{"gene":"RNF25","run_date":"2026-06-10T06:43:37","timeline":{"discoveries":[{"year":2003,"finding":"RNF25 (AO7) interacts with the transactivation domain (TAD) of the NF-κB p65 subunit (RelA) via its C-terminal region, localizes predominantly to the nucleus, and supports NF-κB-dependent transcriptional activation; both the RING finger and C-terminal regions are required for activation, and a RING finger Cys-to-Ser ubiquitination-defective mutant suppresses p65-mediated transactivation.","method":"Yeast two-hybrid screen, in vitro and in vivo co-IP, reporter gene assays, dominant-negative mutant analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal in vitro and in vivo binding confirmed, multiple functional domain mapping experiments, and dominant-negative mutant with clear transcriptional readout in single rigorous study","pmids":["12748188"],"is_preprint":false},{"year":2008,"finding":"RNF25 (AO7) acts as the E3 ubiquitin ligase responsible for ubiquitin-mediated proteasomal degradation of Naked2; TGF-alpha overexpression stabilizes Naked2 by reducing AO7 binding to Naked2 in an EGFR-independent manner.","method":"Co-IP, overexpression/knockdown, proteasomal degradation assay, half-life measurement","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct identification of E3-substrate relationship with co-IP, proteasomal degradation assay, and TGF-alpha protection mechanism in single rigorous study","pmids":["18757723"],"is_preprint":false},{"year":2015,"finding":"RNF25 (AO7) contains a structurally unique UbcH5B-binding region (U5BR) connected by an 11-amino acid linker to its RING domain, forming a clamp that surrounds UbcH5B with unusually high affinity; the U5BR interacts with the backside of UbcH5B distinct from the active site and RING-interacting region. This high-affinity clamp paradoxically decreases the rate of ubiquitination by blocking stimulatory non-covalent ubiquitin binding to the backside of UbcH5B, but enhances ubiquitination when backside binding is abrogated.","method":"Co-crystallization (X-ray crystallography), in vitro ubiquitination assay, mutagenesis at RING-E2 interface","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation by in vitro ubiquitination assay and mutagenesis, multiple orthogonal methods in single rigorous study","pmids":["26475854"],"is_preprint":false},{"year":2016,"finding":"RNF25 physically interacts with Nkd1 and Axin and positively regulates Wnt signaling by disrupting the Nkd1-Axin inhibitory complex; this function is independent of its E3 ubiquitin ligase activity. Knockdown of rnf25 in zebrafish embryos attenuated transcription of Wnt target genes.","method":"Co-IP, in vivo zebrafish knockdown, Wnt reporter assay, E3 ligase-dead mutant analysis","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP interaction data combined with in vivo zebrafish KD and ligase-dead mutant, single lab","pmids":["27007149"],"is_preprint":false},{"year":2018,"finding":"RNF25 mediates NF-κB activation in gefitinib-treated EGFR-mutant NSCLC cells, which induces IL-6 expression and subsequent ERK reactivation, causing drug resistance; depletion of RNF25 sensitizes cells to gefitinib while forced expression augments resistance.","method":"Genome-wide RNAi screen, RNF25 knockdown/overexpression, NF-κB reporter assay, cytokine measurement, ERK phosphorylation analysis","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide screen hit validated by KD/OE with defined pathway readouts (NF-κB, IL-6, ERK), single lab","pmids":["29789542"],"is_preprint":false},{"year":2024,"finding":"PKA senses oxidative stress via redox modification of its β catalytic subunit (PRKACB) at Cys200 and Cys344, leading to phosphorylation of RNF25 at Ser450, which activates RNF25-catalyzed ubiquitin-mediated degradation of E-cadherin (ECAD); RNF25 repression restores ECAD protein expression and inhibits HCC metastasis in vitro and in vivo.","method":"In vitro ubiquitination assay, site-directed mutagenesis, co-IP, redox modification analysis, in vivo tumor metastasis model, RNF25 knockdown","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct identification of E3-substrate (ECAD), PKA-RNF25 phosphorylation axis with mutagenesis, in