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

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.

Evolving specificity of tRNA 3-methyl-cytidine-32 (m3C32) modification: a subset of tRNAsSer requires N6-isopentenylation of A37

“…1D). In this regard, it matches the higher eukaryote m 3 C32 expansion substrates, tRNA Arg CCU and tRNA Arg UCU at positions C35, U36, and t 6 A37 (Thiaville et al 2015b).…”

“…The tRNAs Thr and tRNA Ser GCU share U at position 36, part of the U36-A37-A38 consensus for t 6 A37 (Morin et al 1998;Thiaville et al 2015b), and are known to contain t 6 A37 (Jühling et al 2009). We therefore analyzed existing tRNA modification data (Jühling et al 2009) and found that virtually all known tRNAs with m 3 C32 also carry i 6 A37, t 6 A37 or their derivatives (Fig.…”

“…5E). However, it should be noted that several others that carry t 6 A37, namely tRNAs for Met, Ile, Asn Lys, and Arg (Thiaville et al 2015b), do not contain m 3 C32 even though several have C at position 32 (see Fig. 1C C32, it is the only one with C rather than G in anticodon position 35 (see Fig.…”

Regulation of mRNA Translation in Neurons—A Matter of Life and Death

“…Recently, homozygous mutations in KAE1 (kinase-associated endopeptidase; OSGEP ), which plays a role in the biosynthesis of N 6 -threonylcarbamoyladenosine (t 6 A) modifications of tRNA, have been linked progressive cerebellar atrophy and leukodystrophy (Edvardson et al, 2017). This modification is present at nucleotide 37 in the anticodon stem loop of nearly all tRNAs decoding ANN (where N is any nucleotide) codons, and defects in t 6 A biosynthesis in yeast lead to leaky scanning bypass of start codons, as well as impaired reading frame maintenance (Thiaville et al, 2016). This suggests that certain hypomodified tRNAs can disrupt translation elongation and lead to neuropathology.…”

Transcriptome-wide Analysis of Roles for tRNA Modifications in Translational Regulation

“…A handful of recent studies have carried out transcriptome-wide analysis of translation, using ribosome profiling (Ingolia et al, 2009) to illuminate the roles for specific tRNA-modifying enzymes in translation and proteostasis (Laguesse et al, 2015; Nedialkova and Leidel, 2015; Thiaville et al, 2016; Tuorto et al, 2015; Zinshteyn and Gilbert, 2013). Here, we adopt this approach to systematically study the roles for tRNA modifications in translational regulation in S. cerevisiae , using ribosome profiling to generate ribosome occupancy maps for 57 yeast deletion strains.…”