{"gene":"DDX31","run_date":"2026-04-28T17:46:02","timeline":{"discoveries":[{"year":2003,"finding":"DDX31 was classified as a member of the DEAD-box (DDX) family of putative RNA helicases in humans, characterized by the conserved helicase domain containing eight motifs including the DEAD (Asp-Glu-Ala-Asp) box, establishing it as a putative RNA helicase.","method":"Bioinformatic sequence analysis of helicase domain motifs in the human genome","journal":"Genomics","confidence":"Low","confidence_rationale":"Tier 4 — computational/sequence-based classification only","pmids":["12782131"],"is_preprint":false},{"year":2002,"finding":"DDX31 was identified as a component of the human nucleolar proteome, establishing its nucleolar localization.","method":"Proteomic analysis (mass spectrometry) of HeLa cell nucleoli","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 — organellar proteomics by MS in intact nucleoli","pmids":["12429849"],"is_preprint":false},{"year":2005,"finding":"DDX31 was detected as a constituent of the human nucleolar proteome and its flux was quantitatively tracked in response to metabolic inhibitors, confirming stable nucleolar association.","method":"Quantitative mass spectrometry-based nucleolar proteomics with stable isotope labeling","journal":"Nature","confidence":"Medium","confidence_rationale":"Tier 2 — quantitative organellar proteomics with metabolic perturbation","pmids":["15635413"],"is_preprint":false},{"year":2012,"finding":"DDX31 was identified as an mRNA-binding protein in human HeLa cells through covalent UV crosslinking to RNA followed by oligo(dT) purification, establishing it as a bona fide component of the mRNA-bound proteome.","method":"UV crosslinking and oligo(dT) pulldown coupled with quantitative mass spectrometry (interactome capture)","journal":"Cell","confidence":"Medium","confidence_rationale":"Tier 2 — two complementary UV crosslinking protocols with biochemical and statistical validation","pmids":["22658674","22681889"],"is_preprint":false},{"year":2012,"finding":"DDX31 physically interacts and colocalizes with nucleophosmin (NPM1) in the nucleoli of renal cell carcinoma (RCC) cells. Attenuation of DDX31 or NPM1 expression decreased pre-ribosomal RNA (pre-rRNA) biogenesis. DDX31 knockdown caused NPM1 translocation from nucleoli to the nucleoplasm/cytoplasm, where NPM1 then bound HDM2, reducing HDM2-p53 interaction and leading to p53 stabilization, G1 arrest, and apoptosis. Conversely, ectopic DDX31 overexpression drove proliferation of kidney cells.","method":"Co-immunoprecipitation, colocalization imaging, RNAi knockdown, ectopic overexpression, pre-rRNA biogenesis assay, cell cycle analysis, apoptosis assay","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Co-IP, imaging, functional knockdown/overexpression) establishing mechanistic pathway","pmids":["23019224"],"is_preprint":false},{"year":2018,"finding":"In early muscle-invasive bladder cancer (MIBC) cells, nuclear DDX31 binds mutant p53 (mutp53) and SP1, enhancing mutp53 transcriptional activation to drive migration and invasion. In advanced MIBC cells, cytoplasmic DDX31 binds EGFR and phospho-nucleolin (NCL), activating EGFR-Akt signaling. Blocking the DDX31/NCL interaction downregulated EGFR/Akt signaling and produced antitumor effects in vivo.","method":"Co-immunoprecipitation, subcellular fractionation, RNAi knockdown, overexpression, in vivo tumor models, signaling pathway analysis","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including Co-IP for distinct complexes, in vivo validation, subcellular localization-dependent functional distinction","pmids":["29440146"],"is_preprint":false},{"year":2022,"finding":"OpenCell endogenous tagging revealed DDX31 subcellular localization in human cells, consistent with its nucleolar/nuclear residence.","method":"Genome engineering with endogenous fluorescent tagging combined with confocal live-cell imaging and mass spectrometry","journal":"Science (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 — endogenous tagging with live imaging, systematic approach","pmids":["35271311"],"is_preprint":false},{"year":2023,"finding":"Transactivation domain (TAD) mutants of p53 stabilize EGFR in the cytosol and promote EGFR-mediated AKT signaling by enhancing the interaction between EGFR and AKT via DDX31 in the cytoplasm. This places DDX31 as a scaffold that bridges EGFR and AKT in the context of gain-of-function p53 TAD mutations.","method":"Co-immunoprecipitation, subcellular fractionation, signaling pathway analysis, gene expression profiling across multiple tissue types","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP establishing ternary complex, validated in multiple tissue contexts, but single study","pmids":["36977662"],"is_preprint":false},{"year":2022,"finding":"Inhibition