Biosynthesis of wybutosine, a hyper-modified nucleoside in eukaryotic phenylalanine tRNA
2006; Springer Nature; Volume: 25; Issue: 10 Linguagem: Inglês
10.1038/sj.emboj.7601105
ISSN1460-2075
AutoresAkiko Noma, Yohei Kirino, Yoshiho Ikeuchi, Tsutomu Suzuki,
Tópico(s)Cancer-related gene regulation
ResumoArticle27 April 2006free access Biosynthesis of wybutosine, a hyper-modified nucleoside in eukaryotic phenylalanine tRNA Akiko Noma Akiko Noma Search for more papers by this author Yohei Kirino Yohei Kirino Search for more papers by this author Yoshiho Ikeuchi Yoshiho Ikeuchi Search for more papers by this author Tsutomu Suzuki Corresponding Author Tsutomu Suzuki Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Akiko Noma Akiko Noma Search for more papers by this author Yohei Kirino Yohei Kirino Search for more papers by this author Yoshiho Ikeuchi Yoshiho Ikeuchi Search for more papers by this author Tsutomu Suzuki Corresponding Author Tsutomu Suzuki Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Author Information Akiko Noma, Yohei Kirino, Yoshiho Ikeuchi and Tsutomu Suzuki 1 1Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo, Japan *Corresponding author. Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Tel.: +81 3 5841 8752; Fax: +81 3 3816 0106; E-mail: [email protected] The EMBO Journal (2006)25:2142-2154https://doi.org/10.1038/sj.emboj.7601105 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Wybutosine (yW) is a tricyclic nucleoside with a large side chain found at the 3′-position adjacent to the anticodon of eukaryotic phenylalanine tRNA. yW supports codon recognition by stabilizing codon–anticodon interactions during decoding on the ribosome. To identify genes responsible for yW synthesis from uncharacterized genes of Saccharomyces cerevisiae, we employed a systematic reverse genetic approach combined with mass spectrometry (‘ribonucleome analysis’). Four genes YPL207w, YML005w, YGL050w and YOL141w (named TYW1, TYW2, TYW3 and TYW4, respectively) were essential for yW synthesis. Mass spectrometric analysis of each modification intermediate of yW revealed its sequential biosynthetic pathway. TYW1 is an iron–sulfur (Fe–S) cluster protein responsible for the tricyclic formation. Multistep enzymatic formation of yW from yW-187 could be reconstituted in vitro using recombinant TYW2, TYW3 and TYW4 with S-adenosylmethionine, suggesting that yW synthesis might proceed through sequential reactions in a complex formed by multiple components assembled with the precursor tRNA. This hypothesis is also supported by the fact that plant ortholog is a large fusion protein consisting of TYW2 and TYW3 with the C-terminal domain of TYW4. Introduction A characteristic structural feature of functional RNAs is the presence of post-transcriptional modifications. During RNA processing and maturation, RNA molecules undergo various chemical modifications by RNA-modifying enzymes. Accurate maintenance of RNA modifications is required for various biological functions (Björk, 1995; Suzuki, 2005). Our recent studies revealed that lack of tRNA modification leads to translational defects, which have been associated with human diseases (Kirino and Suzuki, 2004; Kirino et al, 2005). To date, more than 100 different RNA modifications have been reported in RNA molecules from all domains of life (Rozenski et al, 1999). Most have been identified and characterized in tRNA molecules. Many tRNAs have modifications at the first (wobble) position of the anticodon (position 34) and adjacent to the 3′-position of the anticodon (position 37). The wobble modifications at position 34 are important for precise decoding mediated by the codon-anticodon interaction (Suzuki, 2005). Position 37 of the tRNA usually contains a modified purine nucleoside. This modification is often a hyper-modified nucleoside with large molecular weight, such as N6-threonylcarbamoyladenosine (t6A), 2-methylthio-N6-isopentenyladenosine (ms2i6A) or wybutosine (yW). These modifications play a critical role in the stabilization of codon–anticodon pairing through base–stacking interactions and function to maintain the reading frame. yW (whose base is wye, Figure 1A), which contains a tricyclic