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• W3020878785 abstract "Mitochondrial DNA gene expression is coordinately regulated both pre- and post-transcriptionally, and its perturbation can lead to human pathologies. Mitochondrial rRNAs (mt-rRNAs) undergo a series of nucleotide modifications after release from polycistronic mitochondrial RNA precursors, which is essential for mitochondrial ribosomal biogenesis. Cytosine N4-methylation (m4C) at position 839 (m4C839) of the 12S small subunit mt-rRNA was identified decades ago; however, its biogenesis and function have not been elucidated in detail. Here, using several approaches, including immunofluorescence, RNA immunoprecipitation and methylation assays, and bisulfite mapping, we demonstrate that human methyltransferase-like 15 (METTL15), encoded by a nuclear gene, is responsible for 12S mt-rRNA methylation at m4C839 both in vivo and in vitro. We tracked the evolutionary history of RNA m4C methyltransferases and identified a difference in substrate preference between METTL15 and its bacterial ortholog rsmH. Additionally, unlike the very modest impact of a loss of m4C methylation in bacterial small subunit rRNA on the ribosome, we found that METTL15 depletion results in impaired translation of mitochondrial protein-coding mRNAs and decreases mitochondrial respiration capacity. Our findings reveal that human METTL15 is required for mitochondrial function, delineate the evolution of methyltransferase substrate specificities and modification patterns in rRNA, and highlight a differential impact of m4C methylation on prokaryotic ribosomes and eukaryotic mitochondrial ribosomes. Mitochondrial DNA gene expression is coordinately regulated both pre- and post-transcriptionally, and its perturbation can lead to human pathologies. Mitochondrial rRNAs (mt-rRNAs) undergo a series of nucleotide modifications after release from polycistronic mitochondrial RNA precursors, which is essential for mitochondrial ribosomal biogenesis. Cytosine N4-methylation (m4C) at position 839 (m4C839) of the 12S small subunit mt-rRNA was identified decades ago; however, its biogenesis and function have not been elucidated in detail. Here, using several approaches, including immunofluorescence, RNA immunoprecipitation and methylation assays, and bisulfite mapping, we demonstrate that human methyltransferase-like 15 (METTL15), encoded by a nuclear gene, is responsible for 12S mt-rRNA methylation at m4C839 both in vivo and in vitro. We tracked the evolutionary history of RNA m4C methyltransferases and identified a difference in substrate preference between METTL15 and its bacterial ortholog rsmH. Additionally, unlike the very modest impact of a loss of m4C methylation in bacterial small subunit rRNA on the ribosome, we found that METTL15 depletion results in impaired translation of mitochondrial protein-coding mRNAs and decreases mitochondrial respiration capacity. Our findings reveal that human METTL15 is required for mitochondrial function, delineate the evolution of methyltransferase substrate specificities and modification patterns in rRNA, and highlight a differential impact of m4C methylation on prokaryotic ribosomes and eukaryotic mitochondrial ribosomes. Mitochondrial gene expression requires a series of interconnected processes encompassing mitochondrial DNA (mtDNA) replication and repair, mitochondrial RNA transcription, maturation, and mitoribosome assembly (1Costanzo M.C. Fox T.D. Control of mitochondrial gene expression in Saccharomyces cerevisiae.Annu. Rev. Genet. 