{"gene":"OBI1","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2019,"finding":"OBI1 (C13ORF7/RNF219) is associated with the ORC complex and catalyses multi-mono-ubiquitylation of a subset of chromatin-bound ORC3 and ORC5 during S-phase; OBI1 silencing causes defective origin firing (reduced CMG formation) without affecting pre-RC establishment, and expression of non-ubiquitylable ORC3/5 mutants impairs origin firing, identifying ORC3/5 as functionally relevant OBI1 substrates.","method":"Pre-RC proteomic interactome (MS), Co-IP, in vitro ubiquitylation assay, siRNA silencing, CMG formation assay, non-ubiquitylable ORC3/5 mutant expression","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (proteomics, Co-IP, in vitro ubiquitylation, mutant rescue) in a single rigorous study establishing substrate identity and functional consequence","pmids":["31160578"],"is_preprint":false},{"year":2020,"finding":"RNF219 (OBI1) ubiquitin ligase activity mediates α-catenin degradation, which promotes YAP1/β-catenin complex-dependent epigenetic modifications of the LGALS3 promoter, resulting in LGALS3 upregulation in hepatocellular carcinoma cells.","method":"Overexpression/knockdown functional assays, ubiquitin ligase activity assay, chromatin/promoter epigenetic analysis, co-immunoprecipitation","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — mechanistic pathway placed by Co-IP and functional assays in a single lab; multiple methods but full reconstitution not described in abstract","pmids":["33643786"],"is_preprint":false},{"year":2020,"finding":"RNF219 (OBI1) interacts with the CCR4-NOT deadenylase complex and the RNF219–CCR4-NOT complex exhibits deadenylation activity in vitro; RNF219 depletion in mouse embryonic stem cells impairs neuronal lineage commitment and alters expression of 2-cell-specific and neuronal genes.","method":"Co-immunoprecipitation, in vitro deadenylation assay, RNA-seq, siRNA knockdown, ES cell differentiation assay","journal":"Journal of molecular cell biology","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro deadenylation assay plus Co-IP and functional KD phenotype, single lab","pmids":["33104214"],"is_preprint":false},{"year":2022,"finding":"RNF219 (OBI1) co-purifies with the CCR4-NOT complex and both inhibits its deadenylase activity and enhances its capacity to repress translation of a target mRNA in a deadenylation-independent manner, directing the CCR4-NOT complex toward translational repression.","method":"Co-purification, in vitro deadenylase activity assay, translation repression reporter assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro enzymatic assay plus translation assay, single lab, two orthogonal readouts","pmids":["35660762"],"is_preprint":false},{"year":2022,"finding":"RNF219 (OBI1) activates the NF-κB pathway in nasopharyngeal carcinoma cells, evidenced by increased p65 nuclear translocation and upregulation of NF-κB target genes; NF-κB pathway inhibition in RNF219-overexpressing cells reverses RNF219-driven invasion, migration, and proliferation.","method":"Overexpression/knockdown, p65 nuclear translocation assay, NF-κB target gene expression analysis, pharmacological NF-κB inhibition rescue experiment","journal":"Molecular biotechnology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, indirect pathway placement via reporter and pharmacological inhibition, no direct biochemical binding or substrate identified","pmids":["36512252"],"is_preprint":false},{"year":2025,"finding":"The C-terminal part of RNF219 (OBI1) directly binds the CNOT1 DUF3819 domain (pull-down assay) and ubiquitinates CNOT6L in vitro; RNF219 knockdown in HEK293T cells elevates CNOT6L protein levels and increases cell proliferation, suggesting RNF219 suppresses CNOT6L via proteasome-mediated degradation.","method":"Mass spectrometry of immunoprecipitates, pull-down assay (domain mapping), in vitro ubiquitination assay, siRNA knockdown, cell proliferation assay","journal":"FEBS open bio","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — direct binding domain mapped by pull-down, in vitro ubiquitination of substrate confirmed, functional KD phenotype; single lab","pmids":["40598799"],"is_preprint":false},{"year":2025,"finding":"Mutations within the C3HC4 scaffold of the RNF219 (OBI1) RING finger domain drive liquid-liquid phase separation (LLPS) to form condensates that encapsulate the CCR4-NOT complex and inhibit its RNA deadenylation activity; the adjacent coiled-coil 1 (CC1) domain promotes condensate formation, while the wild-type RING finger domain intrinsically suppresses LLPS.","method":"RING domain mutagenesis, live-cell imaging of condensates (LLPS), in vitro deadenylation assay, domain deletion/mutation analysis, cell proliferation assay","journal":"Cell proliferation","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis combined with LLPS imaging and in vitro deadenylation assay, single lab, multiple orthogonal methods","pmids":["40497348"],"is_preprint":false}],"current_model":"OBI1 (RNF219/C13ORF7) is an E3 ubiquitin ligase that promotes DNA replication origin firing by multi-mono-ubiquitylating ORC3 and ORC5 within the ORC complex during S-phase; it also associates with the CCR4-NOT deadenylase complex via its C-terminus binding to the CNOT1 DUF3819 domain, simultaneously inhibiting CCR4-NOT deadenylase activity while enhancing its deadenylation-independent translational repression, and can ubiquitinate the CCR4-NOT subunit CNOT6L to target it for proteasomal degradation; RING domain mutations cause aberrant liquid-liquid phase separation that sequesters CCR4-NOT and further inhibits mRNA deadenylation."},"narrative":{"mechanistic_narrative":"OBI1 (RNF219/C13ORF7) is a RING-type E3 ubiquitin ligase that operates in two distinct cellular arenas: control of DNA replication origin firing and regulation of mRNA fate through the CCR4-NOT deadenylase complex [PMID:31160578, PMID:33104214]. In S-phase, OBI1 associates with the ORC complex and catalyses multi-mono-ubiquitylation of chromatin-bound ORC3 and ORC5; this modification is required for efficient origin firing and CMG helicase assembly without affecting pre-replication complex establishment, and non-ubiquitylable ORC3/5 mutants impair firing, establishing them as functionally relevant substrates [PMID:31160578]. Independently, OBI1 binds the CCR4-NOT complex through its C-terminus, which directly engages the CNOT1 DUF3819 domain [PMID:40598799]; in this context it inhibits CCR4-NOT deadenylase activity while enhancing the complex's deadenylation-independent capacity to repress translation [PMID:35660762], and it ubiquitinates the CCR4-NOT subunit CNOT6L to drive its proteasomal degradation [PMID:40598799]. The integrity of the RING finger is central to this regulation: wild-type RING suppresses liquid-liquid phase separation, whereas mutations in its C3HC4 scaffold drive condensate formation that encapsulates and further inhibits CCR4-NOT [PMID:40497348].","teleology":[{"year":2019,"claim":"Established OBI1's first molecular function by identifying it as an ORC-associated E3 ligase whose ubiquitylation of ORC3/5 is required for replication origin firing, defining how an unstudied RING protein contributes to the cell cycle.","evidence":"Pre-RC proteomic interactome, Co-IP, in vitro ubiquitylation, siRNA, CMG formation assay, and non-ubiquitylable mutant rescue in human cells","pmids":["31160578"],"confidence":"High","gaps":["Ubiquitin chain topology and the readers that interpret ORC3/5 mono-ubiquitylation are not defined","Whether the same catalytic activity links to its mRNA-regulatory functions is unresolved"]},{"year":2020,"claim":"Connected OBI1 ligase activity to oncogenic signaling by showing it degrades α-catenin to enable YAP1/β-catenin-driven epigenetic activation of LGALS3, extending its role beyond replication into transcriptional control in cancer.","evidence":"Overexpression/knockdown, ubiquitin ligase assay, promoter epigenetic analysis and Co-IP in hepatocellular carcinoma cells","pmids":["33643786"],"confidence":"Medium","gaps":["Direct ubiquitylation of α-catenin not reconstituted in abstract","Single tumor-cell context"]},{"year":2020,"claim":"Opened the second functional arena by demonstrating OBI1 physically associates with the CCR4-NOT deadenylase and influences mRNA fate during stem-cell differentiation, implicating it in post-transcriptional control.","evidence":"Co-IP, in vitro deadenylation assay, RNA-seq and ES cell differentiation phenotype after knockdown","pmids":["33104214"],"confidence":"Medium","gaps":["Subunit