tRNA-modifying enzyme mutations induce codon-specific mistranslation and protein aggregation in yeast

Abstract/SummaryProtein 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.