base, is one of the most complexly modified guanosine residues. yW and its derivatives are found at position 37 of eukaryotic and archaeal phenylalanine tRNA (tRNAPhe) (Figure 1B) (Blobstein et al, 1973), with the exception of Bombyx mori (Keith and Dirheimer, 1980) and Drosophila melanogaster (Altwegg and Kubli, 1979), where 1-methylguanosine (m1G) is present. yW does not have any major influence on the phenylalanylation of tRNAPhe (Thiebe and Zachau, 1968), but may stabilize the first base pair of the codon–anticodon duplex in the ribosomal A site by base stacking (Konevega et al, 2004). tRNAPhe that lacks the yW modification has been shown to enhance frameshifting (Carlson et al, 1999) and influence the replication of human immunodeficiency virus (HIV) RNA (Hatfield et al, 1989). In addition, mouse tRNAPhe isolated from neuroblastoma cells has m1G instead of the naturally occurring hydroxywybutosine (OHyW), and tRNAPhe from Ehrlich ascites tumors contains an undermodified OHyW (OHyW-72; OHyW minus 72 Da) (Kuchino et al, 1982). These data imply that disruption of the yW modification may have pathological consequences. Figure 1.Chemical structure of wybutosine (yW) and secondary structure of tRNAPhe. (A) Chemical structure of yW. Carbon and nitrogen atoms in the tricyclic base are numbered. The α-amino-α-carboxypropyl group at C-7 is boxed by a dotted line. (B) Secondary structure of the S. cerevisiae tRNAPhe with modified nucleosides: wybutosine (yW), 2′-O-methylguanosine (Gm), 2′-O-methylcytidine (Cm), pseudouridine (Ψ), 5-methylcytidine (m5C), 7-methylguanosine (m7G), 2-methylguanosine (m2G), N2,N2-dimethylguanosine (m22G), dihydrouridine (D), 1-methyladenosine (m1A) and 5-methyluridine (m5U). The anticodon-containing fragment produced by RNase T1 digestion is shown in gray. Arrows indicate the sites for RNase T1 cleavage. Download figure Download PowerPoint The biosynthesis of yW is a multienzymatic process. yW originates from a genetically encoded guanine (Li et al, 1973; Thiebe and Poralla, 1973), and the presence of m1G37 in tRNAsPhe from neuroblastoma cells, as well as B. mori and D. melanogaster, suggested that m1G is the first precursor in the synthesis of yW. N1-methylation of G37 in tRNAPhe is catalyzed by an S-adenosylmethionine(Ado-Met)-dependent tRNA methylase (TRM5) in the initial step of yW synthesis (Droogmans and Grosjean, 1987). Disruption of the TRM5 gene led to a severe growth defect (Björk et al, 2001), suggesting that m1G37 has a critical role in maintaining the correct reading frame in addition to initiating yW synthesis in tRNAPhe. The GAA anticodon of tRNAPhe is necessary for yW synthesis (Droogmans and Grosjean, 1987), although the anticodon is not required for m1G formation by TRM5 (Brule et al, 2004). Thus, subsequent steps in the multienzymatic process from m1G to yW strictly discriminate tRNAPhe from other m1G-containing tRNAs. Very little is known about the biosynthetic steps from m1G to yW. It has been reported that the α-amino-α-carboxypropyl side chain at the C-7 position of the yW-base (Figure 1A) is transferred from methionine or S-adenosylmethionine in yeast cells (Munch and Thiebe, 1975). However, in Vero cells, the lateral chain of the yW-base is derived from lysine (Pergolizzi et al, 1979). These data imply that the biosynthesis of yW in tRNAPhe might vary from one species to another. Although the number of genes involved in biosynthesis of yW is not known, the genes responsible for yW synthesis must be present among the uncharacterized genes in the Saccharomyces cerevisiae genome. To identify genes responsible for RNA modifications, we have employed a reverse genetic approach combined with mass spectrometry (‘ribonucleome’ analysis) (Soma et al, 2003; Suzuki, 2005; Ikeuchi et al, 2006). This analysis utilizes a series of gene-deletion strains of S. cerevisiae. Total RNA extracted from each strain was analyzed by liquid chromatography-mass spectrometry (LC/MS), to determine whether a particular gene deletion leads to the absence of a specific modified base and, thus, permits us to identify the enzyme or protein responsible for this modification. In the case of essential genes, we analyzed