Defining the Role of Aminoacyl-tRNA Synthetases in Human Health and Disease

项目摘要 氨酰-tRNA合成酶（ARSs）是一种广泛表达的必需酶， 将氨基酸连接到同源的tRNA分子上。重要的是，编码ARS的37个基因座中有34个已经被发现。 涉及无数显性和隐性临床表型，使这些酶成为 人类遗传性疾病现在重要的是系统地评估ARS等位基因在人类疾病中的作用 表型，并确定它们如何影响蛋白质翻译。这些数据将提供深入了解 疾病相关ARS等位基因的分子病理学，其影响广泛的组织。此外，委员会认为， 明确ARS相关疾病的分子机制将允许通过以下方法快速诊断患者： 区分人类群体中的致病性等位基因和非致病性等位基因。我们和其他人已经证明， 疾病相关的ARS等位基因引起tRNA充电的功能丧失效应。但若干 关键的问题仍然存在，包括：什么是由ARS等位基因引起的疾病表型的全谱？ 在人群中有哪些ARS等位基因是致病的？功能丧失 错义ARS变异导致显性周围神经病变？以及功能丧失的ARS变体 影响mRNA加工和蛋白质表达？在这里，我们采用多个既定的和互补的 模型系统--计算系统、生物化学系统、细胞系统、酵母系统、蠕虫系统和小鼠系统--来解决上述问题 问题.我们的努力将包括：（1）研究患者人群，以揭示新发现的ARS变异， 疾病发作;（2）使用蠕虫和小鼠模型深入探究ARS相关表型;（3） 使用大规模平行诱变系统地确定ARS变体对基因功能的影响 和哺乳动物细胞活力测定;（4）测试神经病相关的ARS变体的显性- 体外和体内的负性和毒性功能获得效应;和（5）测试功能丧失、疾病- 通过核糖体谱分析和质谱分析研究相关ARS变体对蛋白质翻译的影响 酵母、蠕虫和小鼠模型。总而言之，本建议书中概述的研究领域将大大改善 我们对某些ARS等位基因如何引起显性和隐性人类疾病表型的理解。

PROJECT ABSTRACT Aminoacyl-tRNA synthetases (ARSs) are a ubiquitously expressed, essential class of enzymes responsible for ligating amino acids to cognate tRNA molecules. Importantly, 34 of the 37 loci encoding an ARS have been implicated in myriad dominant and recessive clinical phenotypes, making these enzymes a major contributor to human inherited disease. It is now important to systematically assess the role of ARS alleles in human disease phenotypes and to determine how they affect protein translation. These data will provide insight into the molecular pathology of disease-associated ARS alleles, which affect a wide range of tissues. Furthermore, defining the molecular mechanisms of ARS-associated disease will allow rapid patient diagnosis through distinguishing pathogenic from non-pathogenic alleles in human populations. We and others have shown that disease-associated ARS alleles cause a loss-of-function effect on tRNA charging. However, a number of critical questions remain, including: What is the full spectrum of disease phenotypes caused by ARS alleles? What is the subset of ARS alleles in human populations that are pathogenic? How do loss-of-function missense ARS variants cause dominant peripheral neuropathy? and How do loss-of-function ARS variants affect mRNA processing and protein expression? Here, we employ multiple established and complementary model systems—computational, biochemical, cellular, yeast, worm, and mouse—to address the above questions. Our efforts will include: (1) studying patient populations to implicate newly identified ARS variants in disease onset; (2) deeply interrogating ARS-related phenotypes using worm and mouse models; (3) systematically determining the effect of ARS variants on gene function using massively parallel mutagenesis and mammalian cell viability assays; (4) testing neuropathy-associated ARS variants for both dominant- negative and toxic gain-of-function effects in vitro and in vivo; and (5) testing loss-of-function, disease- associated ARS variants for an effect on protein translation via ribosomal profiling and mass spectrometry in yeast, worm, and mouse models. In sum, the areas of study outlined in this proposal will dramatically improve our understanding of how certain ARS alleles give rise to dominant and recessive human disease phenotypes.