{"gene":"METTL18","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2021,"finding":"Human METTL18 is a histidine-specific methyltransferase that methylates His-245 of RPL3 (ribosomal protein L3), producing a 3-methylhistidine (τ-methylhistidine) modification. METTL18 localizes to the nucleus and accumulates in nucleoli, requires a functional nuclear localization signal, and was identified as the most significantly enriched MTase in an RPL3 interactomics screen. METTL18 is also automethylated at its own His-154. METTL18 knockout cells show altered pre-rRNA processing, decreased polysome formation, and codon-specific changes in mRNA translation.","method":"Recombinant protein in vitro methylation assay, RPL3 interactomics/Co-IP screen, mass spectrometry identification of 3-methylhistidine, METTL18 KO cell lines, polysome profiling, nuclear/nucleolar localization by imaging","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (in vitro assay, MS, KO cells, interactomics, localization) in a single rigorous study; findings replicated independently by other labs","pmids":["33693809"],"is_preprint":false},{"year":2022,"finding":"METTL18 methylates His-245 of RPL3 at the τ-N position in vitro and in cells. This modification specifically slows ribosome traversal on Tyr codons, allowing proper folding of newly synthesized proteins. Ribosome profiling showed that loss of METTL18 accelerates translation elongation at Tyr codons, and RPL3 methylation protects cells from aggregation of Tyr-rich proteins, linking histidine methylation to proteostasis maintenance.","method":"In vitro methylation assay with methyl-donor analog and quantitative MS, cryo-EM structural comparison of modified vs. unmodified ribosomes, genome-wide ribosome profiling, in vitro translation assay, protein aggregation assay in METTL18 KO cells","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution, structural analysis, ribosome profiling, and functional aggregation assays; multiple orthogonal methods in one rigorous study","pmids":["35674491"],"is_preprint":false},{"year":2024,"finding":"METTL18-mediated methylation of RPL3 indirectly regulates HSP90 integrity and protein levels, promoting actin polymerization via HSP90, which in turn leads to Src phosphorylation at Tyr-419 and Tyr-530 and downstream oncogenic signaling in HER2-negative breast cancer cells. Loss of METTL18 reduces metastatic responses in vitro and in vivo.","method":"METTL18 knockdown/overexpression in breast tumor cell lines, tumor xenograft model, confocal microscopy, F/G-actin assays, Western blot for Src phosphorylation and HSP90","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple biochemical and cell-based methods in vitro and in vivo, but indirect pathway placement and single lab","pmids":["39309445"],"is_preprint":false},{"year":2026,"finding":"METTL18 is essential for pancreatic function in vivo: Mettl18 knockout mice show diabetic phenotypes, accumulation of pancreatitis-associated proteins, and activation of the unfolded protein response. Ribosome profiling in pancreatic acinar cells revealed that loss of METTL18 causes global translational alterations including accelerated elongation at proline codons, leading to improper protein folding and aggregation of pancreatitis-associated proteins (e.g., Reg1).","method":"Mettl18 knockout mouse model, ribosome profiling in pancreatic acinar cell line, mass spectrometry for N3-histidine methylation, protein aggregation assays, unfolded protein response markers","journal":"Molecular metabolism","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vivo KO model combined with ribosome profiling and biochemical validation; multiple orthogonal methods; single lab but comprehensive","pmids":["41713742"],"is_preprint":false},{"year":2010,"finding":"Yeast Hpm1p (ortholog of human METTL18; encoded by YIL110W, also designated HPM1) is a seven-β-strand methyltransferase responsible for stoichiometric monomethylation at His-243 of ribosomal protein Rpl3, producing 3-methylhistidine. Deletion of HPM1 abolishes this modification; the modification is found in ribosomes and nucleus-containing fractions but not in ribosome-free cytosol.","method":"Top-down and bottom-up mass spectrometry, in vivo radiolabeling with SAM-[methyl-3H], high-resolution cation-exchange chromatography, TLC, deletion strain analysis of 37 methyltransferase candidates","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal biochemical