Characterization of cDNA encoding the human tRNA-guanine transglycosylase (TGT) catalytic subunit.

Queuosine (Q) is a 7-deazaguanosine found in the first position of the anticodon of tRNAs that recognize NAU and NAC codons (Tyr, Asn, Asp and His). Eukaryotes synthesize Q by the base-for-base exchange of queuine (Q base) for guanine in the unmodified tRNA, a reaction catalyzed by TGT. A search of the human EST database for sequences with significant homology to the well studied TGT from Escherichia coli identified several candidates for full-length (1.3-1.4 kb) cDNA clones. Three candidate cDNA clones, available from IMAGE Consortium, LLNL, (Lennon et al., 1996, Genomics 33, 151-152) were obtained: IMAGE Clone Id Nos. 611146, 1422928, and 72154. Here we report the complete sequences of these clones. IMAGE:72154 contains an ORF encoding a 44 kDa polypeptide with high homology to bacterial TGTs and was subcloned into the mammalian expression vector pMAMneo-Cat. When this construct was transfected into the TGT-negative cell line, GC(3)/c1 (Gündüz et al., 1992, Biochim. Biophys. Acta 1139, 229-238), it restored the ability of the cells to form Q-containing tRNA. This TGT cDNA sequence is encoded in human chromosome 19 clone CTC-539A10 (GenBank accession no. AC011475), enabling determination of the exon-intron boundaries for the TGT gene. The sequence of IMAGE:611146 is 5'-truncated by 76 bp compared to that from IMAGE:72154 and, except for two differences in the 3'-non-coding region, the remainder of the sequence is identical to that of IMAGE:72154. IMAGE:1422928 is a 1390 bp chimera: the 5'-portion, bp 1-708, is identical to a genomic DNA sequence from chromosome 15 (GenBank accession no. AC067805, bp 148976-149683); the 3'-end, bp 726-1390, is identical to the 3'-end of the TGT cDNA sequence from IMAGE:611146.

Biosynthesis of archaeosine, a novel derivative of 7-deazaguanosine specific to archaeal tRNA, proceeds via a pathway involving base replacement on the tRNA polynucleotide chain.

Archaeosine is a novel derivative of 7-deazaguanosine found in transfer RNAs of most organisms exclusively in the archaeal phylogenetic lineage and is present in the D-loop at position 15. We show that this modification is formed by a posttranscriptional base replacement reaction, catalyzed by a new tRNA-guanine transglycosylase (TGT), which has been isolated from Haloferax volcanii and purified nearly to homogeneity. The molecular weight of the enzyme was estimated to be 78 kDa by SDS-gel electrophoresis. The enzyme can insert free 7-cyano-7-deazaguanine (preQ0 base) in vitro at position 15 of an H. volcanii tRNA T7 transcript, replacing the guanine originally located at that position without breakage of the phosphodiester backbone. Since archaeosine base and 7-aminomethyl-7-deazaguanine (preQ1 base) were not incorporated into tRNA by this enzyme, preQ0 base appears to be the actual substrate for the TGT of H. volcanii, a conclusion supported by characterization of preQ0 base in an acid-soluble extract of H. volcanii cells. Thus, this novel TGT in H. volcanii is a key enzyme for the biosynthetic pathway leading to archaeosine in archaeal tRNAs.

Cloning and characterization of cDNA encoding the rabbit tRNA-guanine transglycosylase 60-kilodalton subunit.

Eukaryotes synthesize queuosine (nucleoside Q) by the irreversible base-for-base exchange of queuine (Q base) for guanine at tRNA position 34, a reaction catalyzed by tRNA-guanine transglycosylase (TGT). The physiological role of Q remains unknown but the tRNA of tumor cells often is undermodified with respect to Q. Toward an understanding of the function of Q in normal and neoplastic cells we have isolated and characterized the cDNA for rabbit TGT. Rabbit erythrocyte TGT was reported previously to be a dimer of 60- and 43-kDa subunits (N. K. Howes and W. R. Farkas, 1978, J. Biol. Chem. 253, 9082-9078). Here we present the cDNA sequence for the apparent 60-kDa subunit; it contains an open reading frame encoding a 493-residue protein. The rabbit TGT 60-kDa subunit shares significant sequence similarity with the deubiquitinating enzyme family (F. R. Papa and M. Hochstrasser, 1993, nature 366, 313-319), especially with sequence elements that include conserved Cys and His residues.

