Glucose starvation induces the accumulation of m¹G37-bearing tRNA^Phe in the nucleus. (A) LC/MS analyses of the total nucleosides in the tRNA^Phe molecules isolated from yeast cells (BY4742) after 0h (uppermost panels), 1h (second panels), 2h (third panels), and 3h (fourth panels) of glucose starvation. As a control, the nucleosides of the tRNA^Phe molecules that were isolated from ΔTYW1 are shown in the lowermost panels. The Left panels show the mass chromatograms of the base-related ion () of monomethylated guanosine (m/z 166), which detect m¹G and m²G. The Right panels show the mass chromatogram of the proton adduct of yWpA (m/z 838). (B) LC/MS RNA fragment analyses of the RNaseA-digested tRNA^Phe molecules. The panels show the mass chromatograms that detect the doubly charged ions of the anticodon-containing fragments that bear m¹G (Left panels, m/z 1,013.65) and yW (Right panels, m/z 1,119.20). The vertical axes display the apparent ratio of m¹G or yW, which is calculated on the basis of the intensity of the m¹G- and yW-containing fragments. (C) The TRM5-GFP (Upper) and TRM5-NES-GFP (Lower) constructs are shown. The photos show the phase contrast (PH), GFP-fluorescence, Hoechst 33342, and merged images for each strain. (D) LC/MS analyses of the total nucleosides in the tRNA^Phe molecules that were isolated from the TRM5-GFP (Upper) and TRM5-NES-GFP (Lower) strains after 4h of glucose starvation. The Left and Right panels show the mass chromatograms of the base-related ion () of monomethylated guanosine (m/z 166) and the proton adducts of yWpA (m/z 838), respectively.

Schematic depiction of the tRNA^Phe maturation pathway in S. cerevisiae. The primary transcript of tRNA^Phe (pri-tRNA^Phe) is end-matured by 5′/3′ trimming and the addition of CCA to form precursor tRNA^Phe (pre-tRNA^Phe). Ten positions are then modified in the nucleus, after which the pre-tRNA^Phe is exported to the cytoplasm, where the intron is removed by the splicing machinery at the mitochondrial outer membrane. The spliced tRNA^Phe is then modified by the cytoplasmic methyltransferases Trm11p/Trm112p and Trm7p, which make the m²G10 and Cm32/Gm34 modifications, respectively. The tRNA^Phe is imported into the nucleus, where G37 is modified into m¹G37 by Trm5p. The postspliced m¹G37-bearing tRNA^Phe is then reexported to the cytoplasm, where yW37 is generated through consecutive reactions catalyzed by Tyw1p, Tyw2p, Tyw3p, and Tyw4p.

Secondary structures of precursor and mature tRNA^Phe from S. cerevisiae and the biosynthesis of wybutosine (yW). (A) Secondary structures of the precursor (Left) and mature (Right) forms of S.cerevisiae cytoplasmic tRNA^Phe. The modifications are shown. With regard to the precursor tRNA^Phe, it is the intron-bearing precursor that accumulates in the mutant rna1 (2). The unmodified positions G10, C32, G34, and G37 are boxed, arrows indicate the splicing sites, and the intron sequence is shown in small letters. In the mature tRNA^Phe, the yW modification is boxed. (B) Pathway of yW biosynthesis in S.cerevisiae tRNA^Phe. The chemical structures of guanine (G), N¹-methylguanine (m¹G), and wybutosine (yW) are shown along with the modification enzymes and the locations in which the modifications occur. Thus, Trm5p methylates G37 in the nucleus to generate m¹G. In the cytoplasm, yW37 is synthesized by sequential reactions catalyzed by Tyw1p, Tyw2p, Tyw3p, and Tyw4p.

Accumulation of nuclear m¹G37-bearing tRNA^Phe after the overexpression of Trz1p. (A) The tagged Trz1p (indicated by the arrow) that was obtained after affinity purification by using IgG sepharose was analyzed by SDS-PAGE. Shown are the whole cell extract (lane 1), the flow through (lane 2), and the eluted IgG sepharose fraction (lane 3). The heavy chain of IgG is asterisked. (B) The RNA components extracted from the tagged Trz1p fraction (lane 2) were separated on denatured PAGE. The pre-tRNAs assigned are indicated. Total RNA (lane 1) is shown for comparison. (C) LC/MS RNA fragment analyses of the RNase A-digested spliced tRNA^Phe molecules that were isolated from the RNAs that coprecipitated with the tagged Trz1p. The Left and Right panels show the mass chromatograms that detect the doubly charged ions of the anticodon-containing fragments that bear m¹G (Left panel, m/z 1,013.65) and yW (Right panel, m/z 1,119.20), respectively. The vertical axes display the apparent ratios of m¹G to yW, which were calculated from the intensities of the m¹G- and yW-containing fragments. The asterisk indicates an unassigned RNA fragment. (D) LC/MS analyses of the total nucleosides in the spliced tRNA^Phe molecules that were isolated from the Trz1p plasmid-harboring yeast strain after it was grown in conditions that repressed (Upper) or induced (Lower) plasmid expression. The left and righthand panels show the mass chromatograms of the base-related ion () of monomethylated guanosine (m/z 166) and the proton adduct of yWpA (m/z 838), respectively.