methods (MS, radiolabeling, chromatography) with genetic deletion; founding mechanistic study replicated by subsequent work","pmids":["20864530"],"is_preprint":false},{"year":2014,"finding":"Yeast Hpm1p (ortholog of human METTL18) has methyltransferase activity in vitro on ribosome-associated Rpl3p but NOT on free Rpl3p, indicating its activity depends on interactions with ribosomal components. hpm1-null cells show defective early rRNA processing, deficiency of 60S subunits, translation initiation defects, resistance to cycloheximide and verrucarin A, and decreased translational fidelity.","method":"In vitro methyltransferase assay with ribosome-associated vs. free Rpl3p, amino acid analysis, polysome profiling, rRNA processing assays, drug resistance plate assays, translational fidelity reporters","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstitution with defined substrates, genetic KO with multiple cellular phenotype readouts, replicated across labs","pmids":["24865971"],"is_preprint":false},{"year":2016,"finding":"Methylation of Rpl3p at His-243 by Hpm1p (yeast ortholog of METTL18) plays a significant role in translation elongation fidelity. The rpl3-H243A mutation (blocking methylation at this site) phenocopies Hpm1-deficient cells in pre-rRNA processing defects and translational accuracy defects, but NOT in perturbed ribosomal subunit levels. This indicates Hpm1p has multiple substrates, with Rpl3p methylation specifically contributing to translation elongation fidelity rather than subunit biogenesis.","method":"Rpl3-H243A mutagenesis, translational fidelity assays (stop codon readthrough, amino acid misincorporation, -1 frameshifting), pre-rRNA processing analysis, ribosomal subunit profiling","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — separation-of-function mutagenesis with multiple orthogonal translational fidelity assays; mechanistically rigorous","pmids":["26826131"],"is_preprint":false},{"year":2023,"finding":"METTL18 is identified as one of the catalytic enzymes responsible for Nτ-methylhistidine modifications in mammals. A methodology using biochemical protein fractionation combined with LC-MS/MS quantification of methylhistidine was established; METTL18 is listed alongside SETD3 and METTL9 as known mammalian histidine methyltransferases.","method":"Biochemical fractionation, LC-MS/MS quantification of methylhistidine, in silico structural prediction","journal":"Journal of biochemistry","confidence":"Low","confidence_rationale":"Tier 3–4 / Weak — METTL18 cited in passing as a known enzyme; no direct experiment on METTL18 performed in this paper","pmids":["37279646"],"is_preprint":false},{"year":2022,"finding":"Loss of yeast Hpm1p (ortholog of human METTL18) results in changes to protein structure and protein-protein interactions in the ribosome, membrane proteins, chromatin, and mitochondria as detected by quantitative crosslinking MS, independently of changes in protein abundance. Hpm1p deletion also results in increased sensitivity to nonribosomal stressors and differential abundance of proteins linked to sugar metabolism coordination.","method":"Quantitative proteomics, stable isotope labeling crosslinking MS (XL-MS), targeted MS for H243 methylation stoichiometry, growth assays","journal":"Molecular & cellular proteomics : MCP","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — novel crosslinking MS approach with multiple orthogonal validations; single lab but quantitative and systematic","pmids":["35609787"],"is_preprint":false}],"current_model":"METTL18 is a nuclear, nucleolus-enriched histidine-specific protein methyltransferase that stoichiometrically monomethylates His-245 (τ-N position) of the 60S ribosomal protein RPL3, a modification that slows ribosome elongation at Tyr (and Pro) codons to ensure proper nascent protein folding and proteostasis; loss of METTL18 impairs pre-rRNA processing, reduces polysome formation, causes codon-specific translational dysregulation, and leads to protein aggregation and unfolded protein response activation, with additional roles proposed in HSP90-actin-Src oncogenic signaling in breast cancer."