1990; 24 (2088182): 91-11310.1146/annurev.ge.24.120190.000515Crossref PubMed Google Scholar, 2Taanman J.W. The mitochondrial genome: structure, transcription, translation and replication.Biochim. Biophys. Acta. 1999; 1410 (10076021): 103-12310.1016/S0005-2728(98)00161-3Crossref PubMed Scopus (916) Google Scholar). The mt-RNAs, especially rRNAs and tRNAs, are subjected to extensive enzyme-mediated modifications, which play key roles in RNA stability, RNA structure, and mitochondrial ribosome assembly (3Pearce S.F. Rebelo-Guiomar P. D'Souza A.R. Powell C.A. Van Haute L. Minczuk M. Regulation of mammalian mitochondrial gene expression: recent advances.Trends Biochem. Sci. 2017; 42 (28285835): 625-63910.1016/j.tibs.2017.02.003Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 4Bohnsack M.T. Sloan K.E. The mitochondrial epitranscriptome: the roles of RNA modifications in mitochondrial translation and human disease.Cell. Mol. Life Sci. 2018; 75: 241-26010.1007/s00018-017-2598-6Crossref PubMed Scopus (51) Google Scholar). Some of these modifications are deposited co-transcriptionally or immediately after transcription, whereas others occur when the rRNA is assembled into the preribosomal particle (3Pearce S.F. Rebelo-Guiomar P. D'Souza A.R. Powell C.A. Van Haute L. Minczuk M. Regulation of mammalian mitochondrial gene expression: recent advances.Trends Biochem. Sci. 2017; 42 (28285835): 625-63910.1016/j.tibs.2017.02.003Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 4Bohnsack M.T. Sloan K.E. The mitochondrial epitranscriptome: the roles of RNA modifications in mitochondrial translation and human disease.Cell. Mol. Life Sci. 2018; 75: 241-26010.1007/s00018-017-2598-6Crossref PubMed Scopus (51) Google Scholar, 5Sergiev P.V. Aleksashin N.A. Chugunova A.A. Polikanov Y.S. Dontsova O.A. Structural and evolutionary insights into ribosomal RNA methylation.Nat. Chem. Biol. 2018; 14 (29443970): 226-23510.1038/nchembio.2569Crossref PubMed Scopus (61) Google Scholar). Prokaryotic and eukaryotic cytoplasmic rRNAs contain more than 30 and 200 modified sites, respectively, but only ∼10 modifications are found in the mitochondrial rRNAs (4Bohnsack M.T. Sloan K.E. The mitochondrial epitranscriptome: the roles of RNA modifications in mitochondrial translation and human disease.Cell. Mol. Life Sci. 2018; 75: 241-26010.1007/s00018-017-2598-6Crossref PubMed Scopus (51) Google Scholar, 5Sergiev P.V. Aleksashin N.A. Chugunova A.A. Polikanov Y.S. Dontsova O.A. Structural and evolutionary insights into ribosomal RNA methylation.Nat. Chem. Biol. 2018; 14 (29443970): 226-23510.1038/nchembio.2569Crossref PubMed Scopus (61) Google Scholar). These modifications are located at the functionally important regions of the mitoribosome, such as the decoding center of the small subunit (SSU), suggesting that these modifications might be preserved because of their essential roles (5Sergiev P.V. Aleksashin N.A. Chugunova A.A. Polikanov Y.S. Dontsova O.A. Structural and evolutionary insights into ribosomal RNA methylation.Nat. Chem. Biol. 2018; 14 (29443970): 226-23510.1038/nchembio.2569Crossref PubMed Scopus (61) Google Scholar, 6Dubin D.T. Methylated nucleotide content of mitochondrial ribosomal RNA from hamster cells.J. Mol. Biol. 1974; 84 (4133889): 257-27310.1016/0022-2836(74)90584-1Crossref PubMed Scopus (40) Google Scholar). The best-characterized example is the TFB1M-mediated dimethylation on the two highly conserved sites, A936 and A937, at the 3′-end of the 12S mt-rRNA, which is necessary for the assembly of the SSU (7Metodiev M.D. Lesko N. Park C.B. Camara Y. Shi Y. Wibom R. Hultenby K. Gustafsson C.M. Larsson N.G. Methylation of 12S rRNA is necessary for in vivo stability of the small subunit of the mammalian mitochondrial ribosome.Cell Metab. 2009; 9 (19356719): 386-39710.1016/j.cmet.2009.03.001Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 8Seidel-Rogol B.L. McCulloch V. Shadel G.S. Human mitochondrial transcription factor B1 methylates ribosomal RNA at a conserved stem-loop.Nat. Genet. 