Title: <scp>NSUN</scp> 3 and <scp>ABH</scp> 1 modify the wobble position of mt‐t <scp>RNA</scp> <sup>Met</sup> to expand codon recognition in mitochondrial translation
Abstract: Article6 August 2016Open Access NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation Sara Haag Sara Haag Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Search for more papers by this author Katherine E Sloan Katherine E Sloan Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Search for more papers by this author Namit Ranjan Namit Ranjan Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Ahmed S Warda Ahmed S Warda Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Search for more papers by this author Jens Kretschmer Jens Kretschmer Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Search for more papers by this author Charlotte Blessing Charlotte Blessing Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Search for more papers by this author Benedikt Hübner Benedikt Hübner Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Search for more papers by this author Jan Seikowski Jan Seikowski Institute for Organic and Biomolecular Chemistry, Georg-August-University, Göttingen, Germany Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Sven Dennerlein Sven Dennerlein Institute for Cellular Biochemistry, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Search for more papers by this author Peter Rehling Peter Rehling Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Institute for Cellular Biochemistry, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Göttingen Centre for Molecular Biosciences, Georg-August-University, Göttingen, Germany Search for more papers by this author Marina V Rodnina Marina V Rodnina Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Claudia Höbartner Corresponding Author Claudia Höbartner [email protected] orcid.org/0000-0001-7063-5456 Institute for Organic and Biomolecular Chemistry, Georg-August-University, Göttingen, Germany Search for more papers by this author Markus T Bohnsack Corresponding Author Markus T Bohnsack [email protected] Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Göttingen Centre for Molecular Biosciences, Georg-August-University, Göttingen, Germany Search for more papers by this author Sara Haag Sara Haag Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Search for more papers by this author Katherine E Sloan Katherine E Sloan Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Search for more papers by this author Namit Ranjan Namit Ranjan Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Ahmed S Warda Ahmed S Warda Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Search for more papers by this author Jens Kretschmer Jens Kretschmer Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Search for more papers by this author Charlotte Blessing Charlotte Blessing Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Search for more papers by this author Benedikt Hübner Benedikt Hübner Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Search for more papers by this author Jan Seikowski Jan Seikowski Institute for Organic and Biomolecular Chemistry, Georg-August-University, Göttingen, Germany Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Sven Dennerlein Sven Dennerlein Institute for Cellular Biochemistry, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Search for more papers by this author Peter Rehling Peter Rehling Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Institute for Cellular Biochemistry, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Göttingen Centre for Molecular Biosciences, Georg-August-University, Göttingen, Germany Search for more papers by this author Marina V Rodnina Marina V Rodnina Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Claudia Höbartner Corresponding Author Claudia Höbartner [email protected] orcid.org/0000-0001-7063-5456 Institute for Organic and Biomolecular Chemistry, Georg-August-University, Göttingen, Germany Search for more papers by this author Markus T Bohnsack Corresponding Author Markus T Bohnsack [email protected] Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany Göttingen Centre for Molecular Biosciences, Georg-August-University, Göttingen, Germany Search for more papers by this author Author Information Sara Haag1,‡, Katherine E Sloan1,‡, Namit Ranjan2,‡, Ahmed S Warda1,‡, Jens Kretschmer1, Charlotte Blessing1, Benedikt Hübner1, Jan Seikowski3,4, Sven Dennerlein5, Peter Rehling4,5,6, Marina V Rodnina2, Claudia Höbartner *,3 and Markus T Bohnsack *,1,6 1Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany 2Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany 3Institute for Organic and Biomolecular Chemistry, Georg-August-University, Göttingen, Germany 4Max Planck Institute for Biophysical Chemistry, Göttingen, Germany 5Institute for Cellular Biochemistry, University Medical Center Göttingen, Georg-August-University, Göttingen, Germany 6Göttingen Centre for Molecular Biosciences, Georg-August-University, Göttingen, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 551 395968; Fax: +49 551 395960; E-mail: [email protected] *Corresponding author. Tel: +49 551 3920906; Fax: +49 551 3921712; E-mail: [email protected] The EMBO Journal (2016)35:2104-2119https://doi.org/10.15252/embj.201694885 See also: F Boos et al (October 2016) [The copyright line of this article was changed on 1 October 2016 after original online publication.] PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mitochondrial gene expression uses a non-universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt-)tRNAMet mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt-tRNAMet to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt-tRNAMet to generate an f5C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilisation of m5C34 mt-tRNAMet in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt-tRNAMet function. Together, our data reveal how modifications in mt-tRNAMet are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNAMet to recognise the different codons encoding methionine. Synopsis RNA methyltransferase NSUN3 acts specifically on mitochondrial tRNAMet, allowing different codons to be recognised by this single tRNA and offering insight on the consequence of reported disease mutations. The RNA methyltransferase NSUN3 introduces a 5-methylcytosine modification at position 34 in the mitochondrial tRNAMet. The m5C34 can be further oxidised by the dioxygenase ABH1/ALKBH1 to generate f5C34 in mt-tRNAMet. These “wobble position” modifications installed by NSUN3 and ABH1 are required for efficient mitochondrial translation. The modification pathway enables mt-tRNAMet to recognise the three codons for methionine used in the non-universal genetic code of mitochondria. NSUN3 requires a stable anticodon stem-loop for mt-tRNAMet methylation, explaining why mutations that disrupt the basepairing can lead to disease. Introduction More than a hundred different chemical modifications of ribonucleosides have been identified in cellular RNAs (Czerwoniec et al, 2009; Motorin & Helm, 2011). Modifications regulate the biogenesis, structure and function of the corresponding RNAs and RNA–protein complexes (RNPs). Many modifications occur in RNAs involved in translation and are therefore likely to affect protein synthesis. Several modified ribonucleosides including 6-methyladenosine (m6A), 5-methylcytidine (m5C), 1-methyladenosine (m1A) and pseudouridine have recently been shown to occur in messenger (m)RNAs and to affect their biogenesis, translation and stability (see e.g. Carlile et al, 2014; Liu & Jia, 2014; Dominissini et al, 2016). Methylated nucleosides can undergo further modification and proteins of the AlkB family of alpha-ketoglutarate and Fe(II)-dependent dioxygenases (ALKBH1-8 and FTO in human cells) can oxidise or even remove modifications in DNA and RNA (Fedeles et al, 2015; Ougland et al, 2015), increasing the dynamics and regulation of RNA modifications and their roles in RNA metabolism. Compared to mRNAs and other cellular RNAs, transfer (t)RNAs and ribosomal (r)RNAs contain the highest proportion of modified nucleosides. The large majority of rRNA modifications are already installed co-transcriptionally by small nucleolar (sno)RNPs, and only few base modifications require the action of lone-standing enzymes (Watkins & Bohnsack, 2012; Sharma & Lafontaine, 2015). tRNAs contain the largest variety of nucleoside modifications, and many of them are suggested to affect tRNA biogenesis and nuclear export, tRNA structure, interaction with aminoacyl-tRNA-sythetases or codon recognition during translation (Agris et al, 2007; Leisegang et al, 2012; Hori, 2014; Duechler et al, 2016; Ranjan & Rodnina, 2016). Many tRNAs contain base modifications of the nucleoside at position 34 of the tRNA anticodon (the “wobble position”). These modifications modulate codon–anticodon basepairing, often allowing one tRNA to recognise several different nucleosides in the third position of the codon. Mutations in enzymes responsible for introducing these “wobble base” modifications or