contacts not mapped","Direct effect on deadenylation rate not yet resolved (later refined)"]},{"year":2022,"claim":"Refined the mechanism of CCR4-NOT regulation by showing OBI1 inhibits deadenylase activity while enhancing deadenylation-independent translational repression, recasting it as a directional modulator of the complex rather than a passive partner.","evidence":"Co-purification, in vitro deadenylase assay and translation repression reporter assay","pmids":["35660762"],"confidence":"Medium","gaps":["Molecular basis for the switch toward translational repression unknown","Physiological mRNA targets not identified"]},{"year":2022,"claim":"Tested whether OBI1 drives oncogenic phenotypes through NF-κB signaling, providing a pathway-level placement in nasopharyngeal carcinoma.","evidence":"Overexpression/knockdown, p65 translocation, NF-κB target analysis and pharmacological inhibition rescue","pmids":["36512252"],"confidence":"Low","gaps":["No direct biochemical binding or substrate linking OBI1 to NF-κB identified","Indirect pathway placement via reporter and inhibitor only","Single cell-line context"]},{"year":2025,"claim":"Mapped the physical interface and a catalytic substrate within CCR4-NOT, showing the OBI1 C-terminus binds the CNOT1 DUF3819 domain and ubiquitinates CNOT6L for degradation, linking complex binding to substrate turnover and proliferation control.","evidence":"MS of immunoprecipitates, domain-mapping pull-down, in vitro ubiquitination and knockdown proliferation assay in HEK293T","pmids":["40598799"],"confidence":"Medium","gaps":["In-cell ubiquitylation and chain type on CNOT6L not shown","Whether CNOT6L turnover accounts for the differentiation phenotype unclear"]},{"year":2025,"claim":"Revealed a phase-separation mechanism whereby RING-finger integrity gates condensate formation, showing C3HC4 mutations drive LLPS that sequesters and inhibits CCR4-NOT while wild-type RING suppresses LLPS.","evidence":"RING mutagenesis, live-cell condensate imaging, in vitro deadenylation and domain deletion in cells","pmids":["40497348"],"confidence":"Medium","gaps":["Physiological triggers of RING-dependent LLPS unknown","Relationship between LLPS regulation and ORC-substrate ubiquitylation undefined"]},{"year":null,"claim":"How OBI1's replication-origin function and its CCR4-NOT/mRNA-regulatory functions are coordinated within a single protein, and whether they share regulatory inputs, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model linking ORC ubiquitylation and post-transcriptional control","Endogenous mRNA targets and physiological condensate regulation not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,5]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,5]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,6]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[0]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[2,3]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[5]}],"complexes":["ORC complex","CCR4-NOT complex"],"partners":["ORC3","ORC5","CNOT1","CNOT6L"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5W0B1","full_name":"ORC ubiquitin ligase 1","aliases":["RING finger protein 219"],"length_aa":726,"mass_kda":81.1,"function":"E3 ubiquitin ligase essential for DNA replication origin activation during S phase (PubMed:31160578). Acts as a replication origin selector which selects the origins to be fired and catalyzes the multi-mono-ubiquitination of a subset of chromatin-bound ORC3 and ORC5 during S-phase (PubMed:31160578)","subcellular_location":"Chromosome","url":"https://www.uniprot.org/uniprotkb/Q5W0B1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/OBI1","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"AHSA1","stoichiometry":0.2},{"gene":"CAPZB","stoichiometry":0.2},{"gene":"DNAJB6","stoichiometry":0.2},{"gene":"DNAJC7","stoichiometry":0.2},{"gene":"HSPA4","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/OBI1","total_profiled":1310},"omim":[{"mim_id":"618405","title":"ZINC FINGER PROTEIN 717; ZNF717","url":"https://www.omim.org/entry/618405"},{"mim_id":"615906","title":"ORC UBIQUITIN LIGASE 1; OBI1","url":"https://www.omim.org/entry/615906"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Nuclear bodies","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/OBI1"},"hgnc":{"alias_symbol":["FLJ13449"],"prev_symbol":["C13orf7","RNF219"]},"alphafold":{"accession":"Q5W0B1","domains":[{"cath_id":"3.30.40.10","chopping":"13-77","consensus_level":"medium","plddt":79.5217,"start":13,"end":77},{"cath_id":"1.20.5","chopping":"218-261","consensus_level":"medium","plddt":94.8375,"start":218,"end":261}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5W0B1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5W0B1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5W0B1-F1-predicted_aligned_error_v6.png","plddt_mean":57.