2003; 33 (12496758): 23-2410.1038/ng1064Crossref PubMed Scopus (134) Google Scholar). The NOP2/Sun RNA Methyltransferase 4 (NSUN4) forms a complex with MTERF4 to catalyze m5C methylation at position 841 in 12S mt-rRNA and to coordinate the mitoribosome assembly (9Metodiev M.D. Spahr H. Loguercio Polosa P. Meharg C. Becker C. Altmueller J. Habermann B. Larsson N.G. Ruzzenente B. NSUN4 is a dual function mitochondrial protein required for both methylation of 12S rRNA and coordination of mitoribosomal assembly.PLoS Genet. 2014; 10 (24516400): e100411010.1371/journal.pgen.1004110Crossref PubMed Scopus (131) Google Scholar, 10Spahr H. Habermann B. Gustafsson C.M. Larsson N.G. Hallberg B.M. Structure of the human MTERF4–NSUN4 protein complex that regulates mitochondrial ribosome biogenesis.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (22949673): 15253-1525810.1073/pnas.1210688109Crossref PubMed Scopus (65) Google Scholar). However, enzymes for m4C and m5U (uracil) methylation in mammalian mitochondrial rRNAs remain to be identified (6Dubin D.T. Methylated nucleotide content of mitochondrial ribosomal RNA from hamster cells.J. Mol. Biol. 1974; 84 (4133889): 257-27310.1016/0022-2836(74)90584-1Crossref PubMed Scopus (40) Google Scholar, 11Dubin D.T. Taylor R.H. Davenport L.W. Methylation status of 13S ribosomal RNA from hamster mitochondria: the presence of a novel riboside, N4-methylcytidine.Nucleic Acids Res. 1978; 5 (724519): 4385-439710.1093/nar/5.11.4385Crossref PubMed Scopus (34) Google Scholar). Mitochondrial diseases may be caused by mutations in mtDNA (12Taylor R.W. Turnbull D.M. Mitochondrial DNA mutations in human disease.Nat. Rev. Genet. 2005; 6 (15861210): 389-40210.1038/nrg1606Crossref PubMed Scopus (1170) Google Scholar), but growing evidence suggests that defects in the nuclear genes involved in mitochondrial RNA modifications can also lead to human mitochondrial diseases. For instance, loss of TFB1M results in mitochondrial dysfunction that leads to impaired insulin secretion and diabetes (13Sharoyko V.V. Abels M. Sun J. Nicholas L.M. Mollet I.G. Stamenkovic J.A. Gohring I. Malmgren S. Storm P. Fadista J. Spegel P. Metodiev M.D. Larsson N.G. Eliasson L. Wierup N. et al.Loss of TFB1M results in mitochondrial dysfunction that leads to impaired insulin secretion and diabetes.Hum. Mol. Genet. 2014; 23 (24916378): 5733-574910.1093/hmg/ddu288Crossref PubMed Scopus (25) Google Scholar). A missense mutation in pseudouridylate synthase 1 (PUS1), which converts uridine to pseudouridine at several mitochondrial tRNA positions, has been reported to be associated with myopathy, lactic acidosis, and sideroblastic anemia (14Fernandez-Vizarra E. Berardinelli A. Valente L. Tiranti V. Zeviani M. Nonsense mutation in pseudouridylate synthase 1 (PUS1) in two brothers affected by myopathy, lactic acidosis and sideroblastic anaemia (MLASA).J. Med. Genet. 2006; 44 (17056637): 173-18010.1136/jmg.2006.045252Crossref PubMed Scopus (95) Google Scholar). Moreover, a defect in the mitochondrial rRNA methyltransferase MRM2 that causes loss of 2′-O-methyl modification at position U1369 in the human mitochondrial 16S rRNA leads to mitochondrial encephalomyopathy, lactic acidosis, and stroke-like clinical syndrome in patients (15Garone C. D'Souza A.R. Dallabona C. Lodi T. Rebelo-Guiomar P. Rorbach J. Donati M.A. Procopio E. Montomoli M. Guerrini R. Zeviani M. Calvo S.E. Mootha V.K. DiMauro S. Ferrero I. et al.Defective mitochondrial rRNA methyltransferase MRM2 causes MELAS-like clinical syndrome.Hum. Mol. Genet. 