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• W2120221796 abstract "tRNA guanine transglycosylase (TGT) enzymes are responsible for the formation of queuosine in the anticodon loop (position 34) of tRNAAsp, tRNAAsn, tRNAHis, and tRNATyr; 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. tRNA guanine transglycosylase (TGT) enzymes are responsible for the formation of queuosine in the anticodon loop (position 34) of tRNAAsp, tRNAAsn, tRNAHis, and tRNATyr; 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. Queuosine (Q 3The abbreviations used are: QqueuosineTGTtRNA guanine transglycosylaseQv1queuine tRNA guanine transglycosylase domain containing 1 variant 1preQ17-aminomethyl-7-deazaguaninefTGTfull-length mouse TGTtTGTtruncated mouse TGTHAhemagglutininMOPS4-morpholinepropanesulfonic acidDAPI4′,6-diamidino-2-phenylindoleESTexpressed sequence tagGSTglutathione S-transferaseRT-PCRreverse transcription-PCR. 3The abbreviations used are: QqueuosineTGTtRNA guanine transglycosylaseQv1queuine tRNA guanine transglycosylase domain containing 1 variant 1preQ17-aminomethyl-7-deazaguaninefTGTfull-length mouse TGTtTGTtruncated mouse TGTHAhemagglutininMOPS4-morpholinepropanesulfonic acidDAPI4′,6-diamidino-2-phenylindoleESTexpressed sequence tagGSTglutathione S-transferaseRT-PCRreverse transcription-PCR.; (7-{[(4,5-cis-dihydroxy-2-cyclo-penten-1-yl)-amino]methyl}-7-deazaguanosine) is a modified 7-deazaguanosine molecule found at the wobble position of transfer RNA that contains a GUN anticodon sequence: tRNATyr, tRNAAsn, tRNAHis, and tRNAAsp (1.Nishimura S. Prog. Nucleic Acid Res. Mol. Biol. 1983; 28: 49-73Crossref PubMed Scopus (122) Google Scholar). The Q-modification is widely distributed in nature in the tRNA of eubacteria, plants, and animals; a notable exception being yeast and plant leaf cells (2.Kasai H. Oashi Z. Harada F. Nishimura S. Oppenheimer N.J. Crain P.F. Liehr J.G. von Minden D.L. McCloskey J.A. Biochemistry. 1975; 14: 4198-4208Crossref PubMed Scopus (193) Google Scholar, 3.Katze J.R. Basile B. McCloskey J.A. Science. 1982; 216: 55-56Crossref PubMed Scopus (87) Google Scholar). Interestingly, Q-modification has also been detected in aspartyl tRNA from mitochondria of rat (4.Randerath E. Agrawal H.P. Randerath K. Cancer Res. 1984; 44: 1167-1171PubMed Google Scholar) and opossum (5.Mörl M. Dörner M. Pääbo S. Nucleic Acids Res. 1995; 23: 3380-3384Crossref PubMed Scopus (57) Google Scholar). In most eukaryotes, the Q molecule can be further modified by the addition of a mannosyl group to Q-tRNAAsp and a galactosyl group to Q-tRNATyr (1.Nishimura S. Prog. Nucleic Acid Res. Mol. Biol. 1983; 28: 49-73Crossref PubMed Scopus (122) Google Scholar). queuosine tRNA guanine transglycosylase queuine tRNA guanine transglycosylase domain containing 1 variant 1 7-aminomethyl-7-deazaguanine full-length mouse TGT truncated mouse TGT hemagglutinin 4-morpholinepropanesulfonic acid 4′,6-diamidino-2-phenylindole expressed sequence tag glutathione S-transferase reverse transcription-PCR. queuosine tRNA guanine transglycosylase queuine tRNA guanine transglycosylase domain containing 1 variant 1 7-aminomethyl-7-deazaguanine full-length mouse TGT truncated mouse TGT hemagglutinin 4-morpholinepropanesulfonic acid 4′,6-diamidino-2-phenylindole expressed sequence tag glutathione S-transferase reverse transcription-PCR. Eubacteria are unique in their ability to synthesize Q. As part of this biosynthetic process, the eubacterial tRNA guanine transglycosylase (TGT) enzyme inserts the Q precursor molecule, 7-aminomethyl-7-deazaguanine (preQ1) into tRNA, which is then converted to Q by two further enzymatic steps at the tRNA level (6.Iwata-Reuyl D. Bioorg. Chem. 2003; 31: 24-43Crossref