{"gene":"THUMPD3","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2021,"finding":"THUMPD3 forms a complex with TRMT112 (multifunctional methyltransferase subunit TRM112-like protein) that is required for N2-methylguanosine (m2G) modification at position 6 of human cytoplasmic tRNAs. THUMPD3 alone cannot methylate tRNAs; TRMT112 is required to activate its methyltransferase activity. The complex recognizes the 3'-CCA end of mature tRNAs and can methylate all 26 tested G6-containing human cytoplasmic tRNAs. THUMPD3-TRMT112 also introduces m2G at position 7 of tRNATrp.","method":"Reverse genetics coupled with RNA-mass spectrometry; in vitro enzymatic assay; co-immunoprecipitation; THUMPD3-knockout cells","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with substrate specificity mapping, reverse genetics, MS-based modification detection, and genetic knockout with phenotypic readout in a single rigorous study","pmids":["34669960"],"is_preprint":false},{"year":2021,"finding":"THUMPD3-knockout human cells exhibit impaired global protein synthesis and reduced cell growth, establishing a functional consequence of loss of tRNA m2G6/7 modification in vivo.","method":"THUMPD3-knockout cell lines; protein synthesis assay; cell growth assay","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean genetic knockout with defined cellular phenotypes (protein synthesis impairment and growth reduction), single lab with multiple readouts","pmids":["34669960"],"is_preprint":false},{"year":2024,"finding":"Depletion of THUMPD3 from lung cancer cells significantly impairs proliferation and migration. Exogenous THUMPD3 expression in normal lung fibroblasts stimulates their proliferation rate. THUMPD3 maintains expression of a pro-tumour extra-domain B (EDB)-containing isoform of Fibronectin-1 (FN1) mRNA; THUMPD3 depletion promotes an alternative splicing event that removes the EDB-encoding exon from FN1. THUMPD3 depletion selectively affects alternative splicing of ECM, cell adhesion molecule, and neurodevelopmental protein-encoding transcripts.","method":"siRNA/shRNA depletion; transcriptome-wide RNA-seq analysis; exogenous overexpression in fibroblasts; proliferation and migration assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined cellular phenotypes and transcriptome-wide splicing analysis, single lab with multiple orthogonal methods","pmids":["39656728"],"is_preprint":false},{"year":2024,"finding":"An optimised PhOxi-seq method (coupled to a novel bioinformatic pipeline) detected THUMPD3-dependent m2G sites throughout the transcriptome—including in low-abundant mRNAs—generating the first database of high-confidence THUMPD3-dependent m2G sites in multiple RNA classes within a human cancer cell line. Non-THUMPD3-controlled m2G sites were also identified, indicating the existence of other m2G writers.","method":"PhOxi-seq (chemical sequencing method for m2G detection) combined with bioinformatic pipeline; enzyme-dependent comparison using THUMPD3-depleted cells","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — novel transcriptome-wide m2G mapping method with enzyme-dependent validation, but preprint and single lab","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 (transcription factor EB) translation via impaired m2G modification of tRNALeu(CAG), thereby inhibiting autophagy and cancer cell growth. Knockdown inhibits pancreatic cancer cell growth in vitro and in vivo.","method":"shRNA knockdown; in vitro cell growth assays; in vivo xenograft models; autophagic flux assays; translation reporter assays; tRNA modification analysis","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown with defined cellular and molecular phenotypes (translation, autophagy), in vitro and in vivo validation, single lab with multiple orthogonal methods","pmids":["41530782"],"is_preprint":false}],"current_model":"THUMPD3 functions as the catalytic subunit of the THUMPD3-TRMT112 heterodimeric m2G methyltransferase complex, which installs N2-methylguanosine at position 6 (and position 7 of tRNATrp) of human cytoplasmic tRNAs by recognizing their 3'-CCA end; TRMT112 is required to activate THUMPD3's catalytic activity, and loss of this modification impairs global protein synthesis (including translation of specific proteins such as TFEB via tRNALeu(CAG)), reduces cell growth, and—through a tRNA modification-independent or downstream mechanism—regulates cancer-relevant alternative splicing of extracellular matrix transcripts including Fibronectin-1."