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 tRNA^Lys_UUU, 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.

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Thiaville PC, Legendre R, Rojas-Benítez D, Baudin-Baillieu A, Hatin I, Chalancon G, Glavic A, Namy O, de Crécy-Lagard V

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