Characterizing the Molecular and Cellular Effects of Disease-Associated Variants in Mitochondrial Seryl-tRNA Synthetase

Abstract

Proteins are essential cellular building blocks used for important processes including enzymatic reactions and maintaining cell structure. Therefore, accurate and efficient protein translation is critical for cellular function. The ligation of amino acids to cognate tRNAs is the first, essential step of protein translation. This reaction is catalyzed by aminoacyl-tRNA synthetases (ARSs), a family of 37 nuclear-encoded enzymes that are highly conserved and essential in all organisms and cell types. Variants in all 37 ARSs have been implicated in human genetic diseases that include: (1) dominant peripheral neuropathies; and (2) recessive multi-system disorders that can have ARS-dependent, tissue-specific phenotypes. To date, we have yet to fully describe the allelic and clinical spectra of ARS-mediated disease, and several questions remain regarding the mechanisms that lead to tissue-specific effects. This observation is particularly applicable to the mitochondrial ARSs, which support the translation of the 13 mitochondrial-encoded proteins, all of which are components of the oxidative phosphorylation pathway. In this dissertation, we: (1) characterize the functional effects of newly-identified ARS variants; (2) test the utility of a novel yeast complementation model to study mitochondrial seryl-tRNA synthetase (SARS2) variants using the orthologous yeast gene; and (3) develop a human cell complementation model to assess the functional impacts of an allelic series of SARS2 variants on protein expression and mitochondrial function.

In Chapter 1, we present the current state of mitochondrial aminoacyl-tRNA synthetase research and pose major questions that remain to be addressed. In Chapter 2, we characterize six newly-identified variants in cysteinyl-tRNA synthetase (CARS1) and threonyl-tRNA synthetase (TARS1) that are associated with multisystem recessive disease phenotypes. We assess each variant for segregation with disease, amino-acid conservation across evolutionarily diverse species, and allele frequency in the general population. We then use yeast complementation assays to provide functional evidence towards supporting the pathogenicity of these variants. These studies expand the allelic and clinical heterogeneity of both CARS1- and TARS1-associated recessive disease. In Chapter 3, we explore yeast as a model system for studying disease-associated variants in mitochondrial seryl-tRNA synthetase (SARS2) by generating a yeast complementation model to study the functional impacts of variants on yeast growth as a proxy for enzyme function. We find that, while wild-type human SARS2 does not support yeast growth in the absence of the yeast ortholog DIA4, we are able to model a subset of variants in the DIA4 open reading frame to assess the functional effects of variants on the yeast ortholog. Finally, in Chapter 4, we develop a human cell complementation model to study an allelic series of pathogenic SARS2 variants that are associated with diverse clinical phenotypes. We use this model to assess transcriptional changes induced by depletion of SARS2 expression. We also characterize variant-dependent effects on protein expression and mitochondrial oxygen consumption, and assess one high-confidence pathogenic allele for effects on post-transcriptional processing. These findings provide insights into genotype:phenotype correlations related to diverse SARS2-associated clinical phenotypes.

Overall, this dissertation expands the allelic and clinical heterogeneity of ARS-mediated recessive diseases, provides insight into understanding the evolutionary differences between orthologous ARS genes, and furthers our understanding of genotype:phenotype correlations in mitochondrial ARS-mediated disease.