The Cellular and Molecular Dynamics of the Queuosine Modification in Transfer RNA: Definition, Modulation, Deficiencies and Effect of the Queuosine Modification System

The presence of the queuosine modification in the wobble position of tRNAasn, tRNasp, tRNAhis, and tRNAtyr is associated with a decrease in cellular growth rate, an increase in the ability to withstand environmental stress, and differentiation of pleuripotent cells into mature phenotypes. The loss of this normal modification is strongly correlated with neoplastic transformation and tumor progression of a wide variety of cancers. The normal system for formation of the queuosine modification in tRNA was studied in human fibroblast cell cultures and in mouse, rat and human liver tissues. The queuosine modification system is shown to be made up of three distinct mechanisms: uptake of the queuine base across the plasma membrane; incorporation of this base into cytoplasmic tRNA; and salvage of the base from products of normal tRNA degradation. The queuine membrane transporter and incorporation enzyme are activated via phosphorylation by protein kinase C and inactivated by the action of a phosphatase. This regulation by phosphorylation integrates the queuosine modification system into a very sensitive eukaryotic cellular switching mechanism already known to produce phenotypic alterations with strong correlations to changes in queuosine levels. A comparative study of two abnormal human adenocarcinoma cell-lines (colon and breast) was performed to assess their queuosine levels and determine the malfunctioning system step(s) for the cause of the observed deficiencies. The 100% queuosine-deficient colon tumor cell-line possessed a null mutation for the queuosine incorporation enzyme, while the 50-60% queuosine-deficient breast tumor cell-line exhibited a strong deficiency in the queuine salvage mechanism. These results demonstrate the potential for determination of even multiple sites of lesions in the modification system that would yield queuosine-deficient tRNA characteristic of tumors. Computational modeling was utilized to determine the biological function for the queuosine modification. Steric, electrostatic, and structural differences were observed for queuosine, queuosine-analogues and guanosine, the nucleosides incorporated into tRNAasp anticodon stem/loop structures, and in triad complexes of tRNAasp with mRNA and tRNAphe. The results of this research identify indistinguishable energetic parameters for complexes of queuosine-modified anticodon loops when paired with an mRNA containing cytosine- or a uridine-ending codon. However, guanosine-containing anticodon loops demonstrate much stronger energetic stability with cytosine-ending codons. The difference in codon bias is shown to be due to the restriction of anticodon loop flexibility by a queuosine-induced extended intraloop hydrogen bonding network and only minimally due to a shift in hydrogen bonding pattern produced by an intraresidue hydrogen bond. A key difference in the physiology of normal and neoplastic cells is in the increased expression of oncodevelopmental genes with respect to those housekeeping genes needed for survival. Sequence analysis of several oncodevelopmental and housekeeping transcripts suggests the presence of a contrasting bias in the usage of queuosine-related codons which end in cytosine or either cytosine or uridine, respectively. In combination with the mechanism proposed for tRNAasp decoding preferences, this codon usage bias suggests a potentially influential role for the queuosine modification system in the translational control of oncodevelopmental gene expression

Dnmt2 in RNA methylation, RNA inheritance, and environmental responses in the mouse