PubMed Scopus (107) Google Scholar). Eukaryotes by contrast salvage queuosine from food and enteric bacteria either as the free base (referred to as queuine) or as queuosine 5′-phosphate subsequent to normal tRNA turnover (7.Gündüz U. Katze J.R. J. Biol. Chem. 1984; 259: 1110-1113Abstract Full Text PDF PubMed Google Scholar). A Q-related molecule, archaeosine, is found at position 15 of the D loop of most archaeal tRNA, where it functions to stabilize the tRNA structure (8.Gregson J.M. Crain P.F. Edmonds C.G. Gupta R. Hashizume T. Phillipson D.W. McCloskey J.A. J. Biol. Chem. 1993; 268: 10076-10086Abstract Full Text PDF PubMed Google Scholar). The enzyme involved in archaeosine biosynthesis is structurally and mechanistically related to the eubacterial TGT but with adaptations necessitated by the differences imposed by its unique substrate and tRNA specificity (9.Ishitani R. Nureki O. Fukai S. Kijimoto T. Nameki N. Watanabe M. Kondo H. Sekine M. Okada N. Nishimura S. Yokoyama S. J. Mol. Biol. 2002; 318: 665-677Crossref PubMed Scopus (57) Google Scholar, 10.Stengl B. Reuter K. Klebe G. Chembiochem. 2005; 6: 1926-1939Crossref PubMed Scopus (57) Google Scholar). The crystal structure of the Zymononas mobilis (Z. mobilis) TGT has been determined and revealed the enzyme to be an irregular (β/α)8 TIM barrel with a C-terminal zinc-binding subdomain (11.Romier C. Reuter K. Suck D. Ficner R. EMBO J. 1996; 15: 2850-2857Crossref PubMed Scopus (107) Google Scholar). Insight into the residues involved in catalysis came from mutational and kinetic analysis of the recombinant Escherichia coli enzyme (12.Garcia G.A. Kittendorf J.D. Bioorg. Chem. 2005; 33: 229-251Crossref PubMed Scopus (33) Google Scholar) and from the Z. mobilis TGT structure as an RNA-bound intermediate complexed to the final preQ1-modified RNA product (13.Xie W. Liu X. Huang R.H. Nat. Struct. Biol. 2003; 10: 781-788Crossref PubMed Scopus (95) Google Scholar). This work showed the essential role of Asp-280 (Z. mobilis numbering) as the active site nucleophile. Asp-102, which was originally ascribed the role of active site nucleophile, functions as a general acid/base during catalysis (12.Garcia G.A. Kittendorf J.D. Bioorg. Chem. 2005; 33: 229-251Crossref PubMed Scopus (33) Google Scholar, 10.Stengl B. Reuter K. Klebe G. Chembiochem. 2005; 6: 1926-1939Crossref PubMed Scopus (57) Google Scholar). Although, the E. coli and Z. mobilis TGT enzymes are monomeric in solution (14.Reuter K. Ficner R. J. Bacteriol. 1995; 177: 5284-5288Crossref PubMed Google Scholar), at high protein concentrations the E. coli enzyme can oligomerize (15.Garcia G.A. Koch K.A. Chong S. J. Mol. Biol. 1993; 231: 489-497Crossref PubMed Scopus (32) Google Scholar), and structural data from the Z. mobilis TGT has shown the formation of a 2:1 complex with tRNA; a possible functional requirement for catalysis (10.Stengl B. Reuter K. Klebe G. Chembiochem. 2005; 6: 1926-1939Crossref PubMed Scopus (57) Google Scholar). In contrast to the eubacterial enzyme, which is a single protein species, purification of the eukaryotic TGT suggested that the catalytically active enzyme is a heterodimeric molecule: subunits of 60 and 43 kDa in rabbit erythrocytes (16.Howes N.K. Farkas W.R. J. Biol. Chem. 1978; 253: 9082-9087Abstract Full Text PDF PubMed Google Scholar), 66 and 32 kDa in bovine liver (17.Slany R.K. Müller S.O. Eur. J. Biochem. 