{"gene":"RNF25","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2003,"finding":"AO7 (RNF25) interacts with the transactivation domain (TAD) of NF-κB p65 subunit via its C-terminal region, is predominantly nuclear, and supports NF-κB-dependent transcription; a RING finger Cys→Ser ubiquitination-defective mutant acts as a dominant negative suppressing p65-mediated transactivation, indicating the ubiquitin ligase activity is required for transcriptional activation.","method":"Yeast two-hybrid screen, in vitro binding assay, co-immunoprecipitation, luciferase reporter assay, dominant-negative mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Y2H, in vitro, in vivo Co-IP, mutagenesis, reporter assay) in a single study","pmids":["12748188"],"is_preprint":false},{"year":2008,"finding":"RNF25 (AO7) is an E3 ubiquitin ligase that ubiquitylates Naked2, targeting it for proteasomal degradation; TGF-alpha stabilizes Naked2 by physically binding its cytoplasmic tail to reduce AO7 binding, protecting it from this ubiquitin-mediated degradation.","method":"Overexpression/knockdown, protein half-life assay, co-immunoprecipitation, proteasome inhibitor treatment","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods identifying substrate and protection mechanism","pmids":["18757723"],"is_preprint":false},{"year":2015,"finding":"RNF25 (AO7) binds the E2 ubiquitin-conjugating enzyme UbcH5B (UBE2D2) with unusually high affinity via a structurally unique UbcH5B-binding region (U5BR) connected by an 11-amino acid linker to its RING domain, forming a clamp that surrounds the E2; the U5BR contacts the backside of UbcH5B, and this high-affinity clamp binding paradoxically decreases ubiquitination rate by blocking stimulatory non-covalent ubiquitin binding to the E2 backside.","method":"Co-crystallization (X-ray structure), in vitro ubiquitination assay, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — crystal structure combined with in vitro ubiquitination assays and mutagenesis","pmids":["26475854"],"is_preprint":false},{"year":2016,"finding":"RNF25 physically interacts with Nkd1 and Axin in an E3 ligase-independent manner to strengthen Wnt signaling by disrupting the Nkd1-Axin inhibitory complex; RNF25 depletion in zebrafish attenuates Wnt target gene transcription and promotes epithelial character in renal mesenchymal cells.","method":"Co-immunoprecipitation, E3 ligase-dead mutant analysis, RNAi knockdown in zebrafish, cell-based Wnt reporter assay","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2/3 — Co-IP with ligase-dead mutant and in vivo zebrafish knockdown, single lab","pmids":["27007149"],"is_preprint":false},{"year":2018,"finding":"RNF25 mediates NF-κB activation in gefitinib-treated NSCLC cells, which in turn transcriptionally induces IL-6 to reactivate ERK signaling, causing drug resistance; depletion of RNF25 sensitizes cells to gefitinib and overexpression augments resistance.","method":"Genome-wide RNAi screen, siRNA knockdown, overexpression, NF-κB reporter assay, cytokine measurement, ERK phosphorylation analysis","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2/3 — genetic screen followed by multiple functional assays in a single lab","pmids":["29789542"],"is_preprint":false},{"year":2024,"finding":"RNF25 catalyzes ubiquitin-mediated proteasomal degradation of E-cadherin (ECAD) in hepatocellular carcinoma cells; PKA senses oxidative stress via redox modification of its β catalytic subunit (PRKACB) at Cys200 and Cys344, and subsequently phosphorylates RNF25 at Ser450 to activate this E3 ligase activity toward ECAD.","method":"In vitro ubiquitination assay, site-directed mutagenesis (Cys200/344 in PRKACB, Ser450 in RNF25), co-immunoprecipitation, protein stability assay, in vivo xenograft metastasis model","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro ubiquitination, phosphosite mutagenesis, redox modification mapping, and in vivo validation","pmids":["38286671"],"is_preprint":false},{"year":2025,"finding":"circSATB1 acts as a scaffold to facilitate RNF25-mediated ubiquitylation and proteasomal degradation of FKBP8, releasing FKBP8's inhibitory effect on mTOR signaling to promote colorectal cancer liver metastasis.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, in vitro and in vivo metastasis assays","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2/3 — Co-IP and ubiquitination assay identifying new substrate and scaffold mechanism, single lab","pmids":["39921520"],"is_preprint":false},{"year":2025,"finding":"RNF25 binds TRIP4 and catalyzes its non-degradative ubiquitination at Lys135, disrupting TRIP4-p65 interactions and liberating p65 to activate NF-κB signaling and upregulate anti-apoptotic effectors (cIAP2, Bcl-2); the NF-κB inhibitor BAY11-7082 directly binds RNF25 to reverse this activity.