Dnmt2 is a highly conserved RNA methyltransferase that is responsible for cytosine-C5 methylation of C38 in the anticodon-loop of tRNAAspGUC, tRNAValAAC, and tRNAGlyGCC. This modification has been shown to contribute to tRNA integrity and function. Additionally, it was demonstrated that RNA-mediated inheritance in mice depends on the presence of Dnmt2. Since the mechanistic role of Dnmt2 in many biological processes remains elusive, the aim of this thesis was to further functionally characterize Dnmt2 in the mouse. A special focus was set on tissues and cells which had a published Dnmt2-/- phenotype. Dnmt2 characterization thus concentrated on tissues and cells involved in inheritance, i.e. sperm as the transmitting cells and early embryos as the target. Phenotypic characterization showed that relevant Dnmt2-/- tissues were morphologically indistinguishable from those of wild type mice. Sperm, testis, and embryonic tissues showed proper differentiation and tissue characteristics. However, high Dnmt2 expression in testis and sperm supported its role in inheritance. Due to Dnmt2’s known role in RNA methylation, RNAs were analysed in depth in the relevant tissues. RNA-Seq experiments illustrated that RNA expression levels in sperm, testis, and embryonic samples showed only minor differences between wild type and Dnmt2-/-. For RNAs that were further investigated, the potential differences did not appear to have an immediate effect on processes associated with these RNAs. Importantly, targeted bisulfite sequencing and Northern blots showed that Dnmt2 is responsible for tRNA methylation and stability in the male germline. Furthermore, a whole-transcriptome bisulfite sequencing approach confirmed that Dnmt2 specifically methylates tRNAAspGUC, tRNAValAAC, and tRNAGlyGCC, and most likely no other RNAs. The methylation and assurance of integrity of these tRNAs is thus suspected to play a pivotal role in RNA-mediated inheritance. For further functional characterization of Dnmt2, a screen of different physiological as well as non-physiological conditions in cell culture and live mice was carried out to define factors that regulate Dnmt2 activity. It was found that depriving cell cultures and live mice of the micronutrient queuine decreases C38 methylation of tRNAAspGUC. In conclusion, this thesis shows that Dnmt2 is a highly specific tRNA methyltransferase, which confers stability to tRNAs in sperm, and responds to environmental stimuli in mice

Modulation of Expression and Antibacterial Targeting of Shigella flexneri VirF.

Shigella flexneri is a human enteropathogen that invades the intestinal mucosa and results in severe bacillary dysentery. With the emergence of multiantibiotic-resistant strains, the development of novel therapeutics against critical S. flexneri target proteins promises a more effective treatment regimen for shigellosis. VirF is the positive regulator of transcription in the Shigella spp. virulence cascade, and ∆virF mutant strains of S. flexneri are avirulent. Although many cellular factors are associated with expression of the VirF protein (i.e., temperature, pH), previous studies demonstrated that the concentration of VirF protein is decreased by 60% in ∆vacC mutant strains of S.flexneri. VacC is tRNA-guanine transglycosylase (TGT), an RNA-modification enzyme that catalyzes the incorporation of the modified nucleoside queuosine (Q) in substrate tRNA. We have studied the VirF protein as a novel antibacterial target in Shigella flexneri, with a focus on both modulation of VirF expression by TGT and expression and activity of the VirF protein itself. We hypothesized that TGT modulates VirF protein expression through queuine modification at both the level of tRNA and the virF mRNA. We report that the modification state of tRNA with queuine and the Q-cognate codon usage (NAU versus NAC codons) in the target mRNA alters the rate of protein expression. VirF mRNA contains an overall bias of 80% for the NAU Q-cognate codons, and it is conceivable that the subtle decrease in VirF protein expression in the S. flexneri (∆vacC) mutant results from the absence of queuine-modified tRNA. In addition, we report that the virF mRNA is itself a substrate for the eubacterial TGT in vitro. Modification of RNA sequences (termed riboswitches) with small molecule metabolites has been shown to modulate either transcription or translation of the target RNA. To measure VirF activity in the presence of small molecules, we report the development of a high-throughput cell-based reporter assay using a virB operator-β-galactosidase fusion. We have screened approximately 42,000 small molecules and report the identification of 7 compounds with low to mid-micromolar IC50 values in the VirF – β-galactosidase assay. Further studies are pending to identify selective VirF inhibitors.Ph.D.Medicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/75913/1/jcutcher_1.pd

The Characterization of tRNA Modifying Enzymes S-adenosylmethionine : tRNA Ribosyltransferase-isomerase (QueA) and a novel Type I GTP cyclohydrolase