2017; 26 (28973171): 4257-426610.1093/hmg/ddx314Crossref PubMed Scopus (27) Google Scholar). In addition, a 11p14.1 microdeletion was identified to be highly associated with attention-deficit/hyperreactive disorder, autism, developmental delay, and obesity (16Shinawi M. Sahoo T. Maranda B. Skinner S.A. Skinner C. Chinault C. Zascavage R. Peters S.U. Patel A. Stevenson R.E. Beaudet A.L. 11p14.1 microdeletions associated with ADHD, autism, developmental delay, and obesity.Am. J. Med. Genet. A. 2011; 155: 1272-128010.1002/ajmg.a.33878Crossref Scopus (62) Google Scholar). Intriguingly, the microdeletion region always encompasses the METTL-family protein METTL15. More recently, a transancestral meta-analysis of genome-wide association studies uncovered a completely novel SNP (rs10835310) in METTL15 associated with childhood obesity (17Bradfield J.P. Vogelezang S. Felix J.F. Chesi A. Helgeland Ø. Horikoshi M. Karhunen V. Lowry E. Cousminer D.L. Ahluwalia T.S. Thiering E. Boh E.T. Zafarmand M.H. Vilor-Tejedor N. Wang C.A. et al.A trans-ancestral meta-analysis of genome-wide association studies reveals loci associated with childhood obesity.Hum. Mol. Genet. 2019; 28: 3327-333810.1093/hmg/ddz161Crossref PubMed Scopus (18) Google Scholar), further implicating the involvement of METTL15 in this human syndrome, although the underlying molecular mechanisms for these associations are still unclear. In this current study, we demonstrate that human METTL15 protein, encoded by a nuclear gene, is localized in mitochondria and is responsible for methylation of the 12S mt-RNA at C839 in vivo and in vitro. Furthermore, we demonstrate that METTL15-dependent modification of 12S mt-rRNA is necessary for the proper function of mitoribosome. Our study reveals that methylation of 12S mt-rRNA m4C839 by METTL15 is an important epitranscriptomic modification, critical for efficient mitochondrial protein synthesis and respiratory function. METTL15 is a member of the methyltransferase like (METTL) family, characterized by the presence of a binding domain for SAM, which is a methyl-group donor for methylation reactions (18Schubert H.L. Blumenthal R.M. Cheng X. Many paths to methyltransfer: a chronicle of convergence.Trends Biochem. Sci. 2003; 28 (31504550): 329-33510.1016/S0968-0004(03)00090-2Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar, 19Martin J.L. McMillan F.M. SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold.Curr. Opin. Struct. Biol. 2002; 12 (12504684): 783-79310.1016/S0959-440X(02)00391-3Crossref PubMed Scopus (405) Google Scholar). Through phylogenetic analysis, we found that METTL15 is highly conserved during evolution and is an ortholog of the bacterial methyltransferase, rsmH (Fig. 1A and Data File S1), which is responsible for the N4-methylation of m4Cm1402 in the 16S rRNA in almost all species of bacteria (Fig. S1A) (20Kimura S. Suzuki T. Fine-tuning of the ribosomal decoding center by conserved methyl-modifications in the Escherichia coli 16S rRNA.Nucleic Acids Res. 2010; 38 (19965768): 1341-135210.1093/nar/gkp1073Crossref PubMed Scopus (108) Google Scholar). Given its similarity with rsmH (Fig. 1A and Data File S1), we asked whether METTL15 is also a m4Cm methyltransferase for rRNA. We first purified the SSU rRNA fragments containing C1402 or its equivalent nucleotide from four representative species and measured the levels of m4Cm by HPLC-MS/MS. We did not detect any meaningful levels of m4Cm in the cytoplasmic SSU rRNAs from fruit fly, zebrafish, or human; however, a varied but significant amount of Cm was readily detectable (Fig. S1B). This finding is consistent with previous reports that the SSU rRNAs of those eukaryotic cells were abundantly modified by 2′-O-methylated cytosine (Fig. S1C) (21Decatur W.A. Fournier M.J. rRNA modifications and ribosome function.Trends