Discovery of a Gene Family Critical to Wyosine Base Formation in a Subset of Phenylalanine-specific Transfer RNAs*

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 (4). Over 80 modifications are known, and their presence vastly expands the structural and chemical diversity of native tRNA (2). (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 (5,9). 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 (4).
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-␣]purine core structure and a strict occurrence in archaeal and eukaryal tRNA Phe (Figs. 1 and 2). Wyosine bases, isolated from different organisms, show variations in ring methylation and side chain structure (15)(16)(17)(18)(19)(20). Generally, archaeal Yb structures are less differentiated then their eukaryotic counterparts. The hydrophobic nature of yW 37 promotes stacking with adjacent bases (A 36 and A 38 ) and restricts the conformational flexibility of the anticodon (21)(22)(23)(24)(25). Removal of yW produced local changes in anticodon conformation, as well as long range perturbations in tRNA Phe tertiary structure (26). These structural changes were accompanied by subtle differences in codon specificity 4 and a modest increase in retroviral ribosomal frameshifting (determined in cell-free extract) (27)(28)(29). Most interesting, the tRNA Phe from mouse neuroblastoma cell lacked Yb but was more efficient than the fully modified tRNA Phe in a cell-free translation system (30 -32). 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 (33,34). NMR studies of 13 C-enriched tRNA Phe 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 N 3 -methyl moieties (35). Conflicting evidence obscures understanding the origin of the 3-amino-3-carboxypropyl side chain (36,37). The in vivo kinetics of Yb biosynthesis of Xenopus laevis were investigated (34). By using the sitespecifically labeled [ 32 P]tRNA Phe transcript in X. laevis oocytes, Droogmans and Grosjean (34) detected N 1 -methylguanosine (m 1 G) 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 (38 -42). In this study, our methodology produced a single "hit" that allowed us to identify a pathway-specific polypeptideencoding gene that acts downstream from the initial modification event (m 1 G 37 ). Deletion of the gene in Saccharomyces cerevisiae produces tRNA Phe in a modification state similar to that in certain mammalian cell types, including some tumor cell lines.

MATERIALS AND METHODS
Strains and Chemicals-Wild-type and deletion strains (⌬YPL207w) of S. cerevisiae were purchased from Open Biosystems (www.openbiosystems.com). Both strains were of the MAT␣ leu2⌬0 met15⌬0 ura3⌬0 genotype. The YPL207w ORF was replaced with a Kan R 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: 5Ј-ggggacaagtttgtacaaaaaagcaggctatggatccaataatggatggttttcgtgtagctgg-3Ј and 5Ј-ctccctcctattcctgcttaagctttacccagctttcttgtacaaagtggtcccc-3Ј. 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 P GAL1 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 tRNA Phe 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) (43). A BLAST E-value of 10 Ϫ10 was set as a threshold for gene identification.
Bulk tRNA Purification-Bulk tRNA was isolated from yeast cells grown in synthetic complete medium (Ϯ uracil) containing 2% galactose. Cultures (2 liters) were grown at 30°C to an absorbance at 600 nm (A 600 ) 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 His 6 -PheRS was overexpressed in E. coli as described previously (44). 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 MgCl 2 , 0.1% ␤-mercaptoethanol, and protease inhibitor mixture (Roche Diagnostics)) and lysed by sonication. The lysate was cleared by centrifugation (18,000 rpm for 30 min). Yeast His 6 -PheRS was purified by batch affinity chromatography using nickelnitrilotriacetic-agarose beads according to the manufacturer's protocol (Qiagen, Valencia, CA) (45). Purified protein was dialyzed into HPLC buffer A (20 mM Tris (pH 7.5) and 0.1% ␤-mercaptoethanol) and loaded on to a Mono Q HR 5/5 column. Yeast His 6 -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 tRNA Phe 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 His 6 -PheRS was finally concentrated to Ͼ3 mg/ml and snap-frozen in liquid N 2 for storage at Ϫ80°C.
Identical reaction conditions were used for large scale phenylalanyl-tRNA Phe 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 tRNA Phe was precipitated from the aqueous layer with ethanol. Phe-tRNA Phe was resuspended in RP1 buffer (10 mM sodium phosphate (pH 4.5), 1 M sodium formate, and 8 mM MgCl 2 ) prior to reverse-phase chromatography.
Thin Layer Chromatography Analysis of Acid-hydrolyzed Wyebutosine-Pure, lyophilized tRNA (100 g) was reconstituted in 500 l of 50 mM sodium phosphate (pH 3.5) and incubated at 37°C for 18 h to hydrolyze wyebutosine from tRNA Phe . The base was then extracted with ethyl acetate (500 l). 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 F 254 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 (19). 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 MgCl 2 ). 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.