temperature-sensitive mutants cultured at the nonpermissive temperature, or expression-controlled strains (Soma et al, 2003). This analysis enables us to identify the enzymes directly responsible for RNA modification, as well as genes that encode nonenzymatic proteins necessary for the biosynthesis of RNA modifications. These include carriers of the metabolic substrates used in the RNA modification and subunit proteins needed for RNA recognition. Here, using the ribonucleome analysis, we identified four new genes responsible for biosynthesis of yW in S. cerevisiae. Chemical structures of the modification intermediates of yW in these deletion strains enabled us to model a biosynthetic pathway of yW. In addition, yW synthesis was partially reconstituted in vitro. This study will enable us to now develop a complete understanding and in vitro reconstitution of yW synthesis. Results Identification of four genes responsible for yW synthesis by ribonucleome analysis Since yW is a nonessential RNA modification, the complete set of S. cerevisiae deletion strains (4829) serves as a parent population to identify genes responsible for yW synthesis. To reduce the size of the starting population, we selected 3482 genes that have orthologs in Shizosaccharomyces pombe, because S. pombe tRNAs possess all the modified nucleosides found in S. cerevisiae tRNAs. Next, we chose 351 genes with ORFs coding for proteins of unknown function in S. cerevisiae (CYGD: http://mips.gsf.de/genre/proj/yeast) (Guldener et al, 2005) and started the ribonucleome analysis with this population. Parallel small-scale cultivation of each deletion strain was performed in deep 24-well plates. We developed a system that enabled us to obtain total RNA from each cultured strain and perform nuclease digestions in a 96-well format. Samples were then automatically analyzed by LC/MS using ion-trap MS to determine which modified nucleosides, if any, were absent. Our LC/MS analysis routinely detects 20 species of modified nucleosides in yeast total RNA, most are from tRNAs and some are from rRNAs or other noncoding RNAs. In the mass chromatogram, yW is detected as a proton adduct form (MH+) of yWpA (m/z 838, RT 46.8 min), which is a P1 nuclease-resistant dimer (Figure 2A). The protonated base fragment (BH2+) related to yW-base (m/z 377) is also detected (Figure 2A). To search for genes responsible for yW synthesis, we examined mass chromatograms of the 351 selected deletion mutants for peaks at m/z 838 and m/z 377 and identified four deletion strains, YPL207w, YML005w, YGL050w and YOL141w, in which yW was absent (Figure 2A). Since other modified nucleosides in these strains were detected normally (data not shown), the absence of yW is not due to nonspecific reduction of RNA modifications. Moreover, as normal amounts of tRNAPhe could be isolated from each deletion strain, the absence of yW is not due to a specific defect in tRNAPhe transcription (data not shown). Figure 2.Mass spectrometric analysis of total nucleosides and tRNAPhe from S. cerevisiae wild-type and mutant cells. (A) LC/MS analysis of the total nucleosides in the wild-type (WT), ΔYPL207w (TYW1), ΔYML005w (TYW2), ΔYGL050w (TYW3) and ΔYOL141w (TYW4). The upper panel is the UV trace at 254 nm. The middle and lower panels are mass chromatograms detecting MH+ of yWpA (m/z 838) and BH2+ of yW-base (m/z 377), respectively. Arrows indicate the retention time for yWpA. (B) LC/MS fragment analyses of RNaseT1-digested tRNAPhe obtained from wild-type and ΔTYW1-4. The graphs on the left describe the mass chromatograms shown by triply charged ions of anticodon-containing fragments containing yW (m/z 1388) from WT, m1G (m/z 1317) from ΔYPL207w (TYW1), yW-187 (m/z 1325) from ΔYML005w (TYW2), and yW-86 (m/z 1359) or yW-14 (m/z 1383) from ΔYGL050w (TYW3) and yW-72 (m/z 1364) from ΔYOL141w (TYW4). RNA sequences, including modifications for each fragment, are indicated. The graphs on the right show the mass spectrum for each anticodon-containing fragment. Charge states are indicated in parentheses. Download figure Download PowerPoint Cell growth of each deletion strain was measured in rich medium as well as in the minimal medium (Supplementary Figure