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, site-directed mutagenesis (K135), NF-κB reporter assay, drug binding assay","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 — ubiquitination site mapping and interaction disruption assay, single lab","pmids":["40765826"],"is_preprint":false},{"year":2025,"finding":"RNF25 protects reversed DNA replication forks from nucleolytic degradation by MRE11 and CtIP by interacting with the replication fork protection factor REV7 and recruiting REV7 to nascent DNA after replication stress; this fork-protective role is independent of RNF25's ubiquitin ligase activity.","method":"Unbiased genetic screen, single-molecule DNA fiber analysis, co-immunoprecipitation, proximity ligation, ssDNA accumulation assay, S-phase accumulation by flow cytometry, ligase-dead mutant analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 — genetic screen, single-molecule fiber assay, Co-IP, and ligase-dead separation-of-function mutant with multiple orthogonal readouts","pmids":["40764480"],"is_preprint":false},{"year":2026,"finding":"RNF25 ubiquitylates ribosomal protein eS31 to suppress GCN2-dependent integrated stress response (ISR) activation caused by azacytidine-damaged mRNA stalling ribosomes, thereby conferring mRNA damage tolerance and preventing cell death.","method":"Genetic screens, ubiquitination assay, ribosome stalling assay, GCN2 pathway reporters, cell viability assays, mass spectrometry identification of eS31 ubiquitylation site","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 — genetic screens with mechanistic follow-up identifying substrate (eS31), pathway (GCN2-ISR), and functional consequence via multiple orthogonal methods","pmids":["41875887"],"is_preprint":false}],"current_model":"RNF25 is a RING finger E3 ubiquitin ligase that binds the E2 enzyme UbcH5B via a unique clamp mechanism, ubiquitylates diverse substrates (Naked2, E-cadherin, FKBP8, TRIP4, ribosomal protein eS31) to control proteasomal degradation or non-degradative signaling, activates NF-κB transcription by interacting with p65 TAD and ubiquitylating TRIP4, modulates Wnt signaling by disrupting the Nkd1-Axin complex independently of its ligase activity, and protects reversed DNA replication forks from MRE11/CtIP-mediated degradation by recruiting REV7—with its fork-protective role separable from ubiquitin conjugation."},"narrative":{"teleology":[{"year":2003,"claim":"Establishing that RNF25 is a nuclear RING finger protein whose ubiquitin ligase activity is required for NF-κB-dependent transcription answered the question of whether this uncharacterized RING protein functions in gene regulation.","evidence":"Yeast two-hybrid screen identifying p65 TAD interaction, Co-IP, luciferase reporter, and dominant-negative RING mutant in mammalian cells","pmids":["12748188"],"confidence":"High","gaps":["Direct ubiquitylation substrate for NF-κB activation was not identified","Whether RNF25 modifies p65 itself or a co-regulator was unknown","In vivo relevance beyond reporter assays not tested"]},{"year":2008,"claim":"Identification of Naked2 as a direct ubiquitylation substrate targeted for proteasomal degradation established RNF25 as a bona fide E3 ligase with a defined substrate and revealed its role in modulating Wnt-pathway-related signaling molecules.","evidence":"Overexpression/knockdown, protein half-life assays, Co-IP, and proteasome inhibitor treatment in mammalian cells","pmids":["18757723"],"confidence":"High","gaps":["Specific ubiquitylation sites on Naked2 were not mapped","Whether TGF-α-mediated protection of Naked2 occurs in vivo was not addressed","Chain type preference of RNF25-catalyzed ubiquitylation remained unknown"]},{"year":2015,"claim":"Crystal structure of the RNF25–UbcH5B complex revealed a unique clamp mechanism whereby a dedicated U5BR contacts the E2 backside, explaining unusually high-affinity E2 engagement and showing that this paradoxically attenuates catalytic rate by occluding non-covalent ubiquitin binding.","evidence":"X-ray co-crystallography, in vitro ubiquitination assays, and site-directed mutagenesis","pmids":["26475854"],"confidence":"High","gaps":["Physiological consequence of the auto-inhibitory clamp mechanism in cells was not tested","Whether RNF25 uses E2 enzymes other than UbcH5B in vivo was not determined","How the clamp is relieved to permit productive ubiquitylation remained unclear"]},{"year":2016,"claim":"Demonstration that RNF25 disrupts the Nkd1–Axin complex to promote Wnt signaling independently of its RING ligase activity revealed a non-catalytic scaffolding function, broadening the mechanistic repertoire of RNF25.","evidence":"Co-IP with ligase-dead mutant, RNAi knockdown in zebrafish embryos, and Wnt reporter assays","pmids":["27007149"],"confidence":"Medium","gaps":["Structural basis for Nkd1 and Axin binding was not resolved","Whether ligase-independent and ligase-dependent functions compete or cooperate was not examined","Single-lab finding without independent replication"]},{"year":2018,"claim":"A genome-wide RNAi screen linked RNF25 to gefitinib