Identification 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 (1,3,(47)(48)(49)(50)(51)(52). Yb is absent from eubacteria and present in many archaeal and eukaryotic phenylalanine-specific tRNAs (Figs. 1 and 2). Significantly, D. melanogaster tRNA Phe harbors N 1 -methylguanosine at position 37 instead of Yb (53). By using this information and a Protein Link Explorer (PLEX) algorithm (43), 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 (54).
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 (54,55). 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 Ͼ 10 Ϫ5 ).
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 ⌬YPL207w 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 tRNA Phe by cleavage of the N-C bond (56)). 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 (R F ϭ 0.47) 5 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 (57). Extracts from the ⌬YPL207w deletion strain lacked the fluorescent material. Thus, the ⌬YPL207w deletion blocks Yb biosynthesis.
RP-HPLC Analysis of tRNA Phe from Wild-type and Gene Deletion Strains of S. cerevisiae-To confirm that the absence of Yb from acidtreated tRNA from the ⌬YPL207w deletion strain correlated with an alteration of the tRNA itself, the chromatographic properties of tRNA Phe from wild-type (tRNA Phe wt and null (tRNA Phe ⌬ypl207w ) strains were compared. Bulk tRNA from each strain was fractionated using RP-HPLC (Vydac C-4 column), and the presence of tRNA Phe was analyzed by testing each fraction for [ 3 H]phenylalanine acceptance (Fig. 4, A-C) (58). Although viable tRNA Phe from each strain eluted as a single peak, 6 they had markedly different retention times (tRNA Phe wt ϭ 49.5 min, tRNA Phe ⌬ypl207w ϭ 18.0 min). The early elution time of tRNA Phe ⌬ypl207w fits with a more hydrophilic, hypomodified state for that tRNA. Similar shifts have been reported for yeast tRNA Phe after removal of yW by acid treatment, as well as for mammalian tRNA Phe isolated from rat hepatomas that were hypomodified (m 1 G) at position 37 (59). Because all tRNA Phe ⌬ypl207w occurs in a single rapidly eluting peak, the ⌬YPL207w 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 ⌬YPL207w deletion strain is due to a deficiency in tRNA Phe modification.

Discovery of Gene Family Critical to Wyosine Biosynthesis
that the phenotype of the ⌬YPL207w 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 ⌬YPL207w 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 tRNA revealed peaks (a and b) at ϳ49.5 and 18.0 min (Fig. 4C). These retention times are the same as those of tRNA Phe wt and tRNA Phe ⌬ypl207w , respectively. Acid treatment of peak b fractions produced a fluorescent, ethyl acetatesoluble compound with an R F (0.47) and mass (377.1) identical to that of wyebutosine. No yW was detected in peak a fractions. The observation of two peaks was presumed to result from partial complementation. (complete complementation was achieved when cells were harvested at higher optical densities (A 600 Ͼ 2.0 (Fig. 4C, inset)). Thus, expression of YPL207w in the ⌬YPL207w deletion strain restores wyebutosine biosynthesis and rules out downstream polar effects caused by Kan R insertion.
Purification and Mass Determination of tRNA Phe and Identification of a Wyebutosine Precursor by LC/MS of tRNA Phe Hydrolysates-Because disruption of YPL207w produced a hypomodified and chromatographically homogenous tRNA Phe species, we inferred that an intermediate or yW precursor had accumulated at position 37. To identify intermediates, transfer RNA Phe wt was obtained at high purity (amino acid acceptance ϭ 1969 Ϯ 53 pmol/A 260 (60)) using the fractionation procedure described above (TABLE ONE). By contrast, tRNA Phe ⌬ypl207w co-eluted with other tRNA species (154 pmol/A 260 ) and had to be esterified with phenylalanine (using yeast PheRS) to increase the retention time (48.5 min) and resolve it (1836 Ϯ 108 pmol/A 260 ) from other tRNA species.
MALDI mass spectroscopy was used for tRNA molecular weight determination ( Tandem liquid chromatography/mass spectroscopy (LC/MS) was used to analyze tRNA nucleoside composition. For this procedure, tRNAs were extensively digested and dephosphorylated, and nucleosides were resolved by RP-HPLC, under a set of standardized conditions   (17,62,63). Mass (100 -600 m/z) and UV absorption (254, 280, 310 nm) data were collected from the eluents and, along with relative retention times, 7 were used to assign nucleoside identities according to the work of Edmonds et al. (62). Absorbance and mass profiles for the tRNA Phe wt hydrolysate (Fig. 5A) were consistent with the known composition of S. cerevisiae tRNA Phe , with masses for 14 of the 15 ribonucleosides expected for the tRNA Phe being observed (5-methyluridine was not detected, whereas inosine (peak 3b) occurred at ϳ36 min and was attributed to adenosine deaminase activity in commercial preparations of nuclease P1. 8 Peak 9 corresponds to yW and does not appear in the spectrum of tRNA Phe ⌬ypl207w (Fig. 5B). The loss of yW coincided with only one other change, a substantial increase in absorbance of the peak for 2Ј-O-methylguanosine (Gm, peak 5*). Extensive mass scanning over the limits of the peak 5* revealed fragment ions not observed for the Gm peak of tRNA Phe wt (5th peak, Fig. 6A). The most abundant new ion is observed at 166.0 m/z, the expected mass of a base-methylated guanosine (Fig. 6B) (62). Methylation of G 37 fits with the molecular weight differences of tRNA Phe wt and tRNA Phe ⌬ypl207w (⌬M m(expected) ϭ 211 Da, ⌬M m(observed) ϭ 208.7 Da; where M m indicates molecular mass). Relative retention time and co-elution with Gm suggests these ions are produced by N 1 -methylguanosine (m 1 G). Only two other base-methylated guanosines are known to occur in RNA-7-methylguanosine (m 7 G) and N 2 -methylguanosine (m 2 G). These ribonucleosides are accounted for in each spectrum as peaks 3a and 6, respectively, and thus can be eliminated as candidate structures. The homogeneous elution of tRNA Phe ⌬ypl207w during RP-HPLC chromatography and the nearly quantitative doubling (1.96-fold) 9 of peak 5 absorbance indicate stoichiometric methylation of G 37 in cells lacking YPL207w.

