Fig 2: Assessment of loss-of-function effects of AARS1 missense variants in yeast complementation assays with low-copy number vector (p413). Haploid yeast with a doxycycline-repressible endogenous ALA1 (the yeast ortholog of AARS1) were transformed with p413 vectors containing wild-type (WT) or mutant AARS1, or a null allele (G757* AARS1); the vector used in each experiment is indicated across the top. (A) Resulting cultures were plated undiluted or diluted (1:10 or 1:100) on media containing doxycycline and grown at 30°C for five days. (B) Images of yeast spots were quantified to assess the relative growth of mutant variants in comparison to wild-type AARS1. The average growth rate across three colony replicates for each mutant was calculated and is depicted as the bar height. Error bars represent standard deviation. Statistical significance was determined by one-way ANOVA with the Geisser-Greenhouse correction and Dunnett’s multiple comparison’s test with individual variances computed for each comparison. Only comparisons that were statistically significant are annotated with a p-value.

Fig 3: Localization and conservation of AARS1 variants implicated in recessive disease. (A) AARS1 functional domains are indicated in blue (aminoacylation domain), purple (tRNA recognition domain), orange (editing domain), and green (C-terminal domain). The positions of the variants are shown across the top and numbers along the bottom indicate amino-acid positions. The exon 1–4 deletion from Family J is not depicted. (B) Conservation of the affected amino-acid residues for all missense variants implicated in recessive disease. The position of each variant is shown along with flanking AARS1 amino-acid residues from multiple, evolutionarily diverse species. The wild-type human amino-acid residue at the position of the affected residue is shown in red for each species.

Fig 4: Protein expression and dominant-negative effects of loss-of-function AARS1 missense variants. (A) AARS1 protein expression in transformed yeast. Western blot analyses were performed on protein lysates isolated from haploid yeast that were transformed with vectors containing the indicated inserts. The vector used in each experiment is across the top and sizes (kDa) are indicated at the left. (B) Band intensity was quantified relative to control PGK1 expression and normalized to wild-type AARS1 protein levels. (C) Haploid yeast with a doxycycline-repressible endogenous ALA1 (the yeast ortholog of AARS1) and containing a wild-type (WT) AARS1 p413 construct were transformed with pAG425 vectors containing the indicated AARS1 allele. Resulting cultures were plated undiluted or diluted (1:10 or 1:100) on media containing doxycycline and grown at 30°C for five days. (D) Images of yeast spots were quantified to assess the relative growth of yeast co-expressing WT and AARS1 mutants in comparison to yeast co-expressing WT AARS1 on both p413 and pAG425. In both (B) and (D), the average intensity for three replicates was calculated and is depicted as the bar height. Error bars represent standard deviation. Statistical significance was determined by one-way ANOVA with the Geisser-Greenhouse correction and Dunnett’s multiple comparison’s test with individual variances computed for each comparison. Only comparisons that were statistically significant are annotated with a p-value.

Fig 5: Assessment of loss-of-function effects of AARS1 missense variants in yeast complementation assays using a high-copy number vector (pAG425). Haploid yeast with a doxycycline-repressible endogenous ALA1 (the yeast ortholog of AARS1) were transformed with pAG425 vectors containing wild-type (WT) or mutant AARS1, or a vector with a null allele (G757* AARS1); the vector used in each experiment is indicated across the top. (A) Resulting cultures were plated undiluted or diluted (1:10 or 1:100) on media containing doxycycline and grown at 30°C for five days. (B) Images of yeast spots were quantified to assess the relative growth of mutant variants in comparison to wild-type AARS1. The average growth rate across three colony replicates for each mutant was calculated and is depicted as the bar height. Error bars represent standard deviation. Statistical significance was determined by one-way ANOVA with the Geisser-Greenhouse correction and Dunnett’s multiple comparison’s test with individual variances computed for each comparison. Only comparisons that were statistically significant are annotated with a p-value.