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

项目总结： RNA外切体，一种基本的分子机器，对几乎每一个 RNA，是一种多亚基复合体，在所有真核生物中都是保守的 结构。最近，在编码rna结构亚单位的基因中发现了突变。 外显体。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 独特的特定于组织的人类疾病，包括神经退行性疾病和发育障碍。 这种与RNA外切体相关的疾病越来越多，可以归类为“外显子病”。这种疾病-- 导致突变的是错义突变，它改变了这些结构保守区域中的单一氨基酸 亚单位。如果氨基酸替换，解释这些疾病的组织特异性性质是具有挑战性的 一般影响这个复合体的分子功能(S)。相反，不同的氨基酸替换可以 具有不同的结果，最终对关键交互和/或综合体的完整性产生不同的影响 干扰RNA靶向/处理。我假设不同的致病氨基酸替换 不同地影响RNA外切体的功能。这里提出的研究将比较体内的 在结构亚单位基因EXOSC2和EXOSC5中发现的两种外生症突变的后果 (由我们的临床合作者鉴定)，使用酿酒酵母。EXOSC5的突变与 小脑变性，而EXOSC2的突变与一种以视网膜炎为特征的新综合征有关 色素变性、听力丧失、过早衰老和轻度智力残疾。我们已经生成了S。 这些疾病相关氨基酸替换的每一个的Cerevisiae模型：EXOSC2氨基酸替换， 酵母同源基因Rrp4中的G226D和与疾病相关的EXOSC5氨基酸替换L191H。 酵母同源基因Rrp45。这些氨基酸替换中的每一个都会导致对温度敏感的生长缺陷 这一点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 通过不同的RNA外体辅助因子的过度表达而受到差异抑制，这些因子与 赋予靶标特异性的复合体。这些初步数据表明，这两种疾病的体内后果截然不同。 突变，说明了研究每种外显体疾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)比较检测RNA外切体复合体的完整性 两种外生体疾病突变模型(AIM 1)；2)评估不同影响的RNA外切体相互作用 使用靶向生化分析和基于发现的遗传筛选的外生体疾病突变模型 (目标2)；和3)使用RNA-Seq来定义由外生症模型氨基酸改变的RNA的谱 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如何 RNA外切体的结构亚基损害了这一重要复合体的功能，为我们提供了对如何 不同的外生体突变会导致不同的临床表现。

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.