The biological role of many nonessential tRNA modifications outside of the anticodon remains elusive despite their evolutionary conservation. We show here that m7G46 methyltransferase Trm8p/Trm82p acts as a hub of synthetic interactions with several tRNA modification enzymes, resulting in temperature-sensitive growth. Analysis of three double mutants indicates reduced levels of tRNA(Val(AAC)), consistent with a role of the corresponding modifications in maintenance of tRNA levels. Detailed examination of a trm8-delta trm4-delta double mutant demonstrates rapid degradation of preexisting tRNA(Val(AAC)) accompanied by its de-aminoacylation. Multiple copies of tRNA(Val(AAC)) suppress the trm8-delta trm4-delta growth defect, directly implicating this tRNA in the phenotype. These results define a rapid tRNA degradation (RTD) pathway that is independent of the TRF4/RRP6-dependent nuclear surveillance pathway. The degradation of an endogenous tRNA species at a rate typical of mRNA decay demonstrates a critical role of nonessential modifications for tRNA stability and cell survival.

[Keywords: Biochemical genomics; protein microarray; proteome; high-throughput expression; galactose lethality; glycosylation] Supplemental material is available at http://www.genesdev.org.

Mature tRNA is normally extensively modified and extremely stable. Recent evidence suggests that hypomodified mature tRNA in yeast can undergo a quality control check by a rapid tRNA decay (RTD) pathway, since mature tRNA Val(AAC) lacking 7-methylguanosine and 5-methylcytidine is rapidly degraded and deacylated at 37°C in a trm8-⌬ trm4-⌬ strain, resulting in temperature-sensitive growth. We show here that components of this RTD pathway include the 5-3 exonucleases Rat1 and Xrn1, and Met22, which likely acts indirectly through Rat1 and Xrn1. Since deletion of MET22 or mutation of RAT1 and XRN1 prevent both degradation and deacylation of mature tRNA Val(AAC) in a trm8-⌬ trm4-⌬ strain and result in healthy growth at 37°C, hypomodified tRNA Val(AAC) is at least partially functional and structurally intact under these conditions. The integrity of multiple mature tRNA species is subject to surveillance by the RTD pathway, since mutations in this pathway also prevent degradation of at least three other mature tRNAs lacking other combinations of modifications. The RTD pathway is the first to be implicated in the turnover of mature RNA species from the class of stable RNAs. These results and the results of others demonstrate that tRNA, like mRNA, is subject to multiple quality control steps.[Keywords: RNA turnover; mature tRNA; TRM8; TRM4; yeast; tRNA Val(AAC) ] Supplemental material is available at http://www.genesdev.org.

SUMMARY Translation elongation efficiency is largely thought of as the sum of decoding efficiencies for individual codons. Here, we find that adjacent codon pairs modulate translation efficiency. Deploying an approach in Saccharomyces cerevisiae that scored the expression of over 35,000 GFP variants in which three adjacent codons were randomized, we identified 17 pairs of adjacent codons associated with reduced expression. For many pairs, codon order is obligatory for inhibition, implying a more complex interaction than a simple additive effect. Inhibition mediated by adjacent codons occurs during translation itself as GFP expression is restored by increased tRNA levels or by non-native tRNAs with exact-matching anticodons. Inhibition operates in endogenous genes, based on analysis of ribosome profiling data. Our findings suggest translation efficiency is modulated by an interplay between tRNAs at adjacent sites in the ribosome, and that this concerted effect needs to be considered in predicting the functional consequences of codon choice.

For the identification of yeast genes specifying biochemical activities, a genomic strategy that is rapid, sensitive, and widely applicable was developed with an array of 6144 individual yeast strains, each containing a different yeast open reading frame (ORF) fused to glutathione S-transferase (GST). For the identification of ORF-associated activities, strains were grown in defined pools, and GST-ORFs were purified. Then, pools were assayed for activities, and active pools were deconvoluted to identify the source strains. Three previously unknown ORF-associated activities were identified with this strategy: a cyclic phosphodiesterase that acts on adenosine diphosphate-ribose 1"-2" cyclic phosphate (Appr>p), an Appr-1"-p-processing activity, and a cytochrome c methyltransferase.