{"gene":"RNF149","run_date":"2026-06-10T06:43:37","timeline":{"discoveries":[{"year":2024,"finding":"RNF149 promotes ubiquitylation-dependent proteasomal degradation of IFNGR1 (interferon gamma receptor 1) in macrophages, thereby restricting inflammation after myocardial infarction. STAT1 activation induces Rnf149 transcription, which in turn destabilizes IFNGR1 to counteract type-II IFN signaling, creating a negative-feedback loop.","method":"Immunoprecipitation/mass spectrometry, loss-of-function (KO, bone marrow transplantation, macrophage-specific knockdown), adenovirus-mediated overexpression, rescue by IFNGR1 deletion, flow cytometry, transcriptome analysis","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal IP/MS, genetic KO + rescue experiments, bone marrow transplantation, multiple orthogonal methods in one study","pmids":["38989590"],"is_preprint":false},{"year":2023,"finding":"RNF149 interacts with PHLPP2 and promotes its E3 ligase-dependent proteasomal degradation, activating the PI3K/AKT signaling pathway and conferring cisplatin resistance in esophageal squamous cell carcinoma.","method":"Co-immunoprecipitation, overexpression/knockdown in vitro and in vivo, AKT siRNA and small-molecule inhibitor rescue, functional drug resistance assays","journal":"Medical oncology (Northwood, London, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, in vitro and in vivo functional rescue, single lab","pmids":["37658961"],"is_preprint":false},{"year":2023,"finding":"RNF149 is a membrane-bound E3 ubiquitin ligase that selectively binds non-translocated (mislocalized) proteins and ubiquitinates them as part of the pre-emptive ER-associated quality control (pEQC) pathway; its dynamic localization may provide a molecular switch to regulate pEQC during ER stress.","method":"Selective binding assays, association with known pEQC components, loss-of-function showing increased ER translocation flux and myeloproliferative neoplasm phenotype, dynamic localization experiments","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional binding assay, loss-of-function phenotype, localization studies, single lab with multiple methods","pmids":["37031316"],"is_preprint":false},{"year":2025,"finding":"RNF149 interacts with IRF3 and promotes its K27-linked ubiquitination at K409 and K33-linked ubiquitination at K366 and K409, leading to IRF3 proteasomal degradation and reduced IFN-β production, thereby dampening innate antiviral immune responses.","method":"Co-immunoprecipitation, overexpression/knockdown functional assays, site-specific ubiquitination mapping (K409, K366), proteasome inhibitor experiments, IFN-β production assays, viral replication assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro ubiquitination with site-specific mutagenesis (K409, K366), co-IP, functional rescue, single lab with multiple orthogonal methods","pmids":["40245000"],"is_preprint":false},{"year":2023,"finding":"RNF149 promotes HCC progression through its E3 ubiquitin ligase activity and was identified to ubiquitinate DNAJC25 as a new substrate.","method":"Overexpression and knockdown in HCC cells, proteomic profiling, E3 ligase activity dependence demonstrated","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — substrate identification with functional overexpression/KD, single lab, limited mechanistic depth reported in abstract","pmids":["37958377"],"is_preprint":false},{"year":2024,"finding":"RNF149 directly interacts with CD63 and ubiquitinates it via K29-linked polyubiquitin chains, leading to CD63 degradation and negative regulation of LPS/TLR4 signal transduction in monocytes.","method":"Immunoprecipitation, confocal microscopy, western blotting, flow cytometry, site-specific ubiquitination (K29 of ubiquitin monomer), knockdown functional assays","journal":"Heliyon","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, confocal localization, site-specific ubiquitin chain characterization, single lab","pmids":["39104473"],"is_preprint":false},{"year":2025,"finding":"RNF149 and RNF170 cooperatively polyubiquitinate DEK at lysine K349 (within residues 270–350) via K48-linked chains, promoting DEK degradation and suppressing the RIPK1-PANoptosis pathway in bronchial epithelial cells.","method":"Mass spectrometry, molecular docking, RNA sequencing, functional ubiquitination assays, siRNA knockdown, in vivo house dust mite asthma model","journal":"Phytomedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-based site identification, K48-linkage characterization, functional rescue, single lab","pmids":["40120540"],"is_preprint":false},{"year":2022,"finding":"RNF149 is expressed as two variant transcripts in rat testis predicting truncated proteins with