Glycosylated queuosines in tRNAs optimize translational rate and post-embryonic growth.

Transfer RNA (tRNA) modifications are critical for protein synthesis. Queuosine (Q), a 7-deaza-guanosine derivative, is present in tRNA anticodons. In vertebrate tRNAs for Tyr and Asp, Q is further glycosylated with galactose and mannose to generate galQ and manQ, respectively. However, biogenesis and physiological relevance of Q-glycosylation remain poorly understood. Here, we biochemically identified two RNA glycosylases, QTGAL and QTMAN, and successfully reconstituted Q-glycosylation of tRNAs using nucleotide diphosphate sugars. Ribosome profiling of knockout cells revealed that Q-glycosylation slowed down elongation at cognate codons, UAC and GAC (GAU), respectively. We also found that galactosylation of Q suppresses stop codon readthrough. Moreover, protein aggregates increased in cells lacking Q-glycosylation, indicating that Q-glycosylation contributes to proteostasis. Cryo-EM of human ribosome-tRNA complex revealed the molecular basis of codon recognition regulated by Q-glycosylations. Furthermore, zebrafish qtgal and qtman knockout lines displayed shortened body length, implying that Q-glycosylation is required for post-embryonic growth in vertebrates.

Queuosine-tRNA promotes sex-dependent learning and memory formation by maintaining codon-biased translation elongation speed.

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 Q-tRNA 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 Q-dependent 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.

Disruption to tRNA Modification by Queuine Contributes to Inflammatory Bowel Disease.

BACKGROUNDS AND AIMS: Transfer RNA (tRNA) is the most extensively modified RNA in cells. Queuosine modification is a fundamental process for ensuring the fidelity and efficiency of translation from RNA to protein. In eukaryotes, Queuosine tRNA (Q-tRNA) modification relies on the intestinal microbial product queuine. However, the roles and potential mechanisms of Q-containing tRNA (Q-tRNA) modifications in inflammatory bowel disease (IBD) are unknown. METHODS: We explored the Q-tRNA modifications and expression of QTRT1 (queuine tRNA-ribosyltransferase 1) in patients with IBD by investigating human biopsies and reanalyzing datasets. We used colitis models, QTRT1 knockout mice, organoids, and cultured cells to investigate the molecular mechanisms of Q-tRNA modifications in intestinal inflammation. RESULTS: QTRT1 expression was significantly downregulated in ulcerative colitis and Crohn's disease patients. The 4 Q-tRNA-related tRNA synthetases (asparaginyl-, aspartyl-, histidyl-, and tyrosyl-tRNA synthetase) were decreased in IBD patients. This reduction was further confirmed in a dextran sulfate sodium-induced colitis model and interleukin-10-deficient mice. Reduced QTRT1 was significantly correlated with cell proliferation and intestinal junctions, including downregulation of ß-catenin and claudin-5 and the upregulation of claudin-2. These alterations were confirmed in vitro by deleting the QTRT1 gene from cells and in vivo using QTRT1 knockout mice. Queuine treatment significantly enhanced cell proliferation and junction activity in cell lines and organoids. Queuine treatment also reduced inflammation in epithelial cells. Moreover, altered QTRT1-related metabolites were found in human IBD. CONCLUSIONS: tRNA modifications play an unexplored novel role in the pathogenesis of intestinal inflammation by altering epithelial proliferation and junction formation. Further investigation of the role of tRNA modifications will uncover novel molecular mechanisms for the prevention and treatment of IBD.

Probing Queuosine Modifications of Transfer RNA in Single Living Cells via Plasmonic Affinity Sandwich Assay.

Queuosine (Q) modification on tRNA plays an essential role in protein synthesis, participating in many tRNA functions such as folding, stability, and decoding. Appropriate analytical tools for the measurement of tRNA Q modifications are essential for the exploration of new roles of Q-modified tRNAs and the rationalization of their exact mechanisms. However, conventional methods for Q modification analysis suffer from apparent disadvantages, such as destructive cells, tedious procedure, and low sensitivity, which much hamper in-depth studies of Q modification-related biological questions. In this study, we developed a new approach called plasmonic affinity sandwich assay that allows for facile and sensitive determination of Q-modified tRNAs in single living cells. This method relies on the combination of plasmon-enhanced Raman scattering detection, base-paring affinity in-cell microextraction, and a set of boronate affinity and molecularly imprinted labeling nanotags for selective recognition of individual Q modifications, including queuosine, galactosyl queuosine (Gal-Q), and mannosyl queuosine (Man-Q). The developed method exhibited high affinity extraction and high specificity recognition. It allowed for the measurement of tRNA Q modifications in not only Q-rich cultured tumor cells but also Q-deficient primary tumor cells. Usefulness of this approach for investigation of the change of the Q modification level in single cells under oxidative stress was demonstrated. Because of its significant advantages over conventional methods, this approach provides a promising analytical tool for the exploration of more roles of Q-modified tRNAs and elucidation of their mechanisms.

