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• W2018032976 abstract "A large number of post-transcriptional base modifications in transfer RNAs have been described (Sprinzl, M., Horn, C., Brown, M., Ioudovitch, A., and Steinberg, S. (1998) Nucleic Acids Res. 26, 148-153). These modifications enhance and expand tRNA function to increase cell viability. The intermediates and genes essential for base modifications in many instances remain unclear. An example is wyebutosine (yW), a fluorescent tricyclic modification of an invariant guanosine situated on the 3′-side of the tRNAPhe anticodon. Although biosynthesis of yW involves several reaction steps, only a single pathway-specific enzyme has been identified (Kalhor, H. R., Penjwini, M., and Clarke, S. (2005) Biochem. Biophys. Res. Commun. 334, 433-440). We used comparative genomics analysis to identify a cluster of orthologous groups (COG0731) of wyosine family biosynthetic proteins. Gene knock-out and complementation studies in Saccharomyces cerevisiae established a role for YPL207w, a COG0731 ortholog that encodes an 810-amino acid polypeptide. Further analysis showed the accumulation of N1-methylguanosine (m1G37) in tRNA from cells bearing a YPL207w deletion. A similar lack of wyosine base and build-up of m1G37 is seen in certain mammalian tumor cell lines. We proposed that the 810-amino acid COG0731 polypeptide participates in converting tRNAPhe-m1G37 to tRNAPhe-yW. A large number of post-transcriptional base modifications in transfer RNAs have been described (Sprinzl, M., Horn, C., Brown, M., Ioudovitch, A., and Steinberg, S. (1998) Nucleic Acids Res. 26, 148-153). These modifications enhance and expand tRNA function to increase cell viability. The intermediates and genes essential for base modifications in many instances remain unclear. An example is wyebutosine (yW), a fluorescent tricyclic modification of an invariant guanosine situated on the 3′-side of the tRNAPhe anticodon. Although biosynthesis of yW involves several reaction steps, only a single pathway-specific enzyme has been identified (Kalhor, H. R., Penjwini, M., and Clarke, S. (2005) Biochem. Biophys. Res. Commun. 334, 433-440). We used comparative genomics analysis to identify a cluster of orthologous groups (COG0731) of wyosine family biosynthetic proteins. Gene knock-out and complementation studies in Saccharomyces cerevisiae established a role for YPL207w, a COG0731 ortholog that encodes an 810-amino acid polypeptide. Further analysis showed the accumulation of N1-methylguanosine (m1G37) in tRNA from cells bearing a YPL207w deletion. A similar lack of wyosine base and build-up of m1G37 is seen in certain mammalian tumor cell lines. We proposed that the 810-amino acid COG0731 polypeptide participates in converting tRNAPhe-m1G37 to tRNAPhe-yW. In all organisms, the functions of tRNAs in translation are enhanced by a series of post-transcriptional modifications (4Björk G.R. Ericson J.U. Gustafsson C.E. Hagervall T.G. Jonsson Y.H. Wikstrom P.M. Annu. Rev. Biochem. 1987; 56: 263-287Crossref PubMed Google Scholar). Over 80 modifications are known, and their presence vastly expands the structural and chemical diversity of native tRNA (2Grosjean H. Sprinzl M. Steinberg S. Biochimie (Paris). 1995; 77: 139-141Crossref PubMed Scopus (111) Google Scholar). (In a putative RNA world, base modifications may have provided a way to diversify the chemical and structural properties of RNAs.) The modification-dependent structural stability and function correlate with increased cellular fitness and viability (5Persson B.C. Mol. Microbiol. 1993; 8: 1011-1016Crossref PubMed Scopus (68) Google Scholar, 9Björk G.R. Durand J.M. Hagervall T.G. Leipuviene R. Lundgren H.K. Nilsson K. Chen P. Qian Q. Urbonavicius J. FEBS Lett. 1999; 452: 47-51Crossref PubMed Scopus (101) Google Scholar). The importance of these modifications is underscored by a large investment of resources for their biosynthesis, with estimates suggesting nearly 1% of some bacterial genomes being dedicated to tRNA modification genes (4Björk G.R. Ericson J.U. Gustafsson C.E. Hagervall T.G. Jonsson Y.H. Wikstrom P.M. Annu. Rev. Biochem. 