either the PA or RING domain; EGFP-tagged constructs show differential subcellular localization of the two variants in mouse F9 and C18-4 cell lines; RNF149 knockdown in neonatal gonocytes reduces PCNA expression and blunts PDGF-BB/17β-estradiol-induced proliferation, supporting a role for RNF149 in gonocyte proliferation.","method":"siRNA knockdown, EGFP-tagged construct transfection and imaging, RT-PCR, Western blotting, mRNA sequence determination","journal":"Frontiers in endocrinology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization by fluorescent tagging, functional KD with specific proliferative readout, single lab with multiple approaches","pmids":["35634494"],"is_preprint":false},{"year":2025,"finding":"RNF149 is a membrane-anchored E3 ubiquitin ligase whose extracellular domain can be engaged by nanobodies; a bispecific NbTAC (anti-RNF149 × anti-ROR1 nanobody) recruits RNF149 to mediate ROR1 degradation in triple-negative breast cancer cells, demonstrating that RNF149 can ubiquitinate membrane-bound substrates brought into proximity.","method":"Phage display nanobody discovery, bispecific NbTAC functional assays, cell-based ROR1 degradation assays, proliferation inhibition assays","journal":"International journal of biological macromolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct cell-based degradation assay with engineered proximity, single lab","pmids":["40609944"],"is_preprint":false},{"year":2026,"finding":"RNF149 interacts with JAK1 and promotes its K27- and K33-linked ubiquitination, leading to JAK1 proteasomal degradation and suppression of type I IFN-mediated JAK-STAT signaling, thereby facilitating EMCV evasion of antiviral immunity.","method":"Co-immunoprecipitation, overexpression/knockout functional assays, ubiquitin linkage characterization (K27, K33), proteasome inhibitor experiments, ISG expression assays, viral replication assays","journal":"Virulence","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, KO, ubiquitin chain type mapping, functional antiviral readouts, single lab","pmids":["42183758"],"is_preprint":false},{"year":2026,"finding":"RNF149 ubiquitinates CDKN2C (p18) in head and neck squamous cell carcinoma cells, as verified by proximity ligation assay and immunoprecipitation; RNF149 knockdown decreases proliferation and alters response to CDK inhibitors.","method":"Proximity ligation assay (PLA), immunoprecipitation, gene knockdown, proliferation assays, cell cycle analysis, 3D culture assays","journal":"Anticancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — PLA and co-IP for substrate identification, functional KD with specific proliferative and drug-response readouts, single lab","pmids":["42049330"],"is_preprint":false}],"current_model":"RNF149 is a membrane-anchored RING-domain E3 ubiquitin ligase that ubiquitinates multiple substrates—including IFNGR1, IRF3, JAK1, PHLPP2, CD63, DEK, DNAJC25, and CDKN2C—via K27-, K29-, K33-, or K48-linked polyubiquitin chains to drive proteasomal degradation, thereby negatively regulating type I/II interferon signaling, TLR4 signaling, and PI3K/AKT activation, while also functioning in pre-emptive ER quality control of mislocalized proteins, with its transcription induced by STAT1 to create a negative-feedback brake on macrophage-driven inflammation."},"narrative":{"mechanistic_narrative":"RNF149 is a membrane-anchored RING-domain E3 ubiquitin ligase that controls the abundance of diverse substrates by directing them to proteasomal degradation, thereby restraining inflammatory and interferon signaling while also promoting tumor cell proliferation [PMID:38989590, PMID:40245000]. In macrophages, STAT1 activation induces RNF149 transcription, and RNF149 in turn ubiquitinates IFNGR1 to drive its degradation, forming a negative-feedback brake on type-II interferon signaling that limits inflammation after myocardial infarction [PMID:38989590]. RNF149 dampens innate antiviral immunity through parallel mechanisms: it ubiquitinates IRF3 via K27- and K33-linked chains to reduce IFN-β production [PMID:40245000], and ubiquitinates JAK1 via K27- and K33-linked chains to suppress type-I IFN JAK-STAT signaling, facilitating viral immune evasion [PMID:42183758]; it also negatively regulates LPS/TLR4 signaling by K29-linked ubiquitination and degradation of CD63 [PMID:39104473]. In cancer contexts RNF149 degrades the AKT phosphatase PHLPP2 to activate PI3K/AKT signaling and confer cisplatin resistance [PMID:37658961], and acts on additional substrates including DNAJC25, DEK (cooperatively with RNF170 via K48 chains), and CDKN2C to support proliferation [PMID:37958377, PMID:40120540, PMID:42049330]. Beyond signaling, RNF149 functions in pre-emptive