genetic alterations in tRNA sequences that affect such modifications are often associated with disease, especially in mitochondrial tRNAs (Lott et al, 2013; Powell et al, 2015). One ribonucleoside modification that has been identified in several tRNAs, in both cytoplasmic and mitochondrial rRNA, in other non-coding RNAs and in mRNAs is 5-methylcytosine (m5C). m5C modifications can be installed by any of the seven proteins of the Nol1/Nop2/SUN domain (NSUN) family and by an enzyme named DNA methyltransferase 2 (DNMT2). DNMT2 mainly catalyses the m5C modification in position 38 of tRNAAsp in human cells (Goll et al, 2006), while the so far characterised NSUN proteins show specificity for tRNAs (NSUN2, NSUN6; Schaefer et al, 2010; Tuorto et al, 2012; Blanco et al, 2014; Haag et al, 2015a) or rRNA (NSUN1/NOP2, NSUN5; Sloan et al, 2013; Tafforeau et al, 2013; Schosserer et al, 2015). NSUN2 can also modify vault RNAs and mRNAs (Hussain et al, 2013), and NSUN4 was described to localise to mitochondria where it was shown to methylate the mitochondrial 12S rRNA in mice (Cámara et al, 2011; Metodiev et al, 2014). NSUN3 was one of the last uncharacterised members of the family, and we show here that this RNA methyltransferase localises to the mitochondrial matrix in human cells. Using in vivo UV cross-linking and analysis of cDNA (CRAC) and 5-azacytidine (5-AzaC) CRAC, we show that NSUN3 specifically interacts with the mitochondrial tRNAMet where it is responsible for introducing a 5-methylcytosine (m5C) modification at the “wobble position”. In addition, we find that the m5C modification can be further oxidised by the alpha-ketoglutarate and Fe(II)-dependent dioxygenase ALKBH1/ABH1, generating a 5-formylcytidine (f5C) at this position. Analysis of mt-tRNAMet synthesised with the different cytosine modifications in the wobble position revealed that codon recognition in an in vitro translation system utilising mitochondrial initiation and elongation factors depends on the modification state of C34 in mt-tRNAMet. In vivo, knock-down of ABH1 abolishes f5C34 formation, while depletion of NSUN3 leads to a decrease in mt-tRNAMet modification. Furthermore, reducing the levels of either NSUN3 or ABH1 leads to a significant decrease in mitochondrial translation in vivo, suggesting important roles for the modifications installed by the two enzymes in mt-tRNAMet function. Interestingly, our data also show that NSUN3 requires the anticodon stem loop for substrate recognition and a pathogenic mutation in the ASL abolishes C34 methylation, implying that lack of this modification can lead to disease. Results NSUN3 localises to the mitochondrial matrix More than 10 years ago computational analysis identified NSUN3 as a member of the Nol1/Nop2/Sun domain (NSUN) family of putative m5C RNA methyltransferases (Bujnicki et al, 2004). NSUN3 was suggested to localise to mitochondria (Rhee et al, 2013); however, the target spectrum and biological function of the protein have remained unknown. To confirm the mitochondrial localisation of NSUN3, we generated a HEK293 cell line stably expressing NSUN3-GFP from a tetracycline-inducible promoter. Confocal fluorescence microscopy revealed that NSUN3-GFP localises to distinct cytoplasmic foci that showed co-localisation with a mitotracker (Fig 1A), indicating a mitochondrial localisation of NSUN3. To determine whether NSUN3 is imported into mitochondria, we performed protease protection assays using a tetracycline-inducible NSUN3-HisPrcFLAG (Hexahistidine-PreScission protease cleavage site-2×FLAG tagged NSUN3) cell line. We isolated mitochondria that were then either left intact, subjected to swelling to rupture the outer mitochondrial membrane and generate mitoplasts or were disrupted using sonication before treatment with different concentrations of proteinase K. While treatment of intact mitochondria led to the degradation of the outer membrane protein TOM70, the intermembrane space domain of TIM23 was digested in mitoplasts. Similar to the matrix-localised domain of TIM44, NSUN3 only became susceptible to proteinase K digestion upon rupture of mitochondria by sonication (Fig 1B), indicating that NSUN3 is localised in the mitochondrial matrix in human cells. Figure 1. NSUN3 localises to the mitochondrial matrix The localisation of NSUN3 was analysed in HEK293 cells stably expressing NSUN3-GFP. NSUN3-GFP (green) and staining with Mitotracker (red) are shown separately and in an overlay with DAPI to indicate nuclei. The scale bar represents 5 μm. To analyse submitochondrial