title: 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.
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Building FSB-301, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan.
authors.forename: Tomonori
authors.initials: T
authors.lastname: Kaneko
authors.forename: Takeo
authors.initials: T
authors.lastname: Suzuki
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Kapushoc
authors.forename: Mary Anne
authors.initials: MA
authors.lastname: Rubio
authors.forename: Jafar
authors.initials: J
authors.lastname: Ghazvini
authors.forename: Kimitsuna
authors.initials: K
authors.lastname: Watanabe
authors.forename: Larry
authors.initials: L
authors.lastname: Simpson
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D000926:Anticodon / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism; D007891:Leishmania / Q000235:genetics*; D013058:Mass Spectrometry; D008928:Mitochondria / Q000235:genetics*; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D012323:RNA Processing, Post-Transcriptional; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism*
chemical_list: D000926:Anticodon; D016053:RNA, Protozoan; D012343:RNA, Transfer