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=OBI1","jax_strain_url":"https://www.jax.org/strain/search?query=OBI1"},"sequence":{"accession":"Q5W0B1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5W0B1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5W0B1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5W0B1"}},"corpus_meta":[{"pmid":"33643786","id":"PMC_33643786","title":"RNF219/α-Catenin/LGALS3 Axis Promotes Hepatocellular Carcinoma Bone Metastasis and Associated Skeletal Complications.","date":"2020","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/33643786","citation_count":37,"is_preprint":false},{"pmid":"31160578","id":"PMC_31160578","title":"The ORC ubiquitin ligase OBI1 promotes DNA replication origin firing.","date":"2019","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/31160578","citation_count":29,"is_preprint":false},{"pmid":"26278557","id":"PMC_26278557","title":"Porcine recombinant factor VIII (Obizur; OBI-1; BAX801): product characteristics and preclinical profile.","date":"2015","source":"Haemophilia : the official journal of the World Federation of Hemophilia","url":"https://pubmed.ncbi.nlm.nih.gov/26278557","citation_count":28,"is_preprint":false},{"pmid":"33104214","id":"PMC_33104214","title":"RNF219 interacts with CCR4-NOT in regulating stem cell differentiation.","date":"2020","source":"Journal of molecular cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/33104214","citation_count":15,"is_preprint":false},{"pmid":"34944854","id":"PMC_34944854","title":"Locus-Specific Methylation of GSTP1, RNF219, and KIAA1539 Genes with Single Molecule Resolution in Cell-Free DNA from Healthy Donors and Prostate Tumor Patients: Application in Diagnostics.","date":"2021","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/34944854","citation_count":14,"is_preprint":false},{"pmid":"35660762","id":"PMC_35660762","title":"RNF219 regulates CCR4-NOT function in mRNA translation and deadenylation.","date":"2022","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/35660762","citation_count":9,"is_preprint":false},{"pmid":"29755337","id":"PMC_29755337","title":"Influence of APOE and RNF219 on Behavioral and Cognitive Features of Female Patients Affected by Mild Cognitive Impairment or Alzheimer's Disease.","date":"2018","source":"Frontiers in aging neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/29755337","citation_count":8,"is_preprint":false},{"pmid":"35260196","id":"PMC_35260196","title":"Data mining of bulk and single-cell RNA sequencing introduces OBI1-AS1 as an astrocyte marker with possible role in glioma recurrence and progression.","date":"2022","source":"Clinical epigenetics","url":"https://pubmed.ncbi.nlm.nih.gov/35260196","citation_count":7,"is_preprint":false},{"pmid":"36512252","id":"PMC_36512252","title":"RNF219 Promotes Nasopharyngeal Carcinoma Progression by Activating the NF-κB Pathway.","date":"2022","source":"Molecular biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/36512252","citation_count":4,"is_preprint":false},{"pmid":"40598799","id":"PMC_40598799","title":"The E3 ubiquitin ligase, RNF219, suppresses CNOT6L expression to exhibit antiproliferative activity.","date":"2025","source":"FEBS open bio","url":"https://pubmed.ncbi.nlm.nih.gov/40598799","citation_count":2,"is_preprint":false},{"pmid":"36672380","id":"PMC_36672380","title":"Locus-Specific Bisulfate NGS Sequencing of GSTP1, RNF219, and KIAA1539 Genes in the Total Pool of Cell-Free and Cell-Surface-Bound DNA in Prostate Cancer: A Novel Approach for Prostate Cancer Diagnostics.","date":"2023","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/36672380","citation_count":2,"is_preprint":false},{"pmid":"40497348","id":"PMC_40497348","title":"RNF219 