{"gene":"THUMPD3","run_date":"2026-04-28T21:42:59","timeline":{"discoveries":[{"year":2021,"finding":"THUMPD3 forms a complex with TRMT112 (multifunctional methyltransferase subunit TRM112-like protein) to catalyze N2-methylguanosine (m2G) formation at position 6 of cytoplasmic tRNAs in human cells. THUMPD3 alone cannot modify tRNAs; TRMT112 interaction is required to activate its methyltransferase activity. In vitro, THUMPD3-TRMT112 methylates all 26 tested G6-containing human cytoplasmic tRNAs by recognizing the characteristic 3'-CCA of mature tRNAs. The complex also introduces m2G at position 7 of tRNATrp.","method":"Reverse genetics coupled with RNA-mass spectrometry; in vitro enzymatic assay; co-immunoprecipitation; THUMPD3-knockout cell lines","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro activity, KO validation, multiple orthogonal methods in a single study","pmids":["34669960"],"is_preprint":false},{"year":2021,"finding":"THUMPD3 knockout cells exhibit impaired global protein synthesis and reduced cell growth, demonstrating a functional consequence of loss of tRNA m2G6/7 modification in human cells.","method":"THUMPD3-knockout cell lines with global protein synthesis assay and growth assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 — clean KO with defined cellular phenotypes (protein synthesis and growth), supported by mechanistic framework in same study","pmids":["34669960"],"is_preprint":false},{"year":2024,"finding":"THUMPD3 depletion in lung cancer cells impairs proliferation and migration, and exogenous THUMPD3 expression in normal lung fibroblasts stimulates their proliferation. THUMPD3 maintains expression of a pro-tumour EDB-containing isoform of Fibronectin-1 (FN1) mRNA; depletion causes alternative splicing that removes the EDB-encoding exon. THUMPD3 depletion selectively and preferentially affects alternative splicing of ECM and cell adhesion molecule transcripts as well as neurodevelopmental protein-encoding transcripts.","method":"siRNA knockdown in lung cancer cells; transcriptome-wide analysis; exogenous overexpression in normal fibroblasts; proliferation and migration assays; RT-PCR validation of alternative splicing","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (transcriptomics, functional assays, OE), single lab","pmids":["39656728"],"is_preprint":false},{"year":2024,"finding":"Using an optimised PhOxi-seq method coupled to a novel bioinformatic pipeline, THUMPD3-dependent m2G sites were detected transcriptome-wide in a human cancer cell line, demonstrating that THUMPD3 modifies not only tRNAs but also low-abundance mRNAs, generating the first database of high-confidence THUMPD3-dependent m2G sites across multiple RNA classes.","method":"PhOxi-seq (chemical sequencing method for m2G detection) with optimised workflow and bioinformatic pipeline applied to human cancer cell line","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 method (transcriptome-wide enzymatic activity mapping), single lab preprint, no independent replication yet","pmids":["bio_10.1101_2024.08.15.608094"],"is_preprint":true},{"year":2026,"finding":"The THUMPD3-TRMT112 m2G tRNA methyltransferase complex promotes pancreatic cancer cell growth and autophagy. Mechanistically, THUMPD3/TRMT112 deficiency suppresses TFEB translation by impairing m2G modification of tRNALeu(CAG), thereby inhibiting autophagic flux and cancer cell proliferation.","method":"Knockdown in pancreatic cancer cells (in vitro and in vivo xenograft); autophagic flux assays; ribosome/translation analysis linking tRNALeu(CAG) m2G modification to TFEB protein levels","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro and in vivo KD with specific mechanistic pathway (tRNALeu(CAG) m2G → TFEB translation → autophagy), single lab","pmids":["41530782"],"is_preprint":false}],"current_model":"THUMPD3 functions as the catalytic subunit of the THUMPD3-TRMT112 heterodimeric tRNA methyltransferase complex, which installs N2-methylguanosine (m2G) at position 6 (and position 7 of tRNATrp) of cytoplasmic tRNAs by recognizing the 3'-CCA terminus; TRMT112 is required to activate THUMPD3's methyltransferase activity, loss of the complex impairs global protein synthesis (including specific codon-biased translation such as TFEB via tRNALeu(CAG)), and THUMPD3 additionally regulates cancer-relevant alternative splicing of extracellular matrix transcripts in a manner that promotes cell proliferation and migration."