of DDX31 in pancreatic ductal adenocarcinoma (PDAC) cells suppressed invasion and migration, with gene set enrichment analysis indicating DDX31 correlates with activation of the MAPK signaling pathway in PDAC.","method":"RNAi knockdown, invasion/migration assays, GSEA pathway analysis","journal":"Frontiers in cell and developmental biology","confidence":"Low","confidence_rationale":"Tier 3 — functional KD with phenotypic readout but pathway placement primarily by computational GSEA, no direct MAPK mechanistic assay","pmids":["35237592"],"is_preprint":false},{"year":2025,"finding":"DDX31 undergoes ubiquitination-mediated proteasomal degradation via TRIM25-mediated K63-linked polyubiquitination. TBC1D14 promotes DDX31 degradation through this ubiquitination pathway, thereby suppressing ribosome biogenesis (rRNA transcription) and reducing EMT and lymph node metastasis in head and neck squamous cell carcinoma. DDX31 thus acts downstream of TBC1D14 in a pathway linking ubiquitin-dependent protein degradation to ribosome biogenesis control.","method":"Proteomic profiling, Co-immunoprecipitation, ubiquitination assays, overexpression/knockdown experiments, in vivo tumor/metastasis models, ribosome biogenesis assays","journal":"International journal of biological macromolecules","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods including ubiquitination assays, in vivo validation, and identification of E3 ligase (TRIM25) and linkage type (K63)","pmids":["40784392"],"is_preprint":false}],"current_model":"DDX31 is a nucleolar DEAD-box RNA helicase and mRNA-binding protein that forms a complex with NPM1 to support pre-rRNA biogenesis and ribosome biogenesis; loss of DDX31 releases NPM1 to the nucleoplasm/cytoplasm where it sequesters HDM2 away from p53, causing p53 stabilization and growth arrest, while in cancer contexts DDX31 also acts as a cytoplasmic scaffold promoting EGFR-AKT signaling (enhanced by gain-of-function p53 TAD mutants and interaction with nucleolin), and its protein level is regulated by TRIM25-mediated K63-linked polyubiquitination and proteasomal degradation downstream of TBC1D14."},"narrative":{"teleology":[{"year":2002,"claim":"Establishing DDX31 as a nucleolar protein resolved where the gene product resides, directing subsequent functional studies toward ribosome biogenesis.","evidence":"Mass spectrometry-based nucleolar proteomics of HeLa cells","pmids":["12429849"],"confidence":"Medium","gaps":["No functional role established; localization alone does not prove function","Single cell line (HeLa) examined"]},{"year":2003,"claim":"Classification of DDX31 within the DEAD-box RNA helicase family provided a molecular activity prediction but no direct enzymatic demonstration.","evidence":"Bioinformatic motif analysis across human genome","pmids":["12782131"],"confidence":"Low","gaps":["No biochemical demonstration of helicase or ATPase activity","Sequence homology alone does not confirm RNA substrate specificity"]},{"year":2012,"claim":"Two advances established DDX31's molecular interactions: UV-crosslinking identified it as a bona fide mRNA-binding protein, while Co-IP and functional studies in RCC cells demonstrated a DDX31–NPM1 nucleolar complex required for pre-rRNA biogenesis, linking DDX31 depletion to NPM1 relocalization, HDM2 sequestration, p53 stabilization, and growth arrest.","evidence":"UV crosslinking/oligo(dT) interactome capture (HeLa); Co-IP, imaging, RNAi, overexpression, pre-rRNA and cell-cycle assays (RCC cells)","pmids":["22658674","22681889","23019224"],"confidence":"High","gaps":["Direct RNA substrates (rRNA vs mRNA) not resolved","Helicase enzymatic activity still not demonstrated biochemically","Whether p53-dependent growth arrest is the sole consequence of DDX31 loss is untested"]},{"year":2018,"claim":"Discovery that cytoplasmic DDX31 scaffolds an EGFR–nucleolin complex to activate AKT signaling in advanced bladder cancer revealed a localization-dependent oncogenic function distinct from its nucleolar ribosome biogenesis role.","evidence":"Co-IP, subcellular fractionation, RNAi, in vivo tumor models in MIBC cells","pmids":["29440146"],"confidence":"High","gaps":["Mechanism driving DDX31 nucleolar-to-cytoplasmic redistribution in cancer progression unknown","Whether DDX31 helicase activity is required for EGFR–AKT scaffolding not tested"]},{"year":2023,"claim":"Identification of DDX31 as the bridge between EGFR and AKT in the context of gain-of-function p53 TAD mutants explained how mutant p53 enhances EGFR signaling and generalized the cytoplasmic scaffold function beyond bladder cancer.","evidence":"Co-IP demonstrating ternary EGFR–DDX31–AKT complex, subcellular fractionation, multi-tissue analysis","pmids":["36977662"],"confidence":"Medium","gaps":["Single