Genetic background and mistranslation frequency determine the impact of mistranslating tRNASerUGG

Abstract

Transfer RNA variants increase the frequency of mistranslation, the misincorporation of an amino acid not specified by the "standard" genetic code, to frequencies approaching 10% in yeast and bacteria. Cells cope with these variants by having multiple copies of each tRNA isodecoder and through pathways that deal with proteotoxic stress. In this study, we define the genetic interactions of the gene encoding tRNASerUGG,G26A, which mistranslates serine at proline codons. Using a collection of yeast temperature-sensitive alleles, we identify negative synthetic genetic interactions between the mistranslating tRNA and 109 alleles representing 91 genes, with nearly half of the genes having roles in RNA processing or protein folding and turnover. By regulating tRNA expression, we then compare the strength of the negative genetic interaction for a subset of identified alleles under differing amounts of mistranslation. The frequency of mistranslation correlated with the impact on cell growth for all strains analyzed; however, there were notable differences in the extent of the synthetic interaction at different frequencies of mistranslation depending on the genetic background. For many of the strains, the extent of the negative interaction with tRNASerUGG,G26A was proportional to the frequency of mistranslation or only observed at intermediate or high frequencies. For others, the synthetic interaction was approximately equivalent at all frequencies of mistranslation. As humans contain similar mistranslating tRNAs, these results are important when analyzing the impact of tRNA variants on disease, where both the individual's genetic background and the expression of the mistranslating tRNA variant need to be considered.

Keywords: Saccharomyces cerevisiae; amino acid substitution; genetic interactions; mistranslation; tRNA.

Fig. 1.

Phenotypic characterization of mistranslating tRNA^Ser_UGG,G26A. a) Cloverleaf representation of tRNA^Ser_UGG,G26A. The anticodon and G26 substitutions from the wild-type tRNA^Ser are shown in red. b) Mass spectrometry-based analysis of the cellular proteome was performed on the control strain with no additional tRNA (CY8611) and the strain expressing mistranslating tRNA^Ser_UGG,G26A (CY8613). Mistranslation frequency was calculated from the number of unique mistranslated peptides for which the non-mistranslated sibling peptide was also observed. Frequency is defined as the counts of peptides with serine substituted for proline divided by all peptides containing proline and expressed as a percentage. Each point represents 1 biological replicate (n = 5). Mistranslation frequency in the strain expressing tRNA^Ser_UGG,G26A is statistically different compared to the control strain (Welch’s t-test; Bonferroni corrected P-value < 0.05). c) Doubling times for the strains described in B were determined from growth curves of the strains diluted to an OD₆₀₀ of approximately 0.1 in synthetic complete media containing clonNAT and grown for 24 h. Doubling time was calculated with the R package “growthcurver” (Sprouffske and Wagner 2016). Each point represents 1 biological replicate (n = 4). Doubling time is statistically different between the strain expressing tRNA^Ser_UGG,G26A and the control strain (Welch’s t-test; Bonferroni corrected P-value < 0.05). d) Strains described in B were transformed with a GFP reporter transcribed from a promoter containing heat shock response elements, grown to saturation in media lacking uracil, diluted 1:100 in the same media and grown for 18 h at 30°C. Cell densities were normalized and fluorescence measured. Each point represents 1 biological replicate (n = 5). Relative heat shock induction is statistically different in the strain expressing tRNA^Ser_UGG,G26A compared to the control strain (Welch’s t-test; Bonferroni corrected P-value < 0.05).

Fig. 2.

Negative genetic interaction network of the mistranslating tRNA^Ser_UGG,G26A. a) Genes validated as having a negative genetic interactions with tRNA^Ser_UGG,G26A are arranged according to their predicted function based on gene descriptions in the yeast genome database (www.yeastgenome.org; last accessed February 2022). b) SAFE analysis of genes that have a negative genetic interaction with tRNA^Ser_UGG,G26A were mapped onto the yeast genetic interaction profile map (Costanzo et al. 2016) using TheCellMap (Usaj et al. 2017). Blue dots represent genes within the local neighborhood of genes validated to have negative genetic interactions with tRNA^Ser_UGG,G26A. Terms in blue boxes are network regions that are significantly enriched (Bonferroni corrected P-value < 0.05). c) SAFE analysis as performed in B with genetic interactions for tRNA^Pro_G3:U70 (yellow) and tRNA^Ser_UGG,G26A (red) from Berg, Zhu, et al. (2021). Terms in boxes represent network regions that are significantly enriched for the, respectively, mistranslating tRNA (Bonferroni corrected P-value < 0.05).

Fig. 3.

Effect of different mistranslation frequencies on growth differs depending on strain background. a) Schematic of the constructs containing wild-type tRNA^Ser [WT-tS], tRNA^Ser_UGG,G26A-GAL1pr [3′-tS(UGG)], GAL1pr-tRNA^Ser_UGG,G26A [5′-tS(UGG)], and tRNA^Ser_UGG,G26A [tS(UGG)] used to regulate proline to serine mistranslation frequency. Mistranslation frequencies were measured by mass spectrometry in Berg, Isaacson, et al. (2021). b) Wild-type BY4742 or the indicated strains from the temperature-sensitive collection were transformed with the constructs described in A. Strains were grown to confluency in media lacking uracil and diluted 1:32 and spotted on media lacking uracil with galactose as the carbon source. The spot intensity of the strain containing the mistranslating tRNA was divided by the intensity of the strain containing the wild-type tRNA^Ser to determine normalized growth. Each point represents 1 biological replicate.

Fig. 4.

Genetic background alters the impact of different frequencies of proline to serine mistranslation. The average normalized growth calculated as in Fig. 3 expressed as a percentage of the growth of the wild-type strain (BY4742) is shown in blue triangles for the three different mistranslating constructs {Low: tRNA^Ser_UGG,G26A-GAL1pr [3′-tS(UGG)], Medium: GAL1pr-tRNA^Ser_UGG,G26A [5′-tS(UGG)] and High: tRNA^Ser_UGG,G26A [tS(UGG)]} for temperature-sensitive strains expressing ctf8-162 or cdc33-e72g (a), arc35-6 or las17-14 (b), act1-4 (c), and cdc20-1 (d). The growth of the wild-type strain, 100%, is plotted as gray dots. Each point is the average of at least 3 biological replicates as in Fig. 3.