METTL15 introduces N4-methylcytidine into human mitochondrial 12S rRNA and is required for mitoribosome biogenesis

Abstract Post-transcriptional RNA modifications, the epitranscriptome, play important roles in modulating the functions of RNA species. Modifications of rRNA are key for ribosome production and function. Identification and characterization of enzymes involved in epitranscriptome shaping is instrumental for the elucidation of the functional roles of specific RNA modifications. Ten modified sites have been thus far identified in the mammalian mitochondrial rRNA. Enzymes responsible for two of these modifications have not been characterized. Here, we identify METTL15, show that it is the main N4-methylcytidine (m4C) methyltransferase in human cells and demonstrate that it is responsible for the methylation of position C839 in mitochondrial 12S rRNA. We show that the lack of METTL15 results in a reduction of the mitochondrial de novo protein synthesis and decreased steady-state levels of protein components of the oxidative phosphorylation system. Without functional METTL15, the assembly of the mitochondrial ribosome is decreased, with the late assembly components being unable to be incorporated efficiently into the small subunit. We speculate that m4C839 is involved in the stabilization of 12S rRNA folding, therefore facilitating the assembly of the mitochondrial small ribosomal subunits. Taken together our data show that METTL15 is a novel protein necessary for efficient translation in human mitochondria.

[1]  M. Minczuk,et al.  MRM2 and MRM3 are involved in biogenesis of the large subunit of the mitochondrial ribosome , 2014, Molecular biology of the cell.

[2]  John G Doench,et al.  A Genome-wide CRISPR Death Screen Identifies Genes Essential for Oxidative Phosphorylation. , 2016, Cell metabolism.

[3]  E. Shoubridge,et al.  A pseudouridine synthase module is essential for mitochondrial protein synthesis and cell viability , 2017, EMBO reports.

[4]  L. Davenport,et al.  Methylation status of 13S ribosomal RNA from hamster mitochondria: the presence of a novel riboside, N4-methylcytidine. , 1978, Nucleic acids research.

[5]  L. Van Haute,et al.  Engineered mtZFNs for Manipulation of Human Mitochondrial DNA Heteroplasmy. , 2016, Methods in molecular biology.

[6]  Justin L Cotney,et al.  Evidence for an Early Gene Duplication Event in the Evolution of the Mitochondrial Transcription Factor B Family and Maintenance of rRNA Methyltransferase Activity in Human mtTFB1 and mtTFB2 , 2006, Journal of Molecular Evolution.

[7]  Martijn A. Huynen,et al.  TEFM (c17orf42) is necessary for transcription of human mtDNA , 2011, Nucleic acids research.

[8]  V. de Crécy-Lagard,et al.  Comparative RNomics and Modomics in Mollicutes: Prediction of Gene Function and Evolutionary Implications , 2007, IUBMB life.

[9]  S. Klimašauskas,et al.  Bisulfite sequencing protocol displays both 5-methylcytosine and N4-methylcytosine. , 1999, Analytical biochemistry.

[10]  S. Douthwaite,et al.  Indigenous and acquired modifications in the aminoglycoside binding sites of Pseudomonas aeruginosa rRNAs , 2013, RNA biology.

[11]  R. Baer,et al.  Methylated regions of hamster mitochondrial ribosomal RNA: structural and functional correlates. , 1981, Nucleic Acids Research.

[12]  P. Herdewijn,et al.  Post-transcriptional modification mapping in the Clostridium acetobutylicum 16S rRNA by mass spectrometry and reverse transcriptase assays , 2007, Nucleic acids research.

[13]  Miao Yu,et al.  A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation , 2013, Nature chemical biology.

[14]  L. Van Haute,et al.  NSUN2 introduces 5-methylcytosines in mammalian mitochondrial tRNAs , 2019, bioRxiv.

[15]  Satoshi Kimura,et al.  Fine-tuning of the ribosomal decoding center by conserved methyl-modifications in the Escherichia coli 16S rRNA , 2009, Nucleic acids research.

[16]  Wayne A. Decatur,et al.  rRNA modifications and ribosome function. , 2002, Trends in biochemical sciences.

[17]  Yitzhak Pilpel,et al.  Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates , 2016, PLoS biology.

[18]  Ruedi Aebersold,et al.  Architecture of the large subunit of the mammalian mitochondrial ribosome , 2013, Nature.

[19]  R. Guymon,et al.  Influence of phylogeny on posttranscriptional modification of rRNA in thermophilic prokaryotes: the complete modification map of 16S rRNA of Thermus thermophilus. , 2006, Biochemistry.

[20]  M. Minczuk,et al.  Human mitochondrial ribosomes can switch structural tRNAs – but when and why? , 2017, RNA biology.

[21]  Ruedi Aebersold,et al.  The complete structure of the large subunit of the mammalian mitochondrial ribosome , 2014, Nature.

[22]  B. Habermann,et al.  NSUN4 Is a Dual Function Mitochondrial Protein Required for Both Methylation of 12S rRNA and Coordination of Mitoribosomal Assembly , 2014, PLoS genetics.

[23]  Felix Krueger,et al.  Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications , 2011, Bioinform..

[24]  G. Skiniotis,et al.  Atomic resolution snapshot of Leishmania ribosome inhibition by the aminoglycoside paromomycin , 2017, Nature Communications.