DISCUSSION
A comparative genomics analysis was used to link YPL207w to yW biosynthesis. The analysis was initially limited to organisms with completely sequenced genomes and fully characterized tRNA Phe . The phylogenetic distribution of Yb and conservation among these organisms (which include Archaea, fungi, plants, and mammals) suggests that it is among the earliest tRNA modifications to occur after branching of Archaea and eukaryotes from eubacteria. Further review of the tRNA data base indicated that in Bombyx mori the tRNA Phe contains m 1 G 37 , and analysis of several other complete genome sequences revealed that Caenorhabditis elegans and Encephalitozoon cuniculi lack COG0731 orthologs. Like D. melanogaster, these organisms are not equipped to synthesize Yb. Phenylalanine-specific tRNAs of eubacteria also lack Yb, but instead have another highly modified purine at position 37 (2-methylthio-N 6 -isopentenyladenosine). The 2-methylthio-N 6 -isopentenyladenosine modification affects anticodon structure, codon recognition, and translational efficiency in a way that is similar to Yb (6, 64). There-fore, tRNA Phe from eubacteria is distinct from that of eukaryotic organisms that harbor m 1 G 37 .
Deletion of YPL207w in yeast blocked wyebutosine production and caused methylated guanosine (G 37 ) to accumulate in tRNA Phe . We suggest that methylation occurs at N 1 of G 37 . This modification (m 1 G 37 ) was reported in several cell types that produce hypomodified tRNA Phe . For example, in mouse neuroblastoma, 85% of tRNA Phe contained m 1 G 37 (65,66). A significant portion of rabbit reticulocyte tRNA Phe also had m 1 G 37 instead of peroxywyebutosine (28). Furthermore, eukaryotic organisms that do not synthesize Yb (e.g. D. melanogaster and B. mori) produce tRNA Phe bearing m 1 G 37 . These data demonstrate the natural occurrence of m 1 G 37 during tRNA Phe biosynthesis and fit well with our assignment.
Droogmans and Grosjean (34) provided direct evidence that tRNA Phe (m 1 G 37 ) is an integral part of yW biosynthesis. They showed that the transcript synthesized with m 1 G 37 is a competent pathway intermediate and established m 1 G 37 as the obligatory first step in Yb biosynthesis in X. laevis oocytes. Subsequently, the enzyme responsible for G 37 methylation was discovered to be an archaeal/eukaryl specific m 1 G 37 methyltransferase (8,67). Deletion of the S. cerevisiae, methyltransferase-  encoding gene (TRM5), prevents formation of both m 1 G 37 and yW (67). Representatives from the m 1 G 37 -methyltransferase gene family are found in all organisms that synthesize Yb. Based on the accumulation of m 1 G in our study, we hypothesize that YPL207w supports a biosynthetic step immediately after synthesis of m 1 G 37 .
Because COG0731 genes occur in organisms that produce "minimalist" wyosine bases such as 4-demethylwyosine (Fig. 1d), the COG0731 gene product may not be involved in formation of eukaryotic Yb side chains. This suggestion is supported by the work of Kalhor et al. (7) which demonstrated that tRNA from S. cerevisiae lacking the TRM12 gene contained 4-demethylwyosine (structure d, Fig. 1) rather than yW (7).

CONCLUSION
In this study, YPL207w was identified as essential for wyebutosine biosynthesis in yeast. A ⌬YPL207w deletion strain produced tRNA Phe that was fully modified except at position 37 (m 1 G 37 ). N 1 -G 37 -methylated tRNA Phe is a putative intermediate of the yW biosynthetic pathway. Currently, it is not known whether the protein translated from the YPL207w ORF directly modifies tRNA Phe -m 1 G or contributes to tRNA Phe -yW biosynthesis by some indirect means. Thus, the biochemical activity of the YPL207w gene product requires further investigation.