},"narrative":{"teleology":[{"year":2021,"claim":"Identification of THUMPD3 as a tRNA m2G6 methyltransferase resolved the long-unknown enzyme responsible for this modification in human cells, establishing that TRMT112 binding is an obligate activator and that substrate recognition depends on the 3′-CCA of mature tRNAs.","evidence":"In vitro reconstitution of THUMPD3–TRMT112 activity on 26 human tRNAs, RNA mass spectrometry, co-immunoprecipitation, and THUMPD3-KO cell lines","pmids":["34669960"],"confidence":"High","gaps":["Structural basis for TRMT112-mediated activation of THUMPD3 is unknown","How THUMPD3 discriminates position 6 from other guanosines in tRNA has not been determined","Whether THUMPD3 modifies non-tRNA substrates was not addressed"]},{"year":2021,"claim":"Demonstrating that THUMPD3 knockout reduces global translation and cell proliferation established a physiological requirement for tRNA m2G modification in protein synthesis.","evidence":"THUMPD3-KO human cell lines assessed by global protein synthesis assay and growth assays","pmids":["34669960"],"confidence":"High","gaps":["Which specific tRNAs or codons are rate-limiting upon m2G loss was not identified","Contribution of translational versus non-translational effects to the growth defect was not separated"]},{"year":2024,"claim":"Discovery that THUMPD3 depletion selectively perturbs alternative splicing of ECM transcripts—notably shifting Fibronectin-1 to an EDB-minus isoform—revealed a cancer-relevant function beyond global translation.","evidence":"siRNA knockdown in lung cancer cells with transcriptome-wide splicing analysis, RT-PCR validation, and proliferation/migration assays","pmids":["39656728"],"confidence":"Medium","gaps":["Whether the splicing effect is a direct consequence of tRNA methylation or an indirect translational effect on splicing regulators is unknown","Findings from a single lab using siRNA; off-target effects not fully excluded","Relevance to non-lung cancer contexts has not been tested"]},{"year":2026,"claim":"Linking THUMPD3–TRMT112 to codon-biased translation of TFEB via tRNALeu(CAG) m2G modification and downstream autophagic flux provided the first codon-resolution mechanism connecting this modification to a specific oncogenic pathway.","evidence":"Knockdown in pancreatic cancer cells with in vivo xenograft, ribosome/translation analysis, and autophagic flux assays","pmids":["41530782"],"confidence":"Medium","gaps":["Single-lab study; the tRNALeu(CAG)–TFEB codon-bias model awaits independent replication","Whether other Leu-codon-enriched transcripts are similarly affected was not systematically assessed","No structural or biochemical data on how m2G at position 6 of tRNALeu(CAG) influences decoding efficiency"]},{"year":null,"claim":"A high-resolution structure of the THUMPD3–TRMT112 complex, identification of the full scope of in vivo RNA substrates beyond tRNAs, and the direct molecular link between tRNA m2G loss and alternative splicing changes remain open questions.","evidence":"","pmids":[],"confidence":"High","gaps":["No crystal or cryo-EM structure of the THUMPD3–TRMT112 complex","Transcriptome-wide m2G mapping has only been reported in preprint form and awaits peer-reviewed confirmation","Mechanism connecting tRNA methylation to alternative splicing regulation is unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,4]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,3]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,4]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[4]}],"complexes":["THUMPD3-TRMT112"],"partners":["TRMT112"],"other_free_text":[]},"mechanistic_narrative":"THUMPD3 is the catalytic subunit of the THUMPD3–TRMT112 heterodimeric tRNA methyltransferase that installs N2-methylguanosine (m2G) at position 6 of cytoplasmic tRNAs (and position 7 of tRNATrp), recognizing the 3′-CCA terminus of mature tRNAs; THUMPD3 alone is catalytically inactive and requires TRMT112 for methyltransferase activity [PMID:34669960]. Loss of THUMPD3 impairs global protein synthesis and cell growth, and in pancreatic cancer the complex promotes TFEB translation via m2G modification of tRNALeu(CAG), thereby sustaining autophagic flux and proliferation [PMID:34669960, PMID:41530782]. THUMPD3 depletion also selectively alters alternative splicing of extracellular matrix and cell-adhesion transcripts, including removal of the EDB-encoding exon of Fibronectin-1, linking its activity to cancer cell migration and proliferation [PMID:39656728]."