Protein synthesis rate and accuracy are essential for bona fide protein synthesis and proteome homeostasis (proteostasis), however the mRNA translation elongation factors that prevent protein mistranslation, misfolding and aggregation are poorly understood. To address this question, we evaluated the role of 70 yeast tRNA modifying enzyme genes on protein aggregation and used mass spectrometry to identify the aggregated and mistranslated proteins. We show that the mitochondrial tRNA-modifying enzyme Slm3 thiolates the cytoplasmic tRNAs at position 34 and that decreased levels of mcm 5 s 2 U34 in SLM3 mutants are compensated by increasing mcm 5 U34, ncm 5 U34 and ncm 5 Um34 levels. In the tRNA gene knockout strains, stress response proteins are overrepresented in protein aggregates and their genes are enriched in codons decoded by tRNAs lacking mcm 5 U34, mcm 5 s 2 U34, ncm 5 U34, ncm 5 Um34, modifications. Increased rates of amino acid misincorporation were detected in the yeast ELP1, SLM3 and TRM9 gene knockout mutants at protein sites that specifically mapped to the codons sites that are decoded by the hypomodified tRNAs, demonstrating that U34 tRNA modifications safeguard the proteome from translational errors, misfolding and cellular proteotoxic stress..

Protein synthesis rate and accuracy are essential for bona fide protein synthesis and proteome homeostasis (proteostasis), however the mRNA translation elongation factors that prevent protein mistranslation, misfolding and aggregation are poorly understood. To address this question, we evaluated the role of 70 yeast tRNA modifying enzyme genes on protein aggregation and used mass spectrometry to identify the aggregated and mistranslated proteins. We show that the mitochondrial tRNA-modifying enzyme Slm3 thiolates the cytoplasmic tRNAs at position 34 and that decreased levels of mcm5s2U34 in SLM3 mutants are compensated by increasing mcm5U34, ncm5U34 and ncm5Um34 levels. In the tRNA gene knockout strains, stress response proteins are overrepresented in protein aggregates and their genes are enriched in codons decoded by tRNAs lacking mcm5U34, mcm5s2U34, ncm5U34, ncm5Um34, modifications. Increased rates of amino acid misincorporation were detected in the yeast ELP1, SLM3 and TRM9 gene knockout mutants at protein sites that specifically mapped to the codons sites that are decoded by the hypomodified tRNAs, demonstrating that U34 tRNA modifications safeguard the proteome from translational errors, misfolding and cellular proteotoxic stress.

It is known that the presence of protein aggregates in biological samples is associated with natural aging processes and age-related diseases. The objective of this technical study was to evaluate the potential of Fourier Transform Infrared Spectroscopy to identify the presence of protein aggregates in Saccharomyces cerevisiae containing high levels of protein aggregates. We acquired ATR-FTIR spectra at mid-infrared range (between 4000 and 600 cm −1 ) and used multivariate analysis to analyze the data. Significant differences between spectra of wild type and mutant strains in the spectral range assigned to proteins were observed. In particular, an increase in β-sheet structures in mutant strains (spectral signals at 1683 and 1628 cm −1 ) was observed, indicating the putative presence of protein aggregates. These results prove the capacity of FTIR to evaluate changes in protein conformation, mainly protein aggregation, in a fast, simple and non-expensive way, producing insights on the possible application of this technique to the detection of protein aggregates in human biological samples.

The occurrence of protein synthesis errors (mistranslation) above the typical mean mistranslation level of 10−4 is mostly deleterious to yeast, zebrafish and mammal cells. Previous yeast studies have shown that mistranslation affects fitness and deregulates genes related to lipid metabolism, but there is no experimental proof that such errors alter yeast lipid profiles. We engineered yeast strains to misincorporate serine at alanine and glycine sites on a global scale and evaluated the putative effects on the lipidome. Lipids from whole cells were extracted and analysed by thin layer chromatography (TLC), liquid chromatography-mass spectrometry(LC-MS) and gas chromatography (GC). Oxidative damage, fatty acid desaturation and membrane fluidity changes were screened to identify putative alterations in lipid profiles in both logarithmic (fermentative) and post-diauxic shift (respiratory) phases. There were alterations in several lipid classes, namely lyso-phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and triglyceride, and in the fatty acid profiles, namely C16:1, C16:0, C18:1 and C18:0. Overall, the relative content of lipid species with saturated FA increased in detriment of those with unsaturated fatty acids. The expression of the OLE1 mRNA was deregulated, but phospholipid fluidity changes were not observed. These data expand current knowledge of mistranslation biology and highlight its putative roles in human diseases.

Protein biosynthesis is a highly accurate biological process essential for life. Amino acid misincorporation errors (mistranslation) normally occur at low levels, but can increase sharply upon amino acid starvation, exposure to drugs, oxidative stress and other physiological perturbations. These processes disrupt protein function and are normally regarded as being deleterious, however, recent work has shown that they can also be regulated to produce advantageous phenotypes in both prokaryotes and eukaryotes. The biology of such unexpected adaptive mistranslation is poorly understood due to technical difficulties in the identification and quantification of amino acid misincorporations. In this mini-review, we describe proteome scale methodologies involving the use of massspectrometry and bioinformatics tools to directly detect and quantify mistranslation events and also indirect functional methods that permit sensitive, flexible and low-cost analysis of site specific amino acid variation.