},"narrative":{"mechanistic_narrative":"THUMPD3 is the catalytic subunit of a tRNA N2-methylguanosine (m2G) methyltransferase that controls global protein synthesis and cell growth [PMID:34669960]. It heterodimerizes with TRMT112, which is required to activate its otherwise inactive catalytic function; the complex recognizes the 3'-CCA end of mature cytoplasmic tRNAs and installs m2G at position 6 of all tested G6-containing tRNAs, as well as m2G7 of tRNATrp [PMID:34669960]. Loss of THUMPD3 in human cells impairs global protein synthesis and reduces cell growth, establishing m2G6/7 as a translation-supporting modification [PMID:34669960]. In cancer, the THUMPD3-TRMT112 complex promotes pancreatic tumor cell growth and autophagy by sustaining m2G modification of tRNALeu(CAG), which is required for efficient TFEB translation [PMID:41530782]. THUMPD3 additionally maintains a pro-tumor EDB-containing isoform of Fibronectin-1 and influences alternative splicing of extracellular matrix and cell adhesion transcripts, and its depletion impairs cancer cell proliferation and migration [PMID:39656728]. Beyond its tRNA m2G writing activity, transcriptome-wide mapping indicates THUMPD3-dependent m2G sites also exist in mRNAs and other RNA classes [PMID:bio_10.1101_2024.08.15.608094], but the mechanism linking THUMPD3 to splicing has not been resolved in the available corpus.","teleology":[{"year":2021,"claim":"Established THUMPD3 as the catalytic m2G writer for human cytoplasmic tRNAs and defined how its activity is switched on and targeted, answering what enzyme installs tRNA m2G6/7 and how substrate specificity is achieved.","evidence":"Co-immunoprecipitation, in vitro enzymatic reconstitution with substrate mapping, RNA-mass spectrometry, and THUMPD3-knockout cells","pmids":["34669960"],"confidence":"High","gaps":["Structural basis for TRMT112-dependent activation not resolved","Mechanism of 3'-CCA end recognition not defined at atomic level","Whether m2G7 of tRNATrp has a distinct functional role unaddressed"]},{"year":2021,"claim":"Linked loss of THUMPD3-catalyzed tRNA m2G to a cellular phenotype, showing the modification supports global translation and proliferation rather than being dispensable.","evidence":"THUMPD3-knockout cell lines with protein synthesis and cell growth assays","pmids":["34669960"],"confidence":"High","gaps":["Which specific transcripts are most translationally sensitive not identified at this stage","Whether the growth defect is fully attributable to translation impairment unresolved"]},{"year":2024,"claim":"Connected THUMPD3 to cancer-relevant alternative splicing, revealing a function beyond bulk translation by showing it maintains a pro-tumor Fibronectin-1 isoform and shapes ECM/adhesion transcript splicing.","evidence":"siRNA/shRNA depletion with transcriptome-wide RNA-seq, exogenous overexpression in fibroblasts, proliferation and migration assays in lung cancer cells","pmids":["39656728"],"confidence":"Medium","gaps":["Mechanism coupling THUMPD3 to splicing (tRNA-dependent vs independent) not established","Direct vs indirect control of FN1 EDB exon inclusion not distinguished","No reciprocal validation that splicing changes drive the proliferation/migration phenotype"]},{"year":2024,"claim":"Extended the THUMPD3 m2G target landscape beyond tRNAs by transcriptome-wide mapping, addressing whether m2G occurs in other RNA classes and whether THUMPD3 is the sole writer.","evidence":"Optimized PhOxi-seq chemical sequencing with a bioinformatic pipeline, enzyme-dependent comparison in THUMPD3-depleted cancer cells (preprint)","pmids":["bio_10.1101_2024.08.15.608094"],"confidence":"Medium","gaps":["Preprint, single lab, not peer-reviewed","Functional consequence of mRNA m2G sites not tested","Identity of the non-THUMPD3 m2G writers not determined"]},{"year":2026,"claim":"Provided a molecular route from THUMPD3 tRNA modification to a cancer phenotype, showing m2G of tRNALeu(CAG) is required for TFEB translation and thus for autophagy and tumor growth.","evidence":"shRNA knockdown, translation reporter assays, autophagic flux assays, tRNA modification analysis, and in vitro plus in vivo xenograft pancreatic cancer models","pmids":["41530782"],"confidence":"Medium","gaps":["Whether TFEB is the principal effector or one of several codon-sensitive targets unclear","Codon-specific decoding mechanism by which m2G affects tRNALeu(CAG) not dissected","Single lab; generality across cancer types not established"]},{"year":null,"claim":"The mechanism by which THUMPD3 influences alternative splicing, and whether this is separable from its tRNA m2G activity, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No direct RNA-binding or spliceosome-interaction data linking THUMPD3 to splicing","Functional role of THUMPD3-dependent mRNA m2G sites untested","Structural model of the THUMPD3-TRMT112 complex absent"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,3]}],"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]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,4]}],"complexes":["THUMPD3-TRMT112 m2G methyltransferase complex"],"partners":["TRMT112"],"other_free_text":[]}},"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 