1987; 56: 263-287Crossref PubMed Google Scholar).Wyebutosine (yW) 3The abbreviations used are: yWwyebutosineHPLChigh pressure liquid chromatographyLC-MSliquid chromatography-mass spectrometryRPreverse-phaseORFopen reading frameYbswyosine basesm1GN1-methylguanosinePheRSphenylalanyl-tRNA synthetaseGm. 2′O-methylguanosine 3The abbreviations used are: yWwyebutosineHPLChigh pressure liquid chromatographyLC-MSliquid chromatography-mass spectrometryRPreverse-phaseORFopen reading frameYbswyosine basesm1GN1-methylguanosinePheRSphenylalanyl-tRNA synthetaseGm. 2′O-methylguanosine of yeast phenylalanine-specific tRNA (tRNAPhe) was one of the earliest tRNA modifications to be discovered (10RajBhandary U.L. Chang S.H. J. Biol. Chem. 1968; 243: 598-608Abstract Full Text PDF PubMed Google Scholar, 11Chang S.H. RajBhandary U.L. J. Biol. Chem. 1968; 243: 592-597Abstract Full Text PDF PubMed Google Scholar, 12RajBhandary U.L. Stuart A. Chang S.H. J. Biol. Chem. 1968; 243: 584-591Abstract Full Text PDF PubMed Google Scholar, 13RajBhandary U.L. Faulkner R.D. Stuart A. J. Biol. Chem. 1968; 243: 575-583Abstract Full Text PDF PubMed Google Scholar, 14RajBhandary U.L. Stuart A. Hoskinson R.M. Khorana H.G. J. Biol. Chem. 1968; 243: 565-574Abstract Full Text PDF PubMed Google Scholar). Wyebutosine is a fluorescent, tricyclic base and a member of the wyosine family of hypermodified guanosines. All wyosine bases (Ybs) are characterized by a 1H-imidazo[1,2-α]purine core structure and a strict occurrence in archaeal and eukaryal tRNAPhe (Figs. 1 and 2). Wyosine bases, isolated from different organisms, show variations in ring methylation and side chain structure (15McCloskey J.A. Liu X.H. Crain P.F. Bruenger E. Guymon R. Hashizume T. Stetter K.O. Nucleic Acids Symp. Ser. 2000; : 267-268Crossref PubMed Scopus (21) Google Scholar, 16Zhou S. Sitaramaiah D. Noon K.R. Guymon R. Hashizume T. McCloskey J.A. Bioorg. Chem. 2004; 32: 82-91Crossref PubMed Scopus (32) Google Scholar, 17McCloskey J.A. Graham D.E. Zhou S. Crain P.F. Ibba M. Konisky J. Soll D. Olsen G.J. Nucleic Acids Res. 2001; 29: 4699-4706Crossref PubMed Scopus (106) Google Scholar, 18Noon K.R. Guymon R. Crain P.F. McCloskey J.A. Thomm M. Lim J. Cavicchioli R. J. Bacteriol. 2003; 185: 5483-5490Crossref PubMed Scopus (92) Google Scholar, 19Nakanishi K. Blobstein S. Funamizu M. Furutachi N. Van Lear G. Grunberger D. Lanks K.W. Weinstein I.B. Nat. New Biol. 1971; 234: 107-109Crossref PubMed Scopus (52) Google Scholar, 20Blobstein S.H. Grunberger D. Weinstein I.B. Nakanishi K. Biochemistry. 1973; 12: 188-193Crossref PubMed Scopus (91) Google Scholar). Generally, archaeal Yb structures are less differentiated then their eukaryotic counterparts.FIGURE 2Phylogenetic occurrence of wyosine bases in sequenced genomes. Refer to Fig. 1 for chemical structures.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The hydrophobic nature of yW37 promotes stacking with adjacent bases (A36 and A38) and restricts the conformational flexibility of the anticodon (21Stuart J.W. Koshlap K.M. Guenther R. Agris P.F. J. Mol. Biol. 2003; 334: 901-918Crossref PubMed Scopus (61) Google Scholar, 22Kan L.S. Ts'o P.O. von der Haar F. Sprinzl M. Cramer F. Biochemistry. 1975; 14: 3278-3291Crossref PubMed Scopus (30) Google Scholar, 23Maelicke A. von der Haar F. Cramer F. Biopolymers. 1973; 12: 27-43Crossref PubMed Scopus (36) Google Scholar, 24Sussman J.L. Holbrook S.R. Warrant R.W. Church G.M. Kim S.H. J. Mol. Biol. 1978; 123: 607-630Crossref PubMed Scopus (300) Google Scholar, 25Holbrook S.R. Sussman J.L. Warrant R.W. Kim S.H. J. Mol. Biol. 1978; 123: 631-660Crossref PubMed Scopus (245) Google Scholar). Removal of yW produced local changes in anticodon conformation, as well as long range perturbations in tRNAPhe tertiary structure (26Krzyzosiak W.J. Ciesiolka J. Nucleic Acids Res. 1983; 11: 6913-6921Crossref PubMed Scopus (14) Google Scholar). These structural changes were accompanied by subtle differences in codon specificity 4It should be noted that these studies are performed with tRNA lacking any base at position 37. 