Absence of tRNA-guanine transglycosylase in a human colon adenocarcinoma cell line.

Queuosine (Q), found exclusively in the first position of the anticodons of tRNA(Asp), tRNA(Asn), tRNA(His) and tRNA(Tyr), is synthesized in eucaryotes by a base-for-base exchange of queuine, the base of Q, for guanine at tRNA position 34. This reaction is catalyzed by the enzyme tRNA-guanine transglycosylase (EC 2.4.2.29). We measured the specific release of queuine from Q-5'-phosphate (queuine salvage) and the extent of tRNA Q modification in 6 human tumors carried as xenografts in immune-deprived mice. Q-deficient tRNA was found in 3 of the tumors but it did not correlate with diminished queuine salvage. The low tRNA Q content of one tumor, the HxGC3 colon adenocarcinoma, prompted us to examine a HxGC3-derived cell line, GC3/M. GC3/M completely lacks Q in its tRNA and measurable tRNA-guanine transglycosylase activity; the first example of a higher eucaryotic cell which lacks this enzyme. Exposure of GC3/M cells to 5-azacytidine induces the transient appearance of Q-positive tRNA. This result suggests that at least one allele of the transglycosylase gene in GC3/M cells may have been inactivated by DNA methylation. In clinical samples, we found Q-deficient tRNA in 10 of 46 solid tumors, including 2 of 13 colonic carcinomas.

Queuine is incorporated into brain transfer RNA.

Pig brain tRNA was assayed for the presence of queuosine in the first position of the anticodon for each of the Q-family of tRNAs (aspartyl, asparaginyl, histidyl and tyrosyl). The brain tRNA was aminoacylated with each of the four amino acids and the aminoacylated tRNA's analyzed by RPC-5 chromatography. The results of this study show that for all four tRNAs of the family, queuine is substituted for guanine in virtually 100% of the anticodons. Therefore, it can be concluded that queuine is able to cross the blood-brain barrier and that brain contains guanine-queuine tRNA transglycosylase, the enzyme responsible for the excision of guanine from the original transcripts of these tRNAs and insertion of queuine. The determination of whether the tRNA contained queuine was made from the elution profile of the RPC-5 chromatograms and the results confirmed by a change in the RPC-5 elution profile when the tRNAs were reacted with BrCN or NaIO4.

Novel salvage of queuine from queuosine and absence of queuine synthesis in Chlorella pyrenoidosa and Chlamydomonas reinhardtii.

Partially purified extracts from Chlorella pyrenoidosa and Chlamydomonas reinhardtii catalyze the cleavage of queuosine (Q), a modified 7-deazaguanine nucleoside found exclusively in the first position of the anticodon of certain tRNAs, to queuine, the base of Q. This is the first report of an enzyme that specifically cleaves a 7-deazapurine riboside. Guanosine is not a substrate for this activity, nor is the epoxide a derivative of Q. We also establish that both algae can incorporate exogenously supplied queuine into their tRNA but lack Q-containing tRNA when cultivated in the absence of queuine, indicating that they are unable to synthesize Q de novo. Although no physiological function for Q has been identified in these algae, Q cleavage to queuine would enable algae to generate queuine from exogenous Q in the wild and also to salvage (and recycle) queuine from intracellular tRNA degraded during the normal turnover process. In mammalian cells, queuine salvage occurs by the specific cleavage of queuine from Q-5'-phosphate. The present data also support the hypothesis that plants, like animals, cannot synthesize Q de novo.

Identifying inhibitors of queuine modification of tRNA in cultured cells.

Altered queuine modification of tRNA has been associated with cellular development, differentiation, and neoplastic transformation. Present methods of evaluating agents for their ability to induce queuine hypomodification of tRNA are tedious, time-consuming, and not readily amenable to examining cell-type or tissue specificity. Therefore, a rapid, small-scale assay was developed to identify agents that alter queuine modification of tRNA in cultured cells. Monolayer cultures (2cm2) of Chinese hamster embryo cells depleted of queuine for 24 h were evaluated for their ability to incorporate [3H]dihydroqueuine into acid precipitable material (tRNA) in the presence and absence of potential inhibitors. Known inhibitors of the queuine modification enzyme tRNA-guanine ribosyltransferase (e.g., 7-methylguanine, 6-thio-guanine, and 8-azaguanine) were very effective in blocking incorporation of the radiolabel, and the dose-dependent results exhibited small standard deviations in independent experiments. The data indicate that the method is rapid, reliable, and potentially useful with a variety of cell types.