},"prefetch_data":{"uniprot":{"accession":"Q9BV44","full_name":"tRNA (guanine(6)-N(2))-methyltransferase THUMP3","aliases":["THUMP domain-containing protein 3","tRNA(Trp) (guanine(7)-N(2))-methyltransferase THUMP3"],"length_aa":507,"mass_kda":57.0,"function":"Catalytic subunit of the THUMPD3-TRM112 methyltransferase complex, that specifically mediates the S-adenosyl-L-methionine-dependent N(2)-methylation of guanosine nucleotide at position 6 (m2G6) in tRNAs (PubMed:34669960, PubMed:37283053). This is one of the major tRNA (guanine-N(2))-methyltransferases (PubMed:37283053). Also catalyzes the S-adenosyl-L-methionine-dependent N(2)-methylation of guanosine nucleotide at position 7 of tRNA(Trp) (PubMed:34669960)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9BV44/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/THUMPD3","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/THUMPD3","total_profiled":1310},"omim":[{"mim_id":"621532","title":"THUMP DOMAIN PROTEIN 3, tRNA GUANOSINE METHYLTRANSFERASE; THUMPD3","url":"https://www.omim.org/entry/621532"},{"mim_id":"621531","title":"tRNA METHYLTRANSFERASE 11; TRMT11","url":"https://www.omim.org/entry/621531"},{"mim_id":"618630","title":"tRNA METHYLTRANSFERASE SUBUNIT 11-2; TRMT112","url":"https://www.omim.org/entry/618630"},{"mim_id":"613792","title":"CHROMOSOME 3pter-p25 DELETION SYNDROME","url":"https://www.omim.org/entry/613792"},{"mim_id":"611751","title":"THUMP DOMAIN PROTEIN 2, tRNA and snRNA GUANOSINE METHYLTRANSFERASE; THUMPD2","url":"https://www.omim.org/entry/611751"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Cytosol","reliability":"Enhanced"},{"location":"Nucleoli","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/THUMPD3"},"hgnc":{"alias_symbol":["DKFZP434F091"],"prev_symbol":[]},"alphafold":{"accession":"Q9BV44","domains":[{"cath_id":"3.30.2130.30","chopping":"35-140_223-290","consensus_level":"medium","plddt":92.4652,"start":35,"end":290},{"cath_id":"3.40.50.150","chopping":"304-490","consensus_level":"high","plddt":92.9455,"start":304,"end":490}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BV44","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BV44-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BV44-F1-predicted_aligned_error_v6.png","plddt_mean":78.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=THUMPD3","jax_strain_url":"https://www.jax.org/strain/search?query=THUMPD3"},"sequence":{"accession":"Q9BV44","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BV44.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BV44/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BV44"}},"corpus_meta":[{"pmid":"34669960","id":"PMC_34669960","title":"THUMPD3-TRMT112 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The THUMPD3-TRMT112 complex methylates all 26 tested G6-containing human cytoplasmic tRNAs by recognizing the characteristic 3'-CCA of mature tRNAs, and also introduces m2G7 of tRNATrp.\",\n      \"method\": \"Reverse genetics coupled with RNA-mass spectrometry; in vitro enzymatic assay; THUMPD3-knockout cells showing impaired global protein synthesis and reduced growth\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution, reverse genetics, mass spectrometry, and KO functional readout in single study\",\n      \"pmids\": [\"34669960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"THUMPD3 depletion in lung cancer cells impairs proliferation and migration, and promotes an alternative splicing event that removes the extra-domain B (EDB)-encoding exon from Fibronectin-1 (FN1) mRNA, switching from a pro-tumour isoform to a non-EDB isoform; THUMPD3 depletion selectively affects alternative splicing of ECM and cell adhesion molecule transcripts as well as neurodevelopmental protein transcripts.\",\n      \"method\": \"shRNA depletion of THUMPD3 in lung cancer cells; transcriptome-wide RNA-seq; RT-PCR validation of FN1 splicing; exogenous THUMPD3 expression in normal lung fibroblasts\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with transcriptome-wide analysis and specific splicing phenotype, single lab\",\n      \"pmids\": [\"39656728\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"The THUMPD3-TRMT112 m2G methyltransferase complex promotes TFEB translation via m2G modification of tRNALeu(CAG); THUMPD3/TRMT112 knockdown suppresses TFEB translation, thereby reducing autophagic flux and inhibiting pancreatic cancer cell proliferation in vitro and in vivo.\",\n      \"method\": \"Knockdown of THUMPD3/TRMT112 in pancreatic cancer cells; in vitro and in vivo proliferation assays; autophagic flux measurement; mechanistic link to tRNALeu(CAG) m2G modification and TFEB translation\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with defined cellular phenotype and mechanistic pathway placement, single lab\",\n      \"pmids\": [\"41530782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PhOxi-seq, an optimised sequencing method, detects THUMPD3-dependent m2G sites throughout the transcriptome including low-abundant mRNAs, generating the first database of high-confidence THUMPD3-dependent m2G sites in multiple RNA classes within a human cancer cell line, and identifies non-THUMPD3-controlled m2G sites.