is a m2G methyltransferase working on a broad range of tRNA substrates.","date":"2021","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/34669960","citation_count":52,"is_preprint":false},{"pmid":"34509934","id":"PMC_34509934","title":"LncRNA THUMPD3-AS1 enhances the proliferation and inflammatory response of chondrocytes in osteoarthritis.","date":"2021","source":"International immunopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/34509934","citation_count":35,"is_preprint":false},{"pmid":"31819483","id":"PMC_31819483","title":"THUMPD3-AS1 Is Correlated With Non-Small Cell Lung Cancer And Regulates Self-Renewal Through miR-543 And ONECUT2.","date":"2019","source":"OncoTargets and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/31819483","citation_count":31,"is_preprint":false},{"pmid":"35538197","id":"PMC_35538197","title":"THUMPD3-AS1 facilitates cell growth and aggressiveness by the miR-218-5p/SKAP1 axis in colorectal cancer.","date":"2022","source":"Cell biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/35538197","citation_count":11,"is_preprint":false},{"pmid":"37705956","id":"PMC_37705956","title":"LncRNA THUMPD3-AS1 promotes invasion and EMT in gastric cancer by regulating the miR-1297/BCAT1 pathway.","date":"2023","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/37705956","citation_count":10,"is_preprint":false},{"pmid":"36017917","id":"PMC_36017917","title":"THUMPD3-AS1 Is Correlated with Gastric Cancer and Regulates Cell Function through miR-1252-3p and CXCL17.","date":"2022","source":"Critical reviews in eukaryotic gene expression","url":"https://pubmed.ncbi.nlm.nih.gov/36017917","citation_count":9,"is_preprint":false},{"pmid":"38963337","id":"PMC_38963337","title":"THUMPD3-AS1 inhibits ovarian cancer cell apoptosis through the miR-320d/ARF1 axis.","date":"2024","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/38963337","citation_count":7,"is_preprint":false},{"pmid":"39656728","id":"PMC_39656728","title":"THUMPD3 regulates alternative splicing of ECM transcripts in human lung cancer cells and promotes proliferation and migration.","date":"2024","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/39656728","citation_count":3,"is_preprint":false},{"pmid":"41168987","id":"PMC_41168987","title":"LncRNA THUMPD3-AS1 Regulates Behavioral and Synaptic Structural Abnormalities in Schizophrenia via miR-485-5p and ARHGAP8.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/41168987","citation_count":2,"is_preprint":false},{"pmid":"40639318","id":"PMC_40639318","title":"LncRNA THUMPD3-AS1 promotes the proliferation and migration of esophageal cancer cells through the miR-29a-3p/ELK1/PRDX4 signaling pathway.","date":"2025","source":"Seminars in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/40639318","citation_count":2,"is_preprint":false},{"pmid":"39925963","id":"PMC_39925963","title":"LncRNA THUMPD3-AS1/microRNA-4465/KPNA2 axis impacts human hepatocellular carcinoma cell phenotypes.","date":"2025","source":"Regenerative therapy","url":"https://pubmed.ncbi.nlm.nih.gov/39925963","citation_count":1,"is_preprint":false},{"pmid":"37766972","id":"PMC_37766972","title":"Erratum: LncRNA THUMPD3-AS1 promotes invasion and EMT in gastric cancer by regulating the miR-1297/BCAT1 pathway.","date":"2023","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/37766972","citation_count":1,"is_preprint":false},{"pmid":"41530782","id":"PMC_41530782","title":"tRNA m2G methyltransferase complex THUMPD3-TRMT112 promotes pancreatic cancer progression and autophagy via modulating TFEB translation.","date":"2026","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/41530782","citation_count":0,"is_preprint":false},{"pmid":"42259465","id":"PMC_42259465","title":"Inhibition of LncRNA THUMPD3-AS1 enhances olaparib-induced autophagy in BRCA mutant ovarian cancer cells via PI3K pathway.","date":"2026","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/42259465","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.08.15.608094","title":"PhOxi-seq detects enzyme-dependent m  <sup>2</sup>  G in multiple RNA types","date":"2024-08-15","source":"bioRxiv","url":"https://doi.org/10.1101/2024.08.15.608094","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9484,"output_tokens":1550,"usd":0.025851,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8336,"output_tokens":2471,"usd":0.051728,"stage2_stop_reason":"end_turn"},"total_usd":0.077579,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2021,\n      \"finding\": \"THUMPD3 forms a complex with TRMT112 (multifunctional methyltransferase subunit TRM112-like protein) that is required for N2-methylguanosine (m2G) modification at position 6 of human cytoplasmic tRNAs. THUMPD3 alone cannot methylate tRNAs; TRMT112 is required to activate its methyltransferase activity. The complex recognizes the 3'-CCA end of mature tRNAs and can methylate all 26 tested G6-containing human cytoplasmic tRNAs. THUMPD3-TRMT112 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 cells\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with substrate specificity mapping, reverse genetics, MS-based modification detection, and genetic knockout with phenotypic readout in a single rigorous study\",\n      \"pmids\": [\"34669960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"THUMPD3-knockout human cells exhibit impaired global protein synthesis and reduced cell growth, establishing a functional consequence of loss of tRNA m2G6/7 modification in vivo.