ER-associated quality control by selectively binding and ubiquitinating non-translocated, mislocalized proteins [PMID:37031316]. Its membrane topology positions an extracellular domain that can be exploited to ubiquitinate proximity-recruited membrane substrates [PMID:40609944].","teleology":[{"year":2022,"claim":"An early functional handle established that RNF149 has distinct domain-defined variants with different subcellular localizations and a role in cell proliferation, before any substrate was known.","evidence":"siRNA knockdown, EGFP-tagged construct imaging, and RT-PCR in rat/mouse gonocyte and germ-cell lines","pmids":["35634494"],"confidence":"Medium","gaps":["No ubiquitination substrate identified","Functional readout limited to proliferation markers (PCNA) without mechanistic targets"]},{"year":2023,"claim":"RNF149 was defined as a membrane-bound E3 ligase that selectively recognizes mislocalized, non-translocated proteins, placing it in the pre-emptive ER quality control pathway.","evidence":"Selective binding assays, association with known pEQC components, and loss-of-function showing increased ER translocation flux","pmids":["37031316"],"confidence":"Medium","gaps":["Specific endogenous mislocalized substrates not enumerated","Mechanism of the localization switch during ER stress unresolved"]},{"year":2023,"claim":"RNF149 was linked to oncogenic signaling by identifying PHLPP2 and DNAJC25 as degradation substrates, connecting its ligase activity to PI3K/AKT activation and tumor progression.","evidence":"Reciprocal co-IP, in vitro/in vivo knockdown and overexpression, AKT inhibitor rescue in esophageal carcinoma, and proteomic substrate profiling in HCC","pmids":["37658961","37958377"],"confidence":"Medium","gaps":["Ubiquitin chain linkage types not characterized for these substrates","Single-lab findings without orthogonal substrate validation"]},{"year":2024,"claim":"The discovery that STAT1 induces RNF149 to degrade IFNGR1 established RNF149 as a transcriptionally driven negative-feedback brake on interferon-gamma signaling in inflammation.","evidence":"IP/MS, genetic KO with bone marrow transplantation, macrophage-specific knockdown, and rescue by IFNGR1 deletion in a myocardial infarction model","pmids":["38989590"],"confidence":"High","gaps":["Ubiquitin linkage type on IFNGR1 not defined","Direct RING-dependent in vitro ubiquitination of IFNGR1 not shown"]},{"year":2024,"claim":"Identification of CD63 as a K29-linkage substrate extended RNF149's negative regulation to TLR4/LPS innate signaling in monocytes.","evidence":"Co-IP, confocal microscopy, site-specific K29 ubiquitin chain characterization, and knockdown functional assays","pmids":["39104473"],"confidence":"Medium","gaps":["Physiological consequences in vivo not established","Single-lab characterization"]},{"year":2025,"claim":"Mapping site-specific IRF3 ubiquitination (K27 at K409; K33 at K366/K409) showed RNF149 directly dampens antiviral IFN-β induction, defining a chain-type-resolved degradation mechanism.","evidence":"Co-IP, site-specific ubiquitination mutagenesis, proteasome inhibitor and IFN-β/viral replication assays","pmids":["40245000"],"confidence":"High","gaps":["In vivo antiviral relevance not fully defined","Relationship between IRF3 and other innate substrates within one pathway unclear"]},{"year":2025,"claim":"RNF149 was shown to cooperate with RNF170 in K48-linked degradation of DEK, linking it to suppression of RIPK1-PANoptosis in airway epithelium.","evidence":"Mass spectrometry, molecular docking, K48-linkage and site mapping (K349), siRNA, and an in vivo house dust mite asthma model","pmids":["40120540"],"confidence":"Medium","gaps":["Division of labor between RNF149 and RNF170 not resolved","Direct in vitro ubiquitination reconstitution not reported"]},{"year":2025,"claim":"Engagement of RNF149's extracellular domain by a bispecific nanobody (NbTAC) to degrade ROR1 demonstrated that RNF149 can ubiquitinate membrane substrates brought into proximity, validating it as a targetable surface ligase.","evidence":"Phage display nanobody discovery, bispecific NbTAC, and cell-based ROR1 degradation/proliferation assays in TNBC","pmids":["40609944"],"confidence":"Medium","gaps":["Endogenous natural membrane substrates not defined by this approach","Mechanism of extracellular-domain-driven recruitment not structurally resolved"]},{"year":2026,"claim":"Identification of JAK1 as a K27/K33-linkage substrate showed RNF149 suppresses type-I IFN JAK-STAT signaling, a mechanism exploited for viral immune evasion.","evidence":"Co-IP, knockout functional assays, K27/K33 linkage mapping, proteasome inhibitor, ISG and EMCV replication