4It should be noted that these studies are performed with tRNA lacking any base at position 37. and a modest increase in retroviral ribosomal frameshifting (determined in cell-free extract) (27Ghosh K. Ghosh H.P. J. Biol. Chem. 1972; 247: 3369-3375Abstract Full Text PDF PubMed Google Scholar, 28Carlson B.A. Kwon S.Y. Chamorro M. Oroszlan S. Hatfield D.L. Lee B.J. Virology. 1999; 255: 2-8Crossref PubMed Scopus (61) Google Scholar, 29Carlson B.A. Mushinski J.F. Henderson D.W. Kwon S.Y. Crain P.F. Lee B.J. Hatfield D.L. Virology. 2001; 279: 130-135Crossref PubMed Scopus (29) Google Scholar). Most interesting, the tRNAPhe from mouse neuroblastoma cell lacked Yb but was more efficient than the fully modified tRNAPhe in a cell-free translation system (30Mazabraud A. FEBS Lett. 1979; 100: 235-240Crossref PubMed Scopus (7) Google Scholar, 31Smith D.W. McNamara A.L. Mushinski J.F. Hatfield D.L. J. Biol. Chem. 1985; 260: 147-151Abstract Full Text PDF PubMed Google Scholar, 32Shugart L. Exp. Gerontol. 1972; 7: 251-262Crossref PubMed Scopus (13) Google Scholar). Although it is unclear if hypomodified tRNAs contribute to tumor-specific properties, these tRNAs support the high levels of translation required by rapidly dividing cells. Thus, despite a good understanding of its role in maintaining anticodon structure, the function of yW in translation is unclear.Although the biosynthesis of wyebutosine has been partially characterized, the genes involved are largely unknown. Several structural components of yW were identified by metabolic labeling experiments. The purine substructure was shown as being derived from the coded guanosine (33Li H.J. Nakanishi K. Grunberger D. Weinstein I.B. Biochem. Biophys. Res. Commun. 1973; 55: 818-823Crossref PubMed Scopus (29) Google Scholar, 34Droogmans L. Grosjean H. EMBO J. 1987; 6: 477-483Crossref PubMed Scopus (46) Google Scholar). NMR studies of 13C-enriched tRNAPhe implicated the methyl group of methionine as a source for carbon-10 (refer to Fig. 1 for numbering), and for the side chain ester and N3-methyl moieties (35Smith C. Schmidt P.G. Petsch J. Agris P.F. Biochemistry. 1985; 24: 1434-1440Crossref PubMed Scopus (25) Google Scholar). Conflicting evidence obscures understanding the origin of the 3-amino-3-carboxypropyl side chain (36Munch H.J. Thiebe R. FEBS Lett. 1975; 51: 257-258Crossref PubMed Scopus (21) Google Scholar, 37Pergolizzi R.G. Engelhardt D.L. Grunberger D. Nucleic Acids Res. 1979; 6: 2209-2216Crossref PubMed Scopus (12) Google Scholar). The in vivo kinetics of Yb biosynthesis of Xenopus laevis were investigated (34Droogmans L. Grosjean H. EMBO J. 1987; 6: 477-483Crossref PubMed Scopus (46) Google Scholar). By using the site-specifically labeled [32P]tRNAPhe transcript in X. laevis oocytes, Droogmans and Grosjean (34Droogmans L. Grosjean H. EMBO J. 1987; 6: 477-483Crossref PubMed Scopus (46) Google Scholar) detected N1-methylguanosine (m1G) and an unknown compound “X” as intermediates, and they suggested that the pathway may involve numerous metabolites.The recent expansion of publicly available bioinformatics tools and data bases has stimulated gene identification and functional assignment. Novel approaches, which combine public genome information with genetic context, have met with success in linking unknown genes to definitive functions (38Reader J.S. Metzgar D. Schimmel P. de Crécy-Lagard V. J. Biol. Chem. 2004; 279: 6280-6285Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 39Van Lanen S.G. Reader J.S. Swairjo M.A. de Crécy-Lagard V. Lee B. Iwata-Reuyl D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4264-4269Crossref PubMed Scopus (88) Google Scholar, 40Bishop A.C. Xu J. Johnson R.C. Schimmel P. de Crécy-Lagard V. J. Biol. Chem. 2002; 277: 25090-25095Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 41Gerlt J.A. Chem. Biol. 2003; 10: 1141-1142Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 42Osterman A. Overbeek R. Curr. Opin. Chem. Biol. 2003; 7: 238-251Crossref PubMed Scopus (261) Google Scholar). In this study, our methodology produced a single “hit” that allowed us to identify a pathway-specific polypeptide-encoding gene that acts downstream from the initial modification event (m1G37). Deletion of the gene in Saccharomyces cerevisiae produces tRNAPhe in a modification state similar to that in certain mammalian