Queuosine is a hypermodified nucleoside located in the wobble position of bacterial and eukaryotic tRNAs coding for Asp, Tyr, His and Asn. The biosynthesis involves the participation of S-adenosyl-methionine:tRNA ribosyltransferase-isomerase (QueA) and a GTP Cyclohydrolase-I. QueA catalyzes the transfer and isomerization of the ribosyl moiety from AdoMet to preQ1 modified tRNA. Substrate analogs of AdoMet were used to elucidate important substrate-enzyme interactions and to test key steps in the proposed chemical mechanism. Replacing AdoMet with SeAdoMet had little effect upon substrate binding but exhibited 30-fold reduction in kcat, consistent with deprotonation at C-5\u27 as the first catalytic step. 7-deazaAdoMet failed to function as a substrate of QueA, but exhibited a Ki that was only slightly higher than the K m for AdoMet, suggesting that N-7 is critical for catalysis but not substrate binding. Neither the 2\u27- or 3\u27-deoxyAdoMet exhibited activity with QueA, however both analogs had a Ki of only 2-fold higher than the Km of AdoMet. Reported here is the identification and characterization of the COG1469 protein family as a novel Type-I GTP cyclohydrolase (GCYH) that catalyzes the conversion of GTP to 7,8-dihydroneopterin-triphosphate in ∼20% of bacteria and most archaea. The COG1469 proteins and the genes that encode them were renamed GCYH-IB and folE-2, respectively, whereas the canonical cyclohydrolase, was renamed GCYH-IA. B. subtilis and N. gonorrhoeae GCYH-IB are homotrimers that were maximally active in the presence of manganese. GDP also functioned as a substrate; however the removal of the γ-phosphate resulted in a ∼30-fold decrease in kcat/Km. Inhibition analysis with N. gonorrhoeae GCYH-IB demonstrated that 8-oxoGTP functioned as a potent inhibitor with a K145-fold lower than the Km for GTP. Although the 7-deazaGTP did not function as a substrate for GCYH-IB, it exhibited a Ki 7-fold higher than the Km for GTP. The Ki for 2\u27-deoxyGTP was 24-fold higher than the Km of GTP. H245 of N. gonorrhoeae GCYH-IB was believed to function analogous to that of H179 of E. coli FolE which facilitates the elimination of C-8 from GTP. However, H245 mutants were still able to catalyze the elimination of C-8, suggesting that H245 is not involved in the deformylation of GTP