resistance in NSCLC by showing it activates NF-κB, which induces IL-6 to reactivate ERK signaling, connecting RNF25's transcriptional function to a clinically relevant drug-resistance pathway.","evidence":"Genome-wide RNAi screen, siRNA/overexpression, NF-κB reporter, cytokine measurement, and ERK phosphorylation analysis in NSCLC cells","pmids":["29789542"],"confidence":"Medium","gaps":["Direct ubiquitylation target mediating NF-κB activation in this context was not identified","In vivo drug-resistance model was not included","Generalizability beyond NSCLC cell lines not tested"]},{"year":2024,"claim":"Discovery that PKA phosphorylates RNF25 at Ser450 in response to oxidative stress to activate its E3 ligase activity toward E-cadherin established a redox-regulated post-translational switch controlling RNF25 substrate selection and EMT-related metastasis.","evidence":"In vitro ubiquitination, phosphosite and Cys mutagenesis, Co-IP, protein stability assays, and in vivo xenograft metastasis model in hepatocellular carcinoma","pmids":["38286671"],"confidence":"High","gaps":["Whether Ser450 phosphorylation affects other RNF25 substrates was not tested","Structural mechanism by which phosphorylation activates ligase activity unknown","Contribution relative to other E-cadherin-targeting E3 ligases not assessed"]},{"year":2025,"claim":"Three independent studies simultaneously expanded the substrate repertoire and revealed a ligase-independent genome-protection role: RNF25 ubiquitylates FKBP8 (scaffolded by circSATB1) to relieve mTOR inhibition, non-degradatively ubiquitylates TRIP4 at K135 to liberate p65 for NF-κB activation, and recruits REV7 to reversed replication forks to protect nascent DNA from MRE11/CtIP degradation without requiring catalytic activity.","evidence":"Co-IP, ubiquitination assays, site-directed mutagenesis, single-molecule DNA fiber analysis, proximity ligation, ligase-dead separation-of-function mutant, drug-binding assay, and in vivo metastasis models","pmids":["39921520","40765826","40764480"],"confidence":"High","gaps":["How REV7 recruitment is coordinated with other fork-protection factors is unknown","Structural basis for RNF25–REV7 interaction not resolved","Whether the circSATB1-scaffolded mechanism operates outside colorectal cancer is untested"]},{"year":2026,"claim":"Identification of ribosomal protein eS31 as an RNF25 substrate linked the E3 ligase to ribosome quality control by showing that eS31 ubiquitylation suppresses GCN2-dependent integrated stress response activation at damaged-mRNA-stalled ribosomes, conferring mRNA damage tolerance.","evidence":"Genetic screens, ubiquitination and ribosome stalling assays, GCN2 pathway reporters, cell viability assays, and mass spectrometry identification of eS31 ubiquitylation site","pmids":["41875887"],"confidence":"High","gaps":["Ubiquitin chain type on eS31 not determined","Whether RNF25's ribosome quality-control function intersects with its NF-κB or fork-protection roles is unknown","Structural context of eS31 ubiquitylation on the ribosome not resolved"]},{"year":null,"claim":"A unifying model explaining how RNF25 partitions between its diverse catalytic and non-catalytic roles — NF-κB activation, substrate-specific proteasomal degradation, replication fork protection, and ribosome quality control — and whether distinct regulatory inputs (phosphorylation, scaffold RNAs, protein partners) route RNF25 to specific substrates and cellular compartments remains to be established.","evidence":"","pmids":[],"confidence":"Low","gaps":["No knockout mouse or organismal loss-of-function phenotype reported","Ubiquitin chain-type specificity for most substrates undetermined","How the auto-inhibitory U5BR clamp is relieved in vivo is not known"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,5,6,7,9]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,4,7]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"GO:0005840","term_label":"ribosome","supporting_discovery_ids":[9]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3,4,7]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[8]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,5,6,9]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,4,7]}],"complexes":[],"partners":["RELA","UBE2D2","NKD1","TRIP4","MAD2L2","FKBP8","NKD2","CDH1"],"other_free_text":[]},"mechanistic_narrative":"RNF25 is a RING finger E3 ubiquitin ligase that operates through both ligase-dependent and ligase-independent mechanisms to regulate transcription, signaling, DNA replication fork integrity, and ribosome quality control. Its RING domain engages the E2 enzyme UbcH5B via a structurally unique high-affinity clamp involving a dedicated UbcH5B-binding region (U5BR) tethered by a short linker, and its catalytic activity drives proteasomal degradation of substrates including Naked2, E-cadherin, and FKBP8, as well as non-degradative ubiquitylation of TRIP4 and ribosomal protein eS31 [PMID:26475854, PMID:18757723, PMID:38286671, PMID:39921520, PMID:40765826, PMID:41875887]. RNF25 activates NF-κB-dependent transcription by binding the p65 transactivation domain and ubiquitylating TRIP4 to liberate p65, and it modulates Wnt signaling independently of its ligase activity by disrupting the inhibitory Nkd1–Axin complex [PMID:12748188, PMID:40765826, PMID:27007149]. Separately from ubiquitin conjugation, RNF25 protects reversed DNA replication forks from MRE11/CtIP-mediated degradation by recruiting the fork-protection factor REV7 to nascent DNA, and it ubiquitylates ribosomal protein eS31 to suppress GCN2-dependent integrated stress response activation upon encounter with damaged-mRNA-stalled ribosomes [PMID:40764480, PMID:41875887]."},"prefetch_data":{"uniprot":{"accession":"Q96BH1","full_name":"E3 ubiquitin-protein ligase RNF25","aliases":["RING finger protein 25","RING finger protein AO7"],"length_aa":459,"mass_kda":51.2,"function":"E3 ubiquitin-protein ligase that plays a key role in the RNF14-RNF25 translation quality control pathway, a pathway that takes place when a ribosome has stalled during translation, and which promotes ubiquitination and degradation of translation factors on stalled ribosomes (PubMed:36638793, PubMed:37651229, PubMed:37951216). Catalyzes ubiquitination of RPS27A in response to ribosome collisions, promoting activation of RNF14 (PubMed:36638793). RNF25 catalyzes ubiquitination of other ribosomal proteins on stalled ribosomes, such as RPL0, RPL1, RPL12, RPS13 and RPS17 (PubMed:36638793). Also involved in ubiquitination and degradation of stalled ETF1/eRF1 (PubMed:36638793, PubMed:37651229). Independently of its function in the response to stalled ribosomes, mediates ubiquitination and subsequent proteasomal degradation of NKD2 (By similarity). May also stimulate transcription mediated by NF-kappa-B via its interaction with RELA/p65 (PubMed:12748188)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q96BH1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RNF25","classification":"Not Classified","n_dependent_lines":110,"n_total_lines":1208,"dependency_fraction":0.09105960264900662},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SCFD1","stoichiometry":0.2},{"gene":"STX18","stoichiometry":0.2},{"gene":"STX5","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/RNF25","total_profiled":1310},"omim":[{"mim_id":"616014","title":"RING FINGER PROTEIN 25; RNF25","url":"https://www.omim.org/entry/616014"},{"mim_id":"164014","title":"RELA PROTOONCOGENE, NFKB SUBUNIT; RELA","url":"https://www.omim.org/entry/164014"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Actin filaments","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RNF25"},"hgnc":{"alias_symbol":["AO7","FLJ13906"],"prev_symbol":[]},"alphafold":{"accession":"Q96BH1","domains":[{"cath_id":"3.10.110.10","chopping":"16-133","consensus_level":"high","plddt":93.6081,"start":16,"end":133},{"cath_id":"3.30.40.10","chopping":"136-185_197-225","consensus_level":"medium","plddt":88.2473,"start":136,"end":225},{"cath_id":"1.20.5","chopping":"228-262","consensus_level":"medium","plddt":88.1091,"start":228,"end":262}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96BH1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96BH1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96BH1-F1-predicted_aligned_error_v6.png","plddt_mean":67.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RNF25","jax_strain_url":"https://www.jax.org/strain/search?query=RNF25"},"sequence":{"accession":"Q96BH1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96BH1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96BH1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96BH1"}},"corpus_meta":[{"pmid":"12748188","id":"PMC_12748188","title":"RING finger protein AO7 supports NF-kappaB-mediated transcription by interacting with the transactivation domain of the p65 subunit.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12748188","citation_count":34,"is_preprint":false},{"pmid":"38286671","id":"PMC_38286671","title":"Oxidative Stress Promotes Liver Cancer Metastasis via RNF25-Mediated E-Cadherin Protein Degradation.","date":"2024","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/38286671","citation_count":33,"is_preprint":false},{"pmid":"26475854","id":"PMC_26475854","title":"Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B.","date":"2015","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26475854","citation_count":29,"is_preprint":false},{"pmid":"29789542","id":"PMC_29789542","title":"RNF25 promotes gefitinib resistance in EGFR-mutant NSCLC cells by inducing NF-κB-mediated ERK reactivation.","date":"2018","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/29789542","citation_count":24,"is_preprint":false},{"pmid":"18757723","id":"PMC_18757723","title":"EGF receptor-independent action of TGF-alpha protects Naked2 from AO7-mediated ubiquitylation and proteasomal