cell types, including some tumor cell lines.MATERIALS AND METHODSStrains and Chemicals—Wild-type and deletion strains (ΔYPL207w) of S. cerevisiae were purchased from Open Biosystems (www.openbiosystems.com). Both strains were of the MATα leu2Δ0 met15Δ0 ura3Δ0 genotype. The YPL207w ORF was replaced with a KanR cassette in the null strain. All chemicals were obtained in high purity from Sigma unless otherwise noted.Plasmids—YPL207w was cloned from S. cerevisiae genomic DNA by PCR (30 cycles, 1 min at 94 °C, 1 min at 55 °C, and 6 min at 68 °C) using the following oligonucleotides: 5′-ggggacaagtttgtacaaaaaagcaggctatggatccaataatggatggttttcgtgtagctgg-3′ and 5′-ctccctcctattcctgcttaagctttacccagctttcttgtacaaagtggtcccc-3′. The gene was incorporated into pYES-DEST52 (Invitrogen) between the att1 and att2 sites by site-specific recombination using manufacturer-suggested protocols. The vector carried the URA3 marker for auxotrophic selection and a PGAL1 for protein expression. The expression vector for yeast phenyalanyl-tRNA synthetase was a gift from Dr. David Tirrell, California Institute of Technology.Phylogenetic Queries—The occurrence of wyosine in tRNAPhe from several organisms was determined from a data base of annotated tRNA sequences. The genomes of organisms containing (Methanococcus jannaschi, Homo sapiens, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Arabidopsis thaliana) or lacking (Drosophila melanogaster, Escherichia coli, and Bacillus subtilis) the wyosine modification were analyzed using the comparative genomics platform, Protein Link Explorer (PLEX) (43Date S.V. Marcotte E.M. Bioinformatics. 2005; 21: 2558-2559Crossref PubMed Scopus (39) Google Scholar). A BLAST E-value of 10-10 was set as a threshold for gene identification.Bulk tRNA Purification—Bulk tRNA was isolated from yeast cells grown in synthetic complete medium (± uracil) containing 2% galactose. Cultures (2 liters) were grown at 30 °C to an absorbance at 600 nm (A600) of 0.6-1.2. Cells were pelleted by centrifugation and rinsed with 10 mm sodium acetate (pH 4.5). The pellet was resuspended in 100 ml of 0.3 m sodium acetate (pH 4.5), 10 mm EDTA before the addition of 100 ml of water-saturated phenol. After 30 min of vigorous shaking, the phases were separated by centrifugation (5,000 rpm, 20 min). Nucleic acids were precipitated from the aqueous phase with 0.4 volumes of isopropyl alcohol. Precipitate was collected as a pellet (30 min at 10,000 rpm) and dissolved in 10 ml of NB1 buffer (100 mm Tris acetate (pH 6.3), 0.5 mm EDTA, 0.75 m KCl). Reconstituted nucleic acids were loaded onto a Nucleobond AX-500 column equilibrated in NB2 buffer (100 mm Tris acetate (pH 6.3), 15% ethanol, and 400 mm KCl). The column was then washed with 30 ml of the same buffer before elution with 12 ml of NB3 buffer (100 mm Tris acetate (pH 6.3), 15% ethanol, and 650 mm KCl). The eluted RNA was precipitated with isopropyl alcohol, washed with 70% ethanol, and lyophilized.Expression and Purification of Yeast Phenylalanyl-tRNA Synthetase— Yeast His6-PheRS was overexpressed in E. coli as described previously (44Datta D. Wang P. Carrico I.S. Mayo S.L. Tirrell D.A. J. Am. Chem. Soc. 2002; 124: 5652-5653Crossref PubMed Scopus (121) Google Scholar). All subsequent purification steps were performed at 4 °C. Cells pelleted from 2 liters of culture were resuspended in 30 ml of lysis buffer (20 mm Tris (pH 7.5), 5 mm MgCl2, 0.1% β-mercaptoethanol, and protease inhibitor mixture (Roche Diagnostics)) and lysed by sonication. The lysate was cleared by centrifugation (18,000 rpm for 30 min). Yeast His6-PheRS was purified by batch affinity chromatography using nickelnitrilotriacetic-agarose beads according to the manufacturer's protocol (Qiagen, Valencia, CA) (45Schmidt J. Wang R. Stanfield S. Reid B.R. Biochemistry. 