Cdk5rap1-mediated 2-methylthio-N(6)-isopentenyladenosine modification is absent from nuclear-derived RNA species

2-Methylthio-N(6)-isopentenyl modification of adenosine (ms(2)i(6)A) is an evolutionally conserved modification that is found in transfer RNAs (tRNAs). We have recently shown that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) specifically converts i(6)A to ms(2)i(6)A at position A37 of four mitochondrial DNA-encoded tRNAs, and that the modification regulates efficient mitochondrial translation and energy metabolism in mammals. Curiously, a previous study reported that ms(2)i(6)A is present abundantly in nuclear-derived RNA species such as microRNAs, but not in tRNA fractions. To fully understand the molecular property of ms(2)i(6)A, the existence of non-canonical ms(2)i(6)A must be carefully validated. In the present study, we examined ms(2)i(6)A in total RNA purified from human and murine ρ0 cells, in which mitochondrial DNA-derived tRNAs were completely depleted. The ms(2)i(6)A was not detected in these cells at all. We generated a monoclonal antibody against ms(2)i(6)A and examined ms(2)i(6)A in murine RNAs using the antibody. The anti-ms(2)i(6)A antibody only reacted with the tRNA fractions and not in other RNA species. Furthermore, immunocytochemistry analysis using the antibody showed the predominant localization of ms(2)i(6)A in mitochondria and co-localization with the mitochondrial elongation factor Tu. Taken together, we propose that ms(2)i(6)A is a mitochondrial tRNA-specific modification and is absent from nuclear-encoded RNA species.