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: 10198947
• 负责人: ANITA H. CORBETT
• 金额: $ 29.97万
• 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 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10198947
• 关键词: Affect; Amino Acid Substitution; Amino Acids; Animal Model; Atrophic; Behavior; Biochemical; Biological Models; Biology; Brain region; Cells; Cerebellum; Chemistry; Child; Cleaved cell; Collaborations; Complex; Couples; Critical Pathways; Data; Defect; Degradation Pathway; Disease; Drosophila genus; Enzymes; Etiology; Eukaryota; Event; Functional disorder; Gene Expression; Genes; Genetic Transcription; Goals; Human; Immunoprecipitation; Impairment; Learning; Life; Link; Longevity; Mass Spectrum Analysis; Mediating; Memory; Messenger RNA; Molecular; Mus; Mutation; Nature; Neurodegenerative Disorders; Neurologic Dysfunctions; Neurons; Pathology; Pathway interactions; Play; Pontine structure; Pontocerebellar hypoplasia; Post-Transcriptional RNA Processing; Proteins; Publishing; RNA; RNA Decay; RNA Degradation; RNA Interference; RNA Processing; RNA Splicing; RNA analysis; Research Design; Ribonucleases; Role; Saccharomycetales; Series; Specificity; Structure; Substrate Specificity; Testing; Tissues; Transcript; Transfer RNA; Work; Yeast Model System; Yeasts; brain morphology; cell growth; cofactor; disease-causing mutation; endonuclease; exosome; experimental study; fly; human disease; in vivo; insight; molecular pathology; mutant; nervous system disorder; neuron development; public health relevance; transcriptome sequencing

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.

项目概要 RNA 的转录后加工是基因表达的关键调控步骤。许多进化论 保守的 RNA 加工酶介导这些关键的转录后事件。该提案的重点是 与脑桥小脑发育不全 (PCH) 相关的分子机制，PCH 是脑桥小脑发育不全的范例 许多神经系统疾病是由编码 RNA 加工因子的基因突变引起的。 PCH是一个 一组以神经发育不全/萎缩为特征的常染色体隐性神经退行性疾病 小脑和脑桥的损伤通常在出生后第一年内致命。 导致 PCH 1 型 (PCH1) 的突变发生在编码 RNA 结构亚基的基因中 外泌体（人 Rrp40、43 和 45），在 RNA 加工和降解中发挥着关键作用。 导致 PCH 2、4 和 5 型 (PCH2/4/5) 的突变位于编码 tRNA 剪接核酸内切酶的基因中 亚基（TSEN2、15、34 和 54）。 TSEN 在 tRNA 加工中具有明确的作用，但在其他方面也有作用 未定义的函数。这些 RNase 复合物的亚基在进化上都是保守的，并且对于 生存能力。 PCH1 突变导致主要发生在保守残基中的单个氨基酸取代。 这些复合物的多个成分的突变导致 PCH 的发现强烈表明 RNA 加工功能障碍是 PCH 病理学的基础。然而，有限的研究评估了 这些氨基酸取代的功能后果。此外，考虑到常见疾病的病因， RNA 外泌体或 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。这些与常见生物学密切相关 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 一组共同的 RNA 靶标。我们之前的合作成果提供了模型研究的原理证明 生物体可以深入了解特定致病氨基酸取代如何损害 RNA 外泌体 功能。在这里，我们利用我们已建立的合作和广泛的初步数据来执行一系列 机制研究的四个目标。目标 1 评估氨基酸变化的功能后果 使用芽殖酵母发生在 PCH 中；目标 2 对小鼠小脑进行生化分析并进行培养 神经元细胞定义RNA外泌体辅助因子，这可能有助于PCH的组织特异性；目的 3 对芽殖酵母和培养神经元细胞进行研究，以确定 TSEN 的常见 RNA 靶点 和RNA外泌体复合物；最后，Aim 4 在果蝇中采用组织特异性 RNAi 来开始 评估神经元对特定 RNA 外泌体辅因子和 TSEN 亚基的需求。长期来看 这项工作的目标是充分定义这些进化上保守的 RNase 复合物的功能，同时 深入了解可能导致 PCH 神经功能障碍的分子机制。