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. 2003 Feb 3;22(3):657-67.
doi: 10.1093/emboj/cdg066.

Wobble modification differences and subcellular localization of tRNAs in Leishmania tarentolae: implication for tRNA sorting mechanism

Tomonori Kaneko  1 , Takeo Suzuki, Stephen T Kapushoc, Mary Anne Rubio, Jafar Ghazvini, Kimitsuna Watanabe, Larry Simpson, Tsutomu Suzuki
Affiliations

Wobble modification differences and subcellular localization of tRNAs in Leishmania tarentolae: implication for tRNA sorting mechanism

Tomonori Kaneko et al. EMBO J. .

Abstract

In Leishmania tarentolae, all mitochondrial tRNAs are encoded in the nuclear genome and imported from the cytosol. It is known that tRNA(Glu)(UUC) and tRNA(Gln)(UUG) are localized in both cytosol and mitochondria. We investigated structural differences between affinity-isolated cytosolic (cy) and mitochondrial (mt) tRNAs for glutamate and glutamine by mass spectrometry. A unique modification difference in both tRNAs was identified at the anticodon wobble position: cy tRNAs have 5-methoxycarbonylmethyl-2- thiouridine (mcm(5)s(2)U), whereas mt tRNAs have 5- methoxycarbonylmethyl-2'-O-methyluridine (mcm(5)Um). In addition, a trace portion (4%) of cy tRNAs was found to have 5-methoxycarbonylmethyluridine (mcm(5)U) at its wobble position, which could represent a common modification intermediate for both modified uridines in cy and mt tRNAs. We also isolated a trace amount of mitochondria-specific tRNA(Lys)(UUU) from the cytosol and found mcm(5)U at its wobble position, while its mitochondrial counterpart has mcm(5)Um. Mt tRNA(Lys) and in vitro transcribed tRNA(Glu) were imported much more efficiently into isolated mitochondria than the native cy tRNA(Glu) in an in vitro importation experiment, indicating that cytosol-specific 2-thiolation could play an inhibitory role in tRNA import into mitochondria.