\",\n      \"method\": \"THUMPD3-knockout cell lines; protein synthesis assay; cell growth assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic knockout with defined cellular phenotypes (protein synthesis impairment and growth reduction), single lab with multiple readouts\",\n      \"pmids\": [\"34669960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Depletion of THUMPD3 from lung cancer cells significantly impairs proliferation and migration. Exogenous THUMPD3 expression in normal lung fibroblasts stimulates their proliferation rate. THUMPD3 maintains expression of a pro-tumour extra-domain B (EDB)-containing isoform of Fibronectin-1 (FN1) mRNA; THUMPD3 depletion promotes an alternative splicing event that removes the EDB-encoding exon from FN1. THUMPD3 depletion selectively affects alternative splicing of ECM, cell adhesion molecule, and neurodevelopmental protein-encoding transcripts.\",\n      \"method\": \"siRNA/shRNA depletion; transcriptome-wide RNA-seq analysis; exogenous overexpression in fibroblasts; proliferation and migration assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined cellular phenotypes and transcriptome-wide splicing analysis, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"39656728\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"An optimised PhOxi-seq method (coupled to a novel bioinformatic pipeline) detected THUMPD3-dependent m2G sites throughout the transcriptome—including in low-abundant mRNAs—generating the first database of high-confidence THUMPD3-dependent m2G sites in multiple RNA classes within a human cancer cell line. Non-THUMPD3-controlled m2G sites were also identified, indicating the existence of other m2G writers.\",\n      \"method\": \"PhOxi-seq (chemical sequencing method for m2G detection) combined with bioinformatic pipeline; enzyme-dependent comparison using THUMPD3-depleted cells\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — novel transcriptome-wide m2G mapping method with enzyme-dependent validation, but preprint and single lab\",\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 (transcription factor EB) translation via impaired m2G modification of tRNALeu(CAG), thereby inhibiting autophagy and cancer cell growth. Knockdown inhibits pancreatic cancer cell growth in vitro and in vivo.\",\n      \"method\": \"shRNA knockdown; in vitro cell growth assays; in vivo xenograft models; autophagic flux assays; translation reporter assays; tRNA modification analysis\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown with defined cellular and molecular phenotypes (translation, autophagy), in vitro and in vivo validation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"41530782\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"THUMPD3 functions as the catalytic subunit of the THUMPD3-TRMT112 heterodimeric m2G methyltransferase complex, which installs N2-methylguanosine at position 6 (and position 7 of tRNATrp) of human cytoplasmic tRNAs by recognizing their 3'-CCA end; TRMT112 is required to activate THUMPD3's catalytic activity, and loss of this modification impairs global protein synthesis (including translation of specific proteins such as TFEB via tRNALeu(CAG)), reduces cell growth, and—through a tRNA modification-independent or downstream mechanism—regulates cancer-relevant alternative splicing of extracellular matrix transcripts including Fibronectin-1.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"THUMPD3 is the catalytic subunit of a tRNA N2-methylguanosine (m2G) methyltransferase that controls global protein synthesis and cell growth [#0, #1]. It heterodimerizes with TRMT112, which is required to activate its otherwise inactive catalytic function; the complex recognizes the 3'-CCA end of mature cytoplasmic tRNAs and installs m2G at position 6 of all tested G6-containing tRNAs, as well as m2G7 of tRNATrp [#0]. Loss of THUMPD3 in human cells impairs global protein synthesis and reduces cell growth, establishing m2G6/7 as a translation-supporting modification [#1]. In cancer, the THUMPD3-TRMT112 complex promotes pancreatic tumor cell growth and autophagy by sustaining m2G modification of tRNALeu(CAG), which is required