity; Mediating; Molecular; Mus; Mutation; Nature; Neurodegenerative Disorders; Neurologic Dysfunctions; Neurons; Pathology; Play; Pontine structure; Pontocerebellar hypoplasia; Post-Transcriptional RNA Processing; RNA; RNA Decay; RNA Degradation; RNA Interference; RNA Processing; RNA Splicing; Research Design; Ribonucleases; Role; Saccharomycetales; Series; Structure; Tissues; Transfer RNA; Work; cofactor; disease-causing mutation; endonuclease; exosome; insight; nervous system disorder; neuron development; public health relevance

Original Project Summary/Abstract for van Hoof, Ambro R01 GM130147 PROJECT SUMMARY Post-transcriptional processing of RNA is a critical regulatory step in gene expression. Many evolutionarily conserved RNA processing enzymes mediate these key post-transcriptional events. This proposal focuses on molecular mechanisms linked to PontoCerebellar Hypoplasia (PCH), which serves as a paradigm for a growing number of neurological diseases caused by mutations in genes encoding RNA processing factors. PCH is a group of autosomal recessive neurodegenerative diseases characterized by hypoplasia/atrophy of the cerebellum and pons that is often fatal within the first year of life. Mutations that cause PCH type 1 (PCH1) occur in genes that encode structural subunits of the RNA exosome (human Rrp40, 43, and 45), which plays critical roles in both RNA processing and degradation. Mutations that cause PCH types 2, 4, and 5 (PCH2/4/5) lie in genes that encode tRNA splicing endonuclease subunits (TSEN2, 15, 34, and 54). TSEN has a well-characterized role in tRNA processing but also other yet undefined functions. The subunits of these RNase complexes are all evolutionarily conserved and essential for viability. PCH1 mutations cause single amino acid substitutions that primarily occur in conserved residues. The discovery that mutations in multiple components of these complexes cause PCH strongly suggests that RNA processing dysfunction underlies PCH pathology. However, limited studies have assessed the functional consequences of these amino acid substitutions. Furthermore, given the common disease etiology, mutations in either the RNA exosome or TSEN complex could impair common RNA targets or classes of RNA targets, but the RNAs affected have not been systematically defined. These links to common biology strongly support our working hypothesis that mutations that cause PCH Types 1/2/4/5 impair the processing of a common set of RNA targets. Our previous collaborative efforts provide proof of principle that studies in model organisms can provide insight into how specific disease-causing amino acid substitutions impair RNA exosome function. Here we draw on our established collaboration and extensive preliminary data to perform a series of mechanistic studies in four aims. Aim 1 assesses the functional consequences of amino acid changes that occur in PCH using budding yeast; Aim 2 employs biochemical analysis in mouse cerebellum and cultured neuronal cells to define RNA exosome cofactors that could contribute to the tissue-specific nature of PCH; Aim 3 couples studies in budding yeast and cultured neuronal cells to identify common RNA targets of the TSEN and RNA exosome complexes; and, finally, Aim 4 employs tissue-specific RNAi in Drosophila to begin to assess the requirement for specific RNA exosome cofactors and TSEN subunits in neurons. The long-term goal of this work is to fully define the function of these evolutionarily conserved RNase complexes while providing insight into molecular mechanisms that could contribute to neurological dysfunction in PCH.

Van Hoof的原始项目摘要/摘要，Ambro R01 GM130147 项目总结 RNA的转录后加工是基因表达调控的关键步骤。许多进化上的 保守的RNA处理酶介导这些关键的转录后事件。这项提案的重点是 与桥小脑发育不全(PCH)相关的分子机制，这是一个生长发育的范例 由编码RNA加工因子的基因突变引起的神经系统疾病的数量。PCH是一种 一组常染色体隐性遗传性神经退行性疾病，其特征是大脑发育不良/萎缩 小脑和脑桥，通常在生命的第一年内是致命的。 导致PCH1型(PCH1)的突变发生在编码RNA结构亚单位的基因中 外切体(人Rrp40、43和45)，在RNA加工和降解中发挥关键作用。 导致PCH2型、4型和5型(PCH2/4/5)的突变存在于编码tRNA剪接内切酶的基因中 亚基(TSEN2、15、34和54)。Tsen在tRNA加工中扮演了一个很好的角色，但也有其他 未定义的函数。这些核糖核酸酶复合体的亚基在进化上都是保守的，对 生存能力。PCH1突变导致单一氨基酸替换，这些替换主要发生在保守残基中。 这些复合体的多个成分的突变导致PCH的发现有力地表明 RNA加工功能障碍是PCH病理的基础。然而，有限的研究评估了 这些氨基酸替换的功能后果。此外，考虑到常见的疾病病因， RNA外切体或Tsen复合体中的突变可能会损害常见的RNA靶标或RNA类别 目标，但受影响的RNA尚未得到系统定义。这些与普通生物学有很强的联系 支持我们的工作假设，即导致PCH类型1/2/4/5的突变会损害 常见的一组RNA靶标。我们之前的合作努力提供了在模型中研究的原则证明 生物体可以洞察特定的致病氨基酸替换如何损害RNA外切体 功能。在这里，我们利用我们已建立的协作和广泛的初步数据来执行一系列 四个目标的机械论研究。目标1评估氨基酸变化的功能后果 使用发芽酵母在PCH中发生；Aim 2使用生化分析在小鼠小脑中进行培养 神经细胞，以确定可能有助于PCH组织特异性的RNA外体辅助因子；目的 在发芽酵母和培养的神经细胞中进行3项联合研究以确定Tsen的共同RNA靶标 和RNA外体复合体；最后，目标4利用果蝇中组织特异性的RNAi开始 评估神经元对特定RNA外体辅因子和Tsen亚单位的需求。长期的 这项工作的目标是充分定义这些进化保守的核糖核酸酶复合体的功能，同时 洞察可能导致PCH神经功能障碍的分子机制。