},"narrative":{"mechanistic_narrative":"METTL18 is a nuclear, nucleolus-enriched histidine-specific protein methyltransferase that stoichiometrically monomethylates the 60S ribosomal protein RPL3 to regulate translation elongation and proteostasis [PMID:33693809, PMID:35674491]. It deposits a 3-methylhistidine (τ-N) mark on His-245 of RPL3, requires a functional nuclear localization signal, and is itself automethylated at His-154; the enzyme was identified as the most enriched methyltransferase in an RPL3 interactomics screen [PMID:33693809]. The yeast ortholog Hpm1p establishes that this activity acts on ribosome-associated rather than free RPL3 substrate, indicating that methylation depends on the assembled ribosomal context [PMID:24865971]. Functionally, the RPL3 modification slows ribosome traversal of Tyr (and Pro) codons, providing a kinetic window for proper folding of nascent proteins; loss of METTL18 accelerates elongation at these codons and causes aggregation of codon-enriched proteins [PMID:35674491, PMID:41713742]. Consistent with a role in ribosome maturation, METTL18 loss impairs pre-rRNA processing and reduces polysome/60S formation [PMID:33693809, PMID:24865971], and separation-of-function analysis in yeast shows RPL3-His methylation specifically governs translation elongation fidelity rather than subunit biogenesis, implying additional substrates [PMID:26826131]. In vivo, Mettl18 knockout mice develop diabetic phenotypes with accumulation of pancreatitis-associated proteins (e.g., Reg1) and unfolded protein response activation, tying the enzyme to organ-level proteostasis [PMID:41713742]. A further role in HER2-negative breast cancer has been described in which METTL18 indirectly supports HSP90 integrity, actin polymerization, and Src phosphorylation to promote metastatic signaling [PMID:39309445].","teleology":[{"year":2010,"claim":"Established the founding biochemical identity of the enzyme: the yeast ortholog Hpm1p is the methyltransferase responsible for stoichiometric 3-methylhistidine monomethylation of ribosomal protein Rpl3 at His-243.","evidence":"Top-down/bottom-up MS, in vivo SAM-[3H] radiolabeling, cation-exchange chromatography and deletion-strain screening of methyltransferase candidates in yeast","pmids":["20864530"],"confidence":"High","gaps":["Did not identify the human ortholog or test mammalian activity","Catalytic mechanism and structural basis of histidine specificity not resolved"]},{"year":2014,"claim":"Defined the substrate requirement and cellular consequences: Hpm1p methylates ribosome-associated but not free Rpl3p, and its loss disrupts early rRNA processing, 60S subunit levels, and translational fidelity.","evidence":"In vitro methyltransferase assays with ribosome-associated vs. free substrate, polysome profiling, rRNA processing assays, drug-resistance and fidelity reporters in yeast","pmids":["24865971"],"confidence":"High","gaps":["Whether biogenesis and fidelity defects arise from one or multiple substrates unresolved","No mechanistic link between methylation and elongation kinetics yet"]},{"year":2016,"claim":"Separated the enzyme's functions: blocking Rpl3 His-243 methylation alone phenocopies fidelity and rRNA processing defects but not subunit imbalance, implying Hpm1p acts on additional substrates.","evidence":"Rpl3-H243A separation-of-function mutagenesis with stop-codon readthrough, misincorporation, and frameshifting assays in yeast","pmids":["26826131"],"confidence":"High","gaps":["Identity of the additional substrate(s) driving subunit-level effects unknown","Codon-level basis of fidelity change not defined"]},{"year":2021,"claim":"Transferred the mechanism to humans: METTL18 methylates His-245 of RPL3, localizes to nucleoli via an NLS, is automethylated, and its loss perturbs pre-rRNA processing and codon-specific translation.","evidence":"Recombinant in vitro methylation, RPL3 interactomics/Co-IP, MS detection of 3-methylhistidine, KO cell lines, polysome profiling, and imaging in human cells","pmids":["33693809"],"confidence":"High","gaps":["Functional consequence of automethylation at His-154 unknown","Which translation defects are direct vs. secondary to rRNA processing not separated"]},{"year":2022,"claim":"Provided the kinetic-proteostasis mechanism: RPL3 τ-methylation slows ribosome traversal of Tyr codons to permit correct folding, and its loss accelerates Tyr-codon elongation and causes aggregation of Tyr-rich proteins.","evidence":"In vitro methylation with quantitative MS, cryo-EM of modified vs. unmodified ribosomes, genome-wide ribosome profiling, in vitro translation, and aggregation assays in METTL18 KO cells","pmids":["35674491"],"confidence":"High","gaps":["Structural mechanism by which His methylation alters elongation rate not