Defining the Genetic and Molecular Mechanisms of tRNA Synthetase Mediated Dominant Neurological Disease

Abstract

The process by which genetic information is translated into protein plays a crucial biological role in cellular functions. Aminoacyl-tRNA synthetases (ARSs) are a ubiquitously expressed, essential family of enzymes that ligate tRNA molecules to cognate amino acids. Mutations in all 37 human ARS loci have been associated with early-onset, multi-system recessive diseases, often impacting the central and peripheral nervous systems, muscles, lungs, and liver. It has been shown through both in vitro aminoacylation and yeast complementation assays that ARS-associated recessive diseases are caused by homozygosity or compound heterozygosity for loss-of-function alleles. On the other hand, only seven ARS loci have been implicated in a dominant neurological condition, often manifesting as axonal peripheral neuropathy. While it has been shown that the majority of dominant ARS-associated disease variants exhibit a loss-of-function effect, research has debated if the pathogenic variants share a common dominant-negative mechanism or if each locus causes a unique toxic gain-of-function effect. In addition, the complete clinical and genetic spectra of dominant and recessive ARS-mediated diseases are not yet fully defined. In this dissertation, we: (1) expand the locus, allelic, and clinical heterogeneity of ARS-associated dominant disease; and (2) investigate a unifying dominant-negative mechanism for dominant ARS-mediated disease.

Alanyl-tRNA synthetase (AARS1, encoding the enzyme for charging alanine to tRNA in the cytoplasm) was previously implicated in dominant neuropathy. Here, we report three newly identified AARS1 variants and demonstrate that they have loss-of-function and dominant-negative properties in a humanized yeast model. The three variants reduce yeast cell growth when co-expressed with wild-type AARS1, suggesting that they are dominantly toxic. By engineering a dimer-reducing variant and introducing it in cis with each of these AARS1 variants, we observed a restoration of normal growth when the double mutants were co-expressed with wild-type AARS1 in yeast. These data suggest that all three alleles exert a dominant-negative mechanism, as disrupting the interaction with the wild-type subunit reverses the growth impairment in yeast.

At the start of this thesis, six ARS enzymes were implicated in dominant neuropathy. Here, we present data showing for the first time that asparagyl-tRNA synthetase (NARS1, encoding the enzyme for charging alanine to tRNA in the cytoplasm) is the seventh ARS enzyme implicated in this phenotype; we identified and characterized four NARS1 variants in patients exhibiting an isolated axonal peripheral neuropathy phenotype. Interestingly, heterozygosity for missense and protein-truncating variants in NARS1 have been previously associated with an early-onset neurodevelopmental syndrome, which include both central and peripheral nervous system involvement. Here, demonstrated that pathogenic NARS1 variants act via a dominant-negative effect, consistent with this being a common mechanism for ARS-related dominant neurological disease. We performed yeast complementation assays to test NARS1 variants in isolation, which revealed loss-of-function effects. To test for dominant-negative properties, we co-expressed mutant human NARS1 with wild-type human NARS1. These studies revealed that NARS1 variants result in expressed proteins that interact with the wild-type subunit, and that the majority of variants repress the ability of the wild-type protein to support yeast cell growth, consistent with a dominant-negative effect.

Overall, this thesis broadens our understanding of the clinical, locus, and allelic spectrum of ARS-related diseases and provides insight on the molecular mechanism of dominant ARS-mediated neurological disease.