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.
Aminoacyl-tRNA synthetases (ARSs) are a family of enzymes that are essential for the production of cellular proteins. To date, genes encoding ARSs have been implicated in many human diseases; however, the process by which these genes cause specific diseases is still unknown. Here, we aim to define how ARS genes cause human disease toward developing effective therapeutics.