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.

项目摘要： RNA外泌体是一种必不可少的分子机，几乎有助于处理和/或衰减 RNA的种类是一种多生产络合物，在所有真核生物中均以序列和序列保守 结构。最近，已经在编码RNA结构亚基的基因中鉴定了突变 外部。尽管这些突变都发生在编码同一复合物组成部分的基因中，但它们会导致 独特的组织特异性人类疾病，包括神经退行性疾病和发育障碍。 越来越多的RNA外泌体连锁疾病的收集可以归类为“外部病变”。疾病 - 引起突变的是错义突变，这些突变会改变这些结构的组成区域中的单个氨基酸 亚基。如果氨基酸取代 通常会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，会影响关键相互作用和/或复合物的完整性，最终 破坏RNA靶向/处理。我假设那是引起疾病的独特氨基酸取代 差异影响RNA外泌体的功能。这里提出的研究将比较体内 在结构亚基基因exosc2和exosc5中鉴定出的两个外瘤突变的后果 （由我们的临床合作者确定），使用酿酒酵母。 exosc5中的突变与 小脑变性，而exosc2中的突变与以视网膜炎为特征的新型综合征有关 色素，听力损失，过早衰老和轻度智力障碍。我们已经生成S. 这些疾病连接的氨基酸取代的酿酒酵母模型：exosc2氨基酸取代， 酵母直系同源物RRP4中的G226D和疾病连接的exosc5氨基酸取代，L191H 酵母直系同源物RRP45。这些氨基酸取代都会导致温度敏感的生长缺陷 可以在酵母遗传学方法中探索。重要的是，我的初步数据表明这些突变 通过不同的RNA外泌体辅助因子的过表达来不同地抑制 复杂以赋予目标特异性。这些初步数据表明了这两个的体内后果明显 突变，说明了研究每种外部病的分子基础的重要性和价值 体内系统中的突变。为了实现这一目标，我将1）检查RNA外泌体复杂完整性比较 两个外肌瘤突变模型（AIM 1）； 2）评估对RNA的不同影响的RNA外泌体相互作用 使用靶向生化测定和基于发现的遗传筛选的外瘤突变模型 （目标2）; 3）雇员RNA-seq定义了由外瘤模型氨基酸改变的RNA频谱 替换（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，提供了有关如何 不同的外病变突变可能导致不同的临床表现。