RING Finger Domain Mutants Drive Phase Separation to Encapsulate CCR4-NOT and Promote Cell Proliferation.","date":"2025","source":"Cell proliferation","url":"https://pubmed.ncbi.nlm.nih.gov/40497348","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.03.16.643544","title":"Distinct Co-Methylation Patterns in African and European Populations and Their Genetic Associations","date":"2025-03-17","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.16.643544","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7831,"output_tokens":1943,"usd":0.026319,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8906,"output_tokens":2779,"usd":0.057002,"stage2_stop_reason":"end_turn"},"total_usd":0.083321,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2019,\n      \"finding\": \"OBI1 (C13ORF7/RNF219) is associated with the ORC complex and catalyses multi-mono-ubiquitylation of a subset of chromatin-bound ORC3 and ORC5 during S-phase; OBI1 silencing causes defective origin firing (reduced CMG formation) without affecting pre-RC establishment, and expression of non-ubiquitylable ORC3/5 mutants impairs origin firing, identifying ORC3/5 as functionally relevant OBI1 substrates.\",\n      \"method\": \"Pre-RC proteomic interactome (MS), Co-IP, in vitro ubiquitylation assay, siRNA silencing, CMG formation assay, non-ubiquitylable ORC3/5 mutant expression\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (proteomics, Co-IP, in vitro ubiquitylation, mutant rescue) in a single rigorous study establishing substrate identity and functional consequence\",\n      \"pmids\": [\"31160578\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RNF219 (OBI1) ubiquitin ligase activity mediates α-catenin degradation, which promotes YAP1/β-catenin complex-dependent epigenetic modifications of the LGALS3 promoter, resulting in LGALS3 upregulation in hepatocellular carcinoma cells.\",\n      \"method\": \"Overexpression/knockdown functional assays, ubiquitin ligase activity assay, chromatin/promoter epigenetic analysis, co-immunoprecipitation\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — mechanistic pathway placed by Co-IP and functional assays in a single lab; multiple methods but full reconstitution not described in abstract\",\n      \"pmids\": [\"33643786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RNF219 (OBI1) interacts with the CCR4-NOT deadenylase complex and the RNF219–CCR4-NOT complex exhibits deadenylation activity in vitro; RNF219 depletion in mouse embryonic stem cells impairs neuronal lineage commitment and alters expression of 2-cell-specific and neuronal genes.\",\n      \"method\": \"Co-immunoprecipitation, in vitro deadenylation assay, RNA-seq, siRNA knockdown, ES cell differentiation assay\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro deadenylation assay plus Co-IP and functional KD phenotype, single lab\",\n      \"pmids\": [\"33104214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF219 (OBI1) co-purifies with the CCR4-NOT complex and both inhibits its deadenylase activity and enhances its capacity to repress translation of a target mRNA in a deadenylation-independent manner, directing the CCR4-NOT complex toward translational repression.\",\n      \"method\": \"Co-purification, in vitro deadenylase activity assay, translation repression reporter assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro enzymatic assay plus translation assay, single lab, two orthogonal readouts\",\n      \"pmids\": [\"35660762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF219 (OBI1) activates the NF-κB pathway in nasopharyngeal carcinoma cells, evidenced by increased p65 nuclear translocation and upregulation of NF-κB target genes; NF-κB pathway inhibition in RNF219-overexpressing cells reverses RNF219-driven invasion, migration, and proliferation.