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Figures

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Fig. 1. Fractionation and isolation of cy and mt tRNAs. (A) Northern analysis of L.tarentolae RNA. Cy and mt RNAs (as indicated) were separated in denaturing 8% polyacrylamide gels. The ethidium bromide-stained gel is shown on the left, with the sizes and positions of several marker RNAs indicated. The results of northern hybridizations with oligonucleotide probes specific for several RNAs are shown: slRNA, the spliced leader RNA; gRNA, the RPS12 block I guide RNA; tRNAGlu(UUC), the glutamic tRNA analyzed in this study. (B) Gel-purified cy and mt tRNAs: cy total tRNA, cy tRNAGlu, mt tRNAGlu, cy tRNAGln, mt tRNAGln, cy tRNALys and mt tRNALys in lanes 1–7, respectively. (C) RNA sequencing of purified cy and mt tRNAsGlu by the method of Donis-Keller (1980) for comparison of the cy (left) and mt (right) tRNA sequences. Each 3′-labeled tRNA was partially digested with an RNase: RNase T1 for G; RNase U2 for A; RNase PhyM for A/U; and RNase CL3 for C. The digests were electrophoresed with an undigested control (–E) and alkaline-treated ladder (Al). Filled circles indicate a band corresponding to position 34 on the alkaline ladder of cy tRNAGlu (U*). No band appears at the same position on the alkaline ladder of mt tRNAGlu, indicating the presence of a 2′-O-methyluridine derivative (U*m).
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Fig. 2. LC/MS nucleoside analysis of tRNAGlu. (A and B) Chromatograms for cy tRNAGlu and mt tRNAGlu, respectively. Top: UV chromatograms for nucleosides. Bottom: mass chromatograms for modified uridines with mass filters at m/z 332.9 and 636.9 to detect, respectively, mcm5s2U in cy tRNAGlu and a dimer form of mcm5Um with the adjacent uridine (mcm5UmpU) in mt tRNAGlu. (C and D) Mass spectra for (C) mcm5s2U in cy tRNAGlu and for (D) mcm5UmpU in mt tRNAGlu. The chemical structure of each nucleoside is shown within the spectrum.
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Fig. 3. APM gel electrophoresis of cy and mt tRNAs visualized by ethidium bromide staining. (A) Cy and mt tRNAsGlu on an APM gel. (B) Cy and mt tRNAsGln and cy and mt tRNALys on a polyacrylamide gel with (left) or without (right) APM. 5′-32P-labeled tRNAs separated on the gels were visualized by the imaging analyzer (Fuji photosystem). Only cy tRNAGlu and cy tRNAGln showed apparent retardation on the APM gel.
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Fig. 4. Comparison of RNA sequences in the anticodon arms of cy and mt tRNAs. The anticodon wobble modifications are mcm5s2U for both cy tRNAGlu and cy tRNAGln, while they are mcm5Um for the mitochondrial counterparts. In addition, a trace amount of tRNAs with mcm5U modification is found in both cy tRNAGlu and cy tRNAGln. For tRNALys, the anticodon wobble modifications are mcm5U and mcm5Um for cy and mt tRNAs, respectively. In tRNALys, Cm modification at position 32 was found in mt tRNA but not in the cytosolic counterpart.
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Fig. 5. Detection of mcm5U in cy tRNAGlu as the modification intermediary for both mcm5s2U and mcm5Um. (A) LC/MS nucleoside analysis chromatograms for cy tRNAGlu: Top: UV trace at 260 nm in the region of 27–36 min. Middle and bottom: mass chromatograms for m/z 316.9 and 332.9 to detect, respectively, mcm5U (RT 29.37) and mcm5s2U (RT 34.05). The longer retention time of each nucleotide in the mass chromatogram compared with that in the UV trace is due to a time lag between the UV detector and mass spectrometer in the LC/MS system. (B) Mass spectrum for mcm5U at RT 29.37 with the chemical structure of mcm5U. The proton adduct and base fragment are detected.
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Fig. 6. (A and B) In vitro importation assays of affinity-purified mt tRNALys and cy tRNAGlu. Both pCp-labeled tRNAs were gel purified and resuspended at the same concentration and specific activity. Lanes 1–5: increasing concentrations (263, 526, 789, 1315 and 2105 fmol) of tRNA incubated with mitochondria and digested with MNase. Lane 6: 1000 fmol of tRNA digested with MNase. Lane 7: 10 fmol of the input RNA (IN). (D and E) In vitro importation assays of cy tRNAGlu and its transcript. Lanes 1–5: increasing concentrations (1000, 2000, 3000, 4000 and 6000 fmol) of RNA incubated with mitochondria and digested with MNase. Lane 6: 1000 fmol of tRNA digested with MNase. Lane 7: 19 fmol of the input RNA (IN). The RNAs were resolved by electrophoresis on 7 M urea/10% acrylamide gels for (A) and (B), or 7 M urea/8% acrylamide gels for (D) and (E). The migration of the full-length RNAs is indicated by an arrow. (C and F) The results showing nuclease protection of the pCp-labeled tRNAs incubated with isolated L.tarentolae mitochondria. Full-length RNAs protected from nuclease digestion were quantitated for native mt tRNALys (diamonds), native cy tRNAGlu (filled circles) and T7-transcribed tRNAGlu (open circles).
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Fig. 7. Schematic depiction of the proposed subcellular distribution mechanism for tRNAs that read NAG codons in L.tarentolae. Precursor tRNAs are transcribed from genes in the nucleus and are exported into the cytosol with the mcm5U modification at the wobble position. Cytosolic tRNA is matured by 2-thiolation of the modification intermediate and localized in the cytosol. A portion of the modification intermediate is imported into the mitochondrion, where 2′-O-methylation of mcm5U follows.
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