study; independent replication in additional systems needed","Structural basis of DDX31-mediated bridging of EGFR and AKT unknown"]},{"year":2025,"claim":"Elucidation of DDX31 protein turnover via TRIM25-mediated K63-linked polyubiquitination downstream of TBC1D14 connected DDX31 abundance control to ribosome biogenesis regulation and metastatic suppression.","evidence":"Ubiquitination assays identifying E3 ligase and linkage type, Co-IP, in vivo metastasis models in HNSCC","pmids":["40784392"],"confidence":"Medium","gaps":["K63-linked ubiquitination typically signals non-degradative fates; mechanism linking K63 chains to proteasomal degradation not clarified","Whether TRIM25 regulation of DDX31 occurs in non-cancer contexts unknown"]},{"year":null,"claim":"The intrinsic RNA helicase/ATPase activity of DDX31 has never been reconstituted in vitro, so whether its DEAD-box enzymatic function is required for ribosome biogenesis or EGFR scaffolding remains an open question.","evidence":"","pmids":[],"confidence":"High","gaps":["No in vitro helicase or ATPase assay reported","Direct RNA substrates (specific rRNA precursors or mRNAs) not identified","No structural information available for DDX31"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,3]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[5,7]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[1,2,4,6]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[5,6]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[5,7]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[4,9]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,7]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[9]}],"complexes":[],"partners":["NPM1","EGFR","NCL","AKT1","TP53","TRIM25","TBC1D14","SP1"],"other_free_text":[]},"mechanistic_narrative":"DDX31 is a nucleolar DEAD-box helicase that functions in ribosome biogenesis and, in cancer contexts, as a cytoplasmic signaling scaffold. In the nucleolus, DDX31 complexes with nucleophosmin (NPM1) to support pre-rRNA biogenesis; its depletion causes NPM1 translocation to the nucleoplasm where NPM1 sequesters HDM2, stabilizing p53 and triggering G1 arrest and apoptosis [PMID:23019224]. When mislocalized to the cytoplasm—as occurs in advanced bladder cancer—DDX31 binds EGFR, phospho-nucleolin, and AKT, serving as a scaffold that activates EGFR–AKT signaling, a function enhanced by gain-of-function p53 transactivation-domain mutants [PMID:29440146, PMID:36977662]. DDX31 protein levels are controlled by TRIM25-mediated K63-linked polyubiquitination and proteasomal degradation downstream of TBC1D14, linking ubiquitin-dependent turnover of DDX31 to regulation of rRNA transcription and metastatic capacity [PMID:40784392]."},"prefetch_data":{"uniprot":{"accession":"Q9H8H2","full_name":"ATP-dependent DNA helicase DDX31","aliases":["DEAD box protein 31","Helicain","Probable ATP-dependent RNA helicase DDX31"],"length_aa":851,"mass_kda":94.1,"function":"May have DNA helicase activity and RNA helicase activity. Probably have ssDNA and RNA dependent ATPase activity (By similarity). Plays a role in ribosome biogenesis and TP53/p53 regulation through its interaction with NPM1 (PubMed:23019224)","subcellular_location":"Nucleus, nucleolus","url":"https://www.uniprot.org/uniprotkb/Q9H8H2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DDX31","classification":"Not Classified","n_dependent_lines":86,"n_total_lines":1208,"dependency_fraction":0.07119205298013245},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"RIOK1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/DDX31","total_profiled":1310},"omim":[{"mim_id":"616533","title":"DEAD-BOX HELICASE 31; DDX31","url":"https://www.omim.org/entry/616533"},{"mim_id":"164040","title":"NUCLEOPHOSMIN/NUCLEOPLASMIN FAMILY, MEMBER 1; NPM1","url":"https://www.omim.org/entry/164040"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoli","reliability":"Supported"},{"location":"Golgi apparatus","reliability":"Additional"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DDX31"},"hgnc":{"alias_symbol":["FLJ13633","FLJ23349","FLJ14578","PPP1R25"],"prev_symbol":[]},"alphafold":{"accession":"Q9H8H2","domains":[{"cath_id":"3.40.50.300","chopping":"227-445","consensus_level":"high","plddt":90.2663,"start":227,"end":445},{"cath_id":"3.40.50.300","chopping":"480-538_553-669_684-747","consensus_level":"high","plddt":88.0569,"start":480,"end":747}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H8H2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H8H2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H8H2-F1-predicted_aligned_error_v6.png","plddt_mean":66.