Biosynthesis of wybutosine, a hyper-modified nucleoside in eukaryotic phenylalanine tRNA

Abstract

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.

Figure 1

Chemical structure of wybutosine (yW) and secondary structure of tRNA^Phe. (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 tRNA^Phe 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.

Figure 2

Mass spectrometric analysis of total nucleosides and tRNA^Phe 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 tRNA^Phe 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.

Figure 3a

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.

Figure 3b

(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.

Figure 3c

(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.

Figure 4

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.

Figure 5

In vitro reconstitution of yW synthesis using recombinant TYW2, TYW3 and TYW4. LC/MS fragment analysis of RNaseT₁-digested tRNA^Phe 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) tRNA^Phe 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) tRNA^Phe 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) tRNA^Phe 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.

Figure 6

Biosynthetic pathway of yW in S. cerevisiae tRNA^Phe. TRM5 methylates G37 of tRNA^Phe 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.

Figure 7

Schematic depiction of a multienzymatic complex of TYW proteins. In S. cerevisiae and in mammals, TYW2, 3 and 4 might coassemble with tRNA^Phe to carry out the sequential reactions of yW synthesis. In plants, a large fusion protein of TYW3-4C-2 and TYW4 might coassemble with tRNA^Phe.