for efficient TFEB translation [#4]. THUMPD3 additionally maintains a pro-tumor EDB-containing isoform of Fibronectin-1 and influences alternative splicing of extracellular matrix and cell adhesion transcripts, and its depletion impairs cancer cell proliferation and migration [#2]. Beyond its tRNA m2G writing activity, transcriptome-wide mapping indicates THUMPD3-dependent m2G sites also exist in mRNAs and other RNA classes [#3], but the mechanism linking THUMPD3 to splicing has not been resolved in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2021,\n      \"claim\": \"Established THUMPD3 as the catalytic m2G writer for human cytoplasmic tRNAs and defined how its activity is switched on and targeted, answering what enzyme installs tRNA m2G6/7 and how substrate specificity is achieved.\",\n      \"evidence\": \"Co-immunoprecipitation, in vitro enzymatic reconstitution with substrate mapping, RNA-mass spectrometry, and THUMPD3-knockout cells\",\n      \"pmids\": [\"34669960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for TRMT112-dependent activation not resolved\",\n        \"Mechanism of 3'-CCA end recognition not defined at atomic level\",\n        \"Whether m2G7 of tRNATrp has a distinct functional role unaddressed\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linked loss of THUMPD3-catalyzed tRNA m2G to a cellular phenotype, showing the modification supports global translation and proliferation rather than being dispensable.\",\n      \"evidence\": \"THUMPD3-knockout cell lines with protein synthesis and cell growth assays\",\n      \"pmids\": [\"34669960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Which specific transcripts are most translationally sensitive not identified at this stage\",\n        \"Whether the growth defect is fully attributable to translation impairment unresolved\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected THUMPD3 to cancer-relevant alternative splicing, revealing a function beyond bulk translation by showing it maintains a pro-tumor Fibronectin-1 isoform and shapes ECM/adhesion transcript splicing.\",\n      \"evidence\": \"siRNA/shRNA depletion with transcriptome-wide RNA-seq, exogenous overexpression in fibroblasts, proliferation and migration assays in lung cancer cells\",\n      \"pmids\": [\"39656728\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism coupling THUMPD3 to splicing (tRNA-dependent vs independent) not established\",\n        \"Direct vs indirect control of FN1 EDB exon inclusion not distinguished\",\n        \"No reciprocal validation that splicing changes drive the proliferation/migration phenotype\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended the THUMPD3 m2G target landscape beyond tRNAs by transcriptome-wide mapping, addressing whether m2G occurs in other RNA classes and whether THUMPD3 is the sole writer.\",\n      \"evidence\": \"Optimized PhOxi-seq chemical sequencing with a bioinformatic pipeline, enzyme-dependent comparison in THUMPD3-depleted cancer cells (preprint)\",\n      \"pmids\": [\"bio_10.1101_2024.08.15.608094\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Preprint, single lab, not peer-reviewed\",\n        \"Functional consequence of mRNA m2G sites not tested\",\n        \"Identity of the non-THUMPD3 m2G writers not determined\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Provided a molecular route from THUMPD3 tRNA modification to a cancer phenotype, showing m2G of tRNALeu(CAG) is required for TFEB translation and thus for autophagy and tumor growth.\",\n      \"evidence\": \"shRNA knockdown, translation reporter assays, autophagic flux assays, tRNA modification analysis, and in vitro plus in vivo xenograft pancreatic cancer models\",\n      \"pmids\": [\"41530782\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether TFEB is the principal effector or one of several codon-sensitive targets unclear\",\n        \"Codon-specific decoding mechanism by which m2G affects tRNALeu(CAG) not dissected\",\n        \"Single lab; generality across cancer types not established\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The mechanism by which THUMPD3 influences alternative splicing, and whether this is separable from its tRNA m2G activity, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No direct RNA-binding or spliceosome-interaction data linking THUMPD3 to splicing\",\n        \"Functional role of THUMPD3-dependent mRNA m2G sites untested\",\n        \"Structural model of the THUMPD3-TRMT112 complex absent\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 3]}\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]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 4]}\n    ],\n    \"complexes\": [\"THUMPD3-TRMT112 m2G methyltransferase complex\"],\n    \"partners\": [\"TRMT112\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}