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

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Oxford University Press

RESUMO

In Leishmania tarentolae, all mitochondrial tRNAs are encoded in the nuclear genome and imported from the cytosol. It is known that tRNAGlu(UUC) and tRNAGln(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 (mcm5s2U), whereas mt tRNAs have 5- methoxycarbonylmethyl-2′-O-methyluridine (mcm5Um). In addition, a trace portion (4%) of cy tRNAs was found to have 5-methoxycarbonylmethyluridine (mcm5U) 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 tRNALys(UUU) from the cytosol and found mcm5U at its wobble position, while its mitochondrial counterpart has mcm5Um. Mt tRNALys and in vitro transcribed tRNAGlu were imported much more efficiently into isolated mitochondria than the native cy tRNAGlu 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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