vitro ubiquitination, and in vivo functional validation, multiple orthogonal methods single lab","pmids":["38286671"],"is_preprint":false},{"year":2025,"finding":"RNF25 binds TRIP4 and catalyzes non-degradative ubiquitination of TRIP4 at lysine 135, disrupting TRIP4-p65 interactions and thereby liberating p65 to activate NF-κB signaling and upregulate anti-apoptotic effectors (cIAP2, Bcl-2); the NF-κB inhibitor BAY11-7082 directly interacts with RNF25 to reverse these effects.","method":"Co-IP, ubiquitination assay, site-directed mutagenesis (K135), p65 interaction assay, NF-κB reporter, BAY11-7082 direct binding assay","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct E3-substrate ubiquitination with site mapping, co-IP for protein interactions, NF-κB pathway readouts, 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; loss of RNF25 causes aberrant ssDNA accumulation and S-phase arrest; this fork protection function is fully separable from RNF25's canonical ubiquitin ligase activity.","method":"Unbiased genetic screen, single-molecule DNA fiber analysis, co-IP, RNF25 knockout cells, ssDNA measurement, mitotic entry assay, ubiquitin ligase-dead mutant","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — unbiased screen validated by DNA fiber analysis, co-IP, KO phenotyping, and ligase-dead mutant separation-of-function, multiple orthogonal methods, published in peer-reviewed journal","pmids":["40764480"],"is_preprint":false},{"year":2025,"finding":"RNF25 acts as a scaffold for the RNF25-FKBP8 complex (facilitated by circSATB1) and mediates ubiquitylation and proteasomal degradation of FKBP8, releasing inhibitory effects on mTOR signaling.","method":"Co-IP, ubiquitination assay, in vitro and in vivo metastasis assays, knockdown/overexpression","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct E3-substrate relationship (RNF25-FKBP8) established by co-IP and ubiquitination assay with mTOR pathway readout, single lab","pmids":["39921520"],"is_preprint":false},{"year":2026,"finding":"RNF25 ubiquitylates ribosomal protein eS31, thereby suppressing GCN2-dependent integrated stress response (ISR) hyperactivation when ribosomes stall on azacytidine-damaged mRNA lesions; loss of RNF25 leads to cytotoxic ISR activation and increased cell death upon azacytidine treatment.","method":"Genetic screens, in vitro ubiquitination assay, ISR activation assays (eIF2α phosphorylation), GCN2 epistasis, RNF25 KO cells","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic screen validated by direct ubiquitination of eS31, ISR pathway epistasis with GCN2, KO phenotyping, multiple orthogonal methods single lab","pmids":["41875887"],"is_preprint":false},{"year":2025,"finding":"RNF25 protects reversed DNA replication forks from nucleolytic degradation by MRE11 and CtIP by interacting with REV7 and recruiting it to nascent DNA after replication stress; this protective role is independent of RNF25 ubiquitin ligase activity (preprint version of PMID:40764480).","method":"Genetic screen, DNA fiber analysis, co-IP, KO cell lines, ubiquitin ligase-dead mutant","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — preprint with multiple methods, subsequently published as peer-reviewed article; confidence limited to Medium pending peer review at time of preprint","pmids":["39829812"],"is_preprint":true}],"current_model":"RNF25 (AO7) is a RING finger E3 ubiquitin ligase that uses a structurally unique clamp mechanism to bind the E2 enzyme UbcH5B/UBE2D2; it ubiquitylates multiple substrates including E-cadherin (PKA-phosphorylation-dependent), Naked2, FKBP8, TRIP4 (non-degradative, K135), and ribosomal protein eS31, and also functions in a ligase-independent manner to protect reversed DNA replication forks by recruiting REV7 and to modulate Wnt signaling by disrupting the Nkd1-Axin complex; collectively, these activities place RNF25 at the intersection of NF-κB transcriptional co-activation, proteasomal substrate degradation, replication stress tolerance, and mRNA damage response."