assays","pmids":["42183758"],"confidence":"Medium","gaps":["Whether IRF3 and JAK1 regulation are coordinate or independent is unresolved","Single-lab characterization"]},{"year":2026,"claim":"RNF149 was shown to ubiquitinate CDKN2C (p18) and modulate CDK-inhibitor response in head and neck carcinoma, extending its substrate range to a cell-cycle regulator.","evidence":"Proximity ligation assay, co-IP, knockdown, proliferation and cell cycle analyses, 3D culture","pmids":["42049330"],"confidence":"Medium","gaps":["Ubiquitin chain linkage and degradation kinetics not characterized","Direct enzymatic ubiquitination not reconstituted"]},{"year":null,"claim":"How RNF149 selects among its many reported substrates and deploys distinct ubiquitin chain linkages (K27/K29/K33/K48) in a context-specific manner remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural basis for substrate or linkage selectivity","No unifying model linking ER quality control to signaling substrate regulation","Adaptor/cofactor requirements for substrate recruitment unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,3,5,6,9]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,5,6,9]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[2]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[5,8]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,3,5,9]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,9]}],"complexes":[],"partners":["IFNGR1","IRF3","JAK1","PHLPP2","CD63","DEK","RNF170","CDKN2C"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8NC42","full_name":"E3 ubiquitin-protein ligase RNF149","aliases":["DNA polymerase-transactivated protein 2","RING finger protein 149","RING-type E3 ubiquitin transferase RNF149"],"length_aa":400,"mass_kda":43.2,"function":"E3 ubiquitin-protein ligase. Ubiquitinates BRAF, inducing its proteasomal degradation","subcellular_location":"Membrane","url":"https://www.uniprot.org/uniprotkb/Q8NC42/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RNF149","classification":"Not Classified","n_dependent_lines":7,"n_total_lines":1208,"dependency_fraction":0.005794701986754967},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CANX","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/RNF149","total_profiled":1310},"omim":[],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RNF149"},"hgnc":{"alias_symbol":["FLJ90504"],"prev_symbol":[]},"alphafold":{"accession":"Q8NC42","domains":[{"cath_id":"3.50.30.30","chopping":"36-194","consensus_level":"high","plddt":91.3014,"start":36,"end":194},{"cath_id":"3.30.40.10","chopping":"250-318","consensus_level":"high","plddt":76.7974,"start":250,"end":318}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NC42","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NC42-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NC42-F1-predicted_aligned_error_v6.png","plddt_mean":74.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RNF149","jax_strain_url":"https://www.jax.org/strain/search?query=RNF149"},"sequence":{"accession":"Q8NC42","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8NC42.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8NC42/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NC42"}},"corpus_meta":[{"pmid":"37891580","id":"PMC_37891580","title":"Spatiotemporal evolution of AML immune microenvironment remodeling and RNF149-driven drug resistance through single-cell multidimensional analysis.","date":"2023","source":"Journal of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37891580","citation_count":14,"is_preprint":false},{"pmid":"38989590","id":"PMC_38989590","title":"RNF149 Destabilizes IFNGR1 in Macrophages to Favor Postinfarction Cardiac Repair.","date":"2024","source":"Circulation research","url":"https://pubmed.ncbi.nlm.nih.gov/38989590","citation_count":12,"is_preprint":false},{"pmid":"37658961","id":"PMC_37658961","title":"RNF149 confers cisplatin resistance in esophageal squamous cell carcinoma via destabilization of PHLPP2 and activating PI3K/AKT signalling.","date":"2023","source":"Medical oncology (Northwood, London, England)","url":"https://pubmed.ncbi.nlm.nih.gov/37658961","citation_count":10,"is_preprint":false},{"pmid":"37031316","id":"PMC_37031316","title":"The role of RNF149 in the pre-emptive quality control substrate ubiquitination.","date":"2023","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/37031316","citation_count":8,"is_preprint":false},{"pmid":"40120540","id":"PMC_40120540","title":"Eupalinolide B targets DEK and PANoptosis through E3 ubiquitin ligases RNF149 and RNF170 to negatively regulate asthma.","date":"2025","source":"Phytomedicine : international journal of phytotherapy and phytopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40120540","citation_count":6,"is_preprint":false},{"pmid":"37958377","id":"PMC_37958377","title":"RNF149 Promotes HCC Progression through Its E3 Ubiquitin Ligase Activity.","date":"2023","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/37958377","citation_count":6,"is_preprint":false},{"pmid":"40245000","id":"PMC_40245000","title":"RNF149 modulates the type I IFN innate antiviral immune responses through degrading IRF3.","date":"2025","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/40245000","citation_count":4,"is_preprint":false},{"pmid":"35634494","id":"PMC_35634494","title":"Role of the Ubiquitin Ligase RNF149 in the Development of Rat Neonatal Gonocytes.","date":"2022","source":"Frontiers in endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/35634494","citation_count":4,"is_preprint":false},{"pmid":"40609944","id":"PMC_40609944","title":"Development of nanobody-based PROTAC (NbTAC) for ROR1 degradation by RNF149.","date":"2025","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/40609944","citation_count":2,"is_preprint":false},{"pmid":"39104473","id":"PMC_39104473","title":"RNF149 negatively regulates LPS/TLR4 signal transduction by ubiquitination-mediated CD63 degradation.","date":"2024","source":"Heliyon","url":"https://pubmed.ncbi.nlm.nih.gov/39104473","citation_count":1,"is_preprint":false},{"pmid":"42049330","id":"PMC_42049330","title":"Ubiquitin Ligase RNF149 Promotes Head and Neck Cancer Growth via Downregulation of CDKN2C.","date":"2026","source":"Anticancer research","url":"https://pubmed.ncbi.nlm.nih.gov/42049330","citation_count":0,"is_preprint":false},{"pmid":"42183758","id":"PMC_42183758","title":"Encephalomyocarditis virus evades IFN-mediated antiviral response by RNF149 targeting JAK1 for ubiquitination and degradation.","date":"2026","source":"Virulence","url":"https://pubmed.ncbi.nlm.nih.gov/42183758","citation_count":0,"is_preprint":false},{"pmid":"42114791","id":"PMC_42114791","title":"Multi-omics identifies RNF149 as a key molecular marker mediating neuron-glia interaction in temporal lobe epilepsy.","date":"2026","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/42114791","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8628,"output_tokens":2761,"usd":0.033649,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9968,"output_tokens":3517,"usd":0.068882,"stage2_stop_reason":"end_turn"},"total_usd":0.102531,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF149 promotes ubiquitylation-dependent proteasomal degradation of IFNGR1 (interferon gamma receptor 1) in macrophages, thereby restricting inflammation after myocardial infarction. STAT1 activation induces Rnf149 transcription, which in turn destabilizes IFNGR1 to counteract type-II IFN signaling, creating a negative-feedback loop.\",\n      \"method\": \"Immunoprecipitation/mass spectrometry, loss-of-function (KO, bone marrow transplantation, macrophage-specific knockdown), adenovirus-mediated overexpression, rescue by IFNGR1 deletion, flow cytometry, transcriptome analysis\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal IP/MS, genetic KO + rescue experiments, bone marrow transplantation, multiple orthogonal methods in one study\",\n      \"pmids\": [\"38989590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RNF149 interacts with PHLPP2 and promotes its E3 ligase-dependent proteasomal degradation, activating the PI3K/AKT signaling pathway and conferring cisplatin resistance in esophageal squamous cell carcinoma.\",\n      \"method\": \"Co-immunoprecipitation, overexpression/knockdown in vitro and in vivo, AKT siRNA and small-molecule inhibitor rescue, functional drug resistance assays\",\n      \"journal\": \"Medical oncology (Northwood, London, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, in vitro and in vivo functional rescue, single lab\",\n      \"pmids\": [\"37658961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RNF149 is a membrane-bound E3 ubiquitin ligase that selectively binds non-translocated (mislocalized) proteins and ubiquitinates them as part of the pre-emptive ER-associated quality control (pEQC) pathway; its dynamic localization may provide a molecular switch to regulate pEQC during ER stress.\",\n      \"method\": \"Selective binding assays, association with known pEQC components, loss-of-function showing increased ER translocation flux and myeloproliferative neoplasm phenotype, dynamic localization experiments\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional binding assay, loss-of-function phenotype, localization studies, single lab with multiple methods\",\n      \"pmids\": [\"37031316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF149 interacts with IRF3 and promotes its K27-linked ubiquitination at K409 and K33-linked ubiquitination at K366 and K409, leading to IRF3 proteasomal degradation and reduced IFN-β production, thereby dampening innate antiviral immune responses.