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DDX31","jax_strain_url":"https://www.jax.org/strain/search?query=DDX31"},"sequence":{"accession":"Q9H8H2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H8H2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H8H2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H8H2"}},"corpus_meta":[{"pmid":"23019224","id":"PMC_23019224","title":"DDX31 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nucleoplasm/cytoplasm where it binds HDM2, reducing HDM2-p53 interaction and stabilizing p53, leading to G1 arrest and apoptosis. DDX31 and NPM1 are also required for pre-ribosomal RNA biogenesis.\",\n      \"method\": \"Co-immunoprecipitation, colocalization imaging, RNAi knockdown with cell-cycle and apoptosis readouts, ectopic overexpression proliferation assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus multiple orthogonal functional readouts (localization, cell cycle, apoptosis, rRNA biogenesis) in a single study\",\n      \"pmids\": [\"23019224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Nuclear DDX31 binds mutant p53 and SP1 to enhance mutant-p53 transcriptional activation, promoting migration and invasion in early muscle-invasive bladder cancer. In advanced disease, cytoplasmic DDX31 binds EGFR and phospho-nucleolin (NCL), activating EGFR-Akt signaling; blocking the DDX31/NCL interaction downregulates EGFR/Akt signaling and suppresses tumor growth in vivo.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, RNAi knockdown, in vivo xenograft with pharmacological inhibition\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Co-IP, fractionation, KD, in vivo rescue) across distinct disease stages\",\n      \"pmids\": [\"29440146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TAD-domain mutant p53 promotes EGFR-mediated signaling by enhancing EGFR interaction with AKT via DDX31 in the cytosol, identifying DDX31 as a scaffold bridging mutant p53-EGFR-AKT complex formation.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, gene expression profiling\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP and fractionation data, single study, mechanistic placement confirmed but limited orthogonal validation of DDX31 specifically\",\n      \"pmids\": [\"36977662\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DDX31 is subject to ubiquitination-dependent proteasomal degradation mediated by TBC1D14, specifically via TRIM25-mediated K63-linked polyubiquitination; DDX31 degradation suppresses ribosome biogenesis and EMT in head and neck squamous cell carcinoma.\",\n      \"method\": \"Proteomic profiling, ubiquitination assay, overexpression/knockdown with RiBi and EMT readouts, in vivo tumor/lymph node metastasis model\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ubiquitination assay identifies specific E3 ligase (TRIM25) and linkage type (K63), supported by in vivo functional data; single study\",\n      \"pmids\": [\"40784392\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DDX31 is a nucleolar DEAD-box RNA helicase that scaffolds a complex with NPM1 to support pre-rRNA biogenesis and sequester NPM1 away from HDM2, thereby suppressing p53 stabilization; it also functions in the cytoplasm by bridging mutant p53/EGFR/AKT complexes to promote oncogenic signaling, and its protein level is regulated by TRIM25-mediated K63-linked ubiquitination and proteasomal degradation downstream of TBC1D14.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries from KEEP papers.\n\n**Classification:**\n\nRetrieved papers:\n1. PMID:23019224 — DDX31/NPM1/p53 in RCC → KEEP\n2. PMID:29440146 — DDX31/mutp53/EGFR in bladder cancer → KEEP\n3. PMID:24608801 — Review mentioning DDX31 in medulloblastoma → KEEP (mentions DDX31 role)\n4. PMID:31233712 — LCN2 interactome; DDX31 only mentioned as survival correlation → EXCLUDE (expression only)\n5. PMID:34175685 — lnc-DDX31 (a lncRNA) in breast cancer → EXCLUDE (case B: alt-locus product)\n6. PMID:36035145 — GBM gene signature; DDX31 in correlation/survival model → EXCLUDE (expression only)\n7. PMID:36977662 — p53 mutants/EGFR; DDX31 mediates TAD mutant EGFR-AKT interaction → KEEP\n8. PMID:31872112 — PfDDX31 in Plasmodium falciparum → EXCLUDE (SYMBOL COLLISION — non-orthologous organism, parasite, fundamentally incompatible cellular context with mammalian DDX31)\n9. PMID:35237592 — DDX31 in PDAC; MAPK pathway, invasion/migration via KD → KEEP\n10. PMID:40784392 — TBC1D14/DDX31 ubiquitination in HNSCC → KEEP\n\nGene2pubmed curated papers:\n- PMID:22658674 — mRNA interactome atlas; DDX31 identified as RBP → KEEP (identifies DDX31 as mRNA-binding protein)\n- PMID:12477932 — cDNA sequencing project → EXCLUDE (no mechanism)\n- PMID:26186194 — BioPlex interactome; may list DDX31 interactions → KEEP (interactome MS)\n- PMID:28514442 — BioPlex 2.0 → KEEP (interactome MS)\n- PMID:26496610 — HeLa interactome → KEEP\n- PMID:22681889 — mRNA-bound proteome; DDX31 as RBP → KEEP\n- PMID:14702039 — cDNA sequencing → EXCLUDE\n- PMID:33961781 — BioPlex 3.0 → KEEP\n- PMID:22939629 — census of protein complexes → KEEP\n- PMID:21873635 — GO annotation → EXCLUDE\n- PMID:29395067 — BioID mRNA granules → KEEP\n- PMID:8125298 — oligo-capping method → EXCLUDE\n- PMID:15489334 — MGC project → EXCLUDE\n- PMID:35271311 — OpenCell localization → KEEP (localization data)\n- PMID:26344197 — metazoan complexes → KEEP\n- PMID:12429849 — nucleolar proteome → KEEP (DDX31 identified in nucleolus)\n- PMID:21145461 — CRL ubiquitin ligase → EXCLUDE (no DDX31 connection evident)\n- PMID:19389623 — PP1 interactome → EXCLUDE\n- PMID:26949251 — Ki-67/heterochromatin → EXCLUDE\n- PMID:12782131 — DDX/DHX gene families classification → KEEP (establishes DDX31 as DEAD-box helicase family member)\n- PMID:22586326 — SIRT7 proteomics; DDX31 may appear → KEEP\n- PMID:27926873 — FOXA1/MLL3 → EXCLUDE (no DDX31 connection)\n- PMID:31871319 — Rho GTPase proximity network → EXCLUDE\n- PMID:24457600 — EED/PRC → EXCLUDE\n- PMID:35140242 — TF interaction networks → EXCLUDE\n- PMID:30021884 — Histone XL-MS → EXCLUDE\n- PMID:32807901 — UFMylation/p53 → EXCLUDE (no DDX31 connection)\n- PMID:34079125 — BioID human cell map → KEEP (localization)\n- PMID:15635413 — nucleolar proteome dynamics → KEEP (DDX31 in nucleolus)\n\nNow extracting discoveries from KEPT papers with actual mechanistic content about DDX31:\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"DDX31 was classified as a member of the DEAD-box (DDX) family of putative RNA helicases in humans, characterized by the conserved helicase domain containing eight motifs including the DEAD (Asp-Glu-Ala-Asp) box, establishing it as a putative RNA helicase.\",\n      \"method\": \"Bioinformatic sequence analysis of helicase domain motifs in the human genome\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — computational/sequence-based classification only\",\n      \"pmids\": [\"12782131\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"DDX31 was identified as a component of the human nucleolar proteome, establishing its nucleolar localization.\",\n      \"method\": \"Proteomic analysis (mass spectrometry) of HeLa cell nucleoli\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — organellar proteomics by MS in intact nucleoli\",\n      \"pmids\": [\"12429849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"DDX31 was detected as a constituent of the human nucleolar proteome and its flux was quantitatively tracked in response to metabolic inhibitors, confirming stable nucleolar association.\",\n      \"method\": \"Quantitative mass spectrometry-based nucleolar proteomics with stable isotope labeling\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — quantitative organellar proteomics with metabolic perturbation\",\n      \"pmids\": [\"15635413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"DDX31 was identified as an mRNA-binding protein in human HeLa cells through covalent UV crosslinking to RNA followed by oligo(dT) purification, establishing it as a bona fide component of the mRNA-bound proteome.\",\n      \"method\": \"UV crosslinking and oligo(dT) pulldown coupled with quantitative mass spectrometry (interactome capture)\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — two complementary UV crosslinking protocols with biochemical and statistical validation\",\n      \"pmids\": [\"22658674\", \"22681889\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"DDX31 physically interacts and colocalizes with nucleophosmin (NPM1) in the nucleoli of renal cell carcinoma (RCC) cells. Attenuation of DDX31 or NPM1 expression decreased pre-ribosomal RNA (pre-rRNA) biogenesis. DDX31 knockdown caused NPM1 translocation from nucleoli to the nucleoplasm/cytoplasm, where NPM1 then bound HDM2, reducing HDM2-p53 interaction and leading to p53 stabilization, G1 arrest, and apoptosis. Conversely, ectopic DDX31 overexpression drove proliferation of kidney cells.\",\n      \"method\": \"Co-immunoprecipitation, colocalization imaging, RNAi knockdown, ectopic overexpression, pre-rRNA biogenesis assay, cell cycle analysis, apoptosis assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Co-IP, imaging, functional knockdown/overexpression) establishing mechanistic pathway\",\n      \"pmids\": [\"23019224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In early muscle-invasive bladder cancer (MIBC) cells, nuclear DDX31 binds mutant p53 (mutp53) and SP1, enhancing mutp53 transcriptional activation to drive migration and invasion. In advanced MIBC cells, cytoplasmic DDX31 binds EGFR and phospho-nucleolin (NCL), activating EGFR-Akt signaling. Blocking the DDX31/NCL interaction downregulated EGFR/Akt signaling and produced antitumor effects in vivo.