Mistranslating tRNA identifies a deleterious S213P mutation in the Saccharomyces cerevisiae eco1-1 allele

Loll-Krippleber

et al. 2022

Preprint

Self Cite

Transfer RNA variants increase the frequency of mistranslation, the mis-incorporation 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 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.

Section: Resultssupporting

confidence: 86%

Section: Methodsmentioning

confidence: 99%

Section: Yeast Strains and Growthmentioning

confidence: 99%

Section: Dna Constructsmentioning

confidence: 99%

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Genetic background and mistranslation frequency determine the impact of mistranslating tRNA^Ser_UGG

Loll-Krippleber

et al. 2022

Preprint

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“…Constructs to integrate mistranslating tRNAs at the HO locus were created using synthetic DNA containing 200 bp up and downstream of the HO translational start as previously described in Zhu et al (2020 ; Supplementary Figure S1; Life Technologies). The construct was cloned into pGEM ® -T Easy (Promega Corp.) as a Not I fragment to create pCB4386.…”

Section: Methodsmentioning

confidence: 99%

The amino acid substitution affects cellular response to mistranslation

G3 Genes|Genomes|Genetics

Ruiz

et al. 2021

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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.

Chemical-genetic interactions with the proline analog L-azetidine-2-carboxylic acid inSaccharomyces cerevisiae

Isaacson

et al. 2020

Preprint

Non-proteinogenic amino acids, such as the proline analog L-azetidine-2-carboxylic acid (AZC), are detrimental to cells because they are mis-incorporated into proteins and lead to proteotoxic stress. Our goal was to identify genes that show chemical-genetic interactions with AZC in Saccharomyces cerevisiae and thus also potentially define the pathways cells use to cope with amino acid mis-incorporation. Screening the yeast deletion and temperature sensitive collections, we found 72 alleles with negative synthetic interactions with AZC treatment and 12 alleles that suppress AZC toxicity. Many of the genes with negative synthetic interactions are involved in protein quality control pathways through the proteasome. Genes involved in actin cytoskeleton organization and endocytosis also had negative synthetic interactions with AZC. Related to this, the number of actin patches per cell increases upon AZC treatment. Many of the same cellular processes were identified to have interactions with proteotoxic stress caused by two other amino acid analogs, canavanine and thialysine, or a mistranslating tRNA variant that mis-incorporates serine at proline codons. Alleles that suppressed AZC-induced toxicity functioned through the amino acid sensing TOR pathway or controlled amino acid permeases required for AZC uptake.