localisation of NSUN3, human mitochondria were isolated and either left untreated, swollen in hypotonic buffer (Mitoplasts) or disrupted by sonication (Sonic.) before treatment with different amounts of proteinase K (PK) where indicated, followed by SDS–PAGE and Western blotting using antibodies against human TIM44, TIM23, TOM70 or FLAG-tagged NSUN3. Note that TIM44 extends into the matrix, while the N-terminus of TIM23 localises to the intermembrane space and TOM70 is largely exposed on the mitochondrial surface. The asterisk indicates a cross-reaction of the TOM70 antibody. Download figure Download PowerPoint NSUN3 associates with mitochondrial tRNAMet To identify NSUN3 target RNAs, we performed UV cross-linking and analysis of cDNA (CRAC; Bohnsack et al, 2012; Sloan et al, 2015) experiments using the NSUN3-HisPrcFLAG cell line; a HEK293 cell line expressing only the HisPrcFLAG tag was used as a control. In addition, cells expressing NSUN3-HisPrcFLAG were treated with the cytidine derivative 5-azacytidine (5-AzaC) as a cross-linking reagent, which is incorporated into nascent RNA and specifically traps m5C RNA methyltransferases on their target nucleotides in a covalent protein–RNA intermediate during the methylation reaction (Fig 2A; Khoddami & Cairns, 2013). Without cross-linking or after UV or 5-AzaC cross-linking in vivo, protein–RNA complexes were purified followed by RNA trimming, radiolabelling and ligation of adaptors to the bound RNA. Protein–RNA complexes were separated by SDS–PAGE, transferred to a membrane and exposed to an X-ray film. Both UV and 5-AzaC cross-linking of NSUN3-HisPrcFLAG resulted in a strong specific signal not observed for the controls (Fig 2B), indicating association of NSUN3 with cellular RNAs. Interacting RNAs were then extracted from the membrane and subjected to RT–PCR to generate a cDNA library for Illumina deep sequencing. Mapping of the obtained sequence reads on the human genome resulted in a strong over-representation of mitochondrial-encoded RNA (mt-RNA). mt-RNA represented 40% and 62% of total reads obtained upon UV or 5-AzaC cross-linking of NSUN3, respectively, compared to only 4% of sequence reads from the HisPrcFLAG control (Figs 2C–E and EV1A), suggesting a specific association of NSUN3 with mitochondrial RNA. As sequences from mitochondrial tRNAs were strongly enriched in the NSUN3-cross-linked fractions (Fig 2D and E, lower panels) compared to the control (Fig 2C, lower panel), we analysed the distribution of reads between the 22 mitochondrial tRNAs. Strikingly, 50 and 95% of the reads mapped to mt-tRNAMet in the NSUN3 UV and 5-AzaC cross-linking experiments, respectively (Fig 2F). In contrast, the data obtained for the HisPrcFLAG control contained only 5% sequencing reads that mapped to mt-tRNAMet, indicating that NSUN3 specifically interacts with this tRNA (Fig 2F). To confirm the specificity of this interaction, we performed 5-AzaC cross-linking using cells expressing the HisPrcFLAG control, NSUN3-HisPrcFLAG and the catalytically inactive NSUN3(C265A)-HisPrcFLAG mutant, in which the catalytic cysteine of the TCT tripeptide that is conserved in motif IV in m5C methyltransferases of the NSUN family is replaced by alanine (C265A). After cross-linking and isolation of complexes via the FLAG-tagged proteins, interacting RNAs were analysed by Northern blotting using probes for the detection of the mitochondrial tRNAs mt-tRNAPro, mt-tRNAGlu and mt-tRNAMet (Fig 2G). While mt-tRNAPro and mt-tRNAGlu could not be detected in any of the eluates, mt-tRNAMet was strongly enriched in the eluate from the NSUN3 wild-type sample, but was not detected in any of the controls, further supporting that mt-tRNAMet specifically interacts with NSUN3. The specific requirement for the conserved catalytic cysteine and the efficient cross-linking of NSUN3 to 5-AzaC containing mt-tRNAMet strongly suggest that NSUN3 is an active m5C RNA methyltransferase that uses the conserved mechanism of the NSUN family to mediate m5C methylation of its substrate mt-tRNAMet in human mitochondria. Figure 2. NSUN3 cross-links to the mitochondrial tRNAMet in vivo A. Structure of 5-azacytidine and formation of a covalent RNA methyltransferase adduct. B. HEK293 cells expressing NSUN3-HisPrcFLAG (NSUN3) or the HisPrcFLAG tag alone (FLAG) were either not cross-linked (−), UV cross-linked (UV) or treated with 5-azacytidine (5-AzaC). The protein–RNA complexes were affinity purified and the bound RNA was trimmed, end-labelled with 32P phosphate and ligated to linkers. Protein–RNA complexes