1995; 230: 221-228Crossref PubMed Scopus (18) Google Scholar), 60 and 34.5 kDa in rat liver (18.Morris R.C. Brooks B.J. Eriotou P. Kelly D.F. Sagar S. Hart K.L. Elliott M.S. Nucleic Acids Res. 1995; 23: 2492-2498Crossref PubMed Scopus (18) Google Scholar), and a homodimer of two 68-kDa proteins in wheat germ (16.Howes N.K. Farkas W.R. J. Biol. Chem. 1978; 253: 9082-9087Abstract Full Text PDF PubMed Google Scholar, 19.Walden Jr., T.L. Howes N. Farkas W.R. J. Biol. Chem. 1982; 257: 13218-13222Abstract Full Text PDF PubMed Google Scholar). A partial amino acid sequence was recovered from two of these active enzyme preparations. The identity of the proteins from bovine liver (17.Slany R.K. Müller S.O. Eur. J. Biochem. 1995; 230: 221-228Crossref PubMed Scopus (18) Google Scholar) could not be assigned at the time of publication. However, our searches show that the peptides from the larger 65-kDa subunit are identical to asparaginyl tRNA synthetase, and those of the smaller 32-kDa subunit correspond to 2,4-dienoyl CoA reductase. A highly pure preparation from rabbit reticulocytes (20.Deshpande K.L. Seubert P.H. Tillman D.M. Farkas W.R. Katze J.R. Arch. Biochem. Biophys. 1996; 326: 1-7Crossref PubMed Scopus (20) Google Scholar) gave peptides with homology to the immunophilin p59, human elongation factor 2 (EF2), and a deubiquitinating enzyme, USP14. It is noteworthy that none of the peptide sequences obtained showed similarity to the eubacterial TGT. The results do suggest, however, that in eukaryotes the TGT activity could be embedded in a multisubunit complex. Most recently, Deshpande and Katze (21.Deshpande K.L. Katze J.R. Gene. 2001; 265: 205-212Crossref PubMed Scopus (10) Google Scholar) identified a cDNA clone encoding a putative TGT catalytic subunit. Cloning the cDNA into a mammalian expression plasmid reconstituted TGT activity in GC3/c1 cells, which are known to be naturally deficient in Q-containing tRNA (22.Gündüz U. Elliott M.S. Seubert P.H. Houghton J.A. Houghton P.J. Trewyn R.W. Katze J.R. Biochim. Biophys. Acta. 1992; 1139: 229-238Crossref PubMed Scopus (21) Google Scholar). In this study, we identify for the first time the composition of the eukaryotic tRNA guanine transglycosylase, reconstitute the catalytic activity in vitro, and examine the intracellular distribution of the active subunits. The E. coli TGT was PCR-amplified from genomic DNA using the primer pair ETF (5′-gcgcatatgaaatttgaactggacaccacc-3′) and ETR (5′-cacctcgagttaatcaacgttcaaaggtggtattc-3′) and cloned into the pET15b plasmid (Novagen) using NdeI and XhoI to generate the ETGT:pET15b plasmid (His tag). The cDNA clones for Qv0 (NM029128; IMAGE: 30105859) and Qv2 (BC017628; IMAGE: 4505816) were purchased from the IMAGE consortium. Primers were designed for the AUG translation start and TGA stop site of Qv0 to search for additional related proteins by reverse transcription-PCR (RT-PCR) leading to the discovery of Qv1, below. Full-length mouse TGT (fTGT) and Qv1 were reverse-transcribed from total kidney RNA of 4-week-old male mice using a fTGT-specific reverse primer FTR (5′-cacctcgagtcatgtgagcatgattcccacagag-3′) and a QTRTD1 reverse primer QR (5′-cacctcgagtgcaaacatctgtctgcaaatgagttc-3′; note the stop codon was converted to an Ala; underlined) according to the Superscript III protocol (Invitrogen) for gene-specific primers. First-strand products were separated from reaction components using a nucleotide removal kit (Qiagen). TGT and Qv1 were amplified by PCR using the aforementioned reverse primers and the forward primers FTF (5′-gacgaattcatggcggcggtaggcagcccaggttc-3′) and QF (5′-cgacatatgatgaagctgagtctcatcaaagtcg-3′), respectively. The fTGT cDNA was cloned into the EcoRI and XhoI restriction sites of pGEX6P1 (Invitrogen) to give the plasmid fTGT:pGEX6P1 (GST tag), whereas Qv1 was cloned into the NdeI and XhoI restriction sites of the pET21a plasmid (Novagen) to produce the plasmid Qv1:pET21a (His tag). A truncated version of the mouse TGT (tTGT) was also produced, missing the coding sequence for the first 16 