},"narrative":{"mechanistic_narrative":"RNF25 (AO7) is a RING-finger E3 ubiquitin ligase that operates at the intersection of NF-κB transcriptional control, targeted protein degradation, and the cellular response to genotoxic and replicative stress [PMID:12748188, PMID:26475854, PMID:40764480]. Structurally, it engages the E2 enzyme UbcH5B through a unique \"clamp\": a dedicated UbcH5B-binding region (U5BR) joined by a short linker to the RING domain, which encircles the E2 with unusually high affinity by contacting its backside; paradoxically this clamp dampens basal ubiquitination by occluding the stimulatory non-covalent ubiquitin-binding surface of UbcH5B [PMID:26475854]. As a ligase RNF25 drives proteasomal turnover of multiple substrates, including Naked2 [PMID:18757723], E-cadherin downstream of redox-activated PKA phosphorylating RNF25 at Ser450 [PMID:38286671], and FKBP8 within a circSATB1-assisted complex that relieves inhibition of mTOR signaling [PMID:39921520], and it also catalyzes non-degradative ubiquitination of TRIP4 at K135 and of ribosomal protein eS31 [PMID:40765826, PMID:41875887]. Through TRIP4 ubiquitination it liberates p65 to activate NF-κB and induce anti-apoptotic effectors, consistent with its original identification as a p65/RelA transactivation-domain partner and NF-κB co-activator [PMID:12748188, PMID:40765826]. RNF25 additionally performs functions fully separable from its ligase activity: it recruits REV7 to nascent DNA to protect reversed replication forks from MRE11/CtIP-mediated degradation [PMID:40764480], and it disrupts the Nkd1-Axin complex to potentiate Wnt signaling [PMID:27007149]. By ubiquitylating eS31, RNF25 restrains GCN2-dependent integrated stress response hyperactivation when ribosomes stall on damaged mRNA [PMID:41875887].","teleology":[{"year":2003,"claim":"Established RNF25 as a nuclear RING-finger protein that physically couples to the NF-κB p65 transactivation domain and is required for p65-driven transcription, defining its first functional context.","evidence":"Yeast two-hybrid, reciprocal co-IP, reporter assays, and a RING/ubiquitination-defective dominant-negative mutant","pmids":["12748188"],"confidence":"High","gaps":["Did not identify a ubiquitination substrate explaining the transcriptional effect","Mechanism linking RING activity to p65 transactivation unresolved at this stage"]},{"year":2008,"claim":"Identified the first direct degradative substrate, showing RNF25 targets Naked2 for proteasomal turnover and that TGF-α stabilizes Naked2 by reducing RNF25 binding.","evidence":"Co-IP, knockdown/overexpression, proteasomal degradation and half-life assays","pmids":["18757723"],"confidence":"High","gaps":["Ubiquitin chain linkage on Naked2 not characterized","E2 partner used in cells not defined here"]},{"year":2015,"claim":"Resolved the structural basis of E2 engagement, revealing a high-affinity clamp around UbcH5B that counterintuitively suppresses ubiquitination by blocking backside ubiquitin binding.","evidence":"Co-crystallography of the RNF25 U5BR/RING-UbcH5B complex with in vitro ubiquitination and interface mutagenesis","pmids":["26475854"],"confidence":"High","gaps":["Physiological consequence of clamp-mediated rate control on specific substrates untested","Whether other E2s are used in vivo unknown"]},{"year":2016,"claim":"Demonstrated a ligase-independent role in Wnt signaling, with RNF25 disrupting the Nkd1-Axin inhibitory complex to potentiate Wnt target gene transcription.","evidence":"Co-IP, zebrafish rnf25 knockdown, Wnt reporter assays, and E3-dead mutant","pmids":["27007149"],"confidence":"Medium","gaps":["Direct structural mechanism of Nkd1-Axin disruption not defined","Single-lab in vivo model"]},{"year":2018,"claim":"Connected RNF25 to therapy resistance, showing it mediates NF-κB activation, IL-6 induction, and ERK reactivation that drives gefitinib resistance in EGFR-mutant NSCLC.","evidence":"Genome-wide RNAi screen with knockdown/overexpression, NF-κB reporter, cytokine and ERK phosphorylation readouts","pmids":["29789542"],"confidence":"Medium","gaps":["Molecular substrate underlying NF-κB activation not identified in this work","Dependence on ligase activity not tested"]},{"year":2024,"claim":"Defined a redox-PKA-RNF25 axis in which oxidative activation of PKA phosphorylates RNF25 at Ser450 to drive E-cadherin degradation and promote HCC