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

Abstract

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..

Elp2 mutations perturb the epitranscriptome and lead to a complex neurodevelopmental phenotype

“…These U 34 modifications tune ribosomal speed during translational elongation, specifically affecting tRNA binding, recognition, rejection, decoding and translocation. Hence, the lack of U 34 modifications leads to changes in the cotranslational folding dynamics of nascent polypeptide chains, promotes misfolding of newly synthesized proteins, and triggers detrimental cellular responses related to protein aggregation in all organisms tested 32,33 . In particular, the translational decoding of A/U-rich codons 34 requires additional stabilization by these modifications due to their weaker codon-anticodon pairing, which would otherwise by default lead to translational infidelity and proteostasis 35 .…”

Elp3‐mediated codon‐dependent translation promotes mTORC2 activation and regulates macrophage polarization

“…Having demonstrated that Elp3 is critical for mitochondrial functions in metabolic reprogramming linked to macrophage polarization, we next explored the underlying molecular mechanisms. Elp3 deficiency leads to the accumulation of multiple proteins in aggregates, both in yeast and in mammalian cells (Nedialkova & Leidel, 2015 ; Rapino et al , 2018 ; Tavares et al , 2020 ). Likewise, Elp3 deficiency caused the accumulation of protein candidates in aggregates from IL‐4‐stimulated BMDMs (Fig EV4C ).…”

“…Elp3 deficiency leads to protein misfolding and aggregation in both yeast and human (Nedialkova & Leidel, 2015 ; Rapino et al , 2018 ). Interestingly, candidates found in these aggregates are enriched in codons decoded by tRNAs lacking mcm 5 s 2 U 34 modifications in Elp3‐deficient yeast (Tavares et al , 2020 ). Therefore, it is tempting to speculate that defects in macrophage polarization seen upon Elp3 deficiency may result, at least in part, from misfolding of a variety of proteins acting downstream of both LPS/IFNγ and IL‐4 signaling cascades.…”

Role of SSD1 in Phenotypic Variation of Saccharomyces cerevisiae Strains Lacking DEG1-Dependent Pseudouridylation

“…Both Deg1 and Elongator have been implicated in the maintenance of protein homeostasis [7,28,29,53,54]. A deficiency in protein homeostasis is thought to account for stress phenotypes of certain combined mutants involving Elongator, tRNA thiolation, and/or Deg1 defects (e.g., temperature sensitivity) [7,28].…”