Preferential import of queuosine-modified tRNAs into Trypanosoma brucei mitochondrion is critical for organellar protein synthesis.

Transfer RNAs (tRNAs) are key players in protein synthesis. To be fully active, tRNAs undergo extensive post-transcriptional modifications, including queuosine (Q), a hypermodified 7-deaza-guanosine present in the anticodon of several tRNAs in bacteria and eukarya. Here, molecular and biochemical approaches revealed that in the protozoan parasite Trypanosoma brucei, Q-containing tRNAs have a preference for the U-ending codons for asparagine, aspartate, tyrosine and histidine, analogous to what has been described in other systems. However, since a lack of tRNA genes in T. brucei mitochondria makes it essential to import a complete set from the cytoplasm, we surprisingly found that Q-modified tRNAs are preferentially imported over their unmodified counterparts. In turn, their absence from mitochondria has a pronounced effect on organellar translation and affects function. Although Q modification in T. brucei is globally important for codon selection, it is more so for mitochondrial protein synthesis. These results provide a unique example of the combined regulatory effect of codon usage and wobble modifications on protein synthesis; all driven by tRNA intracellular transport dynamics.

Analysis of Queuosine tRNA Modification Using APB Northern Blot Assay.

Queuosine (Q) is a hypermodified base that occurs at the wobble position of transfer RNAs (tRNAs) with a GUN anticodon. Q-tRNA modification is widespread among eukaryotes, yet bacteria are the original source of Q. Eukaryotes acquire Q from their diet, or from the gut microbiota (in multicellular organisms). Despite decades of study, the detailed roles of Q-tRNA modification remain to be elucidated, especially regarding its specific mechanisms of action. Here, we describe a method for the fast and reliable detection of Q-tRNA modification levels in individual tRNAs using a few micrograms of total RNA as starting material. The methodology is based on the co-polymerization of boronic acid (N-acryloyl-3-aminophenylboronic acid (APB)) in polyacrylamide gels, and on the interplay between this derivative and free cis-diol groups of the tRNA. During electrophoresis, the cis-diol groups slow down the Q-modified tRNA, which then can be separated from unmodified tRNA and quantified using Northern blot analysis.

Detection and quantification of glycosylated queuosine modified tRNAs by acid denaturing and APB gels.

Queuosine (Q) is a conserved tRNA modification in bacteria and eukaryotes. Eukaryotic Q-tRNA modification occurs through replacing the guanine base with the scavenged metabolite queuine at the wobble position of tRNAs with G34U35N36 anticodon (Tyr, His, Asn, Asp) by the QTRT1/QTRT2 heterodimeric enzyme encoded in the genome. In humans, Q-modification in tRNATyr and tRNAAsp are further glycosylated with galactose and mannose, respectively. Although galactosyl-Q (galQ) and mannosyl-Q (manQ) can be measured by LC/MS approaches, the difficulty of detecting and quantifying these modifications with low sample inputs has hindered their biological investigations. Here we describe a simple acid denaturing gel and nonradioactive northern blot method to detect and quantify the fraction of galQ/manQ-modified tRNA using just microgram amounts of total RNA. Our method relies on the secondary amine group of galQ/manQ becoming positively charged to slow their migration in acid denaturing gels commonly used for tRNA charging studies. We apply this method to determine the Q and galQ/manQ modification kinetics in three human cells lines. For Q-modification, tRNAAsp is modified the fastest, followed by tRNAHis, tRNATyr, and tRNAAsn Compared to Q-modification, glycosylation occurs at a much slower rate for tRNAAsp, but at a similar rate for tRNATyr Our method enables easy access to study the function of these enigmatic tRNA modifications.

Queuosine-modified tRNAs confer nutritional control of protein translation.

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.

Queuosine modification protects cognate tRNAs against ribonuclease cleavage.

