Mammalian NSUN2 introduces 5-methylcytidines into mitochondrial tRNAs

ABSTRACTPost-transcriptional modifications in mitochondrial tRNAs (mt-tRNAs) play critical roles in mitochondrial protein synthesis, which produces respiratory chain complexes. In this study, we used mass spectrometric analysis to map 5-methylcytidine (m5C) at positions 48–50 in eight mouse and six human mt-tRNAs. We also confirmed the absence of m5C in mt-tRNAs isolated fromNsun2knockout (KO) mice, as well as fromNSUN2KO human culture cells. In addition, we successfully reconstituted m5C at positions 48–50 of mt-tRNAin vitrowith NSUN2 protein in the presence ofS-adenosylmethionine (SAM). Although NSUN2 is predominantly localized to the nucleus and introduces m5C into cytoplasmic tRNAs and mRNAs, structured illumination microscopy (SIM) clearly revealed NSUN2 foci inside mitochondria. These observations provide novel insights into the role of NSUN2 in the physiology and pathology of mitochondrial functions.