Biochem. Sci. 2002; 27 (12114023): 344-35110.1016/S0968-0004(02)02109-6Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). In eukaryotic cells, mitochondria has its own ribosomes translating mitochondrial mRNAs, which prompted us to investigate whether METTL15 is a mitoribosome-specific methyltransferase. Indeed, a considerable amount of mitochondrial genome-encoded RNAs, especially 12S and 16S mt-rRNA, but not cytoplasmic RNAs such as 18S rRNAs, are found to be associated with the HA-tagged METTL15 in an RNA immunoprecipitation (RIP) experiment (Fig. 1B). Consistently, immunofluoresence experiments showed that METTL15 is exclusively localized in the mitochondria, which depends on its putative mitochondria-targeting signals (Fig. 1C) (22Fukasawa Y. Tsuji J. Fu S.C. Tomii K. Horton P. Imai K. MitoFates: improved prediction of mitochondrial targeting sequences and their cleavage sites.Mol Cell. Proteomics. 2015; 14 (25670805): 1113-112610.1074/mcp.M114.043083Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), suggesting that METTL15 is a bona fide mitochondria protein that interacts with mitoribosome rRNAs. To unambiguously identify the in vivo methylation sites modified by METTL15, we profiled the mitochondrial RNA methylome in WT and METTL15 knockout (KO) cells using RNA bisulfite sequencing (BS-seq), which detects both m5C and m4C cytosine modifications in RNAs (23Schaefer M. Pollex T. Hanna K. Lyko F. RNA cytosine methylation analysis by bisulfite sequencing.Nucleic Acids Res. 2008; 37 (19059995): e1210.1093/nar/gkn954Crossref PubMed Scopus (217) Google Scholar). The RNA BS-seq revealed that in the absence of METTL15, the methylation level of 12S mt-C839 is drastically decreased from 58% to a near background level (0.9%), which suggests that methylation of 12S mt-C839 may be mediated by METTL15 (Fig. 2, A and B). To validate the BS-seq results, we designed sequence-specific primers to amplify a 145-nucleotide region surrounding C839 from bisulfite-treated RNA samples and employed targeted sequencing (detailed procedures can be found under “Experimental procedures”) to examine the methylation levels of C839. In agreement with the BS-seq result, methylation of C839 was found to almost completely disappear in the METTL15 KO cells (Fig. S2C). Importantly, the methylation level of m4C839 can be fully rescued by WT METTL15 but not a catalytically compromised mutant METTL15 (GA mutant: 108GSGG112 to 108ASAA112) (Fig. S2C) (24Kozbial P.Z. Mushegian A.R. Natural history of S-adenosylmethionine–binding proteins.BMC Struct. Biol. 2005; 5 (16225687): 1910.1186/1472-6807-5-19Crossref PubMed Scopus (172) Google Scholar), which strongly supports the hypothesis that METTL15 is responsible for m4C839 on 12S mt-rRNA in vivo and is consistent with a very recent study (25Haute L.V. Hendrick A.G. D'Souza A.R. Powell C.A. Rebelo-Guiomar P. Harbour M.E. Ding S. Fearnley I.M. Andrews B. Minczuk M. METTL15 introduces N4-methylcytidine into human mitochondrial 12S rRNA and is required for mitoribosome biogenesis.Nucleic Acids Res. 2019; 47 (31665743): 10267-1028110.1093/nar/gkz735Crossref PubMed Scopus (29) Google Scholar). Interestingly, the neighboring methylation site, m5C841, which is catalyzed by NSUN4 (9), is also reduced (but not eliminated) upon METTL15 deletion. In addition, the m5C841 reduction could be fully restored by reintroducing WT METTL15 and partially restored by enzymatically inactive METTL15, suggesting that METTL15 might influence the installation of m5C841 by NSUN4 in both enzymatic activity–dependent and –independent manners (Fig. S2C). Given that both m4C(m) and m5C are able to block the C-to-T transition by bisulfite treatment and therefore cannot be distinguished in