\",\n      \"method\": \"Overexpression/knockdown, p65 nuclear translocation assay, NF-κB target gene expression analysis, pharmacological NF-κB inhibition rescue experiment\",\n      \"journal\": \"Molecular biotechnology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, indirect pathway placement via reporter and pharmacological inhibition, no direct biochemical binding or substrate identified\",\n      \"pmids\": [\"36512252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The C-terminal part of RNF219 (OBI1) directly binds the CNOT1 DUF3819 domain (pull-down assay) and ubiquitinates CNOT6L in vitro; RNF219 knockdown in HEK293T cells elevates CNOT6L protein levels and increases cell proliferation, suggesting RNF219 suppresses CNOT6L via proteasome-mediated degradation.\",\n      \"method\": \"Mass spectrometry of immunoprecipitates, pull-down assay (domain mapping), in vitro ubiquitination assay, siRNA knockdown, cell proliferation assay\",\n      \"journal\": \"FEBS open bio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct binding domain mapped by pull-down, in vitro ubiquitination of substrate confirmed, functional KD phenotype; single lab\",\n      \"pmids\": [\"40598799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Mutations within the C3HC4 scaffold of the RNF219 (OBI1) RING finger domain drive liquid-liquid phase separation (LLPS) to form condensates that encapsulate the CCR4-NOT complex and inhibit its RNA deadenylation activity; the adjacent coiled-coil 1 (CC1) domain promotes condensate formation, while the wild-type RING finger domain intrinsically suppresses LLPS.\",\n      \"method\": \"RING domain mutagenesis, live-cell imaging of condensates (LLPS), in vitro deadenylation assay, domain deletion/mutation analysis, cell proliferation assay\",\n      \"journal\": \"Cell proliferation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis combined with LLPS imaging and in vitro deadenylation assay, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"40497348\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"OBI1 (RNF219/C13ORF7) is an E3 ubiquitin ligase that promotes DNA replication origin firing by multi-mono-ubiquitylating ORC3 and ORC5 within the ORC complex during S-phase; it also associates with the CCR4-NOT deadenylase complex via its C-terminus binding to the CNOT1 DUF3819 domain, simultaneously inhibiting CCR4-NOT deadenylase activity while enhancing its deadenylation-independent translational repression, and can ubiquitinate the CCR4-NOT subunit CNOT6L to target it for proteasomal degradation; RING domain mutations cause aberrant liquid-liquid phase separation that sequesters CCR4-NOT and further inhibits mRNA deadenylation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"OBI1 (RNF219/C13ORF7) is a RING-type E3 ubiquitin ligase that operates in two distinct cellular arenas: control of DNA replication origin firing and regulation of mRNA fate through the CCR4-NOT deadenylase complex [#0, #2]. In S-phase, OBI1 associates with the ORC complex and catalyses multi-mono-ubiquitylation of chromatin-bound ORC3 and ORC5; this modification is required for efficient origin firing and CMG helicase assembly without affecting pre-replication complex establishment, and non-ubiquitylable ORC3/5 mutants impair firing, establishing them as functionally relevant substrates [#0]. Independently, OBI1 binds the CCR4-NOT complex through its C-terminus, which directly engages the CNOT1 DUF3819 domain [#5]; in this context it inhibits CCR4-NOT deadenylase activity while enhancing the complex's deadenylation-independent capacity to repress translation [#3], and it ubiquitinates the CCR4-NOT subunit CNOT6L to drive its proteasomal degradation [#5]. The integrity of the RING finger is central to this regulation: wild-type RING suppresses liquid-liquid phase separation, whereas mutations in its C3HC4 scaffold drive condensate formation that encapsulates and further inhibits CCR4-NOT [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2019,\n      \"claim\": \"Established OBI1's first molecular function by identifying it as an ORC-associated E3 ligase whose ubiquitylation of ORC3/5 is required for replication origin firing, defining how an unstudied RING protein contributes to the cell cycle.\",\n      \"evidence\": \"Pre-RC proteomic interactome, Co-IP, in vitro ubiquitylation, siRNA, CMG formation assay, and non-ubiquitylable mutant rescue in human cells\",\n      \"pmids\": [\"31160578\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain topology and the readers that interpret ORC3/5 mono-ubiquitylation are not defined\", \"Whether the same catalytic activity links to its mRNA-regulatory functions is unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Connected OBI1 ligase activity to oncogenic signaling by showing it degrades \\u03b1-catenin to enable YAP1/\\u03b2-catenin-driven epigenetic activation of LGALS3, extending its role beyond replication into transcriptional control in cancer.