\",\n      \"method\": \"PhOxi-seq transcriptome-wide sequencing with novel bioinformatic pipeline; enzyme-dependent site detection in THUMPD3-depleted vs. control cells\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — novel sequencing technology with enzyme-dependent site mapping, single preprint\",\n      \"pmids\": [\"bio_10.1101_2024.08.15.608094\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"THUMPD3 functions as the catalytic subunit of the THUMPD3-TRMT112 heterodimeric methyltransferase complex that installs N2-methylguanosine (m2G) at positions 6 and 7 of a broad range of human cytoplasmic tRNAs by recognizing their 3'-CCA termini; this m2G modification supports global protein synthesis (including TFEB translation via tRNALeu(CAG)), and THUMPD3 additionally regulates cancer-relevant alternative splicing of ECM transcripts such as Fibronectin-1, affecting cell proliferation and migration.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2021,\n      \"finding\": \"THUMPD3 forms a complex with TRMT112 (multifunctional methyltransferase subunit TRM112-like protein) to catalyze N2-methylguanosine (m2G) formation at position 6 of cytoplasmic tRNAs in human cells. THUMPD3 alone cannot modify tRNAs; TRMT112 interaction is required to activate its methyltransferase activity. In vitro, THUMPD3-TRMT112 methylates all 26 tested G6-containing human cytoplasmic tRNAs by recognizing the characteristic 3'-CCA of mature tRNAs. The complex also introduces m2G at position 7 of tRNATrp.\",\n      \"method\": \"Reverse genetics coupled with RNA-mass spectrometry; in vitro enzymatic assay; co-immunoprecipitation; THUMPD3-knockout cell lines\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro activity, KO validation, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"34669960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"THUMPD3 knockout cells exhibit impaired global protein synthesis and reduced cell growth, demonstrating a functional consequence of loss of tRNA m2G6/7 modification in human cells.\",\n      \"method\": \"THUMPD3-knockout cell lines with global protein synthesis assay and growth assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular phenotypes (protein synthesis and growth), supported by mechanistic framework in same study\",\n      \"pmids\": [\"34669960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"THUMPD3 depletion in lung cancer cells impairs proliferation and migration, and exogenous THUMPD3 expression in normal lung fibroblasts stimulates their proliferation. THUMPD3 maintains expression of a pro-tumour EDB-containing isoform of Fibronectin-1 (FN1) mRNA; depletion causes alternative splicing that removes the EDB-encoding exon. THUMPD3 depletion selectively and preferentially affects alternative splicing of ECM and cell adhesion molecule transcripts as well as neurodevelopmental protein-encoding transcripts.\",\n      \"method\": \"siRNA knockdown in lung cancer cells; transcriptome-wide analysis; exogenous overexpression in normal fibroblasts; proliferation and migration assays; RT-PCR validation of alternative splicing\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (transcriptomics, functional assays, OE), single lab\",\n      \"pmids\": [\"39656728\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Using an optimised PhOxi-seq method coupled to a novel bioinformatic pipeline, THUMPD3-dependent m2G sites were detected transcriptome-wide in a human cancer cell line, demonstrating that THUMPD3 modifies not only tRNAs but also low-abundance mRNAs, generating the first database of high-confidence THUMPD3-dependent m2G sites across multiple RNA classes.\",\n      \"method\": \"PhOxi-seq (chemical sequencing method for m2G detection) with optimised workflow and bioinformatic pipeline applied to human cancer cell line\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 method (transcriptome-wide enzymatic activity mapping), single lab preprint, no independent replication yet\",\n      \"pmids\": [\"bio_10.1101_2024.08.15.608094\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"The THUMPD3-TRMT112 m2G tRNA methyltransferase complex promotes pancreatic cancer cell growth and autophagy. Mechanistically, THUMPD3/TRMT112 deficiency suppresses TFEB translation by impairing m2G modification of tRNALeu(CAG), thereby inhibiting autophagic flux and cancer cell proliferation.