Guanine analog-induced differentiation of human promyelocytic leukemia cells and changes in queuine modification of tRNA.

Treatment of hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient human promyelocytic leukemia (HL-60) cells with 6-thioguanine results in growth inhibition and cell differentiation. 6-Thioguanine is a substrate for the tRNA modification enzyme tRNA-guanine ribosyltransferase, which normally catalyzes the exchange of queuine for guanine in position 1 of the anticodon of tRNAs for asparagine, aspartic acid, histidine, and tyrosine. During the early stages of HGPRT-deficient HL-60 cell differentiation induced by 6-thioguanine, there was a transient decrease in the queuine content of tRNA, and changes in the isoacceptor profiles of tRNA(His) indicate that 6-thioguanine was incorporated into the tRNA in place of queuine. Reversing this structural change in the tRNA anticodon by addition of excess exogenous queuine reversed the 6-thioguanine-induced growth inhibition and differentiation. Similar results were obtained when 8-azaguanine (another inhibitor of queuine modification of tRNA that can be incorporated into the anticodon) replaced 6-thioguanine as the inducing agent. The data suggest a primary role for the change in queuine modification of tRNA in mediating the differentiation of HGPRT-deficient HL-60 cells induced by guanine analogs.

Inhibition of queuine uptake in diploid human fibroblasts by phorbol-12,13-didecanoate. Requirement for a factor derived from early passage cells.

Cell cultures derived from human neonatal foreskins (HF cells) are susceptible to phorbol-12,13-didecanoate- (PDD) induced inhibition of queuine uptake, but this inhibition is pronounced only in early passage HF cells. The present analysis of five different primary cultures demonstrated that, between 10 and 30 population doublings beyond the primary cultures, HF cells gradually became refractile to PDD-induced inhibition of queuine uptake, after which PDD begins to stimulate queuine uptake. Treating late passage HF cells with conditioned medium from early passage HF cells partially restored the PDD-induced inhibition of queuine uptake. This indicates the existence of a factor produced by early passage HF cells that permits PDD to inhibit queuine uptake. The tumor promoter, teleocidin, mimics the effects of PDD on queuine uptake. Both PDD and teleocidin are known to activate protein kinase C; therefore, this kinase may be an intermediary in tumor promoter-induced effects on queuine uptake. Epidermal growth factor, platelet-derived growth factor, and transforming growth factor beta stimulated queuine uptake in both early and late passage HF cells. Growth factor stimulation of uptake was enhanced by PDD in late passage cells but inhibited by PDD in early passage cells. Polyinosinic polycytidylic acid treatment of late passage HF cells partially restored PDD-induced inhibition of queuine uptake. Human recombinant beta-interferon, plus or minus PDD, had no effect on queuine uptake. PDD did not inhibit queuine uptake in the immortal human and non-human cell lines examined.

Queuosine metabolism: possible relation to B-cell activation by C8 derivatives of guanosine.

The observed enhancement of B-lymphocyte activation by 8-bromoguanosine and 8-mercaptoguanosine is hypothesized to occur via a "binding protein" which requires a guanine nucleoside as the syn conformer for productive interaction. In addition, because of the 7-substituent, Q nucleoside also is hypothesized to bind as the syn conformer and, therefore, to be a potential B-lymphocyte activator.

Inhibition of queuine uptake in cultured human fibroblasts by phorbol-12,13-didecanoate.

The modified base queuine is inserted posttranscriptionally into the first position of the anticodon of tyrosine tRNA, histidine tRNA, asparginine tRNA, and aspartic acid tRNA. Phorbol-12,13-didecanoate (PDD) effects a decrease in the queuine content of tRNA in cultured human foreskin fibroblasts. The present data suggest that this results from a PDD-mediated inhibition of queuine uptake. Nonsaturable uptake was observed for tritiated dihydroqueuine (rQT3) for up to 2 hr at 10 to 1000 nM concentrations, while saturation of uptake was observed after 3 to 4 hr. Lineweaver-Burke analysis of concentration versus uptake revealed biphasic uptake kinetics with high and low Km components of approximately 350 and 30 nM, respectively. Competition by queuine of rQT3 uptake indicated that both compounds have equal affinity for the uptake mechanism. PDD inhibited rQT3 uptake but required 30 to 60 min of exposure before the uptake was completely blocked. The rQT3 efflux rate from cells was found to be 3 to 4 times greater than that of uptake, and PDD also inhibited the efflux reaction. The potential inhibitors furosemide, nitrobenzylthioinosine, ouabain, 7-methylguanine, 7-deazaguanine, guanine, guanosine, adenine, adenosine, hypoxanthine, and epidermal growth factor had no effect on rQT3 uptake. However, dipyridamole was immediately effective at reducing rQT3 uptake.

Relationship between a tumor promoter-induced decrease in queuine modification of transfer RNA in normal human cells and the expression of an altered cell phenotype.

With normal human skin fibroblasts in culture, a transient decrease in queuine modification of tRNA precedes a phorbol ester tumor promoter-induced 5- to 10-fold increase in saturation density. Subsequently, an increase in the queuine content of cellular tRNA (to levels comparable to those in untreated cultures) precedes a decrease in saturation density. This reversal of the phorbol ester-induced alteration in tRNA modification occurs in the continued presence of the tumor promoter, and it parallels an increased ability of the cells to salvage queuine from catabolized endogenous tRNA. Addition of exogenous queuine concurrently with the tumor promoter at early passage significantly inhibits the increase in saturation density. The results suggest a role for the decrease in queuine modification of tRNA in mediating the phenotypic change induced by the tumor promoter.

Evidence that the nucleic acid base queuine is incorporated intact into tRNA by animal cells.

Queuine (the base of queuosine, Q) catalytically reduced with tritium or deuterium yields a derivative in which the proton at C-8 (purine numbering system) has been exchanged and the cyclopentene ring has been reduced to a cyclopentane ring. Mouse fibroblast tRNA has been labeled by culturing the cells in medium supplemented with [3H]- and [2H]dihydroqueuine. Such tRNA yields, upon hydrolysis, the nucleoside dihydroqueuosine and a saccharide derivative of dihydroqueuosine. Each product has been identified unambiguously by mass spectrometry and chromatography. Both the 3H- and 2H-labeled material coeluted, and no unlabeled Q nucleoside was found. Therefore, dihydroqueuine is incorporated intact into tRNA in mammalian cells. Furthermore, fractionation of the labeled tRNA on concanavalin A-agarose, which specifically binds the mannosyl-Q-containing tRNAAsp, has shown that the dihydroqueuosine-containing tRNAAsp is mannosylated. This is the first direct evidence that queuine is incorporated intact into mammalian tRNA in vivo.

Substrate and inhibitor specificity of tRNA-guanine ribosyltransferase.

We have tested as inhibitors or substrates of tRNA-guanine ribosyltransferase (EC 2.4.2.29) a number of compounds, including derivatives of 7-deazaguanine, pteridines, purines, pyrimidines and antimalarials. Virtually all purines and pteridines that are inhibitors or substrates of the rabbit reticulocyte enzyme have an amino nitrogen at the 2 position. In addition the 9 position and the oxygen at the 6 position may be important for recognition by the enzyme. Saturation of the double bond in the cyclopentenediol moiety of queuine reduces substrate activity and queuine analogs that lack the cyclopentenediol moiety, such as 7-deazaguanine and 7-aminomethyl-7-deazaguanine, are relatively poor substrates for the enzyme. While adenosine is not an inhibitor, neplanocin A (an adenosine analog in which a cyclopentenediol replaces the ribose moiety) is a poor inhibitor. The incorporation of 7-aminomethyl-7-deazaguanine into the tRNA of L-M cells results in a novel chromatographic form of tRNAAsp, indicating that L-M cells cannot modify this Q precursor (in Escherichia coli) to queuosine. The specific incorporation of 7-deazaguanine and 8-azaguanine into tRNA by L-M cells also results in novel chromatographic forms of tRNAAsp. With intact L-M cells, the enzyme-catalyzed insertion into tRNA of queuine, dihydroqueuine, 7-aminomethyl-7-deazaguanine, or 7-deazaguanine is irreversible, while guanine or 8-azaguanine incorporation is reversible; suggesting that it is the substitution of C-7 for N-7 which prevents the reversible incorporation of queuine into tRNA.

Queuine salvage in mammalian cells. Evidence that queuine is generated from queuosine 5'-phosphate.

Cell-free extracts of the Vero cell line (monkey kidney origin) contain an activity which converts 7-[5-[( (1S,4S,5R)-4, 5-dihydroxy-2-cyclopenten-1-yl)-amino]methyl]-7-deazaguanosine (queuosine) 5'-phosphate into queuine (the base of queuosine). We designate this the queuine salvage activity because it apparently is responsible for the ability of intact Vero cells to salvage queuosine base from tRNA degraded during the normal turnover process. Queuosine-3'-P, mannosylqueuosine-5'-P, or queuosine nucleoside does not support the queuine salvage reaction. Extracts of the L-M cell line (mouse embryo origin) lack the queuine salvage activity, a finding consistent with the inability of intact L-M cells to retrieve queuine subsequent to tRNA turnover (Gündüz, U., and Katze, J. R. (1982) Biochem. Biophys. Res. Commun. 109, 159-167).

Queuine, a modified base incorporated posttranscriptionally into eukaryotic transfer RNA: wide distribution in nature.

Queuine, a modified base found in transfer RNA, appears to be a new dietary factor because (i) previous studies have shown that mice require it for the expression of queuine-containing transfer RNA's but apparently do not synthesize it, and (ii) significant amounts of free queuine are present in common plant and animal food products.

Administration of exogenous queuine is essential for the biosynthesis of the queuosine-containing transfer RNAs in the mouse.

1. Normal mouse liver contains predominantly tRNA that contains queuosine in the first position of the anticodon ((Q+)tRNA). 2. Germ-free mice fed a chemically defined diet devoid of queuine for 1 year have no queuine in all four of the tRNAs that respond to the NAUC codons. 3. The synthesis of (Q+)tRNAs can be induced by injecting queuine, feeding free queuine, or by feeding (Q+)tRNA. 4. When mice that have no (Q+)tRNA are titrated with exogenous queuine, tRNAAsp is modified to the (Q+) state before tRNAHis.

Presence of queuine in Drosophila melanogaster: correlation of free pool with queuosine content of tRNA and effect of mutations in pteridine metabolism.

Queuine, a modified form of 7-deazaguanine present in certain transfer RNAs, is shown to occur in Drosophila melanogaster adults in a free form and its concentration varies as a function of age, nutrition and genotype. In several, but not all mutant strains, the concentrations of queuine and the Q(+) (queuine-containing) form of tRNATyr are correlated. The bioassay employs L-M cells which respond to the presence of queuine by an increase in their Q(+)tRNAAsp that is accompanied by a decrease in the Q(-)tRNAAsp isoacceptors. The increase in Q(+)tRNATyr in Drosophila that occurs on a yeast diet is accompanied by an increase in queuine. Similarly the increase of Q(+)tRNAs with age also is accompanied by an increase in free queuine. In two mutants, brown and sepia, these correlations were either diminished or failed to occur. Indeed, the extract of both mutants inhibited the response of the L-M cells to authentic queuine. When the pteridines that occur at abnormally high levels in sepia were used at 1 x 10(-6)M, the inhibition of the L-M cell assay occurred in the order biopterin greater than pterin greater than sepiapterin. These pteridines were also inhibitory for the purified guanine:tRNA transglycosylase from rabbit but the relative effectiveness then was pterin greater than biopterin greater than sepiapterin. Pterin was competitive with guanine in the enzyme reaction with Ki = 0.9 x 10(-7)M. Also when an extract of sepia was chromatographed on Sephadex G-50, the pteridine-containing fractions only were inhibitory toward the L-M cell assay or the enzyme assay. These results indicate that free queuine occurs in Drosophila but also that certain pteridines may interfere with the incorporation of queuine into RNA.