{"gene":"UBE3D","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2005,"finding":"H10BH (UBE3D) binds UbcH10 (UBE2C) via amino acids 235–257, exhibits self-ubiquitinylation activity dependent on its HECT-like domain, and the C-terminal half (aa 188–389) can bind cyclin B and ubiquitinylate cyclin B in vitro, suggesting it functions as an E3 ubiquitin ligase using UbcH10 as its cognate E2.","method":"Yeast two-hybrid screening, in vitro ubiquitinylation assay, deletion mutagenesis","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution of ubiquitinylation with mutagenesis, but single lab and single study without independent replication","pmids":["15749827"],"is_preprint":false},{"year":2018,"finding":"The yeast ortholog of UBE3D, Yjr141w/Ipa1, belongs to the HECT_2 family; five cysteine residues within the HECT_2 signature and the C-terminus are essential for its activity; Ipa1 interacts with multiple ubiquitin-conjugating enzymes in vivo, localizes to cytosol and nucleus, and influences proteasome activity as evidenced by activation of the Rpn4 regulon and decreased turnover of destabilized proteasome substrates in ipa1 mutants.","method":"Genetic mutant analysis, in vivo interaction assays, proteasome substrate turnover assays, transcriptional reporter (Rpn4 regulon), subcellular localization","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (mutagenesis, genetic epistasis, localization, substrate turnover) in single study in yeast ortholog","pmids":["29519818"],"is_preprint":false},{"year":2022,"finding":"UBE3D stabilizes CPSF73 protein by preventing its ubiquitin-mediated proteasomal degradation; depletion of UBE3D leads to CPSF73 downregulation, a pre-mRNA cleavage defect, and dysregulated gene expression in cells.","method":"siRNA depletion of UBE3D, western blot for CPSF73 protein levels, pre-mRNA cleavage assays, gene expression analysis","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined molecular phenotype (CPSF73 degradation, cleavage defect) using multiple readouts, single lab","pmids":["35992060"],"is_preprint":false},{"year":2022,"finding":"CRISPR/Cas9 knockout of Ube3d in 3T3-L1 cells decreases CPSF73 protein levels and causes global changes in cellular mRNAs consistent with loss of 3'-end processing capacity; overexpression of either UBE3D or CPSF73 rescues the adipogenic differentiation defect and partially restores preadipogenic marker expression, placing UBE3D upstream of CPSF73 in the mRNA 3'-end processing pathway.","method":"CRISPR/Cas9 knockout, overexpression rescue, RNA-seq/global mRNA analysis, western blot, adipocyte differentiation assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with rescue experiment and multiple orthogonal readouts, single lab","pmids":["36519931"],"is_preprint":false},{"year":2022,"finding":"GFP-UBE3D is recruited to DNA double-strand break damage sites via a PCNA-interacting protein (PIP) box interaction with PCNA; UBE3D interacts with KAP1 via residues R377/R378; the AMD-associated V379M mutation abolishes KAP1-UBE3D binding upon oxidation; UBE3D depletion reduces chromatin relaxation and KAP1 recruitment upon DNA damage, implicating UBE3D in homologous recombination repair via heterochromatin de-condensation.","method":"I-SceI-inducible GFP reporter system, immunoprecipitation-mass spectrometry, co-immunoprecipitation, pulldown assays, micrococcal nuclease (MNase) assays, site-directed mutagenesis","journal":"Investigative ophthalmology & visual science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (MS, CoIP, pulldown, MNase, mutagenesis) in single study, single lab","pmids":["36094642"],"is_preprint":false},{"year":2024,"finding":"UBE3D is a cytoplasmic binding partner for CPSF73 and likely uses a conserved cysteine residue to directly coordinate the active-site metal ions of CPSF73, analogous to how BRAT1 engages INTS11; this interaction stabilizes CPSF73 and is required for its nuclear function in pre-mRNA 3'-end processing.","method":"Structural analysis (cryo-EM of paralogous complex as template), binding partner identification, functional complementation analogy with BRAT1-INTS11 complex structure","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — binding partner identification supported by structural analogy and functional evidence, but direct structural validation of UBE3D-CPSF73 complex not fully reported in abstract","pmids":["39032490"],"is_preprint":false},{"year":2025,"finding":"UBE3D interacts with CPSF3 (the mouse homolog of CPSF73/Ysh1), can de-ubiquitinate CPSF3, and deficiency of UBE3D leads to reduced CPSF3 protein levels in both mouse and human cells; RNA-seq of Ube3d knockout embryos shows downregulation of multiple Homeobox genes, and overexpression of dominant-negative CPSF3 partially reduces mRNA levels of several Homeobox genes, placing UBE3D-CPSF3 interaction upstream of Homeobox gene expression.","method":"CRISPR/Cas9 knockout mice, co-immunoprecipitation, de-ubiquitination assay, RNA-seq, dominant-negative overexpression, in situ hybridization, immunofluorescence","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (CoIP, deubiquitination assay, RNA-seq, genetic rescue) in single study, single lab","pmids":["40075082"],"is_preprint":false}],"current_model":"UBE3D is a HECT_2-family E3-like ubiquitin ligase that binds the E2 enzyme UbcH10 and can ubiquitinylate substrates (e.g., cyclin B) in vitro; its primary established cellular role is to stabilize the mRNA 3'-end processing endonuclease CPSF73/CPSF3 by preventing its ubiquitin-mediated proteasomal degradation, thereby maintaining pre-mRNA 3'-end cleavage and polyadenylation capacity—a function conserved from its yeast ortholog Ipa1 (which also modulates proteasome activity)—while additionally participating in DNA double-strand break repair through PCNA-directed recruitment and KAP1-mediated heterochromatin de-condensation."},"narrative":{"mechanistic_narrative":"UBE3D is a HECT_2-family E3-like ubiquitin ligase whose principal characterized role is to stabilize the pre-mRNA 3'-end processing endonuclease CPSF73/CPSF3, thereby maintaining cleavage and polyadenylation capacity and the downstream gene expression programs it controls [PMID:35992060, PMID:36519931, PMID:40075082]. It binds the E2 ubiquitin-conjugating enzyme UbcH10/UBE2C through residues 235–257, displays HECT-dependent self-ubiquitinylation, and ubiquitinylates cyclin B in vitro [PMID:15749827]. Mechanistically, UBE3D is a cytoplasmic binding partner of CPSF73 that uses a conserved cysteine to coordinate the enzyme's active-site metal ions, and its loss—by siRNA depletion or CRISPR knockout—lowers CPSF73/CPSF3 protein, produces pre-mRNA cleavage defects, and globally dysregulates mRNAs; overexpression of UBE3D or CPSF73 rescues the resulting phenotypes, placing UBE3D genetically upstream of CPSF73 [PMID:35992060, PMID:36519931, