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.

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.

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.

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.

Isoaccepting species differences between polysome-bound and total cellular tRNA in SVT2 cells.

Reversed-phase chromatographic comparisons of total cellular tRNAs with tRNAs isolated from a polysome enriched cell fraction establish significant enrichments for specific isoaccepting species of tRNAIle, tRNALeu, tRNALys, tRNAMet and possibly tRNAA la. Similar comparisons of tRNAAsn, tRNAAsp, tRNAHis, tRNAPhe, tRNASer and tRNATyr have been performed as well; however, no prominent differences were observed.

Alterations in SVT2 cell transfer RNAs in response to cell density and serum type.

The chromatographic elution profiles of tRNA-A sn, tRNA-A sp, tRNA-H is and tRNA-T y r from SV40-transformed BALB-3T3 cells grown in fetal calf serum or cald serum-supplemented media have been examined. The relative proportions of certain of the isoaccepting species of these four tRNAs are altered in a similar fashion depending on the serum type. It is suggested that the elution profile alterations reflect the extent of modifications of a specific G residue to the minor nucleoside Q, and that this process differs between untransformed and transformed cells. In addition, cell density appears to influence the Q content of these tRNAs, though other density-dependent tRNA modifications also appear to occur.

Relation of cell type and cell density to the degree of post-transcriptional modification of tRNALys and tRNAPhe.

An examination of the reversed-phase chromatographic profiles of tRNALys and tRNAPhe from SV40-transformed BALB/3T3 cells grown to different cell densities, untransformed BALB/3T3 cells grown to confluency and BALB/c mouse liver indicates that with increasing cell density in culture the degree of the peroxy-Y modification in tRNAPhe and an undetermined modification in tRNALys become more like that of differentiated tissue (liver). Because precursor/product relationships appear to exist among the unmodified and modified forms of the isoaccepting species for each of these tRNAs, the present findings support the view that the often reported differences in tRNA isoaccepting spectra result primarily from differences in post-transcriptional modifications, rather than from different tRNA transcripts.

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.

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.

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.

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.

Relation of cell type and cell density in tissue culture to the isoaccepting spectra of the nucleoside Q containing tRNAs: tRNATyr, tRNAHis, tRNAAsn and tRNAAsp.

An examination, using reversed-phase chromatography and cyanogen bromide treatment, of tRNATyr, tRNAHis, tRNAAsn, and tRNAAsp from SV40-transformed mouse fibroblasts grown to different cell densities, untransformed cells grown to confluence, and mouse liver indicates that: (1) The tissue cultured mouse fibroblasts examined here are hypomodified with respect to nucleoside Q, while liver tRNA is almost completely modified with respect to Q. (2) Cell density and/or proliferative state do not present as major variables in controlling the expression of Q in the present system. (3) SV40 virus transformation is not a major variable controlling the expression of Q in the present system. The present results support previous use of cyanogen bromide effected shifts in chromatographic elution as an assay for nucleoside Q.

Inhibition of nucleoside Q formation in transfer ribonucleic acid during methionine starvation of relaxed-control Escherichia coli.

The elution profiles of Asp-tRNA from unstarved and starved cultures of a relaxed-control (Rel-) strain of Escherichia coli were compared by reversed-phase chromatography. Methionine starvation results in the appearance of several additional species of Asp-tRNA which are not observed with starvation for leucine or histidine. By the criterion of cyanogen bromide-effected shifts in chromatographic elution position, a large portion of the tRNAAsp synthesized in methionine-starved cells lacks the normal Q nucleoside. By the same criterion, virtually all of the tRNAAsp from unstarved, leucine-starved, and histidine-starved cells contain Q. We conclude that methionine starvation prevents the formation of the norma Q nucleoside in Rel- E. coli.

A factor in serum and amniotic fluid is a substrate for the tRNA-modifying enzyme tRNA-guanine transferase.

Q factor, a substance found in animal serum that enables cultured mammalian cells (L-M) to produce tRNA containing queuine (the base of "nucleoside Q", queuosine), has been purified to homogeneity from bovine amniotic fluid. Q factor causes the appearance of Q-containing tRNAAsp in the L-M cells cultivated in serum-free medium, and this was used as an assay to monitor the purification of Q factor. Q factor is a competitive inhibitor of guanine for rabbit reticulocyte tRNA-guanine trnsferase, with a K1 of 4.5 x 10(-8) M. Q factor is inactivated in both the L-M cell and tRNA-guanine transferase assays by treatment with periodate or cyanogen bromide, both of which react with queuine. In L-M cells, nearly complete conversion of Q-free to Q-containing tRNAAsp is observed within 24 hr after addition of pure Q factor to the medium; actinomycin D, cycloheximide, and cycloleucine, inhibitors of RNA synthesis, protein synthesis, and nucleic acid methylation, respectively, do not inhibit this conversion. The product of the reaction, catalyzed by pure rabbit reticulocyte tRNA-guanine transferase, between Q factor and rabbit reticulocyte Q-free tRNAHis is chromatographyically indistinguishable from Q-containing tRNAHis.

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).

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.

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.