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

项目摘要 氨酰-tRNA合成酶(Ars)是一类广泛表达的重要酶。 将氨基酸连接到同源tRNA分子。重要的是，在编码ARS的37个基因座中，有34个是 与无数显性和隐性临床表型有关，使这些酶成为导致 人类遗传性疾病。现在，系统地评估ARS等位基因在人类疾病中的作用是很重要的 并确定它们如何影响蛋白质翻译。这些数据将为我们提供对 与疾病相关的ARS等位基因的分子病理学，影响广泛的组织。此外， 明确ARS相关疾病的分子机制将使患者能够通过 在人类群体中区分致病和非致病等位基因。我们和其他人已经证明了 疾病相关的ARS等位基因会导致tRNA充电功能丧失。然而，一些人 关键问题仍然存在，包括：ARS等位基因引起的疾病表型的全谱是什么？ 人类群体中致病的ARS等位基因亚集是什么？功能丧失是如何 错义ARS变异导致显性周围神经病？功能丧失的ARS变种是如何 影响信使核糖核酸的加工和蛋白质的表达？在这里，我们使用多个既有的和互补的 模型系统--计算、生化、细胞、酵母、蠕虫和鼠标--来解决上述问题 问题。我们的努力将包括：(1)研究患者群体，以发现新发现的ARS变异 疾病发病；(2)利用蠕虫和小鼠模型深入询问ARS相关表型；(3) 应用大规模平行突变系统检测ARS变异对基因功能的影响 和哺乳动物细胞活性分析；(4)检测神经病变相关ARS变异体，以检测显性- 在体外和体内的负性和毒性的功能获得效应；(5)功能丧失、疾病和 相关ARS变异体通过核糖体图谱和质谱学对蛋白质翻译的影响 酵母、蠕虫和老鼠模型。总而言之，这项提案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.