Cdk5rap1-mediated 2-methylthio-N6-isopentenyladenosine modification is absent from nuclear-derived RNA species.

2-Methylthio-N6-isopentenyl modification of adenosine (ms2i6A) 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 i6A to ms2i6A 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 ms2i6A is present abundantly in nuclear-derived RNA species such as microRNAs, but not in tRNA fractions. To fully understand the molecular property of ms2i6A, the existence of non-canonical ms2i6A must be carefully validated. In the present study, we examined ms2i6A in total RNA purified from human and murine ρ0 cells, in which mitochondrial DNA-derived tRNAs were completely depleted. The ms2i6A was not detected in these cells at all. We generated a monoclonal antibody against ms2i6A and examined ms2i6A in murine RNAs using the antibody. The anti-ms2i6A antibody only reacted with the tRNA fractions and not in other RNA species. Furthermore, immunocytochemistry analysis using the antibody showed the predominant localization of ms2i6A in mitochondria and co-localization with the mitochondrial elongation factor Tu. Taken together, we propose that ms2i6A is a mitochondrial tRNA-specific modification and is absent from nuclear-encoded RNA species.