metastasis.","evidence":"In vitro ubiquitination, Ser450 mutagenesis, co-IP, redox analysis, and in vivo metastasis models with RNF25 knockdown","pmids":["38286671"],"confidence":"High","gaps":["Ubiquitin chain type on E-cadherin not specified","Whether Ser450 phosphorylation alters E2 clamp behavior unknown"]},{"year":2025,"claim":"Provided a mechanism for RNF25's NF-κB co-activation: non-degradative K135 ubiquitination of TRIP4 disrupts TRIP4-p65 binding to liberate p65 and upregulate anti-apoptotic genes.","evidence":"Co-IP, ubiquitination with K135 mutagenesis, p65 interaction and NF-κB reporter assays, plus BAY11-7082 direct binding","pmids":["40765826"],"confidence":"Medium","gaps":["Reciprocal validation of TRIP4-p65 displacement in vivo limited","Single-lab study"]},{"year":2025,"claim":"Revealed a ligase-independent genome-protection function: RNF25 recruits REV7 to nascent DNA to shield reversed replication forks from MRE11/CtIP nucleolytic degradation.","evidence":"Unbiased genetic screen, DNA fiber analysis, co-IP, KO phenotyping, and ligase-dead separation-of-function mutant","pmids":["40764480","39829812"],"confidence":"High","gaps":["How RNF25 is itself recruited to stressed forks unknown","Structural basis of RNF25-REV7 interaction undefined"]},{"year":2025,"claim":"Identified FKBP8 as a degradative substrate within a circSATB1-scaffolded RNF25-FKBP8 complex, linking RNF25 to mTOR signaling and metastasis.","evidence":"Co-IP, ubiquitination assay, knockdown/overexpression, and in vitro/in vivo metastasis assays","pmids":["39921520"],"confidence":"Medium","gaps":["Mechanism by which circSATB1 promotes complex formation not detailed","Single-lab study"]},{"year":2026,"claim":"Placed RNF25 in the mRNA damage response, showing it ubiquitylates ribosomal protein eS31 to restrain GCN2-dependent integrated stress response when ribosomes stall on azacytidine-damaged mRNA.","evidence":"Genetic screens, in vitro ubiquitination of eS31, ISR/eIF2α readouts, GCN2 epistasis, and RNF25 KO phenotyping","pmids":["41875887"],"confidence":"High","gaps":["Ubiquitin signal read by the ISR machinery not fully defined","Generality across other ribosome-stalling lesions untested"]},{"year":null,"claim":"How RNF25 partitions between its ligase-dependent and ligase-independent functions, and what governs substrate and effector selection across NF-κB, degradation, fork protection, and stress-response roles, remains unresolved.","evidence":"No single study in the corpus integrates the distinct activities mechanistically","pmids":[],"confidence":"Medium","gaps":["No unifying determinant of substrate choice identified","Interplay between the UbcH5B clamp and physiological ubiquitination outputs not mapped","Regulation of RNF25 localization between nucleus, forks, and cytoplasmic complexes unclear"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,5,8]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,6,9]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,7]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,5,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3,4]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[7]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[9]}],"complexes":["RNF25-UbcH5B (UBE2D2) E3-E2 complex","RNF25-FKBP8 complex (circSATB1-assisted)"],"partners":["RELA","UBE2D2","NKD1","AXIN","TRIP4","REV7","FKBP8","PRKACB"],"other_free_text":[]}},"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":"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":38,"is_preprint":false},{"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":"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":25,"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":6,"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":1,"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":1,"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":1,"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":"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}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9118,"output_tokens":3088,"usd":0.036837,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10417,"output_tokens":3805,"usd":0.073605,"stage2_stop_reason":"end_turn"},"total_usd":0.110442,"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\": 2003,\n      \"finding\": \"RNF25 (AO7) interacts with the transactivation domain (TAD) of the NF-κB p65 subunit (RelA) via its C-terminal region, localizes predominantly to the nucleus, and supports NF-κB-dependent transcriptional activation; both the RING finger and C-terminal regions are required for activation, and a RING finger Cys-to-Ser ubiquitination-defective mutant suppresses p65-mediated transactivation.