the BS-seq analysis (23Schaefer M. Pollex T. Hanna K. Lyko F. RNA cytosine methylation analysis by bisulfite sequencing.Nucleic Acids Res. 2008; 37 (19059995): e1210.1093/nar/gkn954Crossref PubMed Scopus (217) Google Scholar), we turned to an optimized LC–MS/MS method to efficiently separate different forms of methyl cytosines to define the exact type of methylation in C839 (Fig. S2B). As shown in Fig. 2C, m4C was detected in WT cell lines but reduced to a background level in the METTL15 KO cells, indicating that METTL15 may be a m4C methyltransferase for C839. Consistent with the BS-seq data, HPLC-MS/MS analysis also found a modest reduction of m5C at C841 caused by METTL15 depletion, which again points to a potential cross-talk between C839 and C841 methylation. m4C methylation of C839 is mediated by the intrinsic enzymatic activity of METTL15 because reintroduction of WT, but not the catalytically inactive METLL15, back into the METTL15 KO cells restored the methylation level of m4C839 (Fig. 2D and Fig. S2, A–C). Collectively, these findings demonstrate that METTL15 is likely the enzyme responsible for methylation of 12S mt-m4C839 in vivo (Fig. S2D). We next asked whether recombinant METTL15 mediates 12S mt-rRNA methylation at C839. C-terminally FLAG-tagged METTL15 was expressed in 293T cells and purified using an anti-FLAG M2 affinity column (Fig. S3A). Recombinant METTL15 was incubated with 12S mt-rRNA oligonucleotides (nucleotides 832–846) in the presence of d3-SAM (S-(5′-adenosyl)-l-methionine-d3) as a methyl group donor, and the resulting rRNAs oligonucleotides were isolated for LC–MS measurement. As shown in Fig. 3A, m4 methylation of C839 was successfully detected, whereas the catalytically compromised METTL15 failed to mediate C839 methylation. As described before, a main difference between the m4C methylation site of bacterial ribosome and human mitoribosome is that cytosine in bacterial ribosome is mainly m4Cm, whereas in the human mitoribosome at the equivalent cytosine residue, it is m4C without the 2′-O-methylation. This prompted us to determine whether human METTL15 displays any preference for unmodified cytosine versus 2′-O-methylated cytosine. Consistently, unmodified cytosine in the 12S mt-rRNA appears to be a better substrate for human METT15 in vitro (C versus Cm in Fig. 3B). In contrast, rsmH shows a higher apparent activity toward Cm compared with C of 12S mt-rRNAs (solid line in Fig. 3C; Fig. S3C). Interestingly, we found the aromatic amino acid (Trp173) in rsmH, which has been changed to valine (Val242) in the eukaryotic orthologs (METTL15) and might potentially mediate the interaction of 2′-O-methyl group of Cm with rsmH based on the published rsmH structure (Fig. S3B) (26Wei Y. Zhang H. Gao Z.Q. Wang W.J. Shtykova E.V. Xu J.H. Liu Q.S. Dong Y.H. Crystal and solution structures of methyltransferase RsmH provide basis for methylation of C1402 in 16S rRNA.J. Struct. Biol. 2012; 179 (22561317): 29-4010.1016/j.jsb.2012.04.011Crossref PubMed Scopus (15) Google Scholar). These results confirmed that human METTL15 is a bona fide m4C methyltransferase and has a higher activity toward unmodified cytosine than 2′-O-methylated cytosine in vitro. Because METTL15 is localized in mitochondria, we first investigated the effect of METTL15 deletion on mtDNA copy number and transcription of mitochondrial genome-encoded genes. We found METTL15 deletion only causes minor changes of mtDNA copy number and transcription (Fig. S4, A and B). Given that the methylation site lies in the critical region of the mitoribosome, we next asked whether loss of METTL15 affects the function of mitoribosome. We performed mitochondrial ribosome profiling in a 10–30% sucrose gradient to determine