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, RNAi knockdown, overexpression, in vivo tumor models, signaling pathway analysis\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including Co-IP for distinct complexes, in vivo validation, subcellular localization-dependent functional distinction\",\n      \"pmids\": [\"29440146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OpenCell endogenous tagging revealed DDX31 subcellular localization in human cells, consistent with its nucleolar/nuclear residence.\",\n      \"method\": \"Genome engineering with endogenous fluorescent tagging combined with confocal live-cell imaging and mass spectrometry\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — endogenous tagging with live imaging, systematic approach\",\n      \"pmids\": [\"35271311\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Transactivation domain (TAD) mutants of p53 stabilize EGFR in the cytosol and promote EGFR-mediated AKT signaling by enhancing the interaction between EGFR and AKT via DDX31 in the cytoplasm. This places DDX31 as a scaffold that bridges EGFR and AKT in the context of gain-of-function p53 TAD mutations.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, signaling pathway analysis, gene expression profiling across multiple tissue types\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP establishing ternary complex, validated in multiple tissue contexts, but single study\",\n      \"pmids\": [\"36977662\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Inhibition of DDX31 in pancreatic ductal adenocarcinoma (PDAC) cells suppressed invasion and migration, with gene set enrichment analysis indicating DDX31 correlates with activation of the MAPK signaling pathway in PDAC.\",\n      \"method\": \"RNAi knockdown, invasion/migration assays, GSEA pathway analysis\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — functional KD with phenotypic readout but pathway placement primarily by computational GSEA, no direct MAPK mechanistic assay\",\n      \"pmids\": [\"35237592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DDX31 undergoes ubiquitination-mediated proteasomal degradation via TRIM25-mediated K63-linked polyubiquitination. TBC1D14 promotes DDX31 degradation through this ubiquitination pathway, thereby suppressing ribosome biogenesis (rRNA transcription) and reducing EMT and lymph node metastasis in head and neck squamous cell carcinoma. DDX31 thus acts downstream of TBC1D14 in a pathway linking ubiquitin-dependent protein degradation to ribosome biogenesis control.\",\n      \"method\": \"Proteomic profiling, Co-immunoprecipitation, ubiquitination assays, overexpression/knockdown experiments, in vivo tumor/metastasis models, ribosome biogenesis assays\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including ubiquitination assays, in vivo validation, and identification of E3 ligase (TRIM25) and linkage type (K63)\",\n      \"pmids\": [\"40784392\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DDX31 is a nucleolar DEAD-box RNA helicase and mRNA-binding protein that forms a complex with NPM1 to support pre-rRNA biogenesis and ribosome biogenesis; loss of DDX31 releases NPM1 to the nucleoplasm/cytoplasm where it sequesters HDM2 away from p53, causing p53 stabilization and growth arrest, while in cancer contexts DDX31 also acts as a cytoplasmic scaffold promoting EGFR-AKT signaling (enhanced by gain-of-function p53 TAD mutants and interaction with nucleolin), and its protein level is regulated by TRIM25-mediated K63-linked polyubiquitination and proteasomal degradation downstream of TBC1D14.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"DDX31 is a nucleolar DEAD-box RNA helicase that supports pre-ribosomal RNA biogenesis and modulates p53 signaling through compartment-specific protein scaffolding. In the nucleolus, DDX31 sequesters NPM1; loss of DDX31 causes NPM1 redistribution to the nucleoplasm where it engages HDM2, stabilizing p53 and triggering G1 arrest and apoptosis [PMID:23019224]. In the cytoplasm, DDX31 bridges mutant p53, EGFR, and AKT into a signaling complex that activates EGFR–AKT oncogenic signaling, and in the nucleus it cooperates with mutant p53 and SP1 to drive transcriptional programs promoting invasion [PMID:29440146, PMID:36977662]. DDX31 protein levels are regulated by TRIM25-mediated K63-linked polyubiquitination and proteasomal degradation downstream of TBC1D14, coupling its turnover to control of ribosome biogenesis and epithelial–mesenchymal transition [PMID:40784392].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Establishing DDX31 as a nucleolar scaffold for NPM1 resolved how a DEAD-box helicase could link ribosome biogenesis to p53 surveillance: DDX31 retains NPM1 in nucleoli, and its loss redistributes NPM1 to engage HDM2, stabilizing p53 and causing cell-cycle arrest and apoptosis.