\",\n      \"method\": \"Yeast two-hybrid screen, in vitro and in vivo co-IP, reporter gene assays, dominant-negative mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal in vitro and in vivo binding confirmed, multiple functional domain mapping experiments, and dominant-negative mutant with clear transcriptional readout in single rigorous study\",\n      \"pmids\": [\"12748188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"RNF25 (AO7) acts as the E3 ubiquitin ligase responsible for ubiquitin-mediated proteasomal degradation of Naked2; TGF-alpha overexpression stabilizes Naked2 by reducing AO7 binding to Naked2 in an EGFR-independent manner.\",\n      \"method\": \"Co-IP, overexpression/knockdown, proteasomal degradation assay, half-life measurement\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct identification of E3-substrate relationship with co-IP, proteasomal degradation assay, and TGF-alpha protection mechanism in single rigorous study\",\n      \"pmids\": [\"18757723\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RNF25 (AO7) contains a structurally unique UbcH5B-binding region (U5BR) connected by an 11-amino acid linker to its RING domain, forming a clamp that surrounds UbcH5B with unusually high affinity; the U5BR interacts with the backside of UbcH5B distinct from the active site and RING-interacting region. This high-affinity clamp paradoxically decreases the rate of ubiquitination by blocking stimulatory non-covalent ubiquitin binding to the backside of UbcH5B, but enhances ubiquitination when backside binding is abrogated.\",\n      \"method\": \"Co-crystallization (X-ray crystallography), in vitro ubiquitination assay, mutagenesis at RING-E2 interface\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation by in vitro ubiquitination assay and mutagenesis, multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"26475854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RNF25 physically interacts with Nkd1 and Axin and positively regulates Wnt signaling by disrupting the Nkd1-Axin inhibitory complex; this function is independent of its E3 ubiquitin ligase activity. Knockdown of rnf25 in zebrafish embryos attenuated transcription of Wnt target genes.\",\n      \"method\": \"Co-IP, in vivo zebrafish knockdown, Wnt reporter assay, E3 ligase-dead mutant analysis\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP interaction data combined with in vivo zebrafish KD and ligase-dead mutant, single lab\",\n      \"pmids\": [\"27007149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RNF25 mediates NF-κB activation in gefitinib-treated EGFR-mutant NSCLC cells, which induces IL-6 expression and subsequent ERK reactivation, causing drug resistance; depletion of RNF25 sensitizes cells to gefitinib while forced expression augments resistance.\",\n      \"method\": \"Genome-wide RNAi screen, RNF25 knockdown/overexpression, NF-κB reporter assay, cytokine measurement, ERK phosphorylation analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide screen hit validated by KD/OE with defined pathway readouts (NF-κB, IL-6, ERK), single lab\",\n      \"pmids\": [\"29789542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PKA senses oxidative stress via redox modification of its β catalytic subunit (PRKACB) at Cys200 and Cys344, leading to phosphorylation of RNF25 at Ser450, which activates RNF25-catalyzed ubiquitin-mediated degradation of E-cadherin (ECAD); RNF25 repression restores ECAD protein expression and inhibits HCC metastasis in vitro and in vivo.