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 multicellular 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 pools in human cells.

Biochemical and Spectroscopic Studies of Epoxyqueuosine Reductase: A Novel Iron-Sulfur Cluster- and Cobalamin-Containing Protein Involved in the Biosynthesis of Queuosine.

Queuosine is a hypermodified nucleoside present in the wobble position of tRNAs with a 5'-GUN-3' sequence in their anticodon (His, Asp, Asn, and Tyr). The 7-deazapurine core of the base is synthesized de novo in prokaryotes from guanosine 5'-triphosphate in a series of eight sequential enzymatic transformations, the final three occurring on tRNA. Epoxyqueuosine reductase (QueG) catalyzes the final step in the pathway, which entails the two-electron reduction of epoxyqueuosine to form queuosine. Biochemical analyses reveal that this enzyme requires cobalamin and two [4Fe-4S] clusters for catalysis. Spectroscopic studies show that the cobalamin appears to bind in a base-off conformation, whereby the dimethylbenzimidazole moiety of the cofactor is removed from the coordination sphere of the cobalt but not replaced by an imidazole side chain, which is a hallmark of many cobalamin-dependent enzymes. The bioinformatically identified residues are shown to have a role in modulating the primary coordination sphere of cobalamin. These studies provide the first demonstration of the cofactor requirements for QueG.

Queuosine biosynthesis is required for sinorhizobium meliloti-induced cytoskeletal modifications on HeLa Cells and symbiosis with Medicago truncatula.

Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as well, were able to trigger a major reorganization of actin cytoskeleton of cultured HeLa cells in vitro. Cell deformation was associated with an inhibition of the three major small RhoGTPases Cdc42, RhoA and Rac1. Bacterial entry, cytoskeleton rearrangements and modulation of RhoGTPase activity required an intact S. meliloti biosynthetic pathway for queuosine, a hypermodifed nucleoside regulating protein translation through tRNA, and possibly mRNA, modification. We showed that an intact bacterial queuosine biosynthetic pathway was also required for effective nitrogen-fixing symbiosis of S. meliloti with its host plant Medicago truncatula, thus indicating that one or several key symbiotic functions of S. meliloti are under queuosine control. We discuss whether the symbiotic defect of que mutants may originate, at least in part, from an altered capacity to modify plant cell actin cytoskeleton.

Queuosine deficiency in eukaryotes compromises tyrosine production through increased tetrahydrobiopterin oxidation.

Queuosine is a modified pyrrolopyrimidine nucleoside found in the anticodon loop of transfer RNA acceptors for the amino acids tyrosine, asparagine, aspartic acid, and histidine. Because it is exclusively synthesized by bacteria, higher eukaryotes must salvage queuosine or its nucleobase queuine from food and the gut microflora. Previously, animals made deficient in queuine died within 18 days of withdrawing tyrosine, a nonessential amino acid, from the diet (Marks, T., and Farkas, W. R. (1997) Biochem. Biophys. Res. Commun. 230, 233-237). Here, we show that human HepG2 cells deficient in queuine and mice made deficient in queuosine-modified transfer RNA, by disruption of the tRNA guanine transglycosylase enzyme, are compromised in their ability to produce tyrosine from phenylalanine. This has similarities to the disease phenylketonuria, which arises from mutation in the enzyme phenylalanine hydroxylase or from a decrease in the supply of its cofactor tetrahydrobiopterin (BH4). Immunoblot and kinetic analysis of liver from tRNA guanine transglycosylase-deficient animals indicates normal expression and activity of phenylalanine hydroxylase. By contrast, BH4 levels are significantly decreased in the plasma, and both plasma and urine show a clear elevation in dihydrobiopterin, an oxidation product of BH4, despite normal activity of the salvage enzyme dihydrofolate reductase. Our data suggest that queuosine modification limits BH4 oxidation in vivo and thereby potentially impacts on numerous physiological processes in eukaryotes.

Cooperative and directional folding of the preQ1 riboswitch aptamer domain.

