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

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 (m5C) methyltransferase NSun3 and link m5C 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 m5C at the anticodon loop in the mitochondrially encoded transfer RNA methionine (mt-tRNAMet). Further, we demonstrate that m5C deficiency in mt-tRNAMet results in the lack of 5-formylcytosine (f5C) at the same tRNA position. Our findings demonstrate that NSUN3 is necessary for efficient mitochondrial translation and reveal that f5C in human mitochondrial RNA is generated by oxidative processing of m5C.

Post-transcriptional modifications in development and stem cells

“…Similar to m 5 C on DNA, m 5 C in RNA can also be further modified by oxidation processes. Methylation of mitochondrial tRNA Met by NSUN3 is required for the subsequent formation of 5-formylcytosine (f 5 C) at the same tRNA position (Nakano et al, 2016;Van Haute et al, 2016). Recent findings also suggest that TET-mediated oxidation reverses cytosine-5 methylation in vitro and 5-hydroxymethylcytosine (hm 5 C) has been detected in polyAenriched RNA in vivo (Delatte et al, 2016;Fu et al, 2014;Huber et al, 2015).…”

“…1) (Metodiev et al, 2014;Nakano et al, 2016;Schosserer et al, 2015;Sharma et al, 2013;Van Haute et al, 2016). The methylase activity of NSUN7 has not been confirmed; however, its interaction with enhancer RNAs (eRNAs) can influence the transcription of the eRNA-targeted genes (Aguilo et al, 2016).…”

“…The recent development of high-throughput sequencing techniques that allow the detection of N 6 -methyladenosine (m 6 A), 5-methylcytosine (m 5 C) or pseudouridine (Ψ) in low input RNA samples has revived interest in the field and identified functional roles in regulating RNA stability, translation and interactions with other molecules Carlile et al, 2014;Dominissini et al, 2012;Hussain et al, 2013a;Meyer et al, 2012;Squires et al, 2012;Van Haute et al, 2016). The importance of RNA modifications is highlighted by recent studies showing that abnormal RNA modification patterns can have devastating physiological consequences: mutations in RNA-modifying enzymes have been associated with human disorders such as intellectual disability, neuroma-degeneration, obesity and diabetes (Table 1).…”

How RNA modification allows non-conventional decoding in mitochondria

“…1). 3 The related TET proteins, which oxidise m 5 C in DNA, have been shown to form f 5 C and 5-carboxycytosine (ca C could also be detected, 1,3 suggesting that although the majority of mt-tRNA Met is oxidised by ABH1, a portion may remain in the methylated state. It has been discussed that the localization of ABH1 may differ between cell types, raising the possibility that the extent of oxidation of m 5 C34 of mt-tRNA Met may also vary.…”

“…1). 1,2,3 This enzyme is a member of the Nol1/Nop2/SUN domain (NSUN) family, which also contains the RNA methyltransferases NSUN2 and NSUN6 that modify cytoplasmic tRNAs. 4,5 In contrast, NSUN3 localizes to the mitochondrial matrix where it specifically recognizes the anticodon stem loop (ASL) of mt-tRNA Met .…”