degradation.","date":"2008","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/18757723","citation_count":16,"is_preprint":false},{"pmid":"31982684","id":"PMC_31982684","title":"MnCeOX with high efficiency and stability for activating persulfate to degrade AO7 and ofloxacin.","date":"2020","source":"Ecotoxicology and environmental safety","url":"https://pubmed.ncbi.nlm.nih.gov/31982684","citation_count":12,"is_preprint":false},{"pmid":"31195175","id":"PMC_31195175","title":"Efficient degradation of AO7 by ceria-delafossite nanocomposite with non-inert support as a synergistic catalyst in electro-fenton process.","date":"2019","source":"Environmental pollution (Barking, Essex : 1987)","url":"https://pubmed.ncbi.nlm.nih.gov/31195175","citation_count":12,"is_preprint":false},{"pmid":"39921520","id":"PMC_39921520","title":"CircSATB1 Promotes Colorectal Cancer Liver Metastasis through Facilitating FKBP8 Degradation via RNF25-Mediated Ubiquitination.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/39921520","citation_count":10,"is_preprint":false},{"pmid":"27007149","id":"PMC_27007149","title":"Rnf25/AO7 positively regulates wnt signaling via disrupting Nkd1-Axin inhibitory complex independent of its ubiquitin ligase activity.","date":"2016","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/27007149","citation_count":8,"is_preprint":false},{"pmid":"40765826","id":"PMC_40765826","title":"BAY11-7082 Targets RNF25 to Reverse TRIP4 Ubiquitination-dependent NF-κB Activation and Apoptosis Resistance in Renal Cell Carcinoma.","date":"2025","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/40765826","citation_count":4,"is_preprint":false},{"pmid":"40764480","id":"PMC_40764480","title":"The RING finger E3 ligase RNF25 protects DNA replication forks independently of its canonical roles in ubiquitin signaling.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/40764480","citation_count":0,"is_preprint":false},{"pmid":"41991692","id":"PMC_41991692","title":"Mechanical compaction of melamine controls carbon nitride structure and enhances AO7 photodegradation.","date":"2026","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/41991692","citation_count":0,"is_preprint":false},{"pmid":"39829812","id":"PMC_39829812","title":"The RING Finger E3 Ligase RNF25 Protects DNA Replication Forks Independently of its Canonical Roles in Ubiquitin Signaling.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/39829812","citation_count":0,"is_preprint":false},{"pmid":"20825022","id":"PMC_20825022","title":"[Study on the degradation of AO7 by UV/K2S2O8, system: kinetics and pathways].","date":"2010","source":"Huan jing ke xue= Huanjing kexue","url":"https://pubmed.ncbi.nlm.nih.gov/20825022","citation_count":0,"is_preprint":false},{"pmid":"41875887","id":"PMC_41875887","title":"RNF25 confers mRNA damage tolerance by curbing activation of the integrated stress response.","date":"2026","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/41875887","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8355,"output_tokens":2666,"usd":0.032528},"stage2":{"model":"claude-opus-4-6","input_tokens":6008,"output_tokens":2964,"usd":0.15621},"total_usd":0.188738,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"AO7 (RNF25) interacts with the transactivation domain (TAD) of NF-κB p65 subunit via its C-terminal region, is predominantly nuclear, and supports NF-κB-dependent transcription; a RING finger Cys→Ser ubiquitination-defective mutant acts as a dominant negative suppressing p65-mediated transactivation, indicating the ubiquitin ligase activity is required for transcriptional activation.\",\n      \"method\": \"Yeast two-hybrid screen, in vitro binding assay, co-immunoprecipitation, luciferase reporter assay, dominant-negative mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Y2H, in vitro, in vivo Co-IP, mutagenesis, reporter assay) in a single study\",\n      \"pmids\": [\"12748188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"RNF25 (AO7) is an E3 ubiquitin ligase that ubiquitylates Naked2, targeting it for proteasomal degradation; TGF-alpha stabilizes Naked2 by physically binding its cytoplasmic tail to reduce AO7 binding, protecting it from this ubiquitin-mediated degradation.\",\n      \"method\": \"Overexpression/knockdown, protein half-life assay, co-immunoprecipitation, proteasome inhibitor treatment\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods identifying substrate and protection mechanism\",\n      \"pmids\": [\"18757723\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RNF25 (AO7) binds the E2 ubiquitin-conjugating enzyme UbcH5B (UBE2D2) with unusually high affinity via a structurally unique UbcH5B-binding region (U5BR) connected by an 11-amino acid linker to its RING domain, forming a clamp that surrounds the E2; the U5BR contacts the backside of UbcH5B, and this high-affinity clamp binding paradoxically decreases ubiquitination rate by blocking stimulatory non-covalent ubiquitin binding to the E2 backside.