\",\n      \"method\": \"Co-immunoprecipitation, overexpression/knockdown functional assays, site-specific ubiquitination mapping (K409, K366), proteasome inhibitor experiments, IFN-β production assays, viral replication assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro ubiquitination with site-specific mutagenesis (K409, K366), co-IP, functional rescue, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"40245000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RNF149 promotes HCC progression through its E3 ubiquitin ligase activity and was identified to ubiquitinate DNAJC25 as a new substrate.\",\n      \"method\": \"Overexpression and knockdown in HCC cells, proteomic profiling, E3 ligase activity dependence demonstrated\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — substrate identification with functional overexpression/KD, single lab, limited mechanistic depth reported in abstract\",\n      \"pmids\": [\"37958377\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF149 directly interacts with CD63 and ubiquitinates it via K29-linked polyubiquitin chains, leading to CD63 degradation and negative regulation of LPS/TLR4 signal transduction in monocytes.\",\n      \"method\": \"Immunoprecipitation, confocal microscopy, western blotting, flow cytometry, site-specific ubiquitination (K29 of ubiquitin monomer), knockdown functional assays\",\n      \"journal\": \"Heliyon\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, confocal localization, site-specific ubiquitin chain characterization, single lab\",\n      \"pmids\": [\"39104473\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF149 and RNF170 cooperatively polyubiquitinate DEK at lysine K349 (within residues 270–350) via K48-linked chains, promoting DEK degradation and suppressing the RIPK1-PANoptosis pathway in bronchial epithelial cells.\",\n      \"method\": \"Mass spectrometry, molecular docking, RNA sequencing, functional ubiquitination assays, siRNA knockdown, in vivo house dust mite asthma model\",\n      \"journal\": \"Phytomedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-based site identification, K48-linkage characterization, functional rescue, single lab\",\n      \"pmids\": [\"40120540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF149 is expressed as two variant transcripts in rat testis predicting truncated proteins with either the PA or RING domain; EGFP-tagged constructs show differential subcellular localization of the two variants in mouse F9 and C18-4 cell lines; RNF149 knockdown in neonatal gonocytes reduces PCNA expression and blunts PDGF-BB/17β-estradiol-induced proliferation, supporting a role for RNF149 in gonocyte proliferation.\",\n      \"method\": \"siRNA knockdown, EGFP-tagged construct transfection and imaging, RT-PCR, Western blotting, mRNA sequence determination\",\n      \"journal\": \"Frontiers in endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization by fluorescent tagging, functional KD with specific proliferative readout, single lab with multiple approaches\",\n      \"pmids\": [\"35634494\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF149 is a membrane-anchored E3 ubiquitin ligase whose extracellular domain can be engaged by nanobodies; a bispecific NbTAC (anti-RNF149 × anti-ROR1 nanobody) recruits RNF149 to mediate ROR1 degradation in triple-negative breast cancer cells, demonstrating that RNF149 can ubiquitinate membrane-bound substrates brought into proximity.\",\n      \"method\": \"Phage display nanobody discovery, bispecific NbTAC functional assays, cell-based ROR1 degradation assays, proliferation inhibition assays\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct cell-based degradation assay with engineered proximity, single lab\",\n      \"pmids\": [\"40609944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RNF149 interacts with JAK1 and promotes its K27- and K33-linked ubiquitination, leading to JAK1 proteasomal degradation and suppression of type I IFN-mediated JAK-STAT signaling, thereby facilitating EMCV evasion of antiviral immunity.\",\n      \"method\": \"Co-immunoprecipitation, overexpression/knockout functional assays, ubiquitin linkage characterization (K27, K33), proteasome inhibitor experiments, ISG expression assays, viral replication assays\",\n      \"journal\": \"Virulence\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, KO, ubiquitin chain type mapping, functional antiviral readouts, single lab\",\n      \"pmids\": [\"42183758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RNF149 ubiquitinates CDKN2C (p18) in head and neck squamous cell carcinoma cells, as verified by proximity ligation assay and immunoprecipitation; RNF149 knockdown decreases proliferation and alters response to CDK inhibitors.