1971; 10: 3264-3268Crossref PubMed Scopus (52) Google Scholar). Purified protein was dialyzed into HPLC buffer A (20 mm Tris (pH 7.5) and 0.1% β-mercaptoethanol) and loaded on to a Mono Q HR 5/5 column. Yeast His6-PheRS was eluted from the column using linear gradient of NaCl (0-1 m, HPLC buffer A) at a flow rate of 1 ml/min. Fractions containing yeast tRNAPhe amino acylation activity were pooled and dialyzed against storage buffer (20 mm HEPES-HCl (pH 7.5), 100 mm KCl, 2 mm dithiothreitol, and 10% glycerol). Purified yeast His6-PheRS was finally concentrated to >3 mg/ml and snap-frozen in liquid N2 for storage at -80 °C.Aminoacylation of tRNAPhe with Yeast PheRS—Aminoacylation of tRNAPhe was performed in buffer containing 25 mm HEPES-HCl (pH 7.5), 10 mm MgCl2, 100 mm KCl, 2 mm dithiothreitol, 2 mm ATP, 160 μm [3H]phenylalanine (800-5,000 cpm/pmol). The total concentration of tRNAPhe per reaction was limited to 10 μm. Reactions were initiated with enzyme (100 nm yeast His6-PheRS) and incubated for 45 min at 27 °C. [3H]Phenylalanyl-tRNAPhe was quantified as described previously (46Shepard A. Shiba K. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9964-9968Crossref PubMed Scopus (44) Google Scholar).Identical reaction conditions were used for large scale phenylalanyl-tRNAPhe production. However, reactions were quenched with 3 m sodium acetate (0.3 m final (pH 4.5)). Yeast PheRS was then removed by phenol/chloroform extraction before the tRNAPhe was precipitated from the aqueous layer with ethanol. Phe-tRNAPhe was resuspended in RP1 buffer (10 mm sodium phosphate (pH 4.5), 1 m sodium formate, and 8 mm MgCl2) prior to reverse-phase chromatography.Thin Layer Chromatography Analysis of Acid-hydrolyzed Wyebutosine—Pure, lyophilized tRNA (100 μg) was reconstituted in 500 μl of 50 mm sodium phosphate (pH 3.5) and incubated at 37 °C for 18 h to hydrolyze wyebutosine from tRNAPhe. The base was then extracted with ethyl acetate (500 μl). Ethyl acetate was removed under reduced pressure to produce a white residue. The residue was redissolved in a small volume of ethyl acetate and spotted on a Silica Gel 60 F254 TLC plate (EM Science, Gibbstown, NJ). The sample was then chromatographed using the upper layer of a 1-propyl alcohol/ethyl acetate/water (1:4:2) mixture as the mobile phase (19Nakanishi K. Blobstein S. Funamizu M. Furutachi N. Van Lear G. Grunberger D. Lanks K.W. Weinstein I.B. Nat. New Biol. 1971; 234: 107-109Crossref PubMed Scopus (52) Google Scholar). Fluorescent material was visualized by excitation at 300 nm.Fractionation of tRNA by Reverse-phase HPLC—Purified bulk tRNA (1-2 mg) was loaded onto a Vydac C4 semi-preparative HPLC column (catalog number 214TP1010) equilibrated in buffer RP1 (10 mm sodium phosphate (pH 4.5), 1 m sodium formate, and 8 mm MgCl2). A linear gradient to 100% buffer RP2 (10 mm sodium phosphate (pH 4.5) and 15% methanol) was established over 70 min at a flow rate of 4 ml/min. Fractions were collected every 0.5 min and analyzed for phenylalanine acceptance as described above.MALDI—Mass measurements were made using an Applied Biosystems DE system. A salt-tolerant matrix, 2,4,6-trihydroxyacetophenone containing diammonium citrate was used to analyze purified tRNA. Ions were monitored in positive mode. Masses were calculated from an average of 300 scans.Enzymatic Digestion of tRNAPhe—HPLC-purified tRNA (100 μg) was resuspended in 0.1 ml of 10 mm ammonium acetate (pH 5.3). The tRNA was incubated with nuclease P1 (8 units, Sigma) at 45 °C for 2 h. Fresh ammonium bicarbonate was added to a final concentration of 0.1 m before the addition of snake venom phosphodiesterase (0.008 units, Sigma). The mixture was incubated for an additional 2 h at 37°C. Nucleosides were dephosphorylated by incubation with alkaline phosphatase (4 units, New England Biolabs, Beverly, MA) for 1 h at 37°C. Digested tRNA was lyophilized prior to LC-MS analysis.Liquid Chromatography-Mass Spectrometry of tRNAPhe Hydrolysates— LC electrospray ionization mass spectrometry was performed on an Agilent MSD 1100. The system was equipped with a Supelcosil LC-18-S HPLC column (25 cm × 4.6 mm, 5 μm). Prior to chromatography the column was equilibrated in LC-MS buffer A (250 ammonium acetate (pH 6.0)). Nucleosides were eluted using a segmented, linear gradient of LC-MS buffer B (40% acetonitrile). The gradient profile was as follows: 0 min, 0% B; 6 min, 0% B; 8.8 min, 0.2% B; 11.6 min, 0.8% B; 14.4 min, 1.8% B; 17.2 min, 3.2% B; 20 min, 5% B; 50 min, 25% B; 60 min, 50% B; 68 min, 75% B; 74 min, 75% B; 90 min, 100% B; and 96 min, 