\",\n      \"evidence\": \"Overexpression/knockdown, ubiquitin ligase assay, promoter epigenetic analysis and Co-IP in hepatocellular carcinoma cells\",\n      \"pmids\": [\"33643786\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitylation of \\u03b1-catenin not reconstituted in abstract\", \"Single tumor-cell context\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Opened the second functional arena by demonstrating OBI1 physically associates with the CCR4-NOT deadenylase and influences mRNA fate during stem-cell differentiation, implicating it in post-transcriptional control.\",\n      \"evidence\": \"Co-IP, in vitro deadenylation assay, RNA-seq and ES cell differentiation phenotype after knockdown\",\n      \"pmids\": [\"33104214\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Subunit contacts not mapped\", \"Direct effect on deadenylation rate not yet resolved (later refined)\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Refined the mechanism of CCR4-NOT regulation by showing OBI1 inhibits deadenylase activity while enhancing deadenylation-independent translational repression, recasting it as a directional modulator of the complex rather than a passive partner.\",\n      \"evidence\": \"Co-purification, in vitro deadenylase assay and translation repression reporter assay\",\n      \"pmids\": [\"35660762\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis for the switch toward translational repression unknown\", \"Physiological mRNA targets not identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Tested whether OBI1 drives oncogenic phenotypes through NF-\\u03baB signaling, providing a pathway-level placement in nasopharyngeal carcinoma.\",\n      \"evidence\": \"Overexpression/knockdown, p65 translocation, NF-\\u03baB target analysis and pharmacological inhibition rescue\",\n      \"pmids\": [\"36512252\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct biochemical binding or substrate linking OBI1 to NF-\\u03baB identified\", \"Indirect pathway placement via reporter and inhibitor only\", \"Single cell-line context\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mapped the physical interface and a catalytic substrate within CCR4-NOT, showing the OBI1 C-terminus binds the CNOT1 DUF3819 domain and ubiquitinates CNOT6L for degradation, linking complex binding to substrate turnover and proliferation control.\",\n      \"evidence\": \"MS of immunoprecipitates, domain-mapping pull-down, in vitro ubiquitination and knockdown proliferation assay in HEK293T\",\n      \"pmids\": [\"40598799\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In-cell ubiquitylation and chain type on CNOT6L not shown\", \"Whether CNOT6L turnover accounts for the differentiation phenotype unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a phase-separation mechanism whereby RING-finger integrity gates condensate formation, showing C3HC4 mutations drive LLPS that sequesters and inhibits CCR4-NOT while wild-type RING suppresses LLPS.\",\n      \"evidence\": \"RING mutagenesis, live-cell condensate imaging, in vitro deadenylation and domain deletion in cells\",\n      \"pmids\": [\"40497348\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological triggers of RING-dependent LLPS unknown\", \"Relationship between LLPS regulation and ORC-substrate ubiquitylation undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How OBI1's replication-origin function and its CCR4-NOT/mRNA-regulatory functions are coordinated within a single protein, and whether they share regulatory inputs, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model linking ORC ubiquitylation and post-transcriptional control\", \"Endogenous mRNA targets and physiological condensate regulation not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [\"ORC complex\", \"CCR4-NOT complex\"],\n    \"partners\": [\"ORC3\", \"ORC5\", \"CNOT1\", \"CNOT6L\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}