whether there was any difference in the assembly of mitoribosome. The distribution of SSU and large subunit in the sucrose gradient was detected by the presence of 12S and 16S mt-rRNA, respectively. According to the protein complex density, the first peak of 12S rRNA (fraction 8) represents SSU, whereas the first peak of 16S rRNA (fraction 9) represents large the subunit, and the co-fractionated peaks (fractions 12 and 13) represent the mature ribosome. The co-fractionation ratio of 12S and 16S mt-rRNAs in METTL15 KO cells was significantly reduced (compared with factions 12 and 13 in WT cells), thus identifying a major defect in mitoribosome assembly. In addition, the ratio of mRNA encoded by mitochondrial genome was also significantly reduced in the 55S mature monosomes (fractions 12 and 13), indicating compromised translation efficiency, which was consistent with the observed mitoribosome assembly defects (Fig. 4, A–C). Western blotting results of two representative mitochondrial protein-coding genes, COX2 and ND6, showed that the levels of the protein products were also reduced significantly (Fig. S4C and Fig. 4, D and E). Importantly, the translational defects of multiple mitochondria-encoded genes could be rescued by WT METTL15 but not the catalytic mutant (Fig. S4D), suggesting that the impact of METTL15 on mitoribosome is likely to be dependent on N4-methylation of C839. These data thus demonstrate that the methylation mediated by METTL15 is important for the proper function of mitoribosomes and mt-mRNA translation. The most prominent role of mitochondria is to produce ATP through respiration and to regulate cellular metabolism. Most of the ATP synthesized during glucose metabolism is produced in the mitochondria through oxidative phosphorylation (OxPhos) powered by the electron transport chain complex, which consists essentially of ∼70 nuclear-encoded proteins and 13 mtDNA-encoded proteins translated by mitoribosome in mitochondria. To determine the impact of METTL15 loss on respiratory activity, we measured the respiratory activity of METTL15 KO cells using a Seahorse XF96 analyzer. The oxygen consumption rate (OCR) of the METTL15 KO cells was substantially lower than that of WT cells, and this effect depends on the enzymatic activity (Fig. 5, A and B), indicating that METTL15-mediated m4C839 on 12S mt-rRNA is likely to be required for proper oxidative phosphorylation function. After 2 days in culture, the medium of METTL15 KO cells turned yellower, indicating a lower pH and more lactate secretion, although the cell numbers are comparable between WT and KO (Fig. S5A). Consistently, the extracellular acidification rate, which approximates glycolytic activity, was significantly up-regulated in the METTL15 KO cells, likely to compensate for dysfunction of the mitochondria (Fig. 5C). Furthermore, metabolites profiling shows a decline of citrate and α-ketoglutarate, the intermediates of the TCA cycle, which is closely coupled with OxPhos to generate ATP. It is also known that an essential function of respiration in proliferating cells is to support aspartate biosynthesis (27Sullivan L.B. Gui D.Y. Hosios A.M. Bush L.N. Freinkman E. Vander Heiden M.G. Supporting aspartate biosynthesis is an essential function of respiration in proliferating cells.Cell. 2015; 162 (26232225): 552-56310.1016/j.cell.2015.07.017Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 28Wang T. Birsoy K. Hughes N.W. Krupczak K.M. Post Y. Wei J.J. Lander E.S. Sabatini D.M. Identification and characterization of essential genes in the human genome.Science. 