\",\n      \"method\": \"Knockdown in pancreatic cancer cells (in vitro and in vivo xenograft); autophagic flux assays; ribosome/translation analysis linking tRNALeu(CAG) m2G modification to TFEB protein levels\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro and in vivo KD with specific mechanistic pathway (tRNALeu(CAG) m2G → TFEB translation → autophagy), single lab\",\n      \"pmids\": [\"41530782\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"THUMPD3 functions as the catalytic subunit of the THUMPD3-TRMT112 heterodimeric tRNA methyltransferase complex, which installs N2-methylguanosine (m2G) at position 6 (and position 7 of tRNATrp) of cytoplasmic tRNAs by recognizing the 3'-CCA terminus; TRMT112 is required to activate THUMPD3's methyltransferase activity, loss of the complex impairs global protein synthesis (including specific codon-biased translation such as TFEB via tRNALeu(CAG)), and THUMPD3 additionally regulates cancer-relevant alternative splicing of extracellular matrix transcripts in a manner that promotes cell proliferation and migration.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"THUMPD3 is the catalytic subunit of the THUMPD3–TRMT112 heterodimeric methyltransferase complex that installs N2-methylguanosine (m2G) at positions 6 and 7 of human cytoplasmic tRNAs; THUMPD3 alone is catalytically inactive and requires TRMT112 for activation, and the complex recognizes the 3′-CCA terminus of mature tRNAs to methylate all tested G6-containing cytoplasmic tRNA species [PMID:34669960]. Loss of THUMPD3 impairs global protein synthesis and cell growth, and specifically reduces TFEB translation via m2G modification of tRNALeu(CAG), thereby suppressing autophagic flux and pancreatic cancer cell proliferation [PMID:34669960, PMID:41530782]. THUMPD3 depletion in lung cancer cells additionally alters alternative splicing of ECM transcripts, notably shifting Fibronectin-1 from a pro-tumour EDB-containing isoform to a non-EDB isoform, and impairs cell proliferation and migration [PMID:39656728].\",\n  \"teleology\": [\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of THUMPD3 as the obligate catalytic partner of TRMT112 for m2G tRNA methylation resolved which enzyme installs this modification in human cells and showed that THUMPD3 requires TRMT112 for activity.\",\n      \"evidence\": \"In vitro reconstitution of THUMPD3–TRMT112 complex, RNA mass spectrometry, and THUMPD3-knockout cell characterization\",\n      \"pmids\": [\"34669960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of THUMPD3–TRMT112 interaction and substrate recognition not determined\",\n        \"Whether m2G has codon-specific translational effects beyond global protein synthesis was unknown\",\n        \"Possible non-tRNA substrates not systematically mapped\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstration that THUMPD3 depletion reprograms alternative splicing of ECM transcripts (notably FN1 EDB exon exclusion) revealed an unexpected role for THUMPD3 beyond tRNA modification in regulating cancer-relevant mRNA processing.\",\n      \"evidence\": \"shRNA knockdown in lung cancer cells with transcriptome-wide RNA-seq and RT-PCR validation of FN1 splicing\",\n      \"pmids\": [\"39656728\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether the splicing effect is a direct consequence of impaired tRNA modification or an independent function of THUMPD3 is unresolved\",\n        \"No rescue experiment with catalytic-dead THUMPD3 to distinguish methyltransferase-dependent from -independent roles\",\n        \"Single-lab observation not yet independently confirmed\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Linking THUMPD3–TRMT112 to TFEB translation via tRNALeu(CAG) m2G modification connected the tRNA methyltransferase to autophagy regulation and provided a codon-resolution mechanism for how m2G affects specific mRNA translation.\",\n      \"evidence\": \"Knockdown in pancreatic cancer cells with in vitro and in vivo proliferation assays and autophagic flux measurement\",\n      \"pmids\": [\"41530782\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether other Leu(CAG)-codon-enriched transcripts are similarly affected is untested\",\n        \"Direct measurement of tRNALeu(CAG) aminoacylation or ribosome occupancy upon m2G loss not reported\",\n        \"Single-lab finding awaiting independent replication\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The full scope of THUMPD3's non-tRNA substrates, the structural basis for CCA-terminus recognition, and whether its splicing-regulatory function is methyltransferase-dependent remain open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No crystal or cryo-EM structure of THUMPD3–TRMT112 complex available\",\n        \"Transcriptome-wide m2G substrate catalog exists only as preprint and requires peer-reviewed validation\",\n        \"Mechanism linking THUMPD3 to alternative splicing regulation is undefined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [\"THUMPD3-TRMT112\"],\n    \"partners\": [\"TRMT112\"],\n    \"other_free_text\": []\n  }\n}\n```\n\nWait, I need to fix the pathway — I used a GO term instead of Reactome. Let me correct.