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

Zhu

Ruiz

et al. 2021

Preprint

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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.

Section: Methodsmentioning

confidence: 99%

The amino acid substitution affects cellular response to mistranslation

Zhu

Ruiz

et al. 2021

Preprint

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“…Strains from the temperature-sensitive collection are derived from the wild-type MAT a haploid yeast strain BY4741 and described in Costanzo et al (2016) . The strains containing the gene expressing tRNA Ser UGG,G26A (CY8613) were made by integrating modified SUP17 and flanking sequence into Y7092 at the HO locus and selecting for the natMX marker, as previously described in Zhu et al (2020) , using the construct described below. The control strain (CY8611) was made by integrating only the natNT2 marker at the HO locus.…”

Section: Methodsmentioning

confidence: 99%

“…The construct to integrate the gene encoding tRNA Ser UGG,G26A at the HO locus was created using a synthetic DNA containing 200 bp up and downstream of the HO translational start, previously described in Zhu et al (2020) . The construct was cloned into pGEM-T Easy (Promega Corp.) as a Not I fragment to create pCB4386.…”

Section: Methodsmentioning

confidence: 99%

Genetic background and mistranslation frequency determine the impact of mistranslating tRNASerUGG

G3 Genes|Genomes|Genetics

Zhu

Loll-Krippleber

et al. 2022

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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.

“…The threshold frequency at which cells no longer tolerate sustained mistranslation in yeast is approximately 8%, as compared to approximately 10% in E. coli [192,376]. The ability of tRNA variants to effectively fix mutations in protein-encoding genes [for example, 188,380] raises their therapeutic potential in diseases resulting from missense or nonsense mutations [reviewed in 381].…”

Section: Trna-dependent Mistranslationmentioning

confidence: 99%

Transfer RNAs: diversity in form and function

Brandl

2020

RNA Biology

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As the adaptor that decodes mRNA sequence into protein, the basic aspects of tRNA structure and function are central to all studies of biology. Yet the complexities of their properties and cellular roles go beyond the view of tRNAs as static participants in protein synthesis. Detailed analyses through more than 60 years of study have revealed tRNAs to be a fascinatingly diverse group of molecules in form and function, impacting cell biology, physiology, disease and synthetic biology. This review analyzes tRNA structure, biosynthesis and function, and includes topics that demonstrate their diversity and growing importance.