\",\n      \"method\": \"In vitro ubiquitination assay, site-directed mutagenesis, co-IP, redox modification analysis, in vivo tumor metastasis model, RNF25 knockdown\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct identification of E3-substrate (ECAD), PKA-RNF25 phosphorylation axis with mutagenesis, in vitro ubiquitination, and in vivo functional validation, multiple orthogonal methods single lab\",\n      \"pmids\": [\"38286671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF25 binds TRIP4 and catalyzes non-degradative ubiquitination of TRIP4 at lysine 135, disrupting TRIP4-p65 interactions and thereby liberating p65 to activate NF-κB signaling and upregulate anti-apoptotic effectors (cIAP2, Bcl-2); the NF-κB inhibitor BAY11-7082 directly interacts with RNF25 to reverse these effects.\",\n      \"method\": \"Co-IP, ubiquitination assay, site-directed mutagenesis (K135), p65 interaction assay, NF-κB reporter, BAY11-7082 direct binding assay\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct E3-substrate ubiquitination with site mapping, co-IP for protein interactions, NF-κB pathway readouts, 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; loss of RNF25 causes aberrant ssDNA accumulation and S-phase arrest; this fork protection function is fully separable from RNF25's canonical ubiquitin ligase activity.\",\n      \"method\": \"Unbiased genetic screen, single-molecule DNA fiber analysis, co-IP, RNF25 knockout cells, ssDNA measurement, mitotic entry assay, ubiquitin ligase-dead mutant\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — unbiased screen validated by DNA fiber analysis, co-IP, KO phenotyping, and ligase-dead mutant separation-of-function, multiple orthogonal methods, published in peer-reviewed journal\",\n      \"pmids\": [\"40764480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF25 acts as a scaffold for the RNF25-FKBP8 complex (facilitated by circSATB1) and mediates ubiquitylation and proteasomal degradation of FKBP8, releasing inhibitory effects on mTOR signaling.\",\n      \"method\": \"Co-IP, ubiquitination assay, in vitro and in vivo metastasis assays, knockdown/overexpression\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct E3-substrate relationship (RNF25-FKBP8) established by co-IP and ubiquitination assay with mTOR pathway readout, single lab\",\n      \"pmids\": [\"39921520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RNF25 ubiquitylates ribosomal protein eS31, thereby suppressing GCN2-dependent integrated stress response (ISR) hyperactivation when ribosomes stall on azacytidine-damaged mRNA lesions; loss of RNF25 leads to cytotoxic ISR activation and increased cell death upon azacytidine treatment.\",\n      \"method\": \"Genetic screens, in vitro ubiquitination assay, ISR activation assays (eIF2α phosphorylation), GCN2 epistasis, RNF25 KO cells\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic screen validated by direct ubiquitination of eS31, ISR pathway epistasis with GCN2, KO phenotyping, multiple orthogonal methods single lab\",\n      \"pmids\": [\"41875887\"],\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 REV7 and recruiting it to nascent DNA after replication stress; this protective role is independent of RNF25 ubiquitin ligase activity (preprint version of PMID:40764480).\",\n      \"method\": \"Genetic screen, DNA fiber analysis, co-IP, KO cell lines, ubiquitin ligase-dead mutant\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — preprint with multiple methods, subsequently published as peer-reviewed article; confidence limited to Medium pending peer review at time of preprint\",\n      \"pmids\": [\"39829812\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"RNF25 (AO7) is a RING finger E3 ubiquitin ligase that uses a structurally unique clamp mechanism to bind the E2 enzyme UbcH5B/UBE2D2; it ubiquitylates multiple substrates including E-cadherin (PKA-phosphorylation-dependent), Naked2, FKBP8, TRIP4 (non-degradative, K135), and ribosomal protein eS31, and also functions in a ligase-independent manner to protect reversed DNA replication forks by recruiting REV7 and to modulate Wnt signaling by disrupting the Nkd1-Axin complex; collectively, these activities place RNF25 at the intersection of NF-κB transcriptional co-activation, proteasomal substrate degradation, replication stress tolerance, and mRNA damage response.