\",\n      \"evidence\": \"Reciprocal Co-IP, colocalization imaging, RNAi knockdown with cell-cycle, apoptosis, and pre-rRNA biogenesis readouts in renal cell carcinoma cells\",\n      \"pmids\": [\"23019224\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Helicase activity has not been biochemically demonstrated or shown to be required for NPM1 retention or rRNA processing\",\n        \"Structural basis of the DDX31–NPM1 interaction is unknown\",\n        \"Whether DDX31 acts on specific pre-rRNA substrates has not been identified\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrating compartment-specific roles for DDX31 in bladder cancer showed that nuclear DDX31 enhances mutant-p53/SP1 transcription to drive invasion, while cytoplasmic DDX31 activates EGFR–AKT signaling via interaction with phospho-nucleolin and EGFR, establishing DDX31 as a context-dependent oncogenic scaffold.\",\n      \"evidence\": \"Co-IP, subcellular fractionation, RNAi, and in vivo xenograft with pharmacological blockade of DDX31–NCL interaction in bladder cancer models\",\n      \"pmids\": [\"29440146\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The signal or modification governing DDX31 nucleocytoplasmic shuttling is uncharacterized\",\n        \"Whether helicase activity is required for the cytoplasmic signaling function is untested\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identifying DDX31 as the physical bridge between TAD-domain mutant p53, EGFR, and AKT clarified the molecular architecture of the cytoplasmic signaling complex that amplifies EGFR-mediated oncogenic signaling.\",\n      \"evidence\": \"Co-IP and subcellular fractionation with gene expression profiling in mutant-p53 cell models\",\n      \"pmids\": [\"36977662\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"DDX31-specific perturbation within this complex was not extensively validated with orthogonal methods beyond Co-IP\",\n        \"Stoichiometry and domain requirements for the DDX31-mediated scaffold are unresolved\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealing that TRIM25 catalyzes K63-linked polyubiquitination of DDX31 downstream of TBC1D14, targeting it for proteasomal degradation, established the first regulatory axis controlling DDX31 protein abundance and linked it to suppression of ribosome biogenesis and EMT.\",\n      \"evidence\": \"Proteomic profiling, in vitro/in vivo ubiquitination assays, overexpression/knockdown with ribosome biogenesis and EMT readouts, and in vivo metastasis model in HNSCC\",\n      \"pmids\": [\"40784392\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"The specific DDX31 lysine residues targeted by TRIM25 are not mapped\",\n        \"How TBC1D14 recruits TRIM25 to DDX31 mechanistically is unclear\",\n        \"K63-linked ubiquitin chains typically signal non-degradative fates; the mechanism routing K63-ubiquitinated DDX31 to the proteasome needs clarification\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether DDX31 possesses RNA helicase activity, what its RNA substrates are, and the structural determinants of its diverse scaffolding interactions remain open questions central to a complete mechanistic understanding.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No biochemical demonstration of RNA unwinding or ATPase activity for DDX31\",\n        \"No RNA substrates or CLIP-based binding maps have been reported\",\n        \"No structural or domain-deletion studies defining interaction surfaces for NPM1, EGFR, AKT, or mutant p53\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"NPM1\",\n      \"EGFR\",\n      \"AKT1\",\n      \"TP53\",\n      \"NCL\",\n      \"TRIM25\",\n      \"TBC1D14\",\n      \"SP1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"DDX31 is a nucleolar DEAD-box helicase that functions in ribosome biogenesis and, in cancer contexts, as a cytoplasmic signaling scaffold. In the nucleolus, DDX31 complexes with nucleophosmin (NPM1) to support pre-rRNA biogenesis; its depletion causes NPM1 translocation to the nucleoplasm where NPM1 sequesters HDM2, stabilizing p53 and triggering G1 arrest and apoptosis [PMID:23019224]. When mislocalized to the cytoplasm—as occurs in advanced bladder cancer—DDX31 binds EGFR, phospho-nucleolin, and AKT, serving as a scaffold that activates EGFR–AKT signaling, a function enhanced by gain-of-function p53 transactivation-domain mutants [PMID:29440146, PMID:36977662]. DDX31 protein levels are controlled by TRIM25-mediated K63-linked polyubiquitination and proteasomal degradation downstream of TBC1D14, linking ubiquitin-dependent turnover of DDX31 to regulation of rRNA transcription and metastatic capacity [PMID:40784392].