NSUN2 introduces 5-methylcytosines in mammalian mitochondrial tRNAs

Abstract

Expression of human mitochondrial DNA is indispensable for proper function of the oxidative phosphorylation machinery. The mitochondrial genome encodes 22 tRNAs, 2 rRNAs and 11 mRNAs and their post-transcriptional modification constitutes one of the key regulatory steps during mitochondrial gene expression. Cytosine-5 methylation (m5C) has been detected in mitochondrial transcriptome, however its biogenesis has not been investigated in details. Mammalian NOP2/Sun RNA Methyltransferase Family Member 2 (NSUN2) has been characterized as an RNA methyltransferase introducing m5C in nuclear-encoded tRNAs, mRNAs and microRNAs and associated with cell proliferation and differentiation, with pathogenic variants in NSUN2 being linked to neurodevelopmental disorders. Here we employ spatially restricted proximity labelling and immunodetection to demonstrate that NSUN2 is imported into the matrix of mammalian mitochondria. Using three genetic models for NSUN2 inactivation—knockout mice, patient-derived fibroblasts and CRISPR/Cas9 knockout in human cells—we show that NSUN2 is necessary for the generation of m5C at positions 48, 49 and 50 of several mammalian mitochondrial tRNAs. Finally, we show that inactivation of NSUN2 does not have a profound effect on mitochondrial tRNA stability and oxidative phosphorylation in differentiated cells. We discuss the importance of the newly discovered function of NSUN2 in the context of human disease.

Complete chemical structures of human mitochondrial tRNAs

“…We identified m 5 Cs at positions 48-50 in six mt-tRNA species. Recently, we reported that a fraction of NSUN2 is localized in mitochondria and introduces m 5 C in mt-tRNAs 66,90 .…”

Advances in RNA cytosine-5 methylation: detection, regulatory mechanisms, biological functions and links to cancer

“…Moreover, recent evidence has also shown the mapping of m 5 C in mitochondrial transfer RNAs (mt-tRNAs) at position C48-50 within mammalian mitochondria [42]. Originally identified to methylate cytoplasmic tRNAs at the junction of the variable loop and the T stem spanning positions 34, 40 and 48-50, NSUN2 has gradually been found to have diverse RNA substrates, including rRNAs, tRNAs, mRNAs, mt-tRNAs and viral RNAs [9][10][11][43][44][45]. Additionally, NSUN2 exerts multiple functions in the regulation of plant root development, mitochondrial oxidative phosphorylation, protein synthesis and cell cycle progression in response to oxidative stress stimuli [46], and it has also been reported to regulate HIV replication [44], Epstein-Barr virus degradation [45], epidermal differentiation [47] and tumorigenesis [23,48,49].…”

“…With the advances in RNA m 5 C detection techniques, including bisulfite sequencing, m 5 C RNA immunoprecipitation sequencing (m 5 C-RIP-seq), 5-AZAcytidinemediated RNA immunoprecipitation sequencing (Aza-IP-seq) and methylation-individual nucleotide resolution crosslinking immunoprecipitation sequencing (miCLIPseq), over 10,000 potential sites of m 5 C modification were detected within the whole human transcriptome [7], and the existence of m 5 C was found in diverse RNA species from various organisms, not only in ribosomal RNA (rRNA), messenger RNA (mRNA) and transfer RNA (tRNA) but also in viral RNA, vault RNA, small nuclear RNA, small nucleolar RNA, pseudogenes, natural antisense transcripts, enhancer RNA, long noncoding RNA (lncRNA) and microRNA (miRNA) [8][9][10][11][12][13][14][15]. The distribution of m 5 C differs among different organisms, as it has been common to find m 5 C methylation substrates in the tRNA and mRNA of eukaryotes and archaea, while no m 5 C methylation substrates have been detected in bacteria [16].…”

Mitoepigenetics and Its Emerging Roles in Cancer

“…However, NSUN2 can also be imported into mitochondrial matrix and introduces m5C at positions 48, 49, and 50 of several mitochondrial tRNAs, including mt-tRNA Tyr , mt-tRNA His , mt-tRNA Leu(UUR) , mt-tRNA Phe , and mt-tRNA Glu . However, NSUN2 inactivation does not remarkably affect mitochondrial tRNA stability and OxPhos in differentiated cells (Van Haute et al, 2019).…”

“…As cellular organelles present in almost all eukaryotic cells, mitochondria are places where ATP is biosynthesized and are essential for various cellular biological processes, including reactive oxygen species (ROS) generation, intracellular Ca 2+ signaling, heme metabolism, intrinsic apoptosis, mitophagy, metabolism and cell cycle progression (Yien et al, 2014;Picard et al, 2016;van der Bliek et al, 2017;Gómez-Durán et al, 2018). Genetic mutations in mitochondrial DNA (mtDNA) and perturbations in mitochondrial proteins can cause dysfunction of mitochondria that has been shown to be tightly associated with many mitochondrial diseases and cancer (Taylor and Turnbull, 2005;Garone et al, 2012aGarone et al, ,b, 2013Gómez-Durán et al, 2012;Ronchi et al, 2012;Kullar et al, 2017;Chinnery and Gómez-Durán, 2018;Garone and Viscomi, 2018;Andreazza et al, 2019).…”