Transfer RNA supplementation rescues HARS deficiency in a humanized yeast model of Charcot-Marie-Tooth disease

Abstract

Aminoacyl-tRNA synthetases are indispensable enzymes in all cells, ensuring the correct pairing of amino acids to their cognate tRNAs to maintain translation fidelity. Autosomal dominant mutations V133F and Y330C in histidyl-tRNA synthetase (HARS) cause the genetic disorder Charcot-Marie-Tooth type 2W (CMT2W). Treatments are currently restricted to symptom relief, with no therapeutic available that targets the cause of disease. We previously found that histidine supplementation alleviated phenotypic defects in a humanized yeast model of CMT2W caused by HARS V155G and S356N that also unexpectedly exacerbated the phenotype of the two HARS mutants V133F and Y330C. Here, we show that V133F destabilizes recombinant HARS protein, which is rescued in the presence of tRNAHis. HARS V133F and Y330C cause mistranslation and cause changes to the proteome without activating the integrated stress response as validated by mass spectrometry and growth defects that persist with histidine supplementation. The growth defects and reduced translation fidelity caused by V133F and Y330C mutants were rescued by supplementation with human tRNAHis in a humanized yeast model. Our results demonstrate the feasibility of cognate tRNA as a therapeutic that rescues HARS deficiency and ameliorates toxic mistranslation generated by causative alleles for CMT.

Graphical Abstract

Figure 1.

Aminoacylation of tRNA by aaRSs. (A) Aminoacylation and (B) mis-aminoacylation caused by mutations in the catalytic domain. The HARS G⁻¹ identity element on the tRNA is highlighted in green. (B) Mutations in the catalytic domain of an aaRS (red) can lead to mis-aminoacylation of tRNA, resulting in mistranslation at the ribosome. (C) CMT2W causative alleles Y330C and V133F in HARS structure: Human HARS homodimer (PDB ID: 6O76) with the WHEP domain in blue, catalytic domain in tan and tRNA binding domain in purple. Human HARS was superimposed with the crystal structure from Thermus thermophilus in complex with tRNA^His (PDB ID: 4RDX), and the tRNA is shown in dark blue, with the 3′ acceptor stem coloured blue. CMT2W HARS mutants V133F and Y330C depicted in orange. Figure generated with PyMOL (Schrodinger, LLC).

Figure 2.

HARS V133F and Y330C induce a growth phenotype in a humanized yeast model of HARS disease. A) Limited tryptic digestion of HARS proteins. HARS wildtype and mutant proteins were equilibrated with buffer then subjected to digestion by trypsin at 37°C for the time indicated in minutes. Control samples of HARS were incubated without trypsin for 0 or 120 min (C0 and C120). The reactions were quenched with SDS dye and boiling, then separated on a 12% denaturing SDS-polyacrylamide gel followed by staining with Coomassie blue to visualize digest products. The expected mass of 6x-His tagged HARS is approximately 58 kDa. Soluble whole cell RNA from hsHARS, V133F and Y330C mutant yeast was extracted and purified under acidic conditions and separated on an 8M urea gel. (B) Protein thermal stability of recombinant HARS WT or mutants with or without 2 μM tRNA^His minihelix. Purified protein was incubated with tRNA and heated from 25°C to 95°C, measuring fluorescence intensity to follow protein unfolding. The melting temperature (Tm) was determined by fitting fluorescence intensity of three biological replicates per condition to the Boltzmann equation in the Protein Thermal Shift software. Soluble whole cell RNA from hsHARS, V133F, and Y330C mutant yeast was extracted and purified under acidic conditions and separated on an 8 M urea gel. (C) Northern blot and (D) ratio of soluble tRNA^His to 5S RNA of blots for yeast tRNA^His. Blots were imaged using the ChemiDoc MP imaging system and quantified using ImageLab. (E) CD spectra of purified recombinant WT, V133F and Y330C proteins.

Figure 3.

V133F and Y330C change the composition of the proteome in yeast. Heat maps of differentially abundant proteins identified by label-free quantification proteomic analysis between yeast expressing wildtype HARS or (A) V133F or (B) Y330C HARS. Volcano plots of proteins with significant changes shown for (C) V133F and (D) Y330C HARS, with upregulated proteins identified in red and downregulated proteins identified in green. String diagram of proteins (E) downregulated and (F) upregulated in Y330C compared to wildtype. (G) Bar graph of heat shock proteins changed in abundance in Y330C and V133F normalized to wildtype HARS expressing yeast.

Figure 4.

HARS V133F and Y330C lead to mistranslation. Tandem mass spectra are shown for peptides that represent histidine to glutamine mistranslation difference in Y ions highlighted for (A) hsHARS, (C) HARS V133F and (E) HARS Y330C. Mirror plots of the same peptides for (B) HARS V133F and (D) HARS Y330C showing histidine to glutamine mistranslation.

Figure 5.