\n\n```json\n{\n  \"mechanistic_narrative\": \"THUMPD3 is the catalytic subunit of the THUMPD3–TRMT112 heterodimeric methyltransferase complex that installs N2-methylguanosine (m2G) at positions 6 and 7 of human cytoplasmic tRNAs; THUMPD3 alone is catalytically inactive and requires TRMT112 for activation, and the complex recognizes the 3′-CCA terminus of mature tRNAs to methylate all tested G6-containing cytoplasmic tRNA species [PMID:34669960]. Loss of THUMPD3 impairs global protein synthesis and cell growth, and specifically reduces TFEB translation via m2G modification of tRNALeu(CAG), thereby suppressing autophagic flux and pancreatic cancer cell proliferation [PMID:34669960, PMID:41530782]. THUMPD3 depletion in lung cancer cells additionally alters alternative splicing of ECM transcripts, notably shifting Fibronectin-1 from a pro-tumour EDB-containing isoform to a non-EDB isoform, and impairs cell proliferation and migration [PMID:39656728].\",\n  \"teleology\": [\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of THUMPD3 as the obligate catalytic partner of TRMT112 for m2G tRNA methylation resolved which enzyme installs this modification in human cells and showed that THUMPD3 requires TRMT112 for activity.\",\n      \"evidence\": \"In vitro reconstitution of THUMPD3–TRMT112 complex, RNA mass spectrometry, and THUMPD3-knockout cell characterization\",\n      \"pmids\": [\"34669960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of THUMPD3–TRMT112 interaction and substrate recognition not determined\",\n        \"Whether m2G has codon-specific translational effects beyond global protein synthesis was unknown\",\n        \"Possible non-tRNA substrates not systematically mapped\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstration that THUMPD3 depletion reprograms alternative splicing of ECM transcripts (notably FN1 EDB exon exclusion) revealed an unexpected role for THUMPD3 beyond tRNA modification in regulating cancer-relevant mRNA processing.\",\n      \"evidence\": \"shRNA knockdown in lung cancer cells with transcriptome-wide RNA-seq and RT-PCR validation of FN1 splicing\",\n      \"pmids\": [\"39656728\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether the splicing effect is a direct consequence of impaired tRNA modification or an independent function of THUMPD3 is unresolved\",\n        \"No rescue experiment with catalytic-dead THUMPD3 to distinguish methyltransferase-dependent from -independent roles\",\n        \"Single-lab observation not yet independently confirmed\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Linking THUMPD3–TRMT112 to TFEB translation via tRNALeu(CAG) m2G modification connected the tRNA methyltransferase to autophagy regulation and provided a codon-resolution mechanism for how m2G affects specific mRNA translation.\",\n      \"evidence\": \"Knockdown in pancreatic cancer cells with in vitro and in vivo proliferation assays and autophagic flux measurement\",\n      \"pmids\": [\"41530782\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether other Leu(CAG)-codon-enriched transcripts are similarly affected is untested\",\n        \"Direct measurement of tRNALeu(CAG) aminoacylation or ribosome occupancy upon m2G loss not reported\",\n        \"Single-lab finding awaiting independent replication\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The full scope of THUMPD3's non-tRNA substrates, the structural basis for CCA-terminus recognition, and whether its splicing-regulatory function is methyltransferase-dependent remain open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No crystal or cryo-EM structure of THUMPD3–TRMT112 complex available\",\n        \"Transcriptome-wide m2G substrate catalog exists only as preprint and requires peer-reviewed validation\",\n        \"Mechanism linking THUMPD3 to alternative splicing regulation is undefined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [\"THUMPD3-TRMT112\"],\n    \"partners\": [\"TRMT112\"],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"THUMPD3 is the catalytic subunit of the THUMPD3–TRMT112 heterodimeric tRNA methyltransferase that