Global translational impacts of the loss of the tRNA modification t6A in yeast.

Patrick Thiaville,Isabelle Hatin,Diego Rojas-Benitez,Valerie De Crecy-Lagard,Agnes Baudin-Baillieu,Rachel Legendre,Olivier Namy,Guilhem Chalancon,Alvaro Glavic

doi:10.15698/mic2016.01.473

Abstract

The universal tRNA modification t6A is found at position 37 of nearly all tRNAs decoding ANN codons. The absence of t6A37 leads to severe growth defects in baker’s yeast, phenotypes similar to those caused by defects in mcm5s2U34 synthesis. Mutants in mcm5s2U34 can be suppressed by overexpression of tRNALysUUU, but we show t6A phenotypes could not be suppressed by expressing any individual ANN decoding tRNA, and t6A and mcm5s2U are not determinants for each other’s formation. Our results suggest that t6A deficiency, like mcm5s2U deficiency, leads to protein folding defects, and show that the absence of t6A led to stress sensitivities (heat, ethanol, salt) and sensitivity to TOR pathway inhibitors. Additionally, L-homoserine suppressed the slow growth phenotype seen in t6A-deficient strains, and proteins aggregates and Advanced Glycation End-products (AGEs) were increased in the mutants. The global consequences on translation caused by t6A absence were examined by ribosome profiling. Interestingly, the absence of t6A did not lead to global translation defects, but did increase translation initiation at upstream non-AUG codons and increased frame-shifting in specific genes. Analysis of codon occupancy rates suggests that one of the major roles of t6A is to homogenize the process of elongation by slowing the elongation rate at codons decoded by high abundance tRNAs and I34:C3 pairs while increasing the elongation rate of rare tRNAs and G34:U3 pairs. This work reveals that the consequences of t6A absence are complex and multilayered and has set the stage to elucidate the molecular basis of the observed phenotypes.

Highlights

Modifications of the anticodon stem loop (ASL) of transfer RNA are critical for translational speed and accuracy
We found that ANN codons in yeast have in average a higher estimated translational efficiency compared to other codons (Figure 6), suggesting that there is a statistical tendency for ANN-transfer RNA (tRNA) to be in high supply in standard growth conditions
Role of t6A in decoding efficiency varies with the tRNA The codon occupancy results presented in this study suggest that t6A helps rare cognate tRNAs and G:U mismatches compete with Watson:Crick decoding tRNAs and slows decoding by high abundance tRNAs and tRNAs using the wobble U:C base pairings [85]

Summary

INTRODUCTION

Modifications of the anticodon stem loop (ASL) of transfer RNA (tRNA) are critical for translational speed and accuracy. RESULTS mcm5s2U34 or t6A37 are not determinants for each other’s synthesis The similarity of the phenotypes observed in strains deficient in mcm5s2U and t6A synthesis suggests that one of the modifications could be required for the synthesis of the other To test this hypothesis, tRNAs from wild type (BY4741), mcm5s2U-deficient yeast strains (elp3∆, trm9∆, ncs2∆, and ncs6∆) and t6A synthesis mutants (tcs2∆-tcs8∆) were purified and analysed by HPLC. To assess if tRNAs could suppress the slow growth rate seen in mutants of t6A synthesis, an expression plasmid containing tRNALysUUU was transformed into BY4741, tcs2∆, tcs3∆, FIGURE 2: HPLC analysis examining the relationship between mcm5s2U34 and t6A37. Microarray analyses of conditional or point mutations in tcs, tcs, or tcs had been previously reported [38] and in all these studies, an up-regulation of Gcn regulated genes was observed, including genes in the arginine and histidine biosynthesis pathways, the mRNA expression levels of GCN4 itself did not increase. It appears that t6A is helping increase elongation rate of rare tRNAs and G34:U3 pairs and decrease the elongation rate of high abundance and I34:C3 pairs to homogenize the process of elongation

DISCUSSION

Findings

MATERIALS AND METHODS