Loss of Elongator- and KEOPS-Dependent tRNA Modifications Leads to Severe Growth Phenotypes and Protein Aggregation in Yeast.

Leticia Pollo-Oliveira,Akif Ciftci,Maria Martinelli,Raffael Schaffrath,Michael Demott,Peter Dedon,Valérie De Crécy-Lagard,Nick Davis,Roland Klassen,Jo Bacusmo,Thomas Begley

doi:10.3390/biom10020322

Abstract

Modifications found in the Anticodon Stem Loop (ASL) of tRNAs play important roles in regulating translational speed and accuracy. Threonylcarbamoyl adenosine (t6A37) and 5-methoxycarbonyl methyl-2-thiouridine (mcm5s2U34) are critical ASL modifications that have been linked to several human diseases. The model yeast Saccharomyces cerevisiae is viable despite the absence of both modifications, growth is however greatly impaired. The major observed consequence is a subsequent increase in protein aggregates and aberrant morphology. Proteomic analysis of the t6A-deficient strain (sua5 mutant) revealed a global mistranslation leading to protein aggregation without regard to physicochemical properties or t6A-dependent or biased codon usage in parent genes. However, loss of sua5 led to increased expression of soluble proteins for mitochondrial function, protein quality processing/trafficking, oxidative stress response, and energy homeostasis. These results point to a global function for t6A in protein homeostasis very similar to mcm5/s2U modifications.

Highlights

Modifications of the four canonical bases found in the Anticodon Stem Loop (ASL) of tRNAs are critical for optimal decoding of mRNAs [1,2]
S. cerevisiae strains carrying a deletion in ELP3, which encodes the catalytic subunit of the Elongator complex, lack mcm5U in tRNA [28] while strains with deletions of SUA5 encoding the first enzyme of the t6A synthesis pathway, threonylcarbamoyl-AMP synthase, lack t6A [25]
This study showed how the critical tRNA modifications t6A37 and mcm5s2U34 contribute to the maintenance of proteome integrity of the model yeast S. cerevisiae by demonstrating the effects caused by the deficiency of both modifications in the cell

Summary

Introduction

Modifications of the four canonical bases found in the Anticodon Stem Loop (ASL) of tRNAs are critical for optimal decoding of mRNAs [1,2]. In S. cerevisiae, the protein with the longest stretch of codons decoded by t6A containing tRNAs is SWI1 with its stretch of 31 Asn and Thr amino acids starting at position 7 of the protein Because these repeats (90 nts) are longer than the RPFs (28 nts) sequenced, this gene could not be analyzed in the ribosome profiling that compared WT and t6A- strains [36]. We set out to study if the absence of t6A affects the translation of proteins with long stretches of codons decoded by t6A-dependent tRNAs by constructing the plasmid pJMB21, which allowed for the expression of proteins containing an N-terminal HA-tag with a C-terminal GFP fusion. This is consistent with our published ribosome profiling studies [39]. Loss of t6A causes a larger reduction in the translation of proteins enriched in t6A dependent codons such as this SWI1 fragment

Methods

Results

Discussion

Conclusion