A Budding Yeast Model for Human Disease-Mutations in the RNA Exosome

人类疾病 RNA 外泌体突变的萌芽酵母模型

• 批准号: 10160642
• 负责人: Maria Carson Sterrett
• 金额: $ 4.6万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10160642
• 关键词: 5' -exoribonuclease; Affect; Amino Acid Substitution; Amino Acids; Binding; Biochemical; Biochemistry; Biological Assay; Biological Models; Cell physiology; Cells; Cerebellar degeneration; Clinical; Collection; Comparative Study; Complement; Complex; Data; Data Set; Defect; Disease; Eukaryota; Genes; Genetic Models; Genetic Screening; Glycerol; Goals; Growth; Immunoprecipitation; Impairment; Individual; Intellectual functioning disability; Link; Messenger RNA; Missense Mutation; Modeling; Molecular; Molecular Machines; Mutation; Nature; Neurodegenerative Disorders; Northern Blotting; Patients; Phenotype; Premature aging syndrome; Process; RNA; RNA Decay; RNA Processing; Reporting; Retinitis Pigmentosa; Ribosomal RNA; Saccharomyces cerevisiae; Saccharomycetales; Specificity; Structure; Surveys; Symptoms; Syndrome; System; Temperature; Testing; Tissues; Variant; Yeast Model System; Yeasts; base; cofactor; comparative; developmental disease; disease-causing mutation; exosome; genetic approach; hearing impairment; human disease; in vivo; insight; mutant; nervous system disorder; novel; overexpression; transcriptome; transcriptome sequencing; yeast genetics

PROJECT SUMMARY: The RNA exosome, an essential molecular machine that contributes to processing and/or decay of nearly every species of RNA, is a multi-subunit complex that is conserved across all eukaryotes in both sequence and structure. Recently, mutations have been identified in genes that encode structural subunits of the RNA exosome. Although these mutations all occur in genes that encode components of the same complex, they cause distinct, tissue specific human disease, including neurodegenerative diseases and developmental disorders. This growing collection of RNA exosome-linked diseases can be classified as “exosomopathies”. The disease- causing mutations are missense mutations that alter single amino acids in conserved regions of these structural subunits. Explaining the tissue-specific nature of these diseases is challenging if the amino acid substitutions generally affect the molecular function(s) of this complex. Rather, the different amino acid substitutions could have distinct consequences that differentially affect key interactions and/or the integrity of the complex, ultimately disrupting RNA targeting/processing. I hypothesize that distinct disease-causing amino acid substitutions differentially impact the function of the RNA exosome. The studies proposed here will compare the in vivo consequences of two exosomopathy mutations identified in the structural subunit genes EXOSC2 and EXOSC5 (identified by our clinical collaborator), using Saccharomyces cerevisiae. Mutations in EXOSC5 are linked to cerebellar degeneration, while mutations in EXOSC2 are linked to a novel syndrome characterized by retinitis pigmentosa, hearing loss, premature aging and mild intellectual disability. We have already generated S. cerevisiae models for each of these disease-linked amino acid substitutions: EXOSC2 amino acid substitution, G226D in the yeast orthologue Rrp4, and the disease-linked EXOSC5 amino acid substitution, L191H in the yeast orthologue Rrp45. Each of these amino acid substitutions causes a temperature sensitive growth defect that can be exploited in yeast genetics approaches. Importantly, my preliminary data reveal that these mutations are differentially suppressed by overexpression of distinct RNA exosome cofactors, factors that interact with the complex to confer target specificity. These preliminary data suggest distinct in vivo consequences for these two mutations, illustrating the importance and value of studying the molecular underpinnings of each exosomopathy mutation in an in vivo system. To achieve this goal, I will 1) examine RNA exosome complex integrity comparing the two exosomopathy mutant models (Aim 1); 2) assess differentially affected RNA exosome interactions in exosomopathy mutant models using both targeted biochemical assays and discovery-based genetic screens (Aim 2); and 3) employ RNA-Seq to define the spectrum of RNAs altered by exosomopathy-modeled amino acid substitutions (Aim 3). This proposed comparative study will reveal how different amino acid substitutions in structural subunits of the RNA exosome impair the function of this essential complex, providing insight into how different exosomopathy mutations could cause distinct clinical manifestations.

项目概要： RNA外泌体是一种基本的分子机器，它有助于几乎每一个细胞的加工和/或衰变。 RNA是一种多亚基复合物，在所有真核生物中在序列和结构上都是保守的。 结构最近，在编码RNA结构亚基的基因中发现了突变， 外泌体尽管这些突变都发生在编码同一复合体成分的基因中，但它们会导致 不同的、组织特异性的人类疾病，包括神经变性疾病和发育障碍。 这种不断增长的RNA外泌体相关疾病可被归类为“外泌体病”。疾病- 引起突变的是错义突变，其改变这些结构蛋白的保守区域中的单个氨基酸。 亚单位。解释这些疾病的组织特异性性质是具有挑战性的，如果氨基酸取代 通常影响该复合物的分子功能。相反，不同的氨基酸取代可以 具有不同的后果，这些后果最终会对复合体的关键相互作用和/或完整性产生不同的影响 破坏RNA靶向/加工。我假设不同的致病氨基酸替换 差异地影响RNA外泌体的功能。本文提出的研究将比较体内 在结构亚基基因EXOSC 2和EXOSC 5中鉴定的两种外泌体病突变的后果 （由我们的临床合作者鉴定），使用酿酒酵母。EXOSC 5中的突变与 小脑变性，而EXOSC 2突变与一种以视网膜炎为特征的新综合征有关 色素沉着、听力损失、早衰和轻度智力残疾。我们已经生成了S。 这些疾病相关的氨基酸取代中的每一种的酿酒酵母模型：EXOSC 2氨基酸取代， G226 D在酵母直向同源物Rrp 4中，以及疾病相关的EXOSC 5氨基酸取代，L191 H在酵母直向同源物Rrp 4中。 酵母直向同源物Rrp 45。这些氨基酸取代中的每一种都会导致温度敏感性生长缺陷 可以在酵母遗传学方法中加以利用。重要的是，我的初步数据显示这些突变 被不同的RNA外泌体辅因子的过表达差异性地抑制，RNA外泌体辅因子是与 复合物以赋予靶特异性。这些初步数据表明，这两个不同的体内后果 突变，说明了研究每种外泌体病的分子基础的重要性和价值 在体内系统中的突变。为了实现这一目标，我将1）检查RNA外泌体复合物的完整性，比较 两种外泌体病突变体模型（目的1）; 2）评估在 使用靶向生化测定和基于发现的遗传筛选的外泌体病突变体模型 (Aim 2）;和3）采用RNA-Seq来定义由外泌体病模型化的氨基酸改变的RNA谱 替代（目标3）。这项拟议的比较研究将揭示不同的氨基酸取代， RNA外泌体的结构亚基损害了这种基本复合物的功能， 不同的外泌体病突变可引起不同的临床表现。