\",\n      \"method\": \"Co-crystallization (X-ray structure), in vitro ubiquitination assay, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure combined with in vitro ubiquitination assays and mutagenesis\",\n      \"pmids\": [\"26475854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RNF25 physically interacts with Nkd1 and Axin in an E3 ligase-independent manner to strengthen Wnt signaling by disrupting the Nkd1-Axin inhibitory complex; RNF25 depletion in zebrafish attenuates Wnt target gene transcription and promotes epithelial character in renal mesenchymal cells.\",\n      \"method\": \"Co-immunoprecipitation, E3 ligase-dead mutant analysis, RNAi knockdown in zebrafish, cell-based Wnt reporter assay\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — Co-IP with ligase-dead mutant and in vivo zebrafish knockdown, single lab\",\n      \"pmids\": [\"27007149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RNF25 mediates NF-κB activation in gefitinib-treated NSCLC cells, which in turn transcriptionally induces IL-6 to reactivate ERK signaling, causing drug resistance; depletion of RNF25 sensitizes cells to gefitinib and overexpression augments resistance.\",\n      \"method\": \"Genome-wide RNAi screen, siRNA knockdown, overexpression, NF-κB reporter assay, cytokine measurement, ERK phosphorylation analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — genetic screen followed by multiple functional assays in a single lab\",\n      \"pmids\": [\"29789542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF25 catalyzes ubiquitin-mediated proteasomal degradation of E-cadherin (ECAD) in hepatocellular carcinoma cells; PKA senses oxidative stress via redox modification of its β catalytic subunit (PRKACB) at Cys200 and Cys344, and subsequently phosphorylates RNF25 at Ser450 to activate this E3 ligase activity toward ECAD.\",\n      \"method\": \"In vitro ubiquitination assay, site-directed mutagenesis (Cys200/344 in PRKACB, Ser450 in RNF25), co-immunoprecipitation, protein stability assay, in vivo xenograft metastasis model\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro ubiquitination, phosphosite mutagenesis, redox modification mapping, and in vivo validation\",\n      \"pmids\": [\"38286671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"circSATB1 acts as a scaffold to facilitate RNF25-mediated ubiquitylation and proteasomal degradation of FKBP8, releasing FKBP8's inhibitory effect on mTOR signaling to promote colorectal cancer liver metastasis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, in vitro and in vivo metastasis assays\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — Co-IP and ubiquitination assay identifying new substrate and scaffold mechanism, single lab\",\n      \"pmids\": [\"39921520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF25 binds TRIP4 and catalyzes its non-degradative ubiquitination at Lys135, disrupting TRIP4-p65 interactions and liberating p65 to activate NF-κB signaling and upregulate anti-apoptotic effectors (cIAP2, Bcl-2); the NF-κB inhibitor BAY11-7082 directly binds RNF25 to reverse this activity.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, site-directed mutagenesis (K135), NF-κB reporter assay, drug binding assay\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ubiquitination site mapping and interaction disruption assay, single lab\",\n      \"pmids\": [\"40765826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF25 protects reversed DNA replication forks from nucleolytic degradation by MRE11 and CtIP by interacting with the replication fork protection factor REV7 and recruiting REV7 to nascent DNA after replication stress; this fork-protective role is independent of RNF25's ubiquitin ligase activity.\",\n      \"method\": \"Unbiased genetic screen, single-molecule DNA fiber analysis, co-immunoprecipitation, proximity ligation, ssDNA accumulation assay, S-phase accumulation by flow cytometry, ligase-dead mutant analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — genetic screen, single-molecule fiber assay, Co-IP, and ligase-dead separation-of-function mutant with multiple orthogonal readouts\",\n      \"pmids\": [\"40764480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RNF25 ubiquitylates ribosomal protein eS31 to suppress GCN2-dependent integrated stress response (ISR) activation caused by azacytidine-damaged mRNA stalling ribosomes, thereby conferring mRNA damage tolerance and preventing cell death.