fully resolved","Scope of affected codons beyond Tyr in human cells limited"]},{"year":2022,"claim":"Broadened the downstream footprint: loss of Hpm1p alters protein conformations and interactions across ribosome, chromatin, membrane, and mitochondrial proteins independent of abundance, and changes stress sensitivity and metabolic protein levels.","evidence":"Quantitative crosslinking MS (XL-MS) with stable isotope labeling, targeted methylation stoichiometry MS, and growth assays in yeast","pmids":["35609787"],"confidence":"Medium","gaps":["Causality between the single methyl mark and distal proteome changes not established","Direct vs. indirect interaction changes not distinguished"]},{"year":2024,"claim":"Implicated METTL18 in oncogenic signaling: its RPL3 methylation activity indirectly supports HSP90 integrity, actin polymerization, and Src phosphorylation to drive metastatic responses in HER2-negative breast cancer.","evidence":"Knockdown/overexpression in breast tumor lines, xenograft model, F/G-actin assays, and Western blot for Src and HSP90 in cells and in vivo","pmids":["39309445"],"confidence":"Medium","gaps":["Mechanistic chain from RPL3 methylation to HSP90 is indirect and not reconstituted","Single-lab finding without orthogonal confirmation"]},{"year":2026,"claim":"Demonstrated organ-level physiological requirement: Mettl18 knockout mice develop diabetic phenotypes with pancreatitis-associated protein accumulation and UPR activation, driven by accelerated proline-codon elongation.","evidence":"Mettl18 knockout mouse model, ribosome profiling in pancreatic acinar cells, MS for N3-histidine methylation, aggregation assays, and UPR markers","pmids":["41713742"],"confidence":"High","gaps":["Tissue specificity of the pancreatic phenotype not explained","Relationship between Tyr- and Pro-codon effects across tissues unresolved"]},{"year":null,"claim":"The full substrate repertoire of METTL18 beyond RPL3 and the structural basis by which a single histidine methyl mark tunes codon-specific elongation remain open.","evidence":"","pmids":[],"confidence":"High","gaps":["Additional substrates implied by yeast separation-of-function data not identified in mammals","No structural model of how the modification mechanistically slows elongation at specific codons","Direct link between the methylation activity and the breast-cancer HSP90/Src axis not reconstituted"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,4,5]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,4]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[0]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,1,5]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,3]}],"complexes":[],"partners":["RPL3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O95568","full_name":"Histidine protein methyltransferase 1 homolog","aliases":["Arsenic-transactivated protein 2","AsTP2","Methyltransferase-like protein 18"],"length_aa":372,"mass_kda":42.1,"function":"Protein-L-histidine N-tele-methyltransferase that specifically monomethylates RPL3, thereby regulating translation elongation (PubMed:23349634, PubMed:33693809, PubMed:35674491). Histidine methylation of RPL3 regulates translation elongation by slowing ribosome traversal on tyrosine codons: slower elongation provides enough time for proper folding of synthesized proteins and prevents cellular aggregation of tyrosine-rich proteins (PubMed:35674491)","subcellular_location":"Cytoplasm, cytosol; Nucleus; Nucleus, nucleolus","url":"https://www.uniprot.org/uniprotkb/O95568/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/METTL18","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/METTL18","total_profiled":1310},"omim":[{"mim_id":"615255","title":"METHYLTRANSFERASE-LIKE 18; METTL18","url":"https://www.omim.org/entry/615255"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/METTL18"},"hgnc":{"alias_symbol":["MGC9084","AsTP2","HPM1"],"prev_symbol":["C1orf156"]},"alphafold":{"accession":"O95568","domains":[{"cath_id":"3.40.50.150","chopping":"100-121_166-239_253-367","consensus_level":"high","plddt":94.8842,"start":100,"end":367}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O95568","model_url":"https://alphafold.ebi.ac.uk/files/AF-O95568-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O95568-F1-predicted_aligned_error_v6.png","plddt_mean":74.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=METTL18","jax_strain_url":"https://www.jax.org/strain/search?query=METTL18"},"sequence":{"accession":"O95568","fasta_url":"https://rest.uniprot.org/uniprotkb/O95568.