were separated by SDS–PAGE, transferred to nitrocellulose and exposed to an X-ray film. C–E. The UV or 5-AzaC cross-linking and analysis of cDNA (CRAC) experiments with NSUN3-HisPrcFLAG (D, E) or the FLAG control (C) samples were treated as described in (B). The RNA was isolated from the nitrocellulose membrane-bound protein–RNA complexes and converted into cDNA for sequence library production and Illumina deep sequencing. Pie charts present different RNA classes and the relative distribution of Illumina sequence reads that were obtained after mapping of the reads on the human genome. Bar graphs below indicate the distribution of mitochondrial (mt-)tRNA, mt-rRNA and mt-mRNA sequence reads among the reads mapped to the mitochondrial genome. Abbreviations: tRNA, transfer RNA; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA; rRNA, ribosomal RNA; mtRNA, mitochondrial-encoded RNA; miscRNA, miscellaneous RNA; miRNA, microRNA; lncRNA, long non-coding RNA. F. Relative distribution of mitochondrial tRNA sequence reads obtained from the CRAC experiments using UV or 5-AzaC cross-linking with cells expressing the NSUN3-HisPrcFLAG (NSUN3) protein or control cells (FLAG). Only mt-tRNAs that were represented by more than 5% of all mt-tRNAs reads are labelled. G. 5-AzaC cross-linking was performed and RNA associated with wild-type NSUN3, the catalytically inactive NSUN3 mutant (C265A) or the FLAG tag alone was isolated as described in (B). The RNA was analysed by Northern blot using probes against the mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. Inputs (0.1%) are shown on the left and eluates (50%) on the right. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. The cytoplasmic tRNA Met-i and Met-e do not represent substrates of NSUN3 in vivo A, B. The UV or 5-AzaC cross-linking and analysis of cDNA (CRAC) experiments with NSUN3-HisPrcFLAG or FLAG control cells were performed as described for Fig 2. (A) The percentages of the Illumina sequence reads mapped to individual classes of RNA are given graphically for each sample. Abbreviations: tRNA, transfer RNA; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA; rRNA, ribosomal RNA; mtRNA, mitochondrial-encoded RNA; miscRNA, miscellaneous RNA; miRNA, microRNA; lncRNA, long non-coding RNA. (B) The relative distribution of cytoplasmic tRNA sequence reads obtained from the CRAC experiments is shown. Only tRNAs that were represented by more than 5% of all cytoplasmic tRNA reads are labelled. C. In vitro methylation reactions were performed using recombinant His14-MBP-NSUN3 (NSUN3) or the catalytically inactive mutant His14-MBP-NSUN3-C265A (C265A), [3H-methyl]-labelled S-adenosylmethionine as a methyl group donor and in vitro-transcribed mitochondrial mt-tRNAMet, cytoplasmic tRNAiMet and tRNAeMet. The RNA was then separated on a denaturing polyacrylamide gel, stained with ethidium bromide (EtBr) to indicate inputs and exposed to an X-ray film to analyse methylation (3H-Me). D. 5-AzaC cross-linking was performed and RNA-associated with wild-type NSUN3, the catalytic NSUN3 mutant (C265A) or the FLAG tag alone was isolated as described in (A). The RNA was isolated from the purified protein–RNA complexes and analysis by Northern blot using probes against the mt-tRNAMet, mt-tRNAiMet and mt-tRNAeMet. Inputs are shown on the left and eluates on the right. The mt-tRNAMet panel is identical to that shown in Fig 2G. E. The nucleotide sequences of the anticodon stem loops of mt-tRNAMet (left) and tRNAiMet (right) are shown. Download figure Download PowerPoint NSUN3 specifically methylates cytosine 34 in mt-tRNAMet To gain further insight into the catalytic activity of NSUN3, we prepared recombinant NSUN3 protein and the catalytically inactive mutant (NSUN3-C265A) and performed in vitro methylation experiments using in vitro T7 RNA-polymerase transcripts of mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu in the presence of S-[3H-methyl] adenosylmethionine (SAM) as a methyl group donor. NSUN3 efficiently methylated mt-tRNAMet, but not the other transcripts, and the catalytic activity of NSUN3 was abolished by mutation of the catalytic cysteine (Fig 3A). Figure 3. NSUN3 modifies the wobble position of mt-tRNAMet In vitro methylation reactions were performed using recombinant His14-MBP-NSUN3 (NSUN3) or the catalytically inactive mutant His14-MBP-NSUN3-C265A (C265A), 3H-labelled S-adenosylmethionine as a methyl group donor and in vitro-transcribed mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. The RNA was then separated on a denaturing