Conditional accumulation of toxic tRNAs to cause amino acid misincorporation

Abstract

To develop a system for conditional amino acid misincorporation, we engineered tRNAs in the yeast Saccharomyces cerevisiae to be substrates of the rapid tRNA decay (RTD) pathway, such that they accumulate when RTD is turned off. We used this system to test the effects on growth of a library of tRNASer variants with all possible anticodons, and show that many are lethal when RTD is inhibited and the tRNA accumulates. Using mass spectrometry, we measured serine misincorporation in yeast containing each of six tRNA variants, and for five of them identified hundreds of peptides with serine substitutions at the targeted amino acid sites. Unexpectedly, we found that there is not a simple correlation between toxicity and the level of serine misincorporation; in particular, high levels of serine misincorporation can occur at cysteine residues without obvious growth defects. We also showed that toxic tRNAs can be used as a tool to identify sequence variants that reduce tRNA function. Finally, we generalized this method to another tRNA species, and generated conditionally toxic tRNATyr variants in a similar manner. This method should facilitate the study of tRNA biology and provide a tool to probe the effects of amino acid misincorporation on cellular physiology.

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

“…This allows the construction of tRNAs that insert these amino acids at non-cognate codons. Many of these tRNAs have been engineered (for example see Geslain et al 2010;Zimmerman et al 2018;Berg et al 2019). Through analysis similar to that performed here, these mistranslating tRNAs will allow the identification of functionally significant missense mutations in alanine, serine and leucine codons.…”

“…Loss of fidelity at either step can lead to mistranslation. Mistranslation is dramatically increased by tRNA variants that are inaccurately aminoacylated or contain mutations within their anticodon (Geslain et al 2010;Hoffman et al 2017;Lant et al 2017;Berg et al 2017;Zimmerman et al 2018). Nucleotides in the tRNA that are recognized by a specific aaRS are called identity elements and consist of single nucleotides, nucleotide pairs, and structural motifs (de Duve 1988;Hou and Schimmel 1988;Giegé et al 1998).…”

“…One variant, tRNA Ser UGG, G26A, contains a G26A secondary mutation and when expressed from a centromeric plasmid results in a frequency of serine incorporation at proline codons of ~ 5% as determined by mass spectrometry (Berg et al 2019). Zimmerman et al (2018) and Geslain et al (2010) have also demonstrated the possibility of mistranslating serine for a number of amino acids in yeast and mammalian cells.…”

Targeted sequencing reveals expanded genetic diversity of human transfer RNAs

“…Mistranslation is a general feature of the translation process which has a basal error rate of approximately one misincorporated amino acid in every 10 4 to 10 5 codons [55,56]. Many tRNA variants dramatically increase mistranslation in bacterial, fungal, and mammalian cell culture systems [37,39,[57][58][59][60]. For example, we identified a variant of yeast tRNA Pro that results in~5% proteome wide alanine for proline mistranslation with a minimal effect on cell growth in yeast [39].…”

“…However, the anticodon is not an identity element for alanine, leucine, serine and selenocysteine tRNAs. In model systems, changes to the anticodons of these tRNAs result in mistranslation [59,60]. Four specific examples we identified are anticodon mutations in serine tRNAs that change the decoding of serine codons to phenylalanine or asparagine and two anticodon mutations in alanine tRNAs that change the decoding of alanine codons to glycine or threonine.…”