amino acids. This sequence was amplified from the fTGT:pGEX6P1 plasmid using the primer pair tTF (5′-gaccatatgcggctggtcgctgagtgcagtc-3′) and tTR (5′-cacgtcgactcatgtgagcatgattcccacaag-3′). The PCR product was cloned into the NdeI and SalI restriction sites of the pET15b plasmid to yield tTGT:pET15b (His tag). DNA sequencing of all constructs was performed in the forward and reverse direction. The Qv1 sequence had not been previously described and was therefore deposited in the EMBL nucleotide data base under GenBankTM accession number FM985972. For the generation of mammalian expression constructs, the TGT and Qv1 cDNA were cloned into the EcoRI and XhoI sites of pcDNA3.1/Myc-His (Invitrogen), pcDNA3.1/HA (modified plasmid), pCMV.Myc, and pCMV.HA (Clontech). BL21(DE3) tgt::Kmr cells (whose tgt gene was disrupted by the insertion of a group II intron, supplemental Fig. S1) were transformed with ETGT: pET15b, fTGT:pGEX6P1, tTGT:pET15b, and Qv1:pET21a plasmids and cultured in LB medium. Induction of protein expression was performed with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside in 2xYT broth, at 18 °C overnight. His-tagged E. coli TGT, tTGT, and Qv1 proteins were purified by nickel-charged HiTrap chromatography, whereas the GST-tagged fTGT was isolated on a GSTrap FF prepacked column by cleavage of the tag using PreScission Protease (GE Healthcare) according to the manufacturer's instructions. Antisera to TGT and Qv1 were raised in New Zealand White rabbits (Harlan, UK) and purified by NAb Protein A spin columns (Pierce) according to the manufacturer's protocol. Antisera were further purified by counterselection against purified TGT and Qv1 protein bound to Ultralink Biosupport resin (Pierce). Coupling of TGT and Qv1 to resin was carried out overnight in 0.1 m MOPS, 0.6 m sodium citrate, pH 7.5, at room temperature. The resin was quenched with 3 m ethanolamine, washed with 1 m NaCl, and equilibrated with 10 mm Tris, pH 7.4. Antisera were eluted with a 10 mm Tris, pH 7.4 buffer containing 1 mm NaCl. When required, bulk tRNA was isolated from E. coli according to published protocols (23.Yang W.K. Novelli G.D. Methods Enzymol. 1971; 20: 44-55Crossref Scopus (99) Google Scholar). To assess transglycosylase activity, enzyme (2 μg) was added to 150 μl of assay buffer (50 mm Tris-HCl, pH 7.5, 20 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, and 2 units of SUPERasin (Ambion) RNase inhibitor mixture) containing 0.1 absorbance unit of Baker's yeast tRNA (Roche Applied Science) or E. coli tRNA, and 1.8 μm [8-14C]guanine. The reaction was incubated for 2 h at 37 °C before quenching with 450 μl of acid phenol:chloroform, pH 4.5 (Invitrogen) and spun for 3 min at 16,000 × g to separate aqueous and organic layers. The upper (aqueous) phase was loaded onto a 0.5-ml DEAE cellulose (Whatman) spin column and spun at 0.1 × g for 30 s. The flow-through was reloaded on the column another three times. The DEAE was then washed with 2 ml of wash buffer (20 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 200 mm NaCl). Bound tRNA was eluted with 1 ml of elution buffer (20 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 1 m NaCl). Eluate was mixed with 10 volumes of Ecoscint A (National Diagnostics) and scintillation counted. tRNA labeled with [8-14C]guanine in the wobble position of anticodon loop (tRNA*) was produced as described previously (24.Hoops G.C. Townsend L.B. Garcia G.A. Biochemistry. 