\",\n      \"method\": \"Genetic screens, ubiquitination assay, ribosome stalling assay, GCN2 pathway reporters, cell viability assays, mass spectrometry identification of eS31 ubiquitylation site\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — genetic screens with mechanistic follow-up identifying substrate (eS31), pathway (GCN2-ISR), and functional consequence via multiple orthogonal methods\",\n      \"pmids\": [\"41875887\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RNF25 is a RING finger E3 ubiquitin ligase that binds the E2 enzyme UbcH5B via a unique clamp mechanism, ubiquitylates diverse substrates (Naked2, E-cadherin, FKBP8, TRIP4, ribosomal protein eS31) to control proteasomal degradation or non-degradative signaling, activates NF-κB transcription by interacting with p65 TAD and ubiquitylating TRIP4, modulates Wnt signaling by disrupting the Nkd1-Axin complex independently of its ligase activity, and protects reversed DNA replication forks from MRE11/CtIP-mediated degradation by recruiting REV7—with its fork-protective role separable from ubiquitin conjugation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RNF25 is a RING finger E3 ubiquitin ligase that operates through both ligase-dependent and ligase-independent mechanisms to regulate transcription, signaling, DNA replication fork integrity, and ribosome quality control. Its RING domain engages the E2 enzyme UbcH5B via a structurally unique high-affinity clamp involving a dedicated UbcH5B-binding region (U5BR) tethered by a short linker, and its catalytic activity drives proteasomal degradation of substrates including Naked2, E-cadherin, and FKBP8, as well as non-degradative ubiquitylation of TRIP4 and ribosomal protein eS31 [PMID:26475854, PMID:18757723, PMID:38286671, PMID:39921520, PMID:40765826, PMID:41875887]. RNF25 activates NF-κB-dependent transcription by binding the p65 transactivation domain and ubiquitylating TRIP4 to liberate p65, and it modulates Wnt signaling independently of its ligase activity by disrupting the inhibitory Nkd1–Axin complex [PMID:12748188, PMID:40765826, PMID:27007149]. Separately from ubiquitin conjugation, RNF25 protects reversed DNA replication forks from MRE11/CtIP-mediated degradation by recruiting the fork-protection factor REV7 to nascent DNA, and it ubiquitylates ribosomal protein eS31 to suppress GCN2-dependent integrated stress response activation upon encounter with damaged-mRNA-stalled ribosomes [PMID:40764480, PMID:41875887].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Establishing that RNF25 is a nuclear RING finger protein whose ubiquitin ligase activity is required for NF-κB-dependent transcription answered the question of whether this uncharacterized RING protein functions in gene regulation.\",\n      \"evidence\": \"Yeast two-hybrid screen identifying p65 TAD interaction, Co-IP, luciferase reporter, and dominant-negative RING mutant in mammalian cells\",\n      \"pmids\": [\"12748188\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct ubiquitylation substrate for NF-κB activation was not identified\",\n        \"Whether RNF25 modifies p65 itself or a co-regulator was unknown\",\n        \"In vivo relevance beyond reporter assays not tested\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identification of Naked2 as a direct ubiquitylation substrate targeted for proteasomal degradation established RNF25 as a bona fide E3 ligase with a defined substrate and revealed its role in modulating Wnt-pathway-related signaling molecules.\",\n      \"evidence\": \"Overexpression/knockdown, protein half-life assays, Co-IP, and proteasome inhibitor treatment in mammalian cells\",\n      \"pmids\": [\"18757723\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Specific ubiquitylation sites on Naked2 were not mapped\",\n        \"Whether TGF-α-mediated protection of Naked2 occurs in vivo was not addressed\",\n        \"Chain type preference of RNF25-catalyzed ubiquitylation remained unknown\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Crystal structure of the RNF25–UbcH5B complex revealed a unique clamp mechanism whereby a dedicated U5BR contacts the E2 backside, explaining unusually high-affinity E2 engagement and showing that this paradoxically attenuates catalytic rate by occluding non-covalent ubiquitin binding.\",\n      \"evidence\": \"X-ray co-crystallography, in vitro ubiquitination assays, and site-directed mutagenesis\",\n      \"pmids\": [\"26475854\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Physiological consequence of the auto-inhibitory clamp mechanism in cells was not tested\",\n        \"Whether RNF25 uses E2 enzymes other than UbcH5B in vivo was not determined\",\n        \"How the clamp is relieved to permit productive ubiquitylation remained unclear\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstration that RNF25 disrupts the Nkd1–Axin complex to promote Wnt signaling independently of its RING ligase activity revealed a non-catalytic scaffolding function, broadening the mechanistic repertoire of RNF25.