Queuosine-tRNA promotes sex-dependent learning and memory formation by maintaining codon-biased translation elongation speed.
Ontology highlight
ABSTRACT: Queuosine (Q) is a modified nucleoside at the wobble position of specific tRNAs. In mammals, queuosinylation is facilitated by queuine uptake from the gut microbiota and is introduced into tRNA by the QTRT1-QTRT2 enzyme complex. By establishing a Qtrt1 knockout mouse model, we discovered that the loss of QtRNA leads to learning and memory deficits. Ribo-Seq analysis in the hippocampus of Qtrt1-deficient mice revealed not only stalling of ribosomes on Q-decoded codons but also a global imbalance in translation elongation speed between codons that engage in weak and strong interactions with their cognate anticodons. While Qdependent molecular and behavioral phenotypes were identified in both sexes, female mice were affected more severely than males. Proteomics analysis confirmed deregulation of synaptogenesis and neuronal morphology. Together, our findings provide a link between tRNA modification and brain functions and reveal an unexpected role of protein synthesis in sex-dependent cognitive performance.
Project description:Queuosine (Q) is a conserved tRNA modification at the wobble anticodon position of tRNAs that read the codons of amino acids Tyr, His, Asn, and Asp. Q-modification in tRNA plays important roles in the regulation of translation efficiency and fidelity. Queuosine tRNA modification is synthesized de novo in bacteria, whereas the substrate for Q-modification in tRNA in mammals is queuine, the catabolic product of the Q-base of gut bacteria. This gut microbiome dependent tRNA modification may play pivotal roles in translational regulation in different cellular contexts, but extensive studies of Q-modification biology are hindered by the lack of high throughput sequencing methods for its detection and quantitation. Here, we describe a periodate-treatment method of biological RNA samples that enables single base resolution profiling of Q-modification in tRNAs by Nextgen sequencing. Periodate oxidizes the Q-base, which results in specific deletion signatures in the RNA-seq data. Unexpectedly, we found that periodate-treatment also enables the detection of several 2-thio-modifications including τm5s2U, mcm5s2U, cmnm5s2U, and s2C by sequencing in human and E. coli tRNA. We term this method Periodate-dependent analysis of queuosine and thio modification sequencing (PAQS-seq). We assess Q- and 2-thio-modifications at the tRNA isodecoder level, and 2-thio modification changes in stress response. PAQS-seq should be widely applicable in the biological studies of Q- and 2-thio-modifications in mammalian and microbial tRNAs.
Project description:Regulation of protein translation is a key feature of many biological processes. Global protein translation as well as translation at the codon level can be regulated by RNA modifications. These modifications are particularly enriched in tRNAs, where they represent an additional regulatory layer on top of the primary RNA sequence. In eukaryotes, levels of tRNA queuosinylation reflect the bioavailability of the precursor queuine, which is salvaged from the gut microbiota and absorbed in the intestine. We show here that reduced queuine supply results in a strong reduction of tRNA queuosinylation in various cultured human cell lines and mouse tissues. In addition, Dnmt2-dependent tRNA methylation is dynamically modulated by the presence or absence of queuine. Ribosome profiling showed that nutritionally determined queuosine-tRNA (Q-tRNA) levels control the translation speed of Q-tRNA decoded codons. Dysregulation of translation resulted in pronounced endoplasmic reticulum stress and activation of the unfolded protein response, which could be rescued by the addition of queuine. Together, these findings establish a direct link between nutrient availability and the decoding of the transcriptome, thus providing a new perspective in our understanding of translational regulation.
Project description:Cells respond to environmental stress by regulating gene expression at the level of both transcription and translation. The ~50 modified ribonucleotides of the human epitranscriptome contribute to the latter, with mounting evidence that dynamic regulation of tRNA wobble modifications leads to selective translation of stress response proteins from codon-biased genes. Here we show that the response of human HepG2 cells to arsenite exposure is regulated by the availability of queuine, a micronutrient and essential precursor to the wobble modification queuosine (Q) on tRNAs reading GUN codons. Among oxidizing and alkylating agents at equitoxic concentrations, arsenite exposure caused an oxidant-specific increase in Q that correlated with up-regulation of proteins from codon-biased genes involved in energy metabolism. Limiting queuine increased arsenite-induced cell death, altered translation, increased reactive oxygen species levels, and caused mitochondrial dysfunction. In addition to revealing a new epitranscriptomic facet of arsenite toxicity and response, our results highlight the mechanistic links between environmental exposures, stress tolerance, and micronutrients.
Project description:Post-transcriptional modification of tRNAs is critical for protein synthesis. Queuosine (Q), a 7-deaza-guanosine derivative, is present at the first position of anticodons of several tRNA species. In vertebrate tRNAs for Tyr and Asp, Q is further glycosylated with galactose and mannose to generate galactosyl-queuosine (galQ) and mannosyl-queuosine (manQ), respectively. However, the biogenesis and physiological relevance of Q-glycosylation remain poorly understood. Here, we biochemically identified two RNA glycosylases, queuosine-tRNA galactosyltransferase (QTGAL) and queuosine-tRNA mannosyltransferase (QTMAN), and successfully reconstituted galQ and manQ formation on the respective tRNAs in the presence of nucleotide diphosphate sugars. Ribosome profiling analyses of human knockout (KO) cells of QTGAL and QTMAN revealed that Q-glycosylation slowed down elongation at the cognate codons, UAC and GAC (GAU), respectively. Furthermore, protein aggregates were significantly increased in both KO cell lines, indicating that Q-glycosylation contributes to proteostasis of nascent proteins. We determined atomic structures of human cytoplasmic tRNAs for Try and Asp bound to the ribosome A-site, providing the molecular basis of codon recognition regulated by galQ and manQ. Furthermore, zebrafish qtgal and qtman KO lines displayed shortened body length, suggesting Q-glycosylation is required for post-embryonic growth in vertebrates. These findings demonstrate that Q-glycosylation modulates codon-specific translation to optimize the decoding speed in protein synthesis, thereby maintaining correct folding of nascent proteins, proteome integrity, and normal growth of vertebrates.
