Global protein translation as well as translation at the codon level can be regulated by tRNA modifications. In eukaryotes, levels of tRNA queuosinylation reflect the bioavailability of the precursor queuine, which is salvaged from the diet and gut microbiota. We show here that nutritionally determined Q‐tRNA levels promote Dnmt2‐mediated methylation of tRNA Asp and control translational speed of Q‐decoded codons as well as at near‐cognate codons. Deregulation of translation upon queuine depletion results in unfolded proteins that trigger endoplasmic reticulum stress and activation of the unfolded protein response, both in cultured human cell lines and in germ‐free mice fed with a queuosine‐deficient diet. Taken together, our findings comprehensively resolve the role of this anticodon tRNA modification in the context of native protein translation and describe a novel mechanism that links nutritionally determined modification levels to effective polypeptide synthesis and cellular homeostasis.

The accurate definition of an epitranscriptome is endangered by artefacts resulting from RNA degradation after cell death, a ubiquitous yet little investigated process. By tracing RNA marker modifications through tissue preparation protocols, we identified a major blind spot from daily lab routine, that has massive impact on modification analysis in small RNAs. In particular, m6,6A and Am as co-varying rRNA marker modifications, appeared in small RNA fractions following rRNA degradation in vitro and in cellulo. Analysing mouse tissue at different time points post mortem, we tracked the progress of intracellular RNA degradation after cell death, and found it reflected in RNA modification patterns. Differences were dramatic between liver, where RNA degradation commenced immediately after death, and brain, yielding essentially undamaged RNA. RNA integrity correlated with low amounts of co-varying rRNA markers. Thus validated RNA preparations featured differentially modified tRNA populations whose information content allowed a distinction even among the related brain tissues cortex, cerebellum and hippocampus. Inversely, advanced cell death correlated with high rRNA marker content, and correspondingly little with the naïve state of living tissue. Therefore, unless RNA and tissue preparations are executed with utmost care, interpretation of modification patterns in tRNA and small RNA are prone to artefacts.