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 subunitassociated 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 noncanonical ms²i⁶A must be carefully validated. In the present study, we examined ms²i⁶A in total RNA purified from human and murine-0 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 tRNAspecific modification and is absent from nuclearencoded RNA species.