\",\n      \"method\": \"Proximity ligation assay (PLA), immunoprecipitation, gene knockdown, proliferation assays, cell cycle analysis, 3D culture assays\",\n      \"journal\": \"Anticancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — PLA and co-IP for substrate identification, functional KD with specific proliferative and drug-response readouts, single lab\",\n      \"pmids\": [\"42049330\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RNF149 is a membrane-anchored RING-domain E3 ubiquitin ligase that ubiquitinates multiple substrates—including IFNGR1, IRF3, JAK1, PHLPP2, CD63, DEK, DNAJC25, and CDKN2C—via K27-, K29-, K33-, or K48-linked polyubiquitin chains to drive proteasomal degradation, thereby negatively regulating type I/II interferon signaling, TLR4 signaling, and PI3K/AKT activation, while also functioning in pre-emptive ER quality control of mislocalized proteins, with its transcription induced by STAT1 to create a negative-feedback brake on macrophage-driven inflammation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RNF149 is a membrane-anchored RING-domain E3 ubiquitin ligase that controls the abundance of diverse substrates by directing them to proteasomal degradation, thereby restraining inflammatory and interferon signaling while also promoting tumor cell proliferation [#0, #3]. In macrophages, STAT1 activation induces RNF149 transcription, and RNF149 in turn ubiquitinates IFNGR1 to drive its degradation, forming a negative-feedback brake on type-II interferon signaling that limits inflammation after myocardial infarction [#0]. RNF149 dampens innate antiviral immunity through parallel mechanisms: it ubiquitinates IRF3 via K27- and K33-linked chains to reduce IFN-\\u03b2 production [#3], and ubiquitinates JAK1 via K27- and K33-linked chains to suppress type-I IFN JAK-STAT signaling, facilitating viral immune evasion [#9]; it also negatively regulates LPS/TLR4 signaling by K29-linked ubiquitination and degradation of CD63 [#5]. In cancer contexts RNF149 degrades the AKT phosphatase PHLPP2 to activate PI3K/AKT signaling and confer cisplatin resistance [#1], and acts on additional substrates including DNAJC25, DEK (cooperatively with RNF170 via K48 chains), and CDKN2C to support proliferation [#4, #6, #10]. Beyond signaling, RNF149 functions in pre-emptive ER-associated quality control by selectively binding and ubiquitinating non-translocated, mislocalized proteins [#2]. Its membrane topology positions an extracellular domain that can be exploited to ubiquitinate proximity-recruited membrane substrates [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2022,\n      \"claim\": \"An early functional handle established that RNF149 has distinct domain-defined variants with different subcellular localizations and a role in cell proliferation, before any substrate was known.\",\n      \"evidence\": \"siRNA knockdown, EGFP-tagged construct imaging, and RT-PCR in rat/mouse gonocyte and germ-cell lines\",\n      \"pmids\": [\"35634494\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No ubiquitination substrate identified\", \"Functional readout limited to proliferation markers (PCNA) without mechanistic targets\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"RNF149 was defined as a membrane-bound E3 ligase that selectively recognizes mislocalized, non-translocated proteins, placing it in the pre-emptive ER quality control pathway.\",\n      \"evidence\": \"Selective binding assays, association with known pEQC components, and loss-of-function showing increased ER translocation flux\",\n      \"pmids\": [\"37031316\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific endogenous mislocalized substrates not enumerated\", \"Mechanism of the localization switch during ER stress unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"RNF149 was linked to oncogenic signaling by identifying PHLPP2 and DNAJC25 as degradation substrates, connecting its ligase activity to PI3K/AKT activation and tumor progression.\",\n      \"evidence\": \"Reciprocal co-IP, in vitro/in vivo knockdown and overexpression, AKT inhibitor rescue in esophageal carcinoma, and proteomic substrate profiling in HCC\",\n      \"pmids\": [\"37658961\", \"37958377\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin chain linkage types not characterized for these substrates\", \"Single-lab findings without orthogonal substrate validation\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The discovery that STAT1 induces RNF149 to degrade IFNGR1 established RNF149 as a transcriptionally driven negative-feedback brake on interferon-gamma signaling in inflammation.