Structural and functional characterisation of tRNA modifying enzymes

Posttranscriptional and posttranslational modifications are key regulatory mechanisms to expand biological properties of proteins and nucleic acids in living cells. A tremendous number of chemical modifications is found on all RNA species, with the highest diversity in composition and density found in transfer RNA (tRNA). Occurring all over the tRNA body, modifications increase tRNA stability, induce proper folding, and modulate translational fidelity. Non-canonical nucleobases include simple modifications as methylations and alkylations to highly complex extensions as seen in wybutosine and queuosine (Q), of which the latter requires a complete base exchange. Because mammalian tRNA molecules can harbour on average 13 modification at the same time, studying single modifications can be challenging. Especially since modifications exhibit a certain degree of “cross-talk” by influencing each other as seen for Q. Recent studies revealed a link between the hypermodified 7-deaza guanine derivative Q occurring at the wobble base position 34 (Q34) of tRNAAsp and the methylation of cytosine at position 38 (m5C38) introduced by Dnmt2. Here, deposition of m5C38 is stimulated upon prior Q34 modification with an enigmatic underlying biochemical mechanism. Thus, the aim of this study was to gain structural and biochemical insights into RNA modifying enzymes introducing (i) a methyl group to guanine at position 7 (m7G) by TrmB, (ii) methylation of cytosine at position 4 (m5C) by Dnmt2, and (iii) ribosyl transfer and isomerisation to preQ1 by QueA. m7G is found not only as the mRNA-cap structure to protect mRNA from degradation, but also in tRNAs at position 46 in the variable loop introduced by the TrmB/Trm8 enzyme family. Prior to this thesis, several crystal structures of TrmB/Trm8 enzymes have been determined, revealing differences in their biological assembly. In this thesis, the first crystal structure of the homodimeric B. subtilis TrmB in complex with the methyl group donor S-Adenosylmethionine (SAM) and post catalytic product SAH is reported. Analysis of the SAM/SAH crystal structures revealed conserved ligand binding across TrmB/Trm8 enzymes. Structural, biochemical, and computational approaches revealed a 2:2 binding stoichiometry of tRNA to protein, and resulted in the TrmB-tRNAPhe complex model in which two tRNA molecules bind to the homodimeric TrmB. Interestingly, biochemical analysis of TrmB activity at physiological SAM conditions showed a half-of-the sites reactivity, even though each monomer of TrmB is capable of tRNA and ligand binding. Subsequently, the presented biochemical and structural data give valuable insights into TrmB activity and substrate binding. The second part of the thesis focusses on the m5C writer enzyme Dnmt2. Dnmt2 substrate specificity was long enigmatic, as Dnmt2 was first identified as DNA methyltransferase (MTase), however, exhibiting weak methyltransferase activity on DNA. Since the discovery of highly specific MTase activity of Dnmt2 on tRNAAsp, a few more substrates have been identified, including tRNAGly and tRNAVal. However, only tRNAAsp harbours Q at position 34 which was identified to increase Dnmt2 methylation activity. Of the four Q34 tRNAs tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis only the latter harbours a cytosine at position 38, rendering tRNAHis a putative Dnmt2 substrate. Even though m5C38 in tRNAHis could not be observed so far, human Dnmt2 was identified to modify human tRNAHis during the course of this thesis. In contrast to prior work on S. pombe Dnmt2, stimulating effects upon Q modification could not be observed in the human context, implying different functions of Q34 in S. pombe and humans. Furthermore, Dnmt2 activity on tRNAHis was found to be highly pH-dependent rendering tRNAHis from cognate to non-cognate tRNA by shifting the pH from 8.0 to 7.4. The identification of tRNAHis as Dnmt2 substrate gives more insight into Dnmt2 substrate specificity. The S-adenosylmethionine:tRNA ribosyltransferase isomerase (QueA) was the study focus of the last part of this thesis. QueA is of special interest as (i) QueA catalyses the ribosyl-transfer in the penultimate step of Q-biosynthesis, (ii) Q is a major driving force in the virulence of Shigella bacteria, and (iii) QueA inhibition represents and interesting starting point in treating Shigella infection. Prior to this thesis incomplete QueA crystal structures have been determined. During this work, a full length QueA structure model on basis of a QueA crystal structure determined in this thesis was proposed. Investigation of this model enabled the identification of a putative SAM-binding pocket and amino acids possibly involved during catalysis. Furthermore, molecular docking experiments gave rise to a putative QueA-tRNA complex model. The proposed complex model gives valuable insight into QueA activity and provides a model for initial computer-based fragment screening in order to perform structure-based drug design to inhibit Shigella bacterial infection and treat shigellosis. Overall, this thesis provides insights into protein-RNA complexation and substrate specificity by studying complex formation by means of biochemical, structural, and computational methods. Even though, both subunits of the homodimeric TrmB are capable of binding SAM and tRNA, TrmB exhibits at physiological SAM concentration a half-of-the sites reactivity. Furthermore, a connection of enzyme activity to stress response was identified, as human Dnmt2 shows altered enzyme activity on tRNAHis depending on the used pH value. Lastly, the identification of the putative SAM-binding pocket and proposition of a QueA-tRNA complex model represents a valuable starting model for computational and experimental methods in structure-based drug design.2022-05-1

Synthesis of Tritium Labeled Queuine, PreQ1 and Related Azide Probes Toward Examining the Prevalence of Queuine.