1995; 34: 15381-15387Crossref PubMed Scopus (41) Google Scholar). A 150-μl reaction containing 5 absorbance units of tRNA* and 4 μg of enzyme in assay buffer was incubated at 37 °C for 2 h. Reactions were directly loaded onto a DEAE spin column and spun at 0.1 × g for 30 s at 4 °C. The column was washed with 2 ml of wash buffer. Eluate and wash were combined, added to 10 volumes Ecoscint A, and counted by scintillation. Total RNA was extracted from adult male mice and reverse-transcribed using a QTRTD1 reverse primer QSR (5′-gacgtcatctcttggttgcaaccagttg-3′) capable of amplifying all three splice variants or an 18S ribosomal subunit reverse primer 18SR (5′-tgccttccttggatgtgg-3′) according to the Superscript III protocol for gene-specific primers (Invitrogen). First-strand products were separated from reaction components using a nucleotide removal kit (Qiagen). The QTRTD1 splice variants and the 18S subunit were amplified by PCR using the aforementioned reverse primers and the forward primers QSF (5′-ccagctcacactctcatccctagca-3′) and 18SF (5′-cgtctgccctatcaactttc-3′), respectively. Total RNA was size-fractionated, transferred to nylon membrane, and probed using radioactively labeled cDNA for TGT and Qv1 retrieved from the pGEX6P1 and pET21a vectors, respectively. As a loading control, the membrane was stained with methylene blue to visualize the 18S and 28S ribosomal bands. COS-7 monkey kidney cells were from the European Collection of Cell Cultures. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 2 mml-glutamine, 1 mm sodium pyruvate, and 50 units of penicillin/streptomycin in an atmosphere of 5% CO2 at 37 °C. Cells were transfected in OptiMEM (Invitrogen) using Lipofectamine 2000 (Invitrogen) in a ratio of 2 μl to 1 μg of DNA. Total, cytoplasmic, nuclear, membrane, and mitochondrial fractions were isolated using kits (Pierce) as instructed by the manufacturer. Cells were seeded at 1 × 105 in 4-well chamber slides (BD-Falcon). Mitochondrial staining was carried out with 100 nm Mitotracker Red CMXRos (Invitrogen) for 15 min before fixation with 4% (w/v) paraformaldehyde in phosphate-buffered saline. Cells transfected with Myc- and HA-tagged plasmids were incubated with 1:500 mouse monoclonal anti-Myc antisera (Clontech) and rabbit polyclonal anti-HA antisera (Clontech), respectively. Anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 635 (Invitrogen) secondary antibody were used at 1:1000 dilution. For confocal analysis, antisera raised against TGT and Qv1 were used at 1:500 dilution before being probed with 1:1000 anti-rabbit Alexa Fluor 488 (Invitrogen). Nuclei were stained with DAPI (4′,6′-diamidino-2-phenylindole). The coverslips were mounted using Prolong Gold (Invitrogen). Images were taken using the 405-, 488-, and 633-nm laser lines on an Olympus FluoView TM FV1000 confocal laser-scanning microscope (Olympus). The E. coli TGT protein was chosen as bait sequence in protein BLAST (pblast) and translated nucleotide BLAST (tblastn) searches of the mouse nonredundant and EST data bases. Initial searches identified a mouse TGT (NM021888), transcribed from the queuine tRNA ribosyltransferase (QTRT1) gene on chromosome 9, which is equivalent to the previously identified human TGT catalytic subunit (21.Deshpande K.L. Katze J.R. Gene. 2001; 265: 205-212Crossref PubMed Scopus (10) Google Scholar). Further searches, using both the E. coli and mouse TGT proteins as bait, revealed two additional related mouse members of the family (NM029128 and BC017628), and a third was subsequently identified in male mouse kidney (FM985972) by RT-PCR (see “Experimental Procedures”). The three latter proteins arise from alternative spliced transcripts of the queuine tRNA ribosyltransferase domain containing 1 (QTRTD1) gene, which is composed of nine exons and is transcribed from the minus strand of chromosome 16 (supplemental Fig. S2). In accordance with standard convention (25.Eppig J.T. Blake J.A. Bult C.J. Kadin J.A. Richardson J.E. Nucleic