A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations

Abstract

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes that ligate tRNA molecules to their cognate amino acids. Heterozygosity for missense variants or small in-frame deletions in five ARS genes causes axonal peripheral neuropathy, a disorder characterized by impaired neuronal function in the distal extremities. These variants reduce enzyme activity without significantly decreasing protein levels and reside in genes encoding homo-dimeric enzymes. These observations raise the possibility of a dominant-negative effect, in which non-functional mutant ARS subunits dimerize with wild-type ARS subunits and reduce overall ARS activity below 50%, breaching a threshold required for peripheral nerve axons. To test for these dominant-negative properties, we developed a humanized yeast assay to co-express pathogenic human alanyl-tRNA synthetase (AARS1) mutations with wild-type human AARS1. We show that multiple loss-of-function, pathogenic AARS1 variants repress yeast growth in the presence of wild-type human AARS1. This growth defect is rescued when these variants are placed in cis with a mutation that reduces dimerization with the wild-type subunit, demonstrating that the interaction between mutant AARS1 and wild-type AARS1 is responsible for the repressed growth. This demonstrates that neuropathy-associated AARS1 variants exert a dominant-negative effect, which supports a common, loss-of-function mechanism for ARS-mediated dominant peripheral neuropathy.

Comprehensive assessment of recessive, pathogenicAARS1alleles in a humanized yeast model reveals loss-of-function and dominant-negative effects

“…To identify potential dominant-negative AARS1 alleles identified in patients with recessive disease, three complete loss-of-function variants (K81T, E99G, and C901Y AARS1) were assessed for effects on protein expression and for the ability to impact the function of wild-type AARS1. These efforts revealed that one allele (K81T) was expressed at levels comparable to the wild-type protein and resulted in reduced growth when co-expressed with wild-type AARS1, consistent with previously described dominant-negative alleles that cause axonal peripheral neuropathy (Meyer-Schuman et al, 2023). To our knowledge, no single AARS1 variant has been implicated in both recessive and dominant diseases.…”

“…For expression of AARS1 alleles from a high -copy number vector, null (G757* AARS1 , for which no protein product is expressed [(Meyer-Schuman et al, 2023)]), wild-type, or mutant AARS1 in pAG425 expression constructs were individually transformed into a haploid yeast strain, ptetO7- ALA1 (p ALA1 ::kanR-tet07-TATA URA3 ::CMV-tTA MATa ; from the Yeast Tet-Promoters Hughes Collection, Horizon Discovery, accession: YSC1180), and subsequently plated on media lacking leucine (pAG425 harbors the LEU2 gene). Colonies were picked into liquid media lacking leucine and grown at 30°C and shaking at 275 rpm for 48 hours.…”

“…The sequence-verified expression constructs for wild-type and mutated AARS1 were then cloned into either the pAG425 expression vector (pAG425GAL-ccdB; Addgene #14153; pAG425 harbors a galactose-inducible promoter) or the p413 expression vector (ATCC #87370; p413 harbors the constitutive ADH promoter) using Gateway cloning technology (Invitrogen). For expression of AARS1 alleles from a high-copy number vector, null (G757* AARS1, for which no protein product is expressed [(Meyer-Schuman et al, 2023)]), wild-type, or mutant AARS1 in pAG425 expression constructs were individually transformed into a haploid yeast strain, ptetO7-ALA1 (pALA1::kanR-tet07-TATA URA3::CMV-tTA MATa; from the Yeast Tet-Promoters Hughes Collection, Horizon Discovery, accession: YSC1180), and subsequently plated on media lacking leucine (pAG425 harbors the LEU2 gene). Colonies were picked into liquid media lacking leucine and grown at 30°C and shaking at 275 rpm for 48 hours.…”

“…Variants implicated in ARS-mediated dominant peripheral neuropathy affect homodimeric ARS enzymes, demonstrate loss-of-function and/or gain-of-function effects without significantly decreasing protein expression (Griffin et al, 2014;He et al, 2015), and activate the integrated stress response (Spaulding et al, 2021). We and others have presented data indicating a dominant-negative mechanism for neuropathy-associated ARS variants (Mullen et al, 2020;Meyer-Schuman et al, 2023); however, the manner in which this mechanism intersects with the integrated stress response remains unclear. Alanyl-tRNA synthetase 1 (AARS1; MIM: 601065) encodes the cytoplasmic enzyme that charges tRNA Ala with alanine (Antonellis and Green, 2008).…”

A previously unreported NARS1 variant causes dominant distal hereditary motor neuropathy in a French family

“…Yeast has proven to be an effective model system to study neuropathy‐associated aminoacyl‐tRNA synthetase mutations, with the vast majority of pathogenic variants causing loss‐of‐function and dominant negative effects 4,5 . To determine the consequences of the p.(Gly519Arg) variant on NARS1 function, yeast complementation assays were performed.…”