ABSTRACT My long-term professional goal is to study rare human inherited diseases with an emphasis on: (1) defining disease mechanisms and developing therapeutics; and (2) increasing research and awareness through participating in science policy. Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that charge tRNA with cognate amino acids in the cytoplasm and mitochondria; 17 of the 37 nuclear-encoded ARSs act exclusively in the mitochondria. All 17 mitochondrial ARSs have been implicated in human recessive diseases with a broad range of clinical phenotypes, often affecting tissues with high energy demands. Interestingly, certain mitochondrial ARSs cause variable tissue-specific effects, which can depend on the specific genetic variants that a patient carries. One important example of this observation is mitochondrial seryl-tRNA synthetase (SARS2), which has been implicated in disease phenotypes ranging from progressive spastic paresis to HUPRA syndrome (Hyper Uricemia, Pulmonary hypertension, Renal failure in infancy, and Alkalosis). Currently, there are no genetic or molecular explanations for this clinical heterogeneity. Furthermore, the number of known pathogenic variants is limited, resulting in a large gap in our knowledge of the allelic heterogeneity in SARS2-related disease. I will pursue two specific aims to address these issues. Under Aim 1, I will perform massively parallel cell growth assays on all possible variants in the SARS2 open reading frame to assess the functional consequences of each variant. These studies will be performed in haploid (Hap1) cells that harbor: (a) a randomly integrated, 1xFLAG-tagged, doxycycline-inducible wild-type copy of SARS2; and (b) an endogenous SARS2 null allele. I will transduce these cells with lentiviral libraries containing all possible SARS2 variants and will quantitate the frequencies of each allele (via next-generation sequencing) in the presence of doxycycline (i.e., in the presence of wild-type SARS2-1xFLAG) and in the absence of doxycycline (i.e., in the absence of SARS2-1xFLAG). Variants that are reduced in frequency after doxycycline removal will be classified as loss-of-function alleles. Under Aim 2, I will generate clonal Hap1 cell lines that carry individual, known pathogenic SARS2 variants that are associated with distinct clinical phenotypes (i.e., progressive spastic paresis versus HUPRA syndrome). These cell lines will contain an integrated, inducible SARS2-1xFLAG that can be used to maintain cell viability. I will employ these cell lines to assess the effect of each variant on protein translation, mitochondrial function, and interactions with potential SARS2 binding partners. Overall, these studies will: (a) reveal essential regions of the SARS2 protein toward a better understanding of enzyme biology; (b) provide a complete panel of loss-of-function SARS2 variants toward rapid patient diagnosis; and (c) provide insight into the molecular mechanisms of SARS2-related disease towa...