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RNF25 (AO7) is a RING-finger E3 ubiquitin ligase that operates at the intersection of NF-κB transcriptional control, targeted protein degradation, and the cellular response to genotoxic and replicative stress [#0, #2, #7]. Structurally, it engages the E2 enzyme UbcH5B through a unique \\\"clamp\\\": a dedicated UbcH5B-binding region (U5BR) joined by a short linker to the RING domain, which encircles the E2 with unusually high affinity by contacting its backside; paradoxically this clamp dampens basal ubiquitination by occluding the stimulatory non-covalent ubiquitin-binding surface of UbcH5B [#2]. As a ligase RNF25 drives proteasomal turnover of multiple substrates, including Naked2 [#1], E-cadherin downstream of redox-activated PKA phosphorylating RNF25 at Ser450 [#5], and FKBP8 within a circSATB1-assisted complex that relieves inhibition of mTOR signaling [#8], and it also catalyzes non-degradative ubiquitination of TRIP4 at K135 and of ribosomal protein eS31 [#6, #9]. Through TRIP4 ubiquitination it liberates p65 to activate NF-κB and induce anti-apoptotic effectors, consistent with its original identification as a p65/RelA transactivation-domain partner and NF-κB co-activator [#0, #6]. RNF25 additionally performs functions fully separable from its ligase activity: it recruits REV7 to nascent DNA to protect reversed replication forks from MRE11/CtIP-mediated degradation [#7], and it disrupts the Nkd1-Axin complex to potentiate Wnt signaling [#3]. By ubiquitylating eS31, RNF25 restrains GCN2-dependent integrated stress response hyperactivation when ribosomes stall on damaged mRNA [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established RNF25 as a nuclear RING-finger protein that physically couples to the NF-κB p65 transactivation domain and is required for p65-driven transcription, defining its first functional context.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal co-IP, reporter assays, and a RING/ubiquitination-defective dominant-negative mutant\",\n      \"pmids\": [\"12748188\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify a ubiquitination substrate explaining the transcriptional effect\", \"Mechanism linking RING activity to p65 transactivation unresolved at this stage\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified the first direct degradative substrate, showing RNF25 targets Naked2 for proteasomal turnover and that TGF-α stabilizes Naked2 by reducing RNF25 binding.\",\n      \"evidence\": \"Co-IP, knockdown/overexpression, proteasomal degradation and half-life assays\",\n      \"pmids\": [\"18757723\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain linkage on Naked2 not characterized\", \"E2 partner used in cells not defined here\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved the structural basis of E2 engagement, revealing a high-affinity clamp around UbcH5B that counterintuitively suppresses ubiquitination by blocking backside ubiquitin binding.\",\n      \"evidence\": \"Co-crystallography of the RNF25 U5BR/RING-UbcH5B complex with in vitro ubiquitination and interface mutagenesis\",\n      \"pmids\": [\"26475854\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological consequence of clamp-mediated rate control on specific substrates untested\", \"Whether other E2s are used in vivo unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated a ligase-independent role in Wnt signaling, with RNF25 disrupting the Nkd1-Axin inhibitory complex to potentiate Wnt target gene transcription.\",\n      \"evidence\": \"Co-IP, zebrafish rnf25 knockdown, Wnt reporter assays, and E3-dead mutant\",\n      \"pmids\": [\"27007149\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct structural mechanism of Nkd1-Axin disruption not defined\", \"Single-lab in vivo model\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Connected RNF25 to therapy resistance, showing it mediates NF-κB activation, IL-6 induction, and ERK reactivation that drives gefitinib resistance in EGFR-mutant NSCLC.