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Establishing DDX31 as a nucleolar protein resolved where the gene product resides, directing subsequent functional studies toward ribosome biogenesis.\",\n      \"evidence\": \"Mass spectrometry-based nucleolar proteomics of HeLa cells\",\n      \"pmids\": [\"12429849\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No functional role established; localization alone does not prove function\",\n        \"Single cell line (HeLa) examined\"\n      ]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Classification of DDX31 within the DEAD-box RNA helicase family provided a molecular activity prediction but no direct enzymatic demonstration.\",\n      \"evidence\": \"Bioinformatic motif analysis across human genome\",\n      \"pmids\": [\"12782131\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No biochemical demonstration of helicase or ATPase activity\",\n        \"Sequence homology alone does not confirm RNA substrate specificity\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Two advances established DDX31's molecular interactions: UV-crosslinking identified it as a bona fide mRNA-binding protein, while Co-IP and functional studies in RCC cells demonstrated a DDX31–NPM1 nucleolar complex required for pre-rRNA biogenesis, linking DDX31 depletion to NPM1 relocalization, HDM2 sequestration, p53 stabilization, and growth arrest.\",\n      \"evidence\": \"UV crosslinking/oligo(dT) interactome capture (HeLa); Co-IP, imaging, RNAi, overexpression, pre-rRNA and cell-cycle assays (RCC cells)\",\n      \"pmids\": [\"22658674\", \"22681889\", \"23019224\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct RNA substrates (rRNA vs mRNA) not resolved\",\n        \"Helicase enzymatic activity still not demonstrated biochemically\",\n        \"Whether p53-dependent growth arrest is the sole consequence of DDX31 loss is untested\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Discovery that cytoplasmic DDX31 scaffolds an EGFR–nucleolin complex to activate AKT signaling in advanced bladder cancer revealed a localization-dependent oncogenic function distinct from its nucleolar ribosome biogenesis role.\",\n      \"evidence\": \"Co-IP, subcellular fractionation, RNAi, in vivo tumor models in MIBC cells\",\n      \"pmids\": [\"29440146\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism driving DDX31 nucleolar-to-cytoplasmic redistribution in cancer progression unknown\",\n        \"Whether DDX31 helicase activity is required for EGFR–AKT scaffolding not tested\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of DDX31 as the bridge between EGFR and AKT in the context of gain-of-function p53 TAD mutants explained how mutant p53 enhances EGFR signaling and generalized the cytoplasmic scaffold function beyond bladder cancer.\",\n      \"evidence\": \"Co-IP demonstrating ternary EGFR–DDX31–AKT complex, subcellular fractionation, multi-tissue analysis\",\n      \"pmids\": [\"36977662\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single study; independent replication in additional systems needed\",\n        \"Structural basis of DDX31-mediated bridging of EGFR and AKT unknown\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Elucidation of DDX31 protein turnover via TRIM25-mediated K63-linked polyubiquitination downstream of TBC1D14 connected DDX31 abundance control to ribosome biogenesis regulation and metastatic suppression.\",\n      \"evidence\": \"Ubiquitination assays identifying E3 ligase and linkage type, Co-IP, in vivo metastasis models in HNSCC\",\n      \"pmids\": [\"40784392\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"K63-linked ubiquitination typically signals non-degradative fates; mechanism linking K63 chains to proteasomal degradation not clarified\",\n        \"Whether TRIM25 regulation of DDX31 occurs in non-cancer contexts unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The intrinsic RNA helicase/ATPase activity of DDX31 has never been reconstituted in vitro, so whether its DEAD-box enzymatic function is required for ribosome biogenesis or EGFR scaffolding remains an open question.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No in vitro helicase or ATPase assay reported\",\n        \"Direct RNA substrates (specific rRNA precursors or mRNAs) not identified\",\n        \"No structural information available for DDX31\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [1, 2, 4, 6]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5, 6]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [4, 9]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 7]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"NPM1\",\n      \"EGFR\",\n      \"NCL\",\n      \"AKT1\",\n      \"TP53\",\n      \"TRIM25\",\n      \"TBC1D14\",\n      \"SP1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}