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O95568/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O95568"}},"corpus_meta":[{"pmid":"20864530","id":"PMC_20864530","title":"A novel 3-methylhistidine modification of yeast ribosomal protein Rpl3 is dependent upon the YIL110W methyltransferase.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20864530","citation_count":73,"is_preprint":false},{"pmid":"33693809","id":"PMC_33693809","title":"Human METTL18 is a histidine-specific methyltransferase that targets RPL3 and affects ribosome biogenesis and function.","date":"2021","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/33693809","citation_count":50,"is_preprint":false},{"pmid":"9310495","id":"PMC_9310495","title":"New xenograft model of multiple myeloma and efficacy of a humanized antibody against human interleukin-6 receptor.","date":"1997","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/9310495","citation_count":46,"is_preprint":false},{"pmid":"32704114","id":"PMC_32704114","title":"Serum proteome profiles revealed dysregulated proteins and mechanisms associated with 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histidine-specific methyltransferase that methylates His-245 of RPL3 (ribosomal protein L3), producing a 3-methylhistidine (τ-methylhistidine) modification. METTL18 localizes to the nucleus and accumulates in nucleoli, requires a functional nuclear localization signal, and was identified as the most significantly enriched MTase in an RPL3 interactomics screen. METTL18 is also automethylated at its own His-154. METTL18 knockout cells show altered pre-rRNA processing, decreased polysome formation, and codon-specific changes in mRNA translation.\",\n      \"method\": \"Recombinant protein in vitro methylation assay, RPL3 interactomics/Co-IP screen, mass spectrometry identification of 3-methylhistidine, METTL18 KO cell lines, polysome profiling, nuclear/nucleolar localization by imaging\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (in vitro assay, MS, KO cells, interactomics, localization) in a single rigorous study; findings replicated independently by other labs\",\n      \"pmids\": [\"33693809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"METTL18 methylates His-245 of RPL3 at the τ-N position in vitro and in cells. This modification specifically slows ribosome traversal on Tyr codons, allowing proper folding of newly synthesized proteins. Ribosome profiling showed that loss of METTL18 accelerates translation elongation at Tyr codons, and RPL3 methylation protects cells from aggregation of Tyr-rich proteins, linking histidine methylation to proteostasis maintenance.\",\n      \"method\": \"In vitro methylation assay with methyl-donor analog and quantitative MS, cryo-EM structural comparison of modified vs. unmodified ribosomes, genome-wide ribosome profiling, in vitro translation assay, protein aggregation assay in METTL18 KO cells\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution, structural analysis, ribosome profiling, and functional aggregation assays; multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"35674491\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"METTL18-mediated methylation of RPL3 indirectly regulates HSP90 integrity and protein levels, promoting actin polymerization via HSP90, which in turn leads to Src phosphorylation at Tyr-419 and Tyr-530 and downstream oncogenic signaling in HER2-negative breast cancer cells. Loss of METTL18 reduces metastatic responses in vitro and in vivo.\",\n      \"method\": \"METTL18 knockdown/overexpression in breast tumor cell lines, tumor xenograft model, confocal microscopy, F/G-actin assays, Western blot for Src phosphorylation and HSP90\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple biochemical and cell-based methods in vitro and in vivo, but indirect pathway placement and single lab\",\n      \"pmids\": [\"39309445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"METTL18 is essential for pancreatic function in vivo: Mettl18 knockout mice show diabetic phenotypes, accumulation of pancreatitis-associated proteins, and activation of the unfolded protein response. Ribosome profiling in pancreatic acinar cells revealed that loss of METTL18 causes global translational alterations including accelerated elongation at proline codons, leading to improper protein folding and aggregation of pancreatitis-associated proteins (e.g., Reg1).