S4). However, we did not observe any significant differences in growth between wild-type and the deletion strains. Mass spectrometric analysis of the modification intermediates of yW To confirm the absence of yW at position 37 of tRNAPhe and to determine the chemical structure of each modification intermediate, we isolated individual tRNAPhe from each deletion strain. As expected, the nucleoside analysis revealed that the purified tRNAPhe from each deletion strain had no yW nucleoside (Supplementary Figure S1), but other modified nucleosides were present (data not shown). These results demonstrated that the four new genes encode proteins involved in biosynthesis of yW at position 37 of tRNAPhe. In addition, biosyntheses of other modifications in tRNAPhe (Figure 1B) were not affected by the absence of yW, indicating that yW is not required for biosynthesis of other modifications. We have renamed YPL207w, YML005w, YGL050w and YOL141w as TYW1, TYW2, TYW3 and TYW4 (tRNA-yW synthesizing protein 1–4), respectively. To determine the mass of each modification intermediate of yW, the purified tRNAPhe was subjected to RNase T1 digestion and analyzed by LC/MS (T1 mapping), as shown in Figure 2B. In the case of wild-type tRNAPhe, the anticodon-containing RNA fragment (ACmUGmAAyWAΨm5CUGp, MW 4167) produced by RNase T1 digestion (Figure 1B) was clearly observed as a triply charged ion with m/z 1388 (Figure 2B). The mass of the same RNA fragment in tRNAPhe obtained from the ΔTYW1 strain was MW 3956 (m/z 1317), which is the mass of a fragment having m1G37 instead of yW37 (Figure 2B). Appearance of m1G was also confirmed by nucleoside analysis of purified tRNAPhe from the ΔTYW1 strain (Supplementary Figure S2A). The data clearly demonstrate that TYW1 encodes a protein responsible for the second step of yW synthesis (see Figure 6). According to T1 mapping of purified tRNAPhe from the other three deletion strains, ΔTYW2, ΔTYW3 and ΔTYW4 generated the modification intermediates yW-187 (yW minus 187 Da, MW 321), yW-86 or yW-14 (yW minus 86 or 14 Da, MW 422 or 494), and yW-72 (yW minus 72 Da, MW 436), respectively, judging from changes in the mass of each anticodon-containing fragment (Figure 2B). These intermediates were also confirmed by nucleoside analysis of the purified tRNAPhe using LC/MS (Supplementary Figure S2B–D). It is intriguing that tRNAPhe from ΔTYW3 strain has two yW intermediates, yW-86 and yW-14. It is likely that yW-86 is a real intermediate at this step and yW-14 is a product by skipping TYW3-dependent reaction. Judging from the intensity of the mass chromatogram, about 50% of tRNAPhe has yW-14 instead of yW-86. Chemical structures of the modification intermediates of yW To further define the chemical structures of the modification intermediates of yW, nucleosides from each purified mutant tRNAPhe were subjected to LC/MS/MS analysis to observe product ions of the bases produced by collision-induced dissociation (CID). The structures of the compounds can then be determined by referring to the reported MS/MS spectra, since characteristic patterns of the product ions are highly correlated to the chemical structure. When yWpA and its intermediates are ionized as proton adducts (MH+) by electrospray ionization (ESI), base fragments of yW and its derivatives are spontaneously generated in the ion source. The protonated base fragment (BH2+) of the each tRNA was selected as a parent mass and decomposed in collision with helium gas to obtain the spectrum of product ions (Supplementary Figure S3). Most of our data could be interpreted by referring to the CID spectra for chemically synthesized yW derivatives, part of the pioneering work by J McCloskey's group (Zhou et al, 2004a, 2004b). Interpretation of each CID spectrum and determination of chemical structure of each yW intermediate are described in the legend for Supplementary Figure S3. The determined chemical structures for yW-187, yW-86, yW-72 and yW-14 are shown in Figure 6 and Supplementary Figure S3. TYW genes encode highly conserved proteins in eukaryote Using each yeast TYW gene as a query, we have retrieved homologs from S. pombe, human, mouse and Arabidopsis thaliana. The sequence alignments (Figure 