100% B. A flow rate of 0.5 ml/min was maintained during each run/ and the eluate was analyzed in positive ion mode.RESULTSIdentification of COG0731 as a Probable Wyebutosine Synthesis Gene— For the purpose of guiding comparative genomics queries, a compilation of tRNA sequence and modification data were analyzed for the occurrence of wyosine family compounds in organisms with sequenced genomes (1Sprinzl M. Horn C. Brown M. Ioudovitch A. Steinberg S. Nucleic Acids Res. 1998; 26: 148-153Crossref PubMed Scopus (807) Google Scholar, 3Steinberg S. Misch A. Sprinzl M. Nucleic Acids Res. 1993; 21: 3011-3015Crossref PubMed Scopus (233) Google Scholar, 47Keith G. Dirheimer G. Biochem. Biophys. Res. Commun. 1980; 92: 109-115Crossref PubMed Scopus (11) Google Scholar, 48Keith G. Picaud F. Weissenbach J. Ebel J.P. Petrissant G. Dirheimer G. FEBS Lett. 1973; 31: 345-347Crossref PubMed Scopus (53) Google Scholar, 49Guerrier-Takada C. Dirheimer G. Grosjean H. Keith G. FEBS Lett. 1975; 60: 286-289Crossref PubMed Scopus (10) Google Scholar, 50Keith G. Ebel J.P. Dirheimer G. FEBS Lett. 1974; 48: 50-52Crossref PubMed Scopus (29) Google Scholar, 51Martin R. Sibler A.P. Schneller J.M. Keith G. Stahl A.J. Dirheimer G. C. R. Acad. Sci. Hebd. Seances Acad. Sci. D. 1978; 287: 845-848PubMed Google Scholar, 52Sprinzl M. Steegborn C. Hubel F. Steinberg S. Nucleic Acids Res. 1996; 24: 68-72Crossref PubMed Scopus (160) Google Scholar). Yb is absent from eubacteria and present in many archaeal and eukaryotic phenylalanine-specific tRNAs (Figs. 1 and 2). Significantly, D. melanogaster tRNAPhe harbors N1-methylguanosine at position 37 instead of Yb (53Altwegg M. Kubli E. Nucleic Acids Res. 1979; 7: 93-105Crossref PubMed Scopus (16) Google Scholar). By using this information and a Protein Link Explorer (PLEX) algorithm (43Date S.V. Marcotte E.M. Bioinformatics. 2005; 21: 2558-2559Crossref PubMed Scopus (39) Google Scholar), a phylogenetic occurrence query identified genes present in M. jannaschi, H. sapiens, S. cerevisiae, S. pombe, and A. thaliana but not in D. melanogaster, E. coli, or B. subtilis. A single gene family COG0731 fit the desired criteria and belongs to PACE (proteins in Archaea conserved in eukaryotes) Group 22 (54Matte-Tailliez O. Zivanovic Y. Forterre P. Trends Genet. 2000; 16: 533-536Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar).Although the biological role of COG0731 is generally unknown, many characterized PACE-encoding genes are implicated in the organization and processing of genetic material, including rRNA/tRNA maturation and modification (54Matte-Tailliez O. Zivanovic Y. Forterre P. Trends Genet. 2000; 16: 533-536Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 55Armengaud J. Urbonavicius J. Fernandez B. Chaussinand G. Bujnicki J.M. Grosjean H. J. Biol. Chem. 2004; 279: 37142-37152Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Consequently, these genes show correlated expression with genes sharing related biological functions. Analysis of gene expression in yeast (www.yeastgenome.org) revealed that during the cell cycle and in response to DNA-damaging agents, COG0731 family members co-express with genes for ribosome biogenesis, RNA processing, and RNA metabolism (p > 10-5).Yeast Strains Lacking the COG0731 Ortholog Do Not Produce Acilabile Wyebutosine—YPL207w is the S. cerevisiae COG0731 ortholog. To assess the significance of COG0731 to Yb biosynthesis, wild-type and the ΔYPL207w deletion strain of S. cerevisiae were assayed for wyebutosine production. Bulk tRNA from each strain was incubated at pH 3.5 and at 37 °C for several hours (under these conditions the wyebutosine base (yW) is hydrolyzed from tRNAPhe by cleavage of the N-C bond (56Golankiewicz B. Zielonacka-Lis E. Folkman W. Nucleic Acids Res. 1985; 13: 2443-2449Crossref PubMed Scopus (13) Google Scholar)). The liberated base was then isolated by extraction with ethyl acetate, and the extracts were concentrated and spotted on silica gel plates. After thin layer chromatography (Fig. 3), a single fluorescent spot (RF = 0.47) 5A similar RF (0.48) value was reported for yeast yW by Nakanishi et al. (19Nakanishi K. Blobstein S. Funamizu M. Furutachi N. Van Lear G. Grunberger D. Lanks K.W. Weinstein I.B. Nat. New Biol. 1971; 234: 107-109Crossref PubMed Scopus (52) Google Scholar). was detected in extracts prepared from the wild-type strain, and mass spectrometric analysis of this fluorescent material gave a mass of 377.1 Da corresponding to that of yW (57Zhou S. Sitaramaiah D. Pomerantz S.C. Crain P.F. McCloskey J.A. Nucleosides Nucleotides Nucleic Acids. 