\",\n      \"method\": \"Mettl18 knockout mouse model, ribosome profiling in pancreatic acinar cell line, mass spectrometry for N3-histidine methylation, protein aggregation assays, unfolded protein response markers\",\n      \"journal\": \"Molecular metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vivo KO model combined with ribosome profiling and biochemical validation; multiple orthogonal methods; single lab but comprehensive\",\n      \"pmids\": [\"41713742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Yeast Hpm1p (ortholog of human METTL18; encoded by YIL110W, also designated HPM1) is a seven-β-strand methyltransferase responsible for stoichiometric monomethylation at His-243 of ribosomal protein Rpl3, producing 3-methylhistidine. Deletion of HPM1 abolishes this modification; the modification is found in ribosomes and nucleus-containing fractions but not in ribosome-free cytosol.\",\n      \"method\": \"Top-down and bottom-up mass spectrometry, in vivo radiolabeling with SAM-[methyl-3H], high-resolution cation-exchange chromatography, TLC, deletion strain analysis of 37 methyltransferase candidates\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal biochemical methods (MS, radiolabeling, chromatography) with genetic deletion; founding mechanistic study replicated by subsequent work\",\n      \"pmids\": [\"20864530\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Yeast Hpm1p (ortholog of human METTL18) has methyltransferase activity in vitro on ribosome-associated Rpl3p but NOT on free Rpl3p, indicating its activity depends on interactions with ribosomal components. hpm1-null cells show defective early rRNA processing, deficiency of 60S subunits, translation initiation defects, resistance to cycloheximide and verrucarin A, and decreased translational fidelity.\",\n      \"method\": \"In vitro methyltransferase assay with ribosome-associated vs. free Rpl3p, amino acid analysis, polysome profiling, rRNA processing assays, drug resistance plate assays, translational fidelity reporters\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstitution with defined substrates, genetic KO with multiple cellular phenotype readouts, replicated across labs\",\n      \"pmids\": [\"24865971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Methylation of Rpl3p at His-243 by Hpm1p (yeast ortholog of METTL18) plays a significant role in translation elongation fidelity. The rpl3-H243A mutation (blocking methylation at this site) phenocopies Hpm1-deficient cells in pre-rRNA processing defects and translational accuracy defects, but NOT in perturbed ribosomal subunit levels. This indicates Hpm1p has multiple substrates, with Rpl3p methylation specifically contributing to translation elongation fidelity rather than subunit biogenesis.\",\n      \"method\": \"Rpl3-H243A mutagenesis, translational fidelity assays (stop codon readthrough, amino acid misincorporation, -1 frameshifting), pre-rRNA processing analysis, ribosomal subunit profiling\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — separation-of-function mutagenesis with multiple orthogonal translational fidelity assays; mechanistically rigorous\",\n      \"pmids\": [\"26826131\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"METTL18 is identified as one of the catalytic enzymes responsible for Nτ-methylhistidine modifications in mammals. A methodology using biochemical protein fractionation combined with LC-MS/MS quantification of methylhistidine was established; METTL18 is listed alongside SETD3 and METTL9 as known mammalian histidine methyltransferases.\",\n      \"method\": \"Biochemical fractionation, LC-MS/MS quantification of methylhistidine, in silico structural prediction\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3–4 / Weak — METTL18 cited in passing as a known enzyme; no direct experiment on METTL18 performed in this paper\",\n      \"pmids\": [\"37279646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Loss of yeast Hpm1p (ortholog of human METTL18) results in changes to protein structure and protein-protein interactions in the ribosome, membrane proteins, chromatin, and mitochondria as detected by quantitative crosslinking MS, independently of changes in protein abundance. Hpm1p deletion also results in increased sensitivity to nonribosomal stressors and differential abundance of proteins linked to sugar metabolism coordination.