\",\n      \"evidence\": \"Genome-wide RNAi screen with knockdown/overexpression, NF-κB reporter, cytokine and ERK phosphorylation readouts\",\n      \"pmids\": [\"29789542\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular substrate underlying NF-κB activation not identified in this work\", \"Dependence on ligase activity not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined a redox-PKA-RNF25 axis in which oxidative activation of PKA phosphorylates RNF25 at Ser450 to drive E-cadherin degradation and promote HCC metastasis.\",\n      \"evidence\": \"In vitro ubiquitination, Ser450 mutagenesis, co-IP, redox analysis, and in vivo metastasis models with RNF25 knockdown\",\n      \"pmids\": [\"38286671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain type on E-cadherin not specified\", \"Whether Ser450 phosphorylation alters E2 clamp behavior unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided a mechanism for RNF25's NF-κB co-activation: non-degradative K135 ubiquitination of TRIP4 disrupts TRIP4-p65 binding to liberate p65 and upregulate anti-apoptotic genes.\",\n      \"evidence\": \"Co-IP, ubiquitination with K135 mutagenesis, p65 interaction and NF-κB reporter assays, plus BAY11-7082 direct binding\",\n      \"pmids\": [\"40765826\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reciprocal validation of TRIP4-p65 displacement in vivo limited\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a ligase-independent genome-protection function: RNF25 recruits REV7 to nascent DNA to shield reversed replication forks from MRE11/CtIP nucleolytic degradation.\",\n      \"evidence\": \"Unbiased genetic screen, DNA fiber analysis, co-IP, KO phenotyping, and ligase-dead separation-of-function mutant\",\n      \"pmids\": [\"40764480\", \"39829812\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How RNF25 is itself recruited to stressed forks unknown\", \"Structural basis of RNF25-REV7 interaction undefined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified FKBP8 as a degradative substrate within a circSATB1-scaffolded RNF25-FKBP8 complex, linking RNF25 to mTOR signaling and metastasis.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, knockdown/overexpression, and in vitro/in vivo metastasis assays\",\n      \"pmids\": [\"39921520\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which circSATB1 promotes complex formation not detailed\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Placed RNF25 in the mRNA damage response, showing it ubiquitylates ribosomal protein eS31 to restrain GCN2-dependent integrated stress response when ribosomes stall on azacytidine-damaged mRNA.\",\n      \"evidence\": \"Genetic screens, in vitro ubiquitination of eS31, ISR/eIF2α readouts, GCN2 epistasis, and RNF25 KO phenotyping\",\n      \"pmids\": [\"41875887\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin signal read by the ISR machinery not fully defined\", \"Generality across other ribosome-stalling lesions untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RNF25 partitions between its ligase-dependent and ligase-independent functions, and what governs substrate and effector selection across NF-κB, degradation, fork protection, and stress-response roles, remains unresolved.\",\n      \"evidence\": \"No single study in the corpus integrates the distinct activities mechanistically\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying determinant of substrate choice identified\", \"Interplay between the UbcH5B clamp and physiological ubiquitination outputs not mapped\", \"Regulation of RNF25 localization between nucleus, forks, and cytoplasmic complexes unclear\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 5, 8]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 6, 9]},\n      {\"term_id\": \"GO:0061630\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 7]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 5, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3, 4]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"complexes\": [\n      \"RNF25-UbcH5B (UBE2D2) E3-E2 complex\",\n      \"RNF25-FKBP8 complex (circSATB1-assisted)\"\n    ],\n    \"partners\": [\n      \"RELA\",\n      \"UBE2D2\",\n      \"NKD1\",\n      \"AXIN\",\n      \"TRIP4\",\n      \"REV7\",\n      \"FKBP8\",\n      \"PRKACB\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}