[25]  Aaron R D'Souza,et al.  Human mitochondrial ribosomes can switch their structural RNA composition , 2016, Proceedings of the National Academy of Sciences.

[26]  D. Bogenhagen,et al.  Assignment of 2′-O-Methyltransferases to Modification Sites on the Mammalian Mitochondrial Large Subunit 16 S Ribosomal RNA (rRNA)* , 2014, The Journal of Biological Chemistry.

[27]  E. Phizicky,et al.  Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA. , 2002, RNA.

[28]  Jef Rozenski,et al.  The Small Subunit rRNA Modification Database , 2004, Nucleic Acids Res..

[29]  Paulina Kolasinska-Zwierz,et al.  Construction and testing of engineered zinc-finger proteins for sequence-specific modification of mtDNA , 2010, Nature Protocols.

[30]  Pedro Rebelo-Guiomar,et al.  The mammalian mitochondrial epitranscriptome , 2019, Biochimica et biophysica acta. Gene regulatory mechanisms.

[31]  S. Carr,et al.  Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging , 2013, Science.

[32]  D. Bogenhagen,et al.  Mitochondrial Ribosomal RNA (rRNA) Methyltransferase Family Members Are Positioned to Modify Nascent rRNA in Foci near the Mitochondrial DNA Nucleoid* , 2013, The Journal of Biological Chemistry.

[33]  J Ofengand,et al.  Mapping to nucleotide resolution of pseudouridine residues in large subunit ribosomal RNAs from representative eukaryotes, prokaryotes, archaebacteria, mitochondria and chloroplasts. , 1997, Journal of molecular biology.

[34]  Zhili Xu,et al.  A conserved rRNA methyltransferase regulates ribosome biogenesis , 2008, Nature Structural &Molecular Biology.

[35]  S. Pearce,et al.  Mitochondrial transcript maturation and its disorders , 2015, Journal of Inherited Metabolic Disease.

[36]  Victor S Lelyveld,et al.  Mettl1/Wdr4-Mediated m7G tRNA Methylome Is Required for Normal mRNA Translation and Embryonic Stem Cell Self-Renewal and Differentiation. , 2018, Molecular cell.

[37]  Henning Urlaub,et al.  Human METTL16 is a N6‐methyladenosine (m6A) methyltransferase that targets pre‐mRNAs and various non‐coding RNAs , 2017, EMBO reports.

[38]  Michaela Frye,et al.  Deficient methylation and formylation of mt-tRNAMet wobble cytosine in a patient carrying mutations in NSUN3 , 2016, Nature Communications.

[39]  Tsutomu Suzuki,et al.  Trmt61B is a methyltransferase responsible for 1-methyladenosine at position 58 of human mitochondrial tRNAs. , 2012, RNA.

[40]  Yonghong Shi,et al.  Methylation of 12S rRNA is necessary for in vivo stability of the small subunit of the mammalian mitochondrial ribosome. , 2009, Cell metabolism.

[41]  Alexis A. Jourdain,et al.  The Pseudouridine Synthase RPUSD4 Is an Essential Component of Mitochondrial RNA Granules* , 2017, The Journal of Biological Chemistry.

[42]  R. Guymon,et al.  Post-transcriptional modifications in the small subunit ribosomal RNA from Thermotoga maritima, including presence of a novel modified cytidine. , 2007, RNA.

[43]  Pedro Rebelo-Guiomar,et al.  Maturation of selected human mitochondrial tRNAs requires deadenylation , 2017, eLife.

[44]  Alan Brown,et al.  The structure of the human mitochondrial ribosome , 2015, Science.

[45]  I. Fearnley,et al.  Human METTL12 is a mitochondrial methyltransferase that modifies citrate synthase , 2017, FEBS letters.

[46]  E. Shtykova,et al.  Crystal and solution structures of methyltransferase RsmH provide basis for methylation of C1402 in 16S rRNA. , 2012, Journal of structural biology.

[47]  Ruedi Aebersold,et al.  The complete structure of the 55S mammalian mitochondrial ribosome , 2015, Science.

[48]  V. Mootha,et al.  Defective mitochondrial rRNA methyltransferase MRM2 causes MELAS-like clinical syndrome , 2017, Human molecular genetics.

[49]  R. Horvath,et al.  Mitochondrial DNA transcription and translation: clinical syndromes , 2018, Essays in biochemistry.

[50]  Aaron R D'Souza,et al.  Mitochondrial transcription and translation: overview , 2018, Essays in biochemistry.

[51]  M. García-Díaz,et al.  Kinetics and Mechanism of Mammalian Mitochondrial Ribosome Assembly. , 2018, Cell reports.

[52]  B. Lane,et al.  N-4-methyl-2'-O-methyl cytidine and other methyl-substituted nucleoside constituents of Escherichia coli ribosomal and soluble RNA. , 1966, Biochimica et biophysica acta.

[53]  Tsutomu Suzuki,et al.  A complete landscape of post-transcriptional modifications in mammalian mitochondrial tRNAs , 2014, Nucleic acids research.

[54]  H. Fuchs,et al.  Overexpression of the mitochondrial methyltransferase TFB1M in the mouse does not impact mitoribosomal methylation status or hearing , 2015, Human molecular genetics.