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

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.

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; tRNA^Glu(UUC), the glutamic tRNA analyzed in this study. (B) Gel-purified cy and mt tRNAs: cy total tRNA, cy tRNA^Glu, mt tRNA^Glu, cy tRNA^Gln, mt tRNA^Gln, cy tRNA^Lys and mt tRNA^Lys in lanes 1–7, respectively. (C) RNA sequencing of purified cy and mt tRNAs^Glu 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 tRNA^Glu (U*). No band appears at the same position on the alkaline ladder of mt tRNA^Glu, indicating the presence of a 2′-O-methyluridine derivative (U*m).

Fig. 2. LC/MS nucleoside analysis of tRNA^Glu. (A and B) Chromatograms for cy tRNA^Glu and mt tRNA^Glu, 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 tRNA^Glu and a dimer form of mcm⁵Um with the adjacent uridine (mcm⁵UmpU) in mt tRNA^Glu. (C and D) Mass spectra for (C) mcm⁵s²U in cy tRNA^Glu and for (D) mcm⁵UmpU in mt tRNA^Glu. The chemical structure of each nucleoside is shown within the spectrum.

Fig. 3. APM gel electrophoresis of cy and mt tRNAs visualized by ethidium bromide staining. (A) Cy and mt tRNAs^Glu on an APM gel. (B) Cy and mt tRNAs^Gln and cy and mt tRNA^Lys 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 tRNA^Glu and cy tRNA^Gln showed apparent retardation on the APM gel.

Fig. 4. Comparison of RNA sequences in the anticodon arms of cy and mt tRNAs. The anticodon wobble modifications are mcm⁵s²U for both cy tRNA^Glu and cy tRNA^Gln, while they are mcm⁵Um for the mitochondrial counterparts. In addition, a trace amount of tRNAs with mcm⁵U modification is found in both cy tRNA^Glu and cy tRNA^Gln. For tRNA^Lys, the anticodon wobble modifications are mcm⁵U and mcm⁵Um for cy and mt tRNAs, respectively. In tRNA^Lys, Cm modification at position 32 was found in mt tRNA but not in the cytosolic counterpart.

Fig. 5. Detection of mcm⁵U in cy tRNA^Glu as the modification intermediary for both mcm⁵s²U and mcm⁵Um. (A) LC/MS nucleoside analysis chromatograms for cy tRNA^Glu: 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.

Fig. 6. (A and B) In vitro importation assays of affinity-purified mt tRNA^Lys and cy tRNA^Glu. 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 tRNA^Glu 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 tRNA^Lys (diamonds), native cy tRNA^Glu (filled circles) and T7-transcribed tRNA^Glu (open circles).

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.