3A, Figure 3B and Figure 3C) show that these proteins share many conserved regions. Figure 3.Sequence alignments of the TYW proteins. Each TYW protein is aligned with a set of protein homologs from S. cerevisiae (Scere), S. pombe (Spomb), Homo sapiens (Human), Mus musculus (Mouse), A. thaliana (Athal) and O. sativa (Osati). Multiple alignment of each sequence was carried out by Clustal X (Thompson et al, 1997) and displayed by Genedoc multiple sequence alignment editor (Nicholas et al, 1997). White letters in black boxes represent amino-acid residues identical in all species, while white letters in gray boxes represent residues with ∼80% homology. Black letters in gray boxes represent residues with ∼60% homology. (A) Sequence alignment of TYW1 with its homologs. Two conserved domains, flavodoxin-1 domain and Radical-Ado-Met domain, are underlined. The boxed region represents the [4Fe–4S] motif. Positions for site-directed mutagenesis are indicated by arrowheads. Download figure Download PowerPoint Figure 4.(B) Sequence alignment of TYW2 and TYW3 with its homologs. A. thaliana and O. sativa homologs are large fusion proteins including TYW3 and the C-terminal region of TYW4 and TYW2 (TYW3-4C-2). A set of TYW2 homologs and TYW3 homologs are aligned with plant TYW3-4C-2. The Met-10+-like protein family domain in TYW2 is indicated by a line. The C-terminal region of TYW4 in plant TYW3-4C-2 is shaded. Download figure Download PowerPoint Figure 5.(C) Sequence alignment of TYW4 with its homologs. The conserved domain, LCM, is indicated by a line. The boxed regions are Ado-Met-binding motifs, including motif I–III and post-I. The residues important for protein carboxyl methyltransferase activity are indicated by arrowheads. The C-terminal region of TYW4 is shaded. (D) Domain structure of the plant TYW3-4C-2 protein. The plant TYW3-4C-2 protein is a large fusion protein composed of TYW3, the CTD of TYW4 and TYW2. Download figure Download PowerPoint TYW1 (810 aa in S. cerevisiae) and its homologs contain two conserved domains (Figure 3A), a flavodoxin-1 domain in the N-terminus (pfam00258) and a radical-Ado-Met domain in the C-terminus (pfam04055). Flavodoxin is a protein that includes an iron–sulfur (Fe–S) cluster and employs FMN as a cofactor (Wakabayashi et al, 1989). Radical Ado-Met proteins contain a conserved CxxxCxxC motif (Figure 3A), which is responsible for binding a [4Fe–4S] cluster, as a catalytic core for diverse reactions (Sofia et al, 2001). These structural features of the conserved domains suggest that TYW1 is an Fe–S cluster protein responsible for ring formation of the tricyclic base of yW. TYW2 (462 aa in S. cerevisiae) and its homologs contain a conserved Ado-Met-binding motif found in Met-10+-like protein family (lower part of Figure 3B) (Niewmierzycka and Clarke, 1999). In addition, TYW2 was found to be a member of COG2520, which also includes TRM5. The data indicate that TYW2 is an Ado-Met-dependent enzyme. TYW3 (273 aa in S. cerevisiae) was found to be a highly conserved protein (pfam02676/DUF207, COG1590), but its function is unknown. A. thaliana and Oryza sativa homologs of TYW3 have a long extended C-terminal domain (CTD) of about 1000 aa (Figure 3B), and it is interesting to note that the long extended CTD of the plant TYW3 actually contains the CTD of TYW4 as well as the entire sequence of TYW2. Thus, the plant homologs appear to be a large fusion protein including TYW3, the CTD of TYW4 and TYW2, which we have named TYW3-4C-2 (Figure 3B and D). Additionally, the plant TYW4 is a short protein without the CTD (Figure 3C). These data suggest that TYW3-4C-2 acts as a multienzymatic complex for yW synthesis in A. thaliana and O. sativa. Schematic depiction of the topological orientation of TYW2, TYW3, TYW4 and TYW3-4C-2 is shown in Figure 3D. TYW4 (695 aa in S. cerevisiae) has a conserved leucine carboxyl methyltransferase (LCM) domain (pfam04072) in its N-terminal region (Figure 3C). TYW4 has an apparent paralog, PPM1 (protein phosphatase methyltransferase 1), which is a yeast methyltransferase responsible for methylesterification of the C-terminal Leu residue of PP2A (protein phosphatase 2A) (Wu et al, 2000; Kalhor et al, 