\",\n      \"evidence\": \"IP/MS, genetic KO with bone marrow transplantation, macrophage-specific knockdown, and rescue by IFNGR1 deletion in a myocardial infarction model\",\n      \"pmids\": [\"38989590\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin linkage type on IFNGR1 not defined\", \"Direct RING-dependent in vitro ubiquitination of IFNGR1 not shown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identification of CD63 as a K29-linkage substrate extended RNF149's negative regulation to TLR4/LPS innate signaling in monocytes.\",\n      \"evidence\": \"Co-IP, confocal microscopy, site-specific K29 ubiquitin chain characterization, and knockdown functional assays\",\n      \"pmids\": [\"39104473\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological consequences in vivo not established\", \"Single-lab characterization\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mapping site-specific IRF3 ubiquitination (K27 at K409; K33 at K366/K409) showed RNF149 directly dampens antiviral IFN-\\u03b2 induction, defining a chain-type-resolved degradation mechanism.\",\n      \"evidence\": \"Co-IP, site-specific ubiquitination mutagenesis, proteasome inhibitor and IFN-\\u03b2/viral replication assays\",\n      \"pmids\": [\"40245000\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo antiviral relevance not fully defined\", \"Relationship between IRF3 and other innate substrates within one pathway unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"RNF149 was shown to cooperate with RNF170 in K48-linked degradation of DEK, linking it to suppression of RIPK1-PANoptosis in airway epithelium.\",\n      \"evidence\": \"Mass spectrometry, molecular docking, K48-linkage and site mapping (K349), siRNA, and an in vivo house dust mite asthma model\",\n      \"pmids\": [\"40120540\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Division of labor between RNF149 and RNF170 not resolved\", \"Direct in vitro ubiquitination reconstitution not reported\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Engagement of RNF149's extracellular domain by a bispecific nanobody (NbTAC) to degrade ROR1 demonstrated that RNF149 can ubiquitinate membrane substrates brought into proximity, validating it as a targetable surface ligase.\",\n      \"evidence\": \"Phage display nanobody discovery, bispecific NbTAC, and cell-based ROR1 degradation/proliferation assays in TNBC\",\n      \"pmids\": [\"40609944\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous natural membrane substrates not defined by this approach\", \"Mechanism of extracellular-domain-driven recruitment not structurally resolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identification of JAK1 as a K27/K33-linkage substrate showed RNF149 suppresses type-I IFN JAK-STAT signaling, a mechanism exploited for viral immune evasion.\",\n      \"evidence\": \"Co-IP, knockout functional assays, K27/K33 linkage mapping, proteasome inhibitor, ISG and EMCV replication assays\",\n      \"pmids\": [\"42183758\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether IRF3 and JAK1 regulation are coordinate or independent is unresolved\", \"Single-lab characterization\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"RNF149 was shown to ubiquitinate CDKN2C (p18) and modulate CDK-inhibitor response in head and neck carcinoma, extending its substrate range to a cell-cycle regulator.\",\n      \"evidence\": \"Proximity ligation assay, co-IP, knockdown, proliferation and cell cycle analyses, 3D culture\",\n      \"pmids\": [\"42049330\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin chain linkage and degradation kinetics not characterized\", \"Direct enzymatic ubiquitination not reconstituted\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RNF149 selects among its many reported substrates and deploys distinct ubiquitin chain linkages (K27/K29/K33/K48) in a context-specific manner remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural basis for substrate or linkage selectivity\", \"No unifying model linking ER quality control to signaling substrate regulation\", \"Adaptor/cofactor requirements for substrate recruitment unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 3, 5, 6, 9]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 5, 6, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 3, 5, 9]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"IFNGR1\", \"IRF3\", \"JAK1\", \"PHLPP2\", \"CD63\", \"DEK\", \"RNF170\", \"CDKN2C\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}