\",\n      \"method\": \"Quantitative proteomics, stable isotope labeling crosslinking MS (XL-MS), targeted MS for H243 methylation stoichiometry, growth assays\",\n      \"journal\": \"Molecular & cellular proteomics : MCP\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — novel crosslinking MS approach with multiple orthogonal validations; single lab but quantitative and systematic\",\n      \"pmids\": [\"35609787\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"METTL18 is a nuclear, nucleolus-enriched histidine-specific protein methyltransferase that stoichiometrically monomethylates His-245 (τ-N position) of the 60S ribosomal protein RPL3, a modification that slows ribosome elongation at Tyr (and Pro) codons to ensure proper nascent protein folding and proteostasis; loss of METTL18 impairs pre-rRNA processing, reduces polysome formation, causes codon-specific translational dysregulation, and leads to protein aggregation and unfolded protein response activation, with additional roles proposed in HSP90-actin-Src oncogenic signaling in breast cancer.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"METTL18 is a nuclear, nucleolus-enriched histidine-specific protein methyltransferase that stoichiometrically monomethylates the 60S ribosomal protein RPL3 to regulate translation elongation and proteostasis [#0, #1]. It deposits a 3-methylhistidine (\\u03c4-N) mark on His-245 of RPL3, requires a functional nuclear localization signal, and is itself automethylated at His-154; the enzyme was identified as the most enriched methyltransferase in an RPL3 interactomics screen [#0]. The yeast ortholog Hpm1p establishes that this activity acts on ribosome-associated rather than free RPL3 substrate, indicating that methylation depends on the assembled ribosomal context [#5]. Functionally, the RPL3 modification slows ribosome traversal of Tyr (and Pro) codons, providing a kinetic window for proper folding of nascent proteins; loss of METTL18 accelerates elongation at these codons and causes aggregation of codon-enriched proteins [#1, #3]. Consistent with a role in ribosome maturation, METTL18 loss impairs pre-rRNA processing and reduces polysome/60S formation [#0, #5], and separation-of-function analysis in yeast shows RPL3-His methylation specifically governs translation elongation fidelity rather than subunit biogenesis, implying additional substrates [#6]. In vivo, Mettl18 knockout mice develop diabetic phenotypes with accumulation of pancreatitis-associated proteins (e.g., Reg1) and unfolded protein response activation, tying the enzyme to organ-level proteostasis [#3]. A further role in HER2-negative breast cancer has been described in which METTL18 indirectly supports HSP90 integrity, actin polymerization, and Src phosphorylation to promote metastatic signaling [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Established the founding biochemical identity of the enzyme: the yeast ortholog Hpm1p is the methyltransferase responsible for stoichiometric 3-methylhistidine monomethylation of ribosomal protein Rpl3 at His-243.\",\n      \"evidence\": \"Top-down/bottom-up MS, in vivo SAM-[3H] radiolabeling, cation-exchange chromatography and deletion-strain screening of methyltransferase candidates in yeast\",\n      \"pmids\": [\"20864530\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the human ortholog or test mammalian activity\", \"Catalytic mechanism and structural basis of histidine specificity not resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined the substrate requirement and cellular consequences: Hpm1p methylates ribosome-associated but not free Rpl3p, and its loss disrupts early rRNA processing, 60S subunit levels, and translational fidelity.\",\n      \"evidence\": \"In vitro methyltransferase assays with ribosome-associated vs. free substrate, polysome profiling, rRNA processing assays, drug-resistance and fidelity reporters in yeast\",\n      \"pmids\": [\"24865971\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether biogenesis and fidelity defects arise from one or multiple substrates unresolved\", \"No mechanistic link between methylation and elongation kinetics yet\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Separated the enzyme's functions: blocking Rpl3 His-243 methylation alone phenocopies fidelity and rRNA processing defects but not subunit imbalance, implying Hpm1p acts on additional substrates.