2015; 350 (26472758): 1096-110110.1126/science.aac7041Crossref PubMed Scopus (761) Google Scholar). The decline of the aspartate level in METTL15 KO cell is consistent with the compromised respiration function. At the same time, the up-regulated level of lactate suggests that cells use more anaerobic glycolysis to compensate for impaired mitochondrial function (Fig. 5D and S5, A–C). These results indicate that METTL15 is important for maintaining mitochondrial function and cellular metabolic homeostasis. Here we describe the identification of METTL15 as the methyltransferase that generates m4C839 in human 12S mt-rRNA. METTL15 orthologs exist in most eukaryotes, which implies the importance of this methyltransferase for proper mitoribosome functions. Consistent with this hypothesis, mitochondrial translation is inhibited, and oxidative phosphorylation is remarkably compromised in the METTL15 KO cells, identifying an important function of METTL15 in regulating mitochondria functions, likely by methylating 12S mt-rRNA. Ribosomal maturation involves multiple steps of subunit assembly and incorporation of chemical modifications into the rRNA (3Pearce S.F. Rebelo-Guiomar P. D'Souza A.R. Powell C.A. Van Haute L. Minczuk M. Regulation of mammalian mitochondrial gene expression: recent advances.Trends Biochem. Sci. 2017; 42 (28285835): 625-63910.1016/j.tibs.2017.02.003Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The assembly of protein components and rRNAs has been well characterized through high-resolution cryo-EM, whereas the process of modification deposition is still largely unclear (29Klinge S. Woolford Jr., J.L. Ribosome assembly coming into focus.Nat. Rev. Mol. Cell Biol. 2018; 20 (30467428): 116-13110.1038/s41580-018-0078-yCrossref Scopus (112) Google Scholar). For the bacterial 16S rRNA, quantitative analysis of rRNA modifications finds that the modification events seem to occur in a 5′-to-3′ sequential order: from the 5′ body domain, to the 3′ head domain, to the 3′ minor domain (30Popova A.M. Williamson J.R. Quantitative analysis of rRNA modifications using stable isotope labeling and mass spectrometry.J. Am. Chem. Soc. 2014; 136 (24422502): 2058-206910.1021/ja412084bCrossref PubMed Scopus (60) Google Scholar). In this current study, we found that m4C839 methylation appears to precede m5C841 and is important for the nearby m5C841 methylation, suggesting cross-talk between modifications of the two nearby residues. Furthermore, the enzymatically inactive METTL15 can partially restore the m5C841 methylation decrease in METTL15-null cells, raising the question of whether the cross-talk is mediated by physical interactions between METTL15 and the m5C methyltransferase, NSUN4. Undoubtedly, the investigation of how modifications of mt-rRNA are coordinately deposited in an orderly manner will significantly increase our understanding of mitoribosome maturation (31Shi Z. Xu S. Xing S. Yao K. Zhang L. Xue L. Zhou P. Wang M. Yan G. Yang P. Liu J. Hu Z. Lan F. Mettl17, a regulator of mitochondrial ribosomal RNA modifications, is required for the translation of mitochondrial coding genes.FASEB J. 2019; 33 (31487196): 13040-1305010.1096/fj.201901331RCrossref PubMed Scopus (8) Google Scholar). Unlike the universally conserved rRNA modifications (such as m6,6A) (5Sergiev P.V. Aleksashin N.A. Chugunova A.A. Polikanov Y.S. Dontsova O.A. Structural and evolutionary insights into ribosomal RNA methylation.Nat. Chem. Biol. 2018; 14 (29443970): 226-23510.1038/nchembio.2569Crossref PubMed Scopus (61) Google Scholar), the N4-methylation of SSU rRNA is only maintained in prokaryotes and mitochondria of eukaryotic cells. In bacteria, rsmI and rsmH (which is the bacterial METTL15 ortholog) install 2′-O-methylation and N4-methylation, respectively, on the equivalent cytosine to generate m4C" @default.
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