Defining Dominant-negative Effects of Neuropathy-associated tRNA Synthetase Variants

About This Grant

ABSTRACT Aminoacyl-tRNA synthetases (ARSs) are a ubiquitously expressed, essential class of enzymes responsible for ligating amino acids to cognate tRNA molecules. To date, seven genes encoding an ARS have been implicated in dominant axonal neuropathy with extreme clinical heterogeneity. All seven ARSs implicated function as dimers to charge tRNA in the cytoplasm. Our multiple PI team has previously shown that neuropathy- associated ARS mutations cause a loss-of-function effect, which can be detected in enzyme kinetic and yeast complementation assays. We have also shown that ARS missense mutations associated with dominant neuropathy are toxic when over-expressed in yeast and worm model systems, and that dimerization is required for this toxic effect. Combined, these observations strongly support a dominant-negative mechanism where the mutant protein binds to and inhibits the wild-type protein in a heterozygous affected individual. We present here, an innovative pipeline—including genetic, molecular, in vitro, and in vivo approaches—to comprehensively address two critical, unanswered questions: (1) Do neuropathy-associated ARS variants act via a dominant-negative effect? and (2) By what mechanism—and to what degree—do neuropathy-associated ARS variants impact tRNA charging and cell stress pathways? Our initial efforts will focus on a well- characterized, neuropathy associated variant (R329H alanyl-tRNA synthetase [AARS1]). Future efforts will leverage the strategy developed here to study alleles at all seven ARS loci associated with dominant neuropathy. Our efforts will provide an innovative, effective, and efficient strategy to study neuropathy- associated variants at any ARS locus.