Riboswitches are cis-acting RNA fragments that regulate gene expression by sensing cellular levels of the associated small metabolites. In bacteria, the class I preQ(1) riboswitch allows the fine-tuning of queuosine biosynthesis in response to the intracellular concentration of the queuosine anabolic intermediate preQ(1). When binding preQ(1), the aptamer domain undergoes a significant degree of secondary and tertiary structural rearrangement and folds into an H-type pseudoknot. Conformational "switching" of the riboswitch aptamer domain upon recognizing its cognate metabolite plays a key role in the regulatory mechanism of the preQ(1) riboswitch. We investigate the folding mechanism of the preQ(1) riboswitch aptamer domain using all-atom Go̅-model simulations. The folding pathway of such a single domain is found to be cooperative and sequentially coordinated, as the folding proceeds in the 5' → 3' direction. This kinetically efficient folding mechanism suggests a fast ligand-binding response in competition with RNA elongation.

Queuosine formation in eukaryotic tRNA occurs via a mitochondria-localized heteromeric transglycosylase.

tRNA guanine transglycosylase (TGT) enzymes are responsible for the formation of queuosine in the anticodon loop (position 34) of tRNA(Asp), tRNA(Asn), tRNA(His), and tRNA(Tyr); an almost universal event in eubacterial and eukaryotic species. Despite extensive characterization of the eubacterial TGT the eukaryotic activity has remained undefined. Our search of mouse EST and cDNA data bases identified a homologue of the Escherichia coli TGT and three spliced variants of the queuine tRNA guanine transglycosylase domain containing 1 (QTRTD1) gene. QTRTD1 variant_1 (Qv1) was found to be the predominant adult form. Functional cooperativity of TGT and Qv1 was suggested by their coordinate mRNA expression in Northern blots and from their association in vivo by immunoprecipitation. Neither TGT nor Qv1 alone could complement a tgt mutation in E. coli. However, transglycosylase activity could be obtained when the proteins were combined in vitro. Confocal and immunoblot analysis suggest that TGT weakly interacts with the outer mitochondrial membrane possibly through association with Qv1, which was found to be stably associated with the organelle.

Structural Insights into riboswitch control of the biosynthesis of queuosine, a modified nucleotide found in the anticodon of tRNA.

The modified nucleotide queuosine (Q) is almost universally found in the anticodon wobble position of specific tRNAs. In many bacteria, biosynthesis of Q is modulated by a class of regulatory mRNA elements called riboswitches. The preQ(1) riboswitch, found in the 5'UTR of bacterial genes involved in synthesis of the Q precursors preQ(0) and preQ(1), contains the smallest known aptamer domain. We report the solution structure of the preQ(1) riboswitch aptamer domain from Bacillus subtilis bound to preQ(1), which is a unique compact pseudoknot with three loops and two stems that encapsulates preQ(1) at the junction between the two stems. The pseudoknot only forms in the presence of preQ(1), and the 3' A-rich tail of the aptamer domain is an integral part of the pseudoknot. In the absence of preQ(1), the A-rich tail forms part of the antiterminator. These structural studies provide insight into riboswitch transcriptional control of preQ(1) biosynthesis.

P-site pairing subtleties revealed by the effects of different tRNAs on programmed translational bypassing where anticodon re-pairing to mRNA is separated from dissociation.

Programmed ribosomal bypassing occurs in decoding phage T4 gene 60 mRNA. Half the ribosomes bypass a 50 nucleotide gap between codons 46 and 47. Peptidyl-tRNA dissociates from the "take-off" GGA, codon 46, and re-pairs to mRNA at a matched GGA "landing site" codon directly 5' of codon 47 where translation resumes. The system described here allows the contribution of peptidyl-tRNA re-pairing to be measured independently of dissociation. The matched GGA codons have been replaced by 62 other matched codons, giving a wide range of bypassing efficiencies. Codons with G or C in either or both of the first two codon positions yielded high levels of bypassing. The results are compared with those from a complementary study of non-programmed bypassing, where the combined effects of peptidyl-tRNA dissociation and reassociation were measured. The wild-type, GGA, matched codons are the most efficient in their gene 60 context in contrast to the relatively low value in the non-programmed bypassing study.

A minimalist glutamyl-tRNA synthetase dedicated to aminoacylation of the tRNAAsp QUC anticodon.

