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

Abstract

Abstract2-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.

Regulation of mitochondrial iron homeostasis by sideroflexin 2

“…In addition to respiratory complex activities, we examined the level of the 2-methylthio modification of mitochondrial tRNA by mass spectrometry. This modification is mediated by Cdk5 regulatory subunit-associated protein 1 (CDK5RAP1), a mitochondrial iron–sulfur cluster-dependent tRNA-modifying enzyme [ 41 , 42 ]. Similar to complex I activity, the level of the 2-methylthio modification of tRNA did not differ between SFXN2 -KO and control cells (Fig.…”

“…The level of the 2-methylthio- N 6 -isopentenyladenosine modification of mitochondrial tRNA was measured using liquid chromatography-mass spectrometry (Shimadzu LCMS-8050) as previously described [ 42 ]. Briefly, total RNA was purified from control and SFXN2 -KO cells.…”

2-Methylthio Conversion of N6-Isopentenyladenosine in Mitochondrial tRNAs by CDK5RAP1 Promotes the Maintenance of Glioma-Initiating Cells

“…Because the biochemical reaction of CDK5RAP1-mediated 2-methylthio conversion of i 6 A is strictly regulated within the mitochondria and is detected solely in mt-tRNAs (Fakruddin et al., 2017) and because ms 2 i 6 A is predominantly enriched in cell culture medium (∼9.6 times more than the concentration inside cells, Figure S5B), these data suggest that CDK5RAP1 decreases [i 6 A] by promoting the 2-methylthio conversion of i 6 A in the mitochondria and that the consequent ms 2 i 6 A derived from degraded mt-tRNAs is excreted (Figure S5C).…”

S-adenosylmethionine tRNA modification: unexpected/unsuspected implications of former/new players

“…1 ) 49 . Indeed, its main RS activity is performed in the mitochondria 50 , although in previous work, it was hypothesized that this enzyme could have a role in the modification of cytoplasmic tRNA 51 . The most important contributing element to modification dynamics might be the level of intracellular compartmentalization, which is present in all organisms but is the most represented in Eukarya, where two genome-containing compartments - the nucleus and the mitochondria - are found 52 .…”