Acids Res. 2007; 35: D630-D637Crossref PubMed Scopus (88) Google Scholar), the three spliced products have been assigned the names QTRTD1 variant_0 (Qv0), which is missing exon 5, QTRTD1 variant_1 (Qv1), which contains all nine exons, and QTRTD1 variant_2 (Qv2), which in addition to containing all nine exons has retained one-third of intron 6, exploiting a later downstream cryptic donor site to engage with exon 7. The three QTRTD1 splice variants were also identifiable within the Alternative Splicing Data Base (ASD, 26.Thanaraj T.A. Stamm S. Clark F. Riethoven J.J. Le Texier V. Muilu J. Nucleic Acids Res. 2004; 32: D64-D69Crossref PubMed Google Scholar), although Qv1 was present only as a 5′ and 3′ partial clone. Interestingly, four clones in the ASD reveal the existence of exonic isoforms for exon 2 because of alternative splice donor events. In all cases, the extension of exon 2 generates a truncated peptide of only 68 amino acids. These are unlikely to be functional and are not considered further. Alignment of the mouse and Z. mobilis TGT protein sequences (Fig. 1) revealed a high degree of amino acid conservation (42% identity), and it is therefore expected that the eukaryotic TGT adopts a similar (β/α)8 fold (13.Xie W. Liu X. Huang R.H. Nat. Struct. Biol. 2003; 10: 781-788Crossref PubMed Scopus (95) Google Scholar). It is notable from the alignment that the residues involved in catalysis (residue numbering adopted from the Z. mobilis TGT) are highly conserved; the two essential active site aspartates (Asp-102, Asp-280), residues involved in substrate recognition (Ser-103, Asp-156, Gln-203, Gly-230, Leu-231), residues involved in tRNA recognition (Arg-286, Arg-289), and residues responsible for coordinating the zinc ion (Cys-318, Cys-320, Cys-323, and His-349). The three QTRTD1 splice variants Qv0, Qv1, and Qv2 generate protein products of 345, 416, and 271 amino acids, respectively. Aligning the QTRTD1 splice variants with the TGT proteins shows a low overall degree of conservation (Fig. 1). For example, mouse TGT and Qv1 share 23% sequence identity and 41% homology. The potential secondary structure of the QTRTD1 variants was examined using the computer application Porter (27.Pollastri G. McLysaght A. Bioinformatics. 2005; 21: 1719-1720Crossref PubMed Scopus (380) Google Scholar), the results of which are shown below the alignment blocks. Manual adjustments were made to conserve the positioning of the secondary structure elements with respect to the Z. mobilis TGT. It is highly probable given the conserved alternating pattern of secondary elements that the mammalian QTRTD1 variants share a similar (β/α)8 structure to the TGT proteins. Assuming the fidelity of our alignment, the residues known to be important for catalysis in the TGT proteins are not conserved in the N-terminal two-thirds of the QTRTD1 proteins. However, there is a distinct conservation of residues in the C-terminal subdomain involved in zinc binding (Cys-318, Cys-320, Cys-323, and His-349) and a conserved substitution of the active site nucleophile Asp-280 to a glutamic acid residue. A further interesting feature of the alignment is the almost complete conservation of residues in the C terminus of Qv1 and Qv2 with an established role in dimer formation in the Z. mobilis TGT protein (Trp/His-326, Ala-329, Tyr-330, His-333, Leu-334, Glu-339, Leu-341, Leu-345) and a reciprocal presence of dimer-forming residues in the N-terminal β1-α1 region (Ala-49, Thr-50, Lys-52, Leu-74, Pro-78, Phe-92) of mouse TGT (10.Stengl B. Reuter K. Klebe G. Chembiochem. 