The amino acid substitution affects cellular response to mistranslation

Abstract

Mistranslation, the mis-incorporation of an amino acid not specified by the “standard” genetic code, occurs in all organisms. tRNA variants that increase mistranslation arise spontaneously and engineered tRNAs can achieve mistranslation frequencies approaching 10% in yeast and bacteria. Interestingly, human genomes contain tRNA variants with the potential to mistranslate. Cells cope with increased mistranslation through multiple mechanisms, though high levels cause proteotoxic stress. The goal of this study was to compare the genetic interactions and the impact on transcriptome and cellular growth of two tRNA variants that mistranslate at a similar frequency but create different amino acid substitutions in Saccharomyces cerevisiae. One tRNA variant inserts alanine at proline codons whereas the other inserts serine for arginine. Both tRNAs decreased growth rate, with the effect being greater for arginine to serine than for proline to alanine. The tRNA that substituted serine for arginine resulted in a heat shock response. In contrast, heat shock response was minimal for proline to alanine substitution. Further demonstrating the significance of the amino acid substitution, transcriptome analysis identified unique up- and downregulated genes in response to each mistranslating tRNA. Number and extent of negative synthetic genetic interactions also differed depending upon type of mistranslation. Based on the unique responses observed for these mistranslating tRNAs, we predict that the potential of mistranslation to exacerbate diseases caused by proteotoxic stress depends on the tRNA variant. Furthermore, based on their unique transcriptomes and genetic interactions, different naturally occurring mistranslating tRNAs have the potential to negatively influence specific diseases.

Genetic background and mistranslation frequency determine the impact of mistranslating tRNASerUGG

“…Other factors are extrinsic to the tRNA. These include the number of competing tRNAs that buffer the mistranslating tRNA (Zimmerman et al 2018), the environment in which cells expressing the mistranslating tRNA (Berg et al 2021b) are found and the genetic background of the organism. Genetic background contributes to the impact of a mistranslating tRNA in numerous ways.…”

“…Peptides were analyzed on a hybrid quadrupole orbitrap mass spectrometry (Orbitrap Exploris 480; Thermo Fisher Scientific) equipped with an Easy1200 nanoLC system (Thermo Fisher Scientific) as previously described in Berg et al (2021b).…”

“…In a previous screen looking at negative synthetic genetic interactions with tRNA variants that mistranslate alanine at proline codons (tRNA Pro G3:U70; Hoffman et al 2017a) and serine at arginine codons (tRNA Ser UCU,G26A; Berg et al 2021b), we identified 10 and 47 negative genetic interactions, respectively. While these tRNA variants mistranslated at lower frequency (~ 3%) than tRNA Ser UGG,G26A making specific comparison of genetic interactions difficult, it is possible to compare enriched pathways with a SAFE analysis (Figure 2C).…”

“…We previously demonstrated that the negative genetic interactions with mistranslating tRNAs depend on the amino acid substitution (Berg et al 2021b). At similar frequencies of mistranslation, a tRNA variant substituting serine at arginine codons has more genetic interactions than one substituting alanine at proline codons.…”

Synthetic lethality between toxic amino acids, retrograde target genes and chaperones in Saccharomyces cerevisiae

“…However, it is not clear that HSF1 is a regulatory factor in the amino acid starvation response. Furthermore, the cellular response to mistranslation is affected by which amino acid is being mistranslated - for example, yeast cells carrying a tRNA that substitutes serine for arginine results in a heat shock response, but proline to alanine substitution results in a minimal heat shock response [20]. Thus, the amino acid stress imposed by arginine deprivation and canavanine may be a specific response due to the inherent properties of arginine rather than an example of a more general toxic amino acid analog response.…”