Supplementation with human tRNA^His is not toxic and improves growth of yeast strains expressing hsHARS. (A) Structure and sequence differences from Saccharomyces cerevisiae and human tRNA^His. (B) Spotting yeast growth assay at 30°C and (C) quantification of wildtype yeast overexpressing tRNA^His. Yeast strains (BY-4742) with no plasmid, high copy number vector p426, and p426 encoding human tRNA^His for yeast expression were grown in YPD medium, normalized to A₆₀₀ = 1.0 and serially diluted 1:1, 1:4, 1:42, 1:43 and 1:44. Three biological replicates were incubated at 30°C for 3 days. (D) Spotting assay at 30°C and (E) quantification of humanized yeast. ΔHTS1 yeast strains (BY-4742) with a plasmid expressing hsHARS and a plasmid expressing human tRNA^His were grown in SD Leu- or SD Ura- Leu-, respectively, normalized to A₆₀₀ = 1.0 and serially diluted as previously described. Three biological replicates each were spotted on complete SD medium with either 20 or 200 mg/L histidine and incubated at 30°C for 4 days. Spotting plates were imaged using the ChemiDoc MP imaging system and quantified using ImageJ. (F) Volcano plot of label free proteomic analysis comparing wildtype humanized HARS with tRNA supplemented humanized HARS yeast. (G) String maps of differentially expressed proteins in hsHARS yeast cells supplemented with tRNA show downregulated proteins in tRNA supplemented cells compared to unsupplemented cells.

Figure 6.

tRNA^His supplementation restores growth in HARS V133F and Y330C dependent yeast. Growth curve under (A) normal (20 mg/L) and (B) high (200 mg/L) histidine conditions and (C) doubling time of haploid ΔHTS1 yeast (BY-4742) with high (p425) copy number plasmids expressing wildtype or mutant human HARS. Yeast cultures were grown at 30°C in SD Leu⁻ for non-supplemented or SD Ura⁻ Leu⁻ medium for tRNA^His-supplemented cultures in a 96-well plate for 48 h with 10-min read intervals, with three biological and three technical replicates each. The error bars represent one standard deviation of the mean. (D) Northern blot for S. cerevisiae tRNA^His and human tRNA^His from yeast cultures used for growth curve experiment under normal (20 mg/L) histidine conditions. Soluble whole cell RNA from hsHARS, V133F and Y330C mutant yeast was extracted and purified under acidic conditions and separated on an acidic 8 M urea gel. A probe for the 5S RNA was used as loading control. (E) Spotting yeast growth assay at 30°C and (F) quantification. ΔHTS1 yeast strains (BY-4742) with a plasmid expressing hsHARS and a plasmid expressing human tRNA^His were grown in SD Leu⁻ or SD Ura⁻ Leu⁻, respectively, normalized to A₆₀₀= 1.0 and serially diluted as previously described. Three biological replicates each were spotted on complete SD medium with 20 mg/L histidine and incubated at 30°C for 4 days. Spotting plates were imaged using the ChemiDoc MP imaging system and quantified using ImageJ. Error bars represent one standard deviation of the mean.

Figure 7.

tRNA^His supplementation reduces proteome disturbances in HARS V133F and Y330C expressing cells. Volcano plots of differentially expressed proteins identified by label-free quantitative proteomic analysis between wildtype HARS and (A) V133F or (B) Y330C HARS following tRNA^His supplementation of all strains with upregulated proteins identified in red and downregulated proteins in green. (C) Number of proteins significantly changed in abundance by at least 2-fold, as identified by label-free quantification, between wildtype HARS and V133F or Y330C HARS, before and after tRNA^His supplementation. (D) Heat map of proteins with a role in amino acid metabolism that are up (red) and downregulated (green) compared to wildtype HARS with and without tRNA supplementation.

Figure 8.

tRNA^His supplementation alleviates mistranslation in V133F and Y330C expressing yeast. (A) Total spectral counts of mistranslation events in whole proteome samples from humanized HARS WT, V133F and Y330C yeast. Error bars show one standard deviation of the mean. Quantification of the area under the isotopic of the of mistranslated peptide relative to the wildtype peptide normalized to the unsupplemented sample fraction of each peptide for (B) hsHARS, (C) V133F and (D)Y330C. (E) Mistranslation counts separated by type of mistranslation in untreated and tRNA-supplemented cells. Western blots for YFP-HARS of whole cell lysates from (F) unsupplemented and (G) tRNA^His supplemented yeast samples were separated into total [W], soluble [S] and insoluble [P] protein fractions, separated on a 12% SDS-denaturing polyacrylamide gel then transferred to PVDF membranes and blotted with anti-GFP. Ratios of the soluble to insoluble fraction were (H) quantified with normalization to the wildtype sample before and after tRNA^His supplementation; one standard deviation shown by error bars. Mirror plots of (I) HARS V133F with the difference in Y ions highlighted for (J) glutamine mis-incorporation at a histidine codons.

Figure 9.

tRNA^His supplementation rescues disease-causing CMT mutants in a yeast model. (A) Reduced HARS stability in HARS V133F and Y330C lead to mistranslation, the accumulation of insoluble HARS and changes to the proteome focused on amino acid metabolism in a humanized yeast model of CMT. Supplementation with human tRNA^His restores V133F CMT-HARS protein structure and alleviates mistranslation in both V133F and Y330C as well as insoluble HARS accumulation in cells.