2005; 6: 1926-1939Crossref PubMed Scopus (57) Google Scholar). The splicing pattern of the QTRTD1 gene gives rise to significant insertions and deletions. The Qv0 sequence is missing a 71-amino acid segment (Lys-120 to Val-191) relative to Qv1 and Qv2. The purpose of this region is at present unclear. However, it is intriguing that it contains a consensus leucine-rich nuclear export signal (Fig. 1, boxed region, SLLFLDSCLRLQEES), which not only adheres to the accepted form Φ-X2–3-Φ-X2–3-Φ-X-Φ (Φ = L, I, V, F, M; X is any amino acid), but the region is rich in glutamate, aspartate, and serine and occurs at the end of a putative α-helix at the border of secondary structure elements; features typical of a leucine-rich export signal (28.la Cour T. Kiemer L. Mølgaard A. Gupta R. Skriver K. Brunak S. Protein Eng. Des. Sel. 2004; 17: 527-536Crossref PubMed Scopus (621) Google Scholar, 29.Kutay U. Güttinger S. Trends Cell Biol. 2005; 15: 121-124Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). An insertion of 41 amino acids in Qv0 and Qv1 is seen relative to the TGT proteins (at Asp-297). This occurs within the zinc-binding subdomain and immediately C-terminal to the last helix (α12 in Z. mobilis TGT) of the (β/α)8 motif. Qv2 is notable in that although it has the longest coding sequence its transcript produces the shortest protein because of the presence of an early stop site, consequently leading to a complete loss of the terminal zinc-binding domain. The expression pattern of splice variants is often specific to a particular developmental stage, tissue, or disease state. To determine the expression pattern of the QTRTD1 splice variants in adult mouse, primers were designed to exon 2 and exon 7, which could amplify all three splice variants, yielding amplicons of 551 bp, 757 bp, and 1.6 kb for Qv0, Qv1, and Qv2, respectively (Fig. 2A). It was observed that the Qv1 transcript is present in adult mouse brain, heart, kidney, liver, lung, skeletal muscle, spleen, and testis (upper panel). By contrast, no product could be detected for Qv0 or Qv2. Amplification from the 18S ribosomal subunit (125 bp) acted as a loading control (lower panel). We next examined the mRNA expression of TGT and Qv1 in mouse by Northern blot analysis (Fig. 2B). The message for both proteins was found to exist in all tissues examined, giving a hybridization band at 1405 bp for TGT (upper panel) and 3125 bp for Qv1 (middle panel), which reasonably correlates with the predicted sizes of 1334 bp and 3031 bp for the TGT and Qv1 cDNA sequences, respectively. In the case of Qv1 a second fainter hybridizing band, migrating above the main species, was observable in all tissues with the exception of testis. Unexpectedly, the blot analysis revealed a correlation between the relative tissue expression levels of the TGT and Qv1 transcripts. These data indicate that the transcription of both proteins may be coordinately regulated, and by extension that the proteins may function in a cooperative manner. A search of eukaryotic species for the expression of TGT and Qv1 demonstrated that both proteins are invariably found together (results not shown), further hinting their cooperative function. The only eukaryotic species found not to contain a Qv1 homologue was the Baker's yeast, Saccharomyces cerevisiae. Curiously, Baker's yeast is also the only eukaryote from which the TGT gene was absent. The observation that the transcript levels of TGT and Qv1 correlate in mouse tissue and that the proteins are found together in eukaryotes led us to hypothesize that they may account for the heterodimeric tRNA guanine transglycosylase activity observed from protein purification studies. Constructs that add an N- or C-terminal Myc or HA epitope were developed for TGT and Qv1. Initial transient transfection studies showed that the N-terminal tags interfered with either