Project description:Cells respond to environmental stress by regulating gene expression at the level of both transcription and translation. The ~50 modified ribonucleotides of the human epitranscriptome contribute to the latter, with mounting evidence that dynamic regulation of tRNA wobble modifications leads to selective translation of stress response proteins from codon-biased genes. Here we show that the response of human HepG2 cells to arsenite exposure is regulated by the availability of queuine, a micronutrient and essential precursor to the wobble modification queuosine (Q) on tRNAs reading GUN codons. Among oxidizing and alkylating agents at equitoxic concentrations, arsenite exposure caused an oxidant-specific increase in Q that correlated with up-regulation of proteins from codon-biased genes involved in energy metabolism. Limiting queuine increased arsenite-induced cell death, altered translation, increased reactive oxygen species levels, and caused mitochondrial dysfunction. In addition to revealing a new epitranscriptomic facet of arsenite toxicity and response, our results highlight the mechanistic links between environmental exposures, stress tolerance, and micronutrients.
Project description:Queuosine (Q) is a complex tRNA modification found at position 34 of four tRNAs with a GUN anticodon, and it regulates the translational efficiency and fidelity of the respective codons that differ at the Wobble position. In bacteria, the biosynthesis of Q involves two precursors, preQ0 and preQ1, whereas eukaryotes directly obtain Q from bacterial sources. The study of queuosine has been challenging due to the limited availability of high-throughput methods for its detection and analysis. Here, we have employed direct RNA sequencing using nanopore technology to detect the modification of tRNAs with Q and Q precursors. These modifications were detected with high accuracy on synthetic tRNAs as well as on tRNAs extracted from Schizosaccharomyces pombe and Escherichia coli by comparing unmodified to modified tRNAs using the tool JACUSA2. Furthermore, we present an improved protocol for the alignment of raw sequence reads that gives high specificity and recall for tRNAs ex cellulo that, by nature, carry multiple modifications. Altogether, our results show that such 7-deazaguanine-derivatives are readily detectable using direct RNA sequencing. This advancement opens up new possibilities for investigating these modifications in native tRNAs, furthering our understanding of their biological function.
Project description:The genetic code is an abstraction of how mRNA codons and tRNA anticodons molecularly interact during protein synthesis; the stability and regulation of this interaction remains largely unexplored. Here, we quantitatively characterized the expression of mRNA and tRNA genes across developing mouse tissues. Substantial fractions of both gene sets dynamically change expression from early organogenesis to adult tissues. mRNA codon pools are highly stable over development and reflect the genomic background; in contrast, changes in transcription at specific tRNA genes are coordinated across anticodon families to produce a stable isoacceptor output. During development, the pools of mRNA codons and tRNA anticodons are invariant and highly correlated, revealing a stable molecular interaction interlocking transcription and translation.
Project description:Proteins begin to fold as they emerge from translating ribosomes. The kinetics of ribosome transit along a given mRNA can influence nascent chain folding, but the extent to which individual codon translation rates impact proteome integrity remains unknown. Here, we show that slower decoding of discrete codons elicits widespread protein aggregation in vivo. Using ribosome profiling, we find that loss of anticodon wobble uridine (U34) modifications in a subset of tRNAs leads to ribosome pausing at their cognate codons in S. cerevisiae and C. elegans. Yeast cells lacking U34 modifications exhibit gene expression hallmarks of proteotoxic stress and accumulate aggregates of endogenous proteins with key cellular functions. Moreover, these cells are severely compromised in clearing stress-induced protein aggregates. Overexpression of hypomodified tRNAs alleviates ribosome pausing, concomitantly restoring protein homeostasis. Our findings demonstrate that modified U34 is an evolutionarily conserved accelerator of decoding and reveal an unanticipated role for tRNA anticodon modifications in maintaining proteome integrity. Ribosome profiling of wild-type and tRNA modification-deficient yeast and nematodes. Yeast samples were generated in various growth conditions (rich medium versus stress induced by treatment with diamide or rapamycin) and paired mRNA-Seq was performed on a subset of samples. Dataset contains three biological replicates for yeast samples and two biological replicates for nematode samples.
Project description:Eukaryotic transfer RNAs (tRNA) contain on average 13 modifications that perform a wide range of roles in translation and in the generation of tRNA fragments that regulate gene expression. Queuosine (Q) modification occurs in the wobble anticodon position of tRNAs for amino acids His, Asn, Tyr, and Asp. In eukaryotes, Q modification is fully dependent on diet or on gut microbiome in multi-cellular organisms. Despite decades of study, cellular roles of Q modification remain to be fully elucidated. Here we show that in human cells, Q modification specifically protects its cognate tRNAHis and tRNAAsn against cleavage by ribonucleases. We generated cell lines that contain completely depleted or fully Q-modified tRNAs. Using these resources, we found that Q modification significantly reduces angiogenin cleavage of its cognate tRNAs in vitro. Q modification does not change the cellular abundance of the cognate full-length tRNAs, but alters the cellular content of their fragments in vivo in the absence and presence of stress. Our results provide a new biological aspect of Q modification and a mechanism of how Q modification alters small RNA pool in human cells.
Project description:The genetic code is an abstraction of how mRNA codons and tRNA anticodons molecularly interact during protein synthesis; the stability and regulation of this interaction remains largely unexplored. Here, we quantitatively characterized the expression of m