Escherichia coli encodes YadB, a protein displaying 34% identity with the catalytic core of glutamyl-tRNA synthetase but lacking the anticodon-binding domain. We show that YadB is a tRNA modifying enzyme that evidently glutamylates the queuosine residue, a modified nucleoside at the wobble position of the tRNA(Asp) QUC anticodon. This conclusion is supported by a variety of biochemical data and by the inability of the enzyme to glutamylate tRNA(Asp) isolated from an E.coli tRNA-guanosine transglycosylase minus strain deprived of the capacity to exchange guanosine 34 with queuosine. Structural mimicry between the tRNA(Asp) anticodon stem and the tRNA(Glu) amino acid acceptor stem in prokaryotes encoding YadB proteins indicates that the function of these tRNA modifying enzymes, which we rename glutamyl-Q tRNA(Asp) synthetases, is conserved among prokaryotes.

Yeast asparagine (Asn) tRNA without Q base promotes eukaryotic frameshifting more efficiently than mammalian Asn tRNAs with or without Q base.

In this study, we compare the efficiency of Asn tRNA from mammalian sources with and without the highly modified queuosine (Q) base in the wobble position of its anticodon and Asn tRNA from yeast, which naturally lacks Q base, to promote frameshifting. Interestingly, no differences in the ability of the two mammalian Asn tRNAs to promote frameshifting were observed, while yeast tRNA(ASn)(-Q) promoted frameshifting more efficiently than its mammalian counterparts in both rabbit reticulocyte lysates and wheat germ extracts. The shiftability of yeast Asn tRNA is therefore not due, or at least not completely, to the lack of Q base and most likely the shiftiness resides in structural differences elsewhere in the molecule. However, we cannot absolutely rule out a role of Q base in frameshifting as wheat germ extracts and a lysate depleted of most of its tRNA and supplemented with calf liver tRNA contain both Asn tRNA with or without Q base.

Determination of queuosine modification system deficiencies in cultured human cells.

Queuosine-deficient tRNAs are often observed in neoplastic cells. In order to determine possible sites for malfunction of the multistep queuosine modification system, comprehensive studies were performed on two human neoplastic cell lines, the HxGC(3) colon adenocarcinoma and the MCF-7 breast adenocarcinoma, which are 100 and 50-60% queuosine deficient, respectively. These results were compared with data obtained from normal human fibroblast (HFF) cultures which maintain 100% queuosine-modified tRNA populations. Queuine uptake in all three cell types was similar and each demonstrated activation by protein kinase C (PKC). However, incorporation of queuine into tRNA by tRNA:guanine ribosyltransferase (TGRase; E.C. 2.4.2.24) and PKC-catalyzed activation of this enzyme occurred only in HFF and MCF-7 cells. The HxGC(3) cell line exhibited no TGRase activity as was expected. Treatment with 5-azacytidine (5-azaC) induced TGRase activity to a level 20% of that in HFF and MCF-7 cells; however, this 5-azaC-induced TGRase activity was not regulated by PKC. Salvage of the queuine base from tRNA degradation products has been shown in mammalian cells and was measured in the HFF cells. However, salvage activity in the MCF-7 cell line was deficient. Therefore, it was shown by direct measurements that the HxGC(3) cell line is completely lacking in queuosine-modified tRNA due to loss of functional TGRase, while the MCF-7 cell line has an inefficient queuine salvage mechanism resulting in a significant deficiency of queuosine-modified tRNA. These techniques can be applied to any cultured cell types to determine specific lesions of the queuosine modification system, which have been suggested to be associated with neoplastic progression.

Transfer and isomerization of the ribose moiety of AdoMet during the biosynthesis of queuosine tRNAs, a new unique reaction catalyzed by the QueA protein from Escherichia coli.

The enzyme QueA of E coli is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine. The enzyme catalyzes the synthesis of an epoxycyclopentane moiety and transfers this compound to specific tRNAs containing the queuosine precursor 7-(aminomethyl)-7-deazaguanine (preQ1). S-adenosylmethionine (AdoMet) is the sole cofactor that is required for this reaction (Slany et al, 1993, Biochemistry 32, 7811-7817). To proof that the ribose moiety of AdoMet is the precursor of the epoxycyclopentane moiety, labeled AdoMet, was generated from different types of 3H ATP and methionine by the AdoMet synthetase enzyme (MetK) from E coli. The resulting 3H labeled AdoMet was directly used as the cofactor for the QueA reaction. Using [2,5', 8-3H]ATP, containing tritium at C5' of the ribose ring, resulted in an incorporation of radioactivity into preQ1 tRNA, whereas this was not the case when [2,8-3H]ATP was applied. A model for the reaction catalyzed by the S-adenosylmethionine:tRNA ribosyltransferase-isomerase QueA is proposed.