Queuine is a modified nucleotide known to occur in the anticodon of four tRNAs. The queuine modification occurs across all eukaryotes and eubacteria with few exceptions, but its function remains unclear. Prior in vitro work demonstrated that the modification can be incorporated into RNA species other than presently known tRNAs. Queuine is unusual in that, unlike the majority of modified nucleotides that result from changes to genetically encoded bases, it is incorporated into RNA by transglycosylation. Base modification by transglycosylation is unusual and represents an interesting point of entry for study. Due to this method of incorporation the modification can be studied with small molecule probes. Tritium-labeled queuine and preQ1 were prepared to study the differences between the eukaryotic and eubacterial version of the enzyme responsible for the incorporation of queuine, tRNA guanine transglycosylase. A concise, convergent synthesis of queuine was developed that is the shortest route to date. PreQ1, the precursor to queuine incorporated by eubacteria, was used to investigate the prevalence of base modification in E. coli. Three cell lines were utilized to conduct the in vivo experiments of this study: a ΔqueC knockout of E. coli that is unable to synthesize preQ1 so that labeled compound would be incorporated exclusively, a Δtgt knockout strain of E. coli that is unable to incorporate preQ1 and a wild-type E. coli strain. As the modified nucleotide occurs through incorporation of a specially synthesized nucleotide, a study of the sites of modification by prepared probes is possible. The syntheses of two novel azide congeners were undertaken for this purpose. The evaluation of their interaction with tRNA guanine transglycosylase was undertaken to determine if they are substrates of the enzyme. The tritium labeled preQ1 allowed for a general evaluation of the prevalence. That determined the four known tRNAs are the main site of incorporation in vivo, while many other RNAs are substrates in vitro. The azide probes were generated and interact with E. coli TGT, but were not substrates. In summary, we have gained a better understanding of queuine modification of RNAs and have developed tools that will aide in future studies.Ph.D.Medicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91387/1/afb_1.pd

Analysis of Helicobacter pylori VacA-containing vacuoles and VacA intracellular trafficking

The human pathogen Helicobacter pylori colonizes half of the global population. Residing at the stomach epithelium, it contributes to the development of diseases like gastritis, duodenal and gastric ulcers, and gastric cancer. It has evolved a range of mechanisms to aid in colonization and persistence, manipulating the host immune response to avoid clearance. A major factor in this is the secreted vacuolating cytotoxin VacA which has a variety of effects on host cells. VacA is endocytosed and forms anion-selective channels in the endosome membrane, causing the compartment to swell. The resulting VacA-containing vacuoles (VCVs) can take up most of the cellular cytoplasm. Even though vacuolation is VacA's most prominent and namesake effect, the purpose of the vacuoles is still unknown. VacA exerts influence on the host immune response in various ways, both pro- and anti- inflammatorily. Most importantly, it disrupts calcium signaling in T-lymphocytes, inhibiting T-cell activation and proliferation and thereby suppressing the host immune response. Furthermore, VacA is transported to mitochondria, where it activates the mitochondrial apoptosis pathway. Within the cell, VacA has only been shown to localize to endocytic compartments/VCVs and mitochondria. Considering its diverse effects, however, the existence of other cellular sites of action seems plausible. In this study, the VCV proteome was comprehensively analyzed for the first time in order to investigate VCV function. To this end, three different strategies for VCV purification from T-cells were devised and tested. Eventually, VCVs were successfully isolated via immunomagnetic separation, using a VacA-specific primary antibody and a secondary antibody coupled to magnetic beads. The purified vacuoles were then measured by mass spectrometry, revealing not only proteins of the endocytic system, but also proteins usually localized in other cellular compartments. This apparent recruitment of proteins involved in all kinds of cellular pathways indicates a central function of VCVs in VacA intoxication effects. In a global evaluation, the VCV proteome exhibited an enrichment of proteins implicated in immune response, cell death, and cellular signaling; all of these are processes that VacA is known to influence. One of the individual proteins contained in the sample was STIM1, a calcium sensor normally residing in the endoplasmic reticulum (ER) that is important in store- operated calcium entry (SOCE). This corroborates the findings of a concurrent report, in which VacA severely influenced SOCE and colocalized with STIM1. A direct interaction of STIM1 with VacA was examined in a pull-down assay, but could be neither shown nor excluded. Immunofluorescence experiments conducted in HeLa cells confirmed the presence of VacA in the ER and also found it to traffic to the Golgi apparatus, identifying these two cellular compartments as novel VacA target structures. The exact route of VacA transport remains unclear, but the involvement of both the ER and the Golgi suggests the possibility of retrograde trafficking, analogous to other bacterial toxins like shiga and cholera toxins. In summary, the elucidation of the VCV proteome and the discovery of the ER and the Golgi apparatus as VacA target structures have generated intriguing starting points for future studies. The detection of many proteins implicated in VacA intoxication effects in the VCV proteome leads to the proposal of VCVs as signaling hubs that may coordinate the complex meshwork of VacA effects. Further investigation of individual proteins is expected to help greatly in illuminating this matter