polyacrylamide gel, stained with ethidium bromide (EtBr) to indicate inputs and exposed to an X-ray film to analyse methylation (3H-Me). The distribution of Illumina sequence reads along the mt-tRNAMet sequence obtained from CRAC experiments with NSUN3 after UV (light grey) or 5-AzaC cross-linking (dark grey) is given as reads per million mapped reads. The position of the anticodon is indicated by a bar. Cloverleaf scheme of the mt-tRNAMet sequence. Nucleosides that were exchanged in the mutational analysis shown in the following panels are marked with arrows, and the nucleotide positions in the tRNA are given. In vitro methylation assays were performed as described in (A) with His14-MBP-NSUN3 and in vitro-transcribed wild-type mt-tRNAMet and cytidine mutants of the anticodon stem and loop region indicated in (C). Two exposure times of X-ray films are shown 16 h (short) and 3 days (long). In vitro methylation assay of in vitro-transcribed mt-tRNAMet and chemically synthesised mt-tRNAMet containing an m5C modification at the wobble position. The experiment and analysis were performed as described in (A). Download figure Download PowerPoint Besides the strong enrichment of reads from mt-tRNAMet in the CRAC data sets, we had observed that reads mapping to the cytoplasmic tRNAs that mediate incorporation of methionine during translation initiation (tRNAiMet) and elongation (tRNAeMet) were over-represented in the NSUN3 cross-linking data (8% of reads mapped to cytoplasmic tRNA were tRNAMet reads in FLAG control; 18% after UV and 79% after 5-AzaC cross-linking; Fig EV1B). We therefore tested whether NSUN3 could methylate transcripts of tRNAiMet and tRNAeMet in in vitro methyltransferase assays. While mt-tRNAMet was methylated very efficiently by NSUN3, only very weak or no methylation was observed for the tRNAiMet and tRNAeMet transcripts, respectively (Fig EV1C). To analyse possible interactions between NSUN3 and tRNAiMet or tRNAeMet in vivo, we performed 5-AzaC cross-linking and immunoprecipitation experiments using HEK293 cells expressing the HisPrcFLAG tag alone, wild-type or mutant (C265A) NSUN3-HisPrcFLAG and analysed the co-precipitation of tRNAs by Northern blotting. While mt-tRNAMet was strongly enriched with wild-type NSUN3, no association of the cytoplasmic tRNAiMet or tRNAeMet could be detected (Fig EV1D), indicating that NSUN3 does not specifically bind cytoplasmic tRNAs in vivo and that the interactions observed in the 5-AzaC CRAC likely occurred after cell lysis due to similar sequences of the anticodon stem loop of tRNAiMet and mt-tRNAMet (Fig EV1E). Together with the mitochondrial localisation of NSUN3 (Fig 1), these data indicate that NSUN3 can weakly recognise the tRNAiMet as a substrate in vitro, but that mt-tRNAMet, rather than tRNAiMet, represents its genuine methylation substrate in vivo. In order to identify which region of mt-tRNAMet interacts with NSUN3, we analysed the distribution of reads obtained by NSUN3 cross-linking to mt-tRNAMet. Analysis of both UV and 5-AzaC cross-linking experiments showed that the highest read density was obtained with sequences corresponding to the anticodon stem loop (ASL) of mt-tRNAMet (Fig 3B) suggesting that the NSUN3 target residue lies within this region. As NSUN3 is a member of the cytosine methyltransferase family of NSUN proteins, we generated in vitro transcripts of mt-tRNAMet in which each cytosine present in the ASL was individually mutated to an adenosine (ASL loop cytosines) or uracil (cytosines in the stem of the ASL; Fig 3C). Although mutation of several cytosines affected NSUN3-mediated methylation in in vitro methylation assays, only mutation of cytosine 34 abolished the modification (Fig 3D), suggesting that the C34 wobble nucleotide is the NSUN3 target in mt-tRNAMet. This conclusion was confirmed by a lack of methylation when chemically synthesised mt-tRNAMet containing an m5C34 was treated with NSUN3 in methylation assays (Fig 3E), supporting the finding that NSUN3 generates an m5C moiety at position 34 in mt-tRNAMet. Among the mt-tRNAMet mutants (Fig 3D), the C39U mutant, which has previously been identified in patients with mitochondrial dysfunction (Lott et al, 2013; Tang et al, 2013), was a particularly poor substrate for NSUN3, suggesting that this residue might be critical for methylation or that a stable stem in the ASL could be required for NSUN3 recognition. To distinguish between these possibilities, we generated a series of ASL mutants where individual cytosines in the stem were either replaced by u