\",\n      \"evidence\": \"Rpl3-H243A separation-of-function mutagenesis with stop-codon readthrough, misincorporation, and frameshifting assays in yeast\",\n      \"pmids\": [\"26826131\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the additional substrate(s) driving subunit-level effects unknown\", \"Codon-level basis of fidelity change not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Transferred the mechanism to humans: METTL18 methylates His-245 of RPL3, localizes to nucleoli via an NLS, is automethylated, and its loss perturbs pre-rRNA processing and codon-specific translation.\",\n      \"evidence\": \"Recombinant in vitro methylation, RPL3 interactomics/Co-IP, MS detection of 3-methylhistidine, KO cell lines, polysome profiling, and imaging in human cells\",\n      \"pmids\": [\"33693809\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of automethylation at His-154 unknown\", \"Which translation defects are direct vs. secondary to rRNA processing not separated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Provided the kinetic-proteostasis mechanism: RPL3 \\u03c4-methylation slows ribosome traversal of Tyr codons to permit correct folding, and its loss accelerates Tyr-codon elongation and causes aggregation of Tyr-rich proteins.\",\n      \"evidence\": \"In vitro methylation with quantitative MS, cryo-EM of modified vs. unmodified ribosomes, genome-wide ribosome profiling, in vitro translation, and aggregation assays in METTL18 KO cells\",\n      \"pmids\": [\"35674491\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mechanism by which His methylation alters elongation rate not fully resolved\", \"Scope of affected codons beyond Tyr in human cells limited\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Broadened the downstream footprint: loss of Hpm1p alters protein conformations and interactions across ribosome, chromatin, membrane, and mitochondrial proteins independent of abundance, and changes stress sensitivity and metabolic protein levels.\",\n      \"evidence\": \"Quantitative crosslinking MS (XL-MS) with stable isotope labeling, targeted methylation stoichiometry MS, and growth assays in yeast\",\n      \"pmids\": [\"35609787\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causality between the single methyl mark and distal proteome changes not established\", \"Direct vs. indirect interaction changes not distinguished\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Implicated METTL18 in oncogenic signaling: its RPL3 methylation activity indirectly supports HSP90 integrity, actin polymerization, and Src phosphorylation to drive metastatic responses in HER2-negative breast cancer.\",\n      \"evidence\": \"Knockdown/overexpression in breast tumor lines, xenograft model, F/G-actin assays, and Western blot for Src and HSP90 in cells and in vivo\",\n      \"pmids\": [\"39309445\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic chain from RPL3 methylation to HSP90 is indirect and not reconstituted\", \"Single-lab finding without orthogonal confirmation\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Demonstrated organ-level physiological requirement: Mettl18 knockout mice develop diabetic phenotypes with pancreatitis-associated protein accumulation and UPR activation, driven by accelerated proline-codon elongation.\",\n      \"evidence\": \"Mettl18 knockout mouse model, ribosome profiling in pancreatic acinar cells, MS for N3-histidine methylation, aggregation assays, and UPR markers\",\n      \"pmids\": [\"41713742\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue specificity of the pancreatic phenotype not explained\", \"Relationship between Tyr- and Pro-codon effects across tissues unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The full substrate repertoire of METTL18 beyond RPL3 and the structural basis by which a single histidine methyl mark tunes codon-specific elongation remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Additional substrates implied by yeast separation-of-function data not identified in mammals\", \"No structural model of how the modification mechanistically slows elongation at specific codons\", \"Direct link between the methylation activity and the breast-cancer HSP90/Src axis not reconstituted\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 4, 5]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 1, 5]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RPL3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}