Deficient methylation and formylation of mt-tRNA wobble cytosine in a patient carrying mutations in NSUN3

(2016) Deficient methylation and formylation of mt-tRNAMet wobble cytosine in a patient carrying mutations in NSUN3. This version is made available in accordance with publisher policies. Please cite only the published version using the reference above. Austria + The authors contributed equally. Abstract Epitranscriptome modifications are required for structure and function of RNA and defects in these pathways have been associated with human disease. Here, we identify the RNA target for the previously uncharacterized 5-methylcytosine (m 5 C) methyltransferase NSun3 and link m 5 C RNA modifications with energy metabolism. Using whole exome sequencing, we identified loss-of-function mutations in NSUN3 in a patient presenting with combined mitochondrial respiratory chain complex deficiency. Patient-derived fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of NSun3. We show that NSun3 is required for deposition of m 5 C at the anticodon loop in the mitochondrially-encoded transfer RNA methionine (mt-tRNA Met). Further, we demonstrate that m 5 C deficiency in mt-tRNA Met results in the lack of 5-formylcytosine (f 5 C) at the same tRNA position. Our findings demonstrate that NSUN3 is necessary for efficient mitochondrial translation and reveal that f 5 C in human mitochondrial RNA is generated by oxidative processing of m 5 C.