2001). Thus, YOL141w (TYW4) had been previously named PPM2 (Kalhor et al, 2001; Wei et al, 2001), but its biochemical function was never demonstrated directly. The PPM family has three highly conserved Ado-Met-binding motifs (motifs I, II and III). This finding strongly suggests that, using Ado-Met as a methyl donor, TYW4 catalyzes methylation of the carboxyl group of the methionine moiety in yW-72 to convert it to yW-58 (see Figure 6). Biogenesis of an Fe–S cluster is required for TYW1 function in yW synthesis The S. cerevisiae NFS1 gene encodes a cysteine desulfurase involved in Fe–S cluster biogenesis. To test whether yW synthesis requires biosynthesis of an Fe–S cluster, we analyzed total nucleosides from S. cerevisiae YN101 cells in which expression of NFS1 is controlled under the GAL1 promoter (Nakai et al, 2004). We previously demonstrated that inhibition of NFS1 expression resulted in a severe reduction of thio-modifications in both mitochondrial and cytoplasmic tRNAs (Nakai et al, 2004). When cells were cultured in galactose-containing medium, yWpA was clearly observed (Figure 4A). However, when the expression of NFS1 was downregulated by culturing cells in glucose-containing medium, yWpA was severely reduced (Figure 4A). These data demonstrated that biosynthesis of Fe–S clusters by NFS1 is absolutely required for yW synthesis. The Fe–S cluster in TYW1 is likely to be critical for the formation of the tricyclic base of yW. Figure 6.Fe–S cluster in TYW1 involved in yW synthesis. (A) LC/MS analysis of the total nucleosides in the YN101 cells in which expression of NFS1 is controlled under the GAL1 promoter. The left- and right-hand graphs describe the LC/MS chromatograms of yWpA from YN101 cultured with galactose and glucose, respectively. The upper panels are the UV trace at 254 nm. The lower panels are mass chromatograms detecting the yW-base (m/z 377). The relative amount of yWpA in both conditions was normalized to the pseudouridine (Ψ) content. (B) Complementation test of ΔTYW1 by introducing a series of mutant plasmid pTYW1 in which critical residue for Fe–S cluster is mutated. Total nucleosides from each strain were analyzed by LC/MS to detect yWpA. Merged mass chromatograms of yWpA (m/z 838) and yW-base (m/z 377) are shown. Arrows indicate the retention time for yWpA. yWpA was restored by introducing wild-type pTYW1 or pTYW1-E532A. Other mutant plasmids did not complement yW synthesis in ΔTYW1 strain. Download figure Download PowerPoint The radical-Ado-Met domain in the C-terminal region of TYW1 has a conserved C479xxxC483xxC486 motif (Figure 3A) for [4Fe–4S] cluster binding. In addition, E532 and E550 are candidate residues for GGE motif in the radical Ado-Met domain (Pierrel et al, 2004). To clarify functional importance of these conserved residues, site-directed mutagenesis of Cys479, Cys483, Cys486, Glu532 and Glu550 was carried out. The plasmid expressing TYW1 (pTYW1) with each mutation was introduced into ΔTYW1 strain to test complementation activity of each mutant for yW synthesis. As shown in Figure 4B, yW in the ΔTYW1 strain was restored by the introduction of wild-type pTYW1. No activity for yW synthesis could be observed when pTYW1 with C479A, C483A, C486A or E550A mutation was introduced. E532A mutation did not affect TYW1 functionality. These results revealed that three conserved Cys in the CxxxCxxC motif as well as E550 in the GGE motif is critical for yW synthesis. Taken together with the result of NFS1 data, biogenesis of an Fe–S cluster in TYW1 is required for TYW1 function in yW synthesis. Reconstitution of yW synthesis in vitro using recombinant TYW2, TYW3 and TYW4 Next, we performed in vitro reconstitution of yW synthesis using recombinant TYW proteins. A hexahistidine-tagged TYW2, TYW3 and TYW4 were expressed in S. cerevisiae and purified using an Ni+-chelating column. tRNAPhe having yW intermediate purified from each deletion strain was employed as a substrate for each step of yW synthesis in vitro. In vitro reconstitution of the tRNAPhe having yW-187 by recombinant TYW2 was carried out in the presence or absence of Ado-Met. As shown in Figure 5A, in the absence of Ado-Met, the substrate tRNA yielded an