The absence of the queuosine tRNA modification leads to pleiotropic phenotypes revealing perturbations of metal and oxidative stress homeostasis in Escherichia coli K12

Queuosine (Q) is a conserved hypermodification of the wobble base of tRNA containing GUN anticodons but the physiological consequences of Q deficiency are poorly understood in bacteria. This work combines transcriptomic, proteomic and physiological studies to characterize a Q-deficient Escherichia coli K12 MG1655 mutant. The absence of Q led to an increased resistance to nickel and cobalt, and to an increased sensitivity to cadmium, compared to the wild-type (WT) strain. Transcriptomic analysis of the WT and Q-deficient strains, grown in the presence and absence of nickel, revealed that the nickel transporter genes (nikABCDE) are downregulated in the Q- mutant, even when nickel is not added. This mutant is therefore primed to resist to high nickel levels. Downstream analysis of the transcriptomic data suggested that the absence of Q triggers an atypical oxidative stress response, confirmed by the detection of slightly elevated reactive oxygen species (ROS) levels in the mutant, increased sensitivity to hydrogen peroxide and paraquat, and a subtle growth phenotype in a strain prone to accumulation of ROS.This work was funded by the National Institute of General Medical Sciences (NIGMS) grant GM70641, by the National Institute of Environmental Health Sciences (NIEHS) grant ES002109, by the National Science Foundation (NSF) grant CHE-2002950, by the National Research Foundation of Singapore under the Singapore-MIT Alliance for Research and Technology Antimicrobial Resistance Interdisciplinary Research Group, and by Stellate Therapeutics.Peer reviewe

Active Site Chemistry of the NADPH-Dependent:7-Cyano-7-Deazaguanine (PreQ0) Nitrile Oxidoreductase, an Enzyme involved in Queuosine Biosynthesis

Queuosine (Q) is a modified nucleoside found at the wobble position of bacterial and eukaryotic transfer RNAs (tRNAs) that are specific for the amino acids tyrosine, histidine, aspartate and asparagine. A recently discovered enzyme in the biosynthetic pathway of Q, the NADPH-dependent 7-cyano-7-deazaguanine oxidoreductase (QueF), carries out the two-fold, four-electron reduction of Q precursor preQ0 to preQ1 and represents the first example of the enzymatic conversion of the nitrile functional group to an amine. Presented herein are kinetic, spectroscopic, mutational, biophysical, and isotope labeling studies directed at the elucidation of the chemical and kinetic mechanisms of this new class of protein. Steady-state kinetic analysis using a NADPH-linked continuous assay provided the kinetic parameters Km(NADPH) = 19 ± 2 μM and kcat = 0.69 ± 0.02 min-1. To determine the kinetic parameters of preQ0, a fluorescence assay that was used to follow the formation of NADP+ as an alkaline degradation product was optimized, and this method gave the kinetic constants Km (preQ0) = 0.237 ± 0.045 μM, and k cat = 0.66 ± 0.04 min-1. Titrations of enzyme with preQ0, inactivation and protection studies with iodoacetamide, and site-directed mutagenesis of a conserved cysteine residue (Cys55 in Bacillus subtilis), followed by the biochemical and biophysical analysis of the resulting protein products suggest covalent catalysis is employed by QueF, with Cys55 serving as the catalytic nucleophile to form a covalent thioimide adduct. The mechanism of hydride transfer from NADPH to preQ0 was addressed using isotope labeling studies. The data obtained from 1 H-NMR, ESI-MS and steady-state kinetic analysis of QueF suggests that the protein promotes the stereospecific transfer of the pro-R hydride of NADPH. Mutational and kinetic studies are also described for the substitution of a conserved glutamate (Glu97 in B. subtilis). As evidenced by 25-fold to 280-fold increases in Km (preQ 0) values, the results show this residue is critical for preQ0 recognition and binding. In addition, the kinetics revealed 13-fold to 20-fold decreases in kcat, suggesting substitution of Glu97 also impacts chemistry at the active site. These data provide insights into the active-site chemistry of the QueF mediated nitrile reduction and a chemical mechanism consistent with our results is proposed