2004; 23: 41-50Crossref PubMed Scopus (12) Google Scholar). Extracts from the ΔYPL207w deletion strain lacked the fluorescent material. Thus, the ΔYPL207w deletion blocks Yb biosynthesis.FIGURE 3Thin layer chromatography analysis of acid hydrolyzed wyebutosine. Bulk tRNA (100 μg) was purified from wild-type (WT) and YPL207w-deleted (ΔYPL207w) strains of S. cerevisiae. yW was hydrolyzed from tRNA with acid and extracted with ethyl acetate. Concentrated samples were spotted and developed using the upper layer of an ethyl acetate/propyl alcohol/H2O (4:2:1) mixture. Compounds were excited with UV irradiation (300 nm).View Large Image Figure ViewerDownload Hi-res image Download (PPT)RP-HPLC Analysis of tRNAPhe from Wild-type and Gene Deletion Strains of S. cerevisiae—To confirm that the absence of Yb from acid-treated tRNA from the ΔYPL207w deletion strain correlated with an alteration of the tRNA itself, the chromatographic properties of tRNAPhe from wild-type (tRNAPhewt and null (tRNAPheΔypl207w) strains were compared. Bulk tRNA from each strain was fractionated using RP-HPLC (Vydac C-4 column), and the presence of tRNAPhe was analyzed by testing each fraction for [3H]phenylalanine acceptance (Fig. 4, A-C) (58Xue H. Shen W. Wong J.T. J. Chromatogr. 1993; 613: 247-255Crossref PubMed Scopus (16) Google Scholar). Although viable tRNAPhe from each strain eluted as a single peak, 6The high specific activity of [3H]phenylalanine (5,000 cpm/pmol) used during labeling would have enabled detection of rare tRNAPhe species present at less than 0.5% the quantity of the major tRNAPhe peak. they had markedly different retention times (tRNAPhewt = 49.5 min, tRNAPheΔypl207w = 18.0 min). The early elution time of tRNAPheΔypl207w fits with a more hydrophilic, hypomodified state for that tRNA. Similar shifts have been reported for yeast tRNAPhe after removal of yW by acid treatment, as well as for mammalian tRNAPhe isolated from rat hepatomas that were hypomodified (m1G) at position 37 (59Grunberger D. Weinstein I.B. Mushinski J.F. Nature. 1975; 253: 66-67Crossref PubMed Scopus (36) Google Scholar). Because all tRNAPheΔypl207w occurs in a single rapidly eluting peak, the ΔYPL207w deletion appears to block completely the synthesis of yW in S. cerevisiae. These results confirm that the absence of yW from acid-treated tRNA from ΔYPL207w deletion strain is due to a deficiency in tRNAPhe modification.FIGURE 4Reverse-phase liquid chromatography of bulk tRNA. Bulk tRNA (1 mg) was loaded onto a Vydac C4 semipreparative HPLC column equilibrated in 10 mm sodium phosphate (pH 4.5), 1 m sodium formate, and 8 mm MgCl2. A linear gradient to 10 mm sodium phosphate (pH 4.5) and 15% methanol was established over 70 min at 4 ml/min. Fractions were collected every 0.5 min and analyzed for [3H]phenylalanine acceptance as described under “Materials and Methods.” Absorbance at 260 nm and [3H]phenylalanine incorporation are indicated as solid and dotted lines, respectively. Profiles of tRNA isolated from wild-type (A), YPL207w-deleted (B), and complementation strains (C) are presented. Inset, tRNA from complementation strains harvested at A600 >2.0.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Complementation in the Null Strain by Expression of YPL207w in Trans—Aware that polar effects may result from disruption of YPL207w with KanR, we turned to genetic complementation to provide evidence that the phenotype of the ΔYPL207w strain is because of a single gene disruption. For these experiments, the 810-amino acid ORF of YPL207w was cloned into the pYES-DEST52 expression vector. The ΔYPL207w deletion strain was transformed with the recombinant vector and then grown under conditions that ensured continuous gene expression. Total tRNA was isolated, and chromatography of the" @default.