anticodon-containing RNA fragment whose mass is unchanged (ACmUGmAA(yW-187)AΨm5CUGp, MW 3980). In the presence of Ado-Met, yW-187 in tRNAPhe was clearly converted to yW-86 (101 Da increase). No other product, such as yW-173 (14 Da increase), was detected. The data revealed that recombinant TYW2 has an activity to transfer α-amino-α-carboxypropyl group from Ado-Met to the side chain at the C-7 position of the yW-base. This result is consistent with a previous observation that α-amino-α-carboxypropyl group of yW originates from methionine or Ado-Met in yeast cells (Munch and Thiebe, 1975). Figure 7.In vitro reconstitution of yW synthesis using recombinant TYW2, TYW3 and TYW4. LC/MS fragment analysis of RNaseT1-digested tRNAPhe whose yW is partially reconstituted by recombinant TYW2 (A), TYW3 (B) and TYW4 (C) in the presence or absence of Ado-Met. All mass chromatograms are produced by integration of triply (−3), quadruply (−4) and quintuply (−5) charged ions of anticodon-containing fragments. (A) tRNAPhe having yW-187 treated by recombinant TYW2 in the absence (left panels) or in the presence (right panels) of Ado-Met. The top and bottom graphs show mass chromatographs for anticodon-containing fragments containing yW-187 (m/z 1325+994+795) and yW-86 (m/z 1359+1019+815), respectively. (B) tRNAPhe having yW-86 (and yW-14) treated by recombinant TYW3 in the absence (left panels) or in the presence (right panels) of Ado-Met. The top, middle and bottom graphs show mass chromatographs for anticodon-containing fragments containing yW-86 (m/z 1359+1019+815), yW-72 (m/z 1364+1023+818) and yW-14 (m/z 1383+1037+829), respectively. (C) tRNAPhe having yW-72 treated by recombinant TYW4 in the absence (left panels) or in the presence (middle and right panels) of Ado-Met. Experiment in the right panels was carried out by highly purified recombinant TYW4. The top, middle and bottom graphs show mass chromatographs for anticodon-containing fragments containing yW-72 (m/z 1364+1023+818), yW-58 (m/z 1368+1026+821) and yW (m/z 1388+1041+832), respectively. Deconvolution spectra of the mass spectra (RT 34–36 min) for the anticodon-containing fragments having yW-72 reconstituted in the absence (left bottom) or presence (right bottom) of Ado-Met. The fragment with yW-58 was clearly detected in the presence of Ado-Met. Download figure Download PowerPoint Figure 8.Biosynthetic pathway of yW in S. cerevisiae tRNAPhe. TRM5 methylates G37 of tRNAPhe to produce m1G37, utilizing Ado-Met as a methyl donor. TYW1, an Fe–S cluster protein, may catalyze tricyclic formation of yW using Ado-Met and FMN as cofactors. TYW2 transfers α-amino-α-carboxypropyl group from Ado-Met to the side chain at the C-7 position of the yW-187 to produce yW-86. TYW3 methylates N-4 position of yW-86 in Ado-Met-dependent manner to yield yW-72. Finally, TYW4, an Ado-Met-dependent carboxymethyltransferase, methylates the α-carboxy group of yW-72 to form yW-58, which triggers methoxycarbonylation of α-amino group of yW-58 to complete yW. TYW4 is likely responsible for the last two steps. Substrate for methoxycarbonylation and requirement of partner protein still remain to be investigated. Download figure Download PowerPoint In ΔTYW3 strain, tRNAPhe has two yW intermediates, yW-86 and yW-14 (Figure 2B). As shown in Figure 5B, recombinant TYW3 clearly methylated yW-86 to yield yW-72 in Ado-Met-dependent manner. Thus, TYW3 is an N-4 methyltransferase for yW synthesis. On the other hand, yW-14 was not methylated by TYW3 (no yW product is obtained). This suggests that complete synthesis of the side chain at C-7 position inhibits N-4 methylation by TYW3. The strong homology between TYW4 and PPM1 prompted us to test the evolutionary link between RNA methylase and protein methylase. tRNAPhe having yW-72 purified from ΔTYW4 cells was employed as a substrate for the yW reconstitution. In the presence of Ado-Met (Figure 5C, middle panels), UV trace of the chromatogram showed that about 60% of the anticodon-containing RNA fragment with yW-72 was converted to yield the fragments with yW-58 and yW in which the mass had increased by 14 and 72 Da, respectively. We cou
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