\",\n      \"evidence\": \"Co-IP with ligase-dead mutant, RNAi knockdown in zebrafish embryos, and Wnt reporter assays\",\n      \"pmids\": [\"27007149\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Structural basis for Nkd1 and Axin binding was not resolved\",\n        \"Whether ligase-independent and ligase-dependent functions compete or cooperate was not examined\",\n        \"Single-lab finding without independent replication\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"A genome-wide RNAi screen linked RNF25 to gefitinib resistance in NSCLC by showing it activates NF-κB, which induces IL-6 to reactivate ERK signaling, connecting RNF25's transcriptional function to a clinically relevant drug-resistance pathway.\",\n      \"evidence\": \"Genome-wide RNAi screen, siRNA/overexpression, NF-κB reporter, cytokine measurement, and ERK phosphorylation analysis in NSCLC cells\",\n      \"pmids\": [\"29789542\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct ubiquitylation target mediating NF-κB activation in this context was not identified\",\n        \"In vivo drug-resistance model was not included\",\n        \"Generalizability beyond NSCLC cell lines not tested\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery that PKA phosphorylates RNF25 at Ser450 in response to oxidative stress to activate its E3 ligase activity toward E-cadherin established a redox-regulated post-translational switch controlling RNF25 substrate selection and EMT-related metastasis.\",\n      \"evidence\": \"In vitro ubiquitination, phosphosite and Cys mutagenesis, Co-IP, protein stability assays, and in vivo xenograft metastasis model in hepatocellular carcinoma\",\n      \"pmids\": [\"38286671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether Ser450 phosphorylation affects other RNF25 substrates was not tested\",\n        \"Structural mechanism by which phosphorylation activates ligase activity unknown\",\n        \"Contribution relative to other E-cadherin-targeting E3 ligases not assessed\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Three independent studies simultaneously expanded the substrate repertoire and revealed a ligase-independent genome-protection role: RNF25 ubiquitylates FKBP8 (scaffolded by circSATB1) to relieve mTOR inhibition, non-degradatively ubiquitylates TRIP4 at K135 to liberate p65 for NF-κB activation, and recruits REV7 to reversed replication forks to protect nascent DNA from MRE11/CtIP degradation without requiring catalytic activity.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, site-directed mutagenesis, single-molecule DNA fiber analysis, proximity ligation, ligase-dead separation-of-function mutant, drug-binding assay, and in vivo metastasis models\",\n      \"pmids\": [\"39921520\", \"40765826\", \"40764480\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How REV7 recruitment is coordinated with other fork-protection factors is unknown\",\n        \"Structural basis for RNF25–REV7 interaction not resolved\",\n        \"Whether the circSATB1-scaffolded mechanism operates outside colorectal cancer is untested\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identification of ribosomal protein eS31 as an RNF25 substrate linked the E3 ligase to ribosome quality control by showing that eS31 ubiquitylation suppresses GCN2-dependent integrated stress response activation at damaged-mRNA-stalled ribosomes, conferring mRNA damage tolerance.\",\n      \"evidence\": \"Genetic screens, ubiquitination and ribosome stalling assays, GCN2 pathway reporters, cell viability assays, and mass spectrometry identification of eS31 ubiquitylation site\",\n      \"pmids\": [\"41875887\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Ubiquitin chain type on eS31 not determined\",\n        \"Whether RNF25's ribosome quality-control function intersects with its NF-κB or fork-protection roles is unknown\",\n        \"Structural context of eS31 ubiquitylation on the ribosome not resolved\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A unifying model explaining how RNF25 partitions between its diverse catalytic and non-catalytic roles — NF-κB activation, substrate-specific proteasomal degradation, replication fork protection, and ribosome quality control — and whether distinct regulatory inputs (phosphorylation, scaffold RNAs, protein partners) route RNF25 to specific substrates and cellular compartments remains to be established.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No knockout mouse or organismal loss-of-function phenotype reported\",\n        \"Ubiquitin chain-type specificity for most substrates undetermined\",\n        \"How the auto-inhibitory U5BR clamp is relieved in vivo is not known\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 5, 6, 7, 9]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 4, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005840\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3, 4, 7]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 5, 6, 9]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 4, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"RELA\",\n      \"UBE2D2\",\n      \"NKD1\",\n      \"TRIP4\",\n      \"MAD2L2\",\n      \"FKBP8\",\n      \"NKD2\",\n      \"CDH1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}