installs N2-methylguanosine (m2G) at position 6 of cytoplasmic tRNAs (and position 7 of tRNATrp), recognizing the 3′-CCA terminus of mature tRNAs; THUMPD3 alone is catalytically inactive and requires TRMT112 for methyltransferase activity [PMID:34669960]. Loss of THUMPD3 impairs global protein synthesis and cell growth, and in pancreatic cancer the complex promotes TFEB translation via m2G modification of tRNALeu(CAG), thereby sustaining autophagic flux and proliferation [PMID:34669960, PMID:41530782]. THUMPD3 depletion also selectively alters alternative splicing of extracellular matrix and cell-adhesion transcripts, including removal of the EDB-encoding exon of Fibronectin-1, linking its activity to cancer cell migration and proliferation [PMID:39656728].\",\n  \"teleology\": [\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of THUMPD3 as a tRNA m2G6 methyltransferase resolved the long-unknown enzyme responsible for this modification in human cells, establishing that TRMT112 binding is an obligate activator and that substrate recognition depends on the 3′-CCA of mature tRNAs.\",\n      \"evidence\": \"In vitro reconstitution of THUMPD3–TRMT112 activity on 26 human tRNAs, RNA mass spectrometry, co-immunoprecipitation, and THUMPD3-KO cell lines\",\n      \"pmids\": [\"34669960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for TRMT112-mediated activation of THUMPD3 is unknown\",\n        \"How THUMPD3 discriminates position 6 from other guanosines in tRNA has not been determined\",\n        \"Whether THUMPD3 modifies non-tRNA substrates was not addressed\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrating that THUMPD3 knockout reduces global translation and cell proliferation established a physiological requirement for tRNA m2G modification in protein synthesis.\",\n      \"evidence\": \"THUMPD3-KO human cell lines assessed by global protein synthesis assay and growth assays\",\n      \"pmids\": [\"34669960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Which specific tRNAs or codons are rate-limiting upon m2G loss was not identified\",\n        \"Contribution of translational versus non-translational effects to the growth defect was not separated\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery that THUMPD3 depletion selectively perturbs alternative splicing of ECM transcripts—notably shifting Fibronectin-1 to an EDB-minus isoform—revealed a cancer-relevant function beyond global translation.\",\n      \"evidence\": \"siRNA knockdown in lung cancer cells with transcriptome-wide splicing analysis, RT-PCR validation, and proliferation/migration assays\",\n      \"pmids\": [\"39656728\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether the splicing effect is a direct consequence of tRNA methylation or an indirect translational effect on splicing regulators is unknown\",\n        \"Findings from a single lab using siRNA; off-target effects not fully excluded\",\n        \"Relevance to non-lung cancer contexts has not been tested\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Linking THUMPD3–TRMT112 to codon-biased translation of TFEB via tRNALeu(CAG) m2G modification and downstream autophagic flux provided the first codon-resolution mechanism connecting this modification to a specific oncogenic pathway.\",\n      \"evidence\": \"Knockdown in pancreatic cancer cells with in vivo xenograft, ribosome/translation analysis, and autophagic flux assays\",\n      \"pmids\": [\"41530782\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab study; the tRNALeu(CAG)–TFEB codon-bias model awaits independent replication\",\n        \"Whether other Leu-codon-enriched transcripts are similarly affected was not systematically assessed\",\n        \"No structural or biochemical data on how m2G at position 6 of tRNALeu(CAG) influences decoding efficiency\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A high-resolution structure of the THUMPD3–TRMT112 complex, identification of the full scope of in vivo RNA substrates beyond tRNAs, and the direct molecular link between tRNA m2G loss and alternative splicing changes remain open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No crystal or cryo-EM structure of the THUMPD3–TRMT112 complex\",\n        \"Transcriptome-wide m2G mapping has only been reported in preprint form and awaits peer-reviewed confirmation\",\n        \"Mechanism connecting tRNA methylation to alternative splicing regulation is unresolved\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"complexes\": [\n      \"THUMPD3-TRMT112\"\n    ],\n    \"partners\": [\n      \"TRMT112\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}