• W2018032976 created "2016-06-24" @default.
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• W2018032976 date "2005-11-01" @default.
• W2018032976 modified "2023-10-17" @default.
• W2018032976 title "Discovery of a Gene Family Critical to Wyosine Base Formation in a Subset of Phenylalanine-specific Transfer RNAs" @default.
• W2018032976 cites W1544760751 @default.
• W2018032976 cites W1556249430 @default.
• W2018032976 cites W1556273208 @default.
• W2018032976 cites W1556382451 @default.
• W2018032976 cites W1558136913 @default.
• W2018032976 cites W1568503582 @default.
• W2018032976 cites W1588089207 @default.
• W2018032976 cites W1595004313 @default.
• W2018032976 cites W1595094981 @default.
• W2018032976 cites W1605039020 @default.
• W2018032976 cites W1605114604 @default.
• W2018032976 cites W1820306814 @default.
• W2018032976 cites W1967239446 @default.
• W2018032976 cites W1968120233 @default.
• W2018032976 cites W1970320320 @default.
• W2018032976 cites W1976391391 @default.
• W2018032976 cites W1981077282 @default.
• W2018032976 cites W1993782002 @default.
• W2018032976 cites W1994960595 @default.
• W2018032976 cites W1996135412 @default.
• W2018032976 cites W1998816181 @default.
• W2018032976 cites W2004357323 @default.
• W2018032976 cites W2004580361 @default.
• W2018032976 cites W2008450625 @default.
• W2018032976 cites W2010060726 @default.
• W2018032976 cites W2010355629 @default.
• W2018032976 cites W2012065571 @default.
• W2018032976 cites W2014576793 @default.
• W2018032976 cites W2016706267 @default.
• W2018032976 cites W2017340633 @default.
• W2018032976 cites W2017966321 @default.
• W2018032976 cites W2021590319 @default.
• W2018032976 cites W2021836392 @default.
• W2018032976 cites W2022455510 @default.
• W2018032976 cites W2025065034 @default.
• W2018032976 cites W2029474258 @default.
• W2018032976 cites W2031045177 @default.
• W2018032976 cites W2036384145 @default.
• W2018032976 cites W2038550383 @default.
• W2018032976 cites W2038852639 @default.
• W2018032976 cites W2043738469 @default.
• W2018032976 cites W2048245383 @default.
• W2018032976 cites W2063834373 @default.
• W2018032976 cites W2068880413 @default.
• W2018032976 cites W2070455330 @default.
• W2018032976 cites W2071190573 @default.
• W2018032976 cites W2075315521 @default.
• W2018032976 cites W2075919384 @default.
• W2018032976 cites W2078412971 @default.
• W2018032976 cites W2079048673 @default.
• W2018032976 cites W2080059290 @default.
• W2018032976 cites W2080161964 @default.
• W2018032976 cites W2085286256 @default.
• W2018032976 cites W2087158203 @default.
• W2018032976 cites W2087350229 @default.
• W2018032976 cites W2091353246 @default.
• W2018032976 cites W2095421602 @default.
• W2018032976 cites W2096894622 @default.
• W2018032976 cites W2110850678 @default.
• W2018032976 cites W2127776675 @default.
• W2018032976 cites W2130880846 @default.
• W2018032976 cites W2141032258 @default.
• W2018032976 cites W4211164196 @default.
• W2018032976 cites W4234887284 @default.
• W2018032976 cites W4238022135 @default.
• W2018032976 cites W4252182636 @default.
• W2018032976 doi "https://doi.org/10.1074/jbc.m506939200" @default.
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