the expression or the stability of the TGT protein and, therefore, could not be used. This was not the case with the Qv1 constructs. To examine the ability of the proteins to self-associate or co-associate, combinations of N- and C-terminally tagged forms of both proteins were transiently transfected into COS-7 cells, and lysates were immunoprecipitated using HA-specific monoclonal antibody (Fig. 3A). Our results showed that TGT protein, which had been tagged by a Myc epitope (TGT-Myc) or HA epitope (TGT-HA) on the C terminus, is unable to self-associate (left hand upper panel, lane 1). A weak self-association was, however, observed for Qv1 (middle upper panel, lane 1) that had been tagged on the C terminus with a Myc epitope (Qv1-Myc) and on the N terminus with an HA epitope (HA-Qv1). By contrast, an intense signal, indicative of strong interaction, was observed by immunoprecipitation of the TGT-Myc and HA-Qv1 proteins (right hand upper panel, lane 1). Confocal microscopy of TGT-Myc and HA-Qv1 showed similar intracellular localization (Fig. 3B). A conspicuous feature of the confocal image is that the TGT-Myc (upper panel) and HA-Qv1 (middle panel) proteins are absent from the nucleus, as visualized by DAPI stain (lower panel). These data support the possibility that these proteins interact, as suggested earlier by the co-immunoprecipitation experiments. There are no reports describing the successful overexpression of eukaryotic TGT in E. coli. Indeed, it has been stated that the cDNA for the human TGT catalytic subunit is toxic to E. coli (21.Deshpande K.L. Katze J.R. Gene. 2001; 265: 205-212Crossref PubMed Scopus (10) Google Scholar), and it has been shown that expression of E. coli TGT in a naturally Q-deficient E. coli stain, B105, results in loss of viability (30.Dineshkumar T.K. Thanedar S. Subbulakshmi C. Varshney U. Microbiology. 2002; 148: 3779-3787Crossref PubMed Scopus (12) Google Scholar). Despite these reservations, we developed a strain of E. coli (BL21 (DE3) tgt::Kmr) that are deficient in Q to overproduce the mouse TGT and Qv1 proteins; thereby negati" @default.
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• W2120221796 cites W1541728962 @default.
• W2120221796 cites W1562507949 @default.
• W2120221796 cites W1587615515 @default.
• W2120221796 cites W1587840460 @default.
• W2120221796 cites W1603916956 @default.
• W2120221796 cites W1644684322 @default.
• W2120221796 cites W1802135738 @default.
• W2120221796 cites W1816974754 @default.
• W2120221796 cites W1966026670 @default.
• W2120221796 cites W1967650372 @default.
• W2120221796 cites W1969096185 @default.
• W2120221796 cites W1978077199 @default.
• W2120221796 cites W1979416766 @default.
• W2120221796 cites W1986684758 @default.
• W2120221796 cites W1996320649 @default.
• W2120221796 cites W2001629378 @default.
• W2120221796 cites W2017710483 @default.
• W2120221796 cites W2017994420 @default.
• W2120221796 cites W2039442850 @default.
• W2120221796 cites W2042203338 @default.
• W2120221796 cites W2046192291 @default.
• W2120221796 cites W2051077894 @default.
• W2120221796 cites W2052933178 @default.
• W2120221796 cites W2057306271 @default.
• W2120221796 cites W2059081846 @default.
• W2120221796 cites W2060379792 @default.
• W2120221796 cites W2062120671 @default.
• W2120221796 cites W2077956480 @default.
• W2120221796 cites W2091244719 @default.
• W2120221796 cites W2091540783 @default.
• W2120221796 cites W2119483030 @default.
• W2120221796 cites W2136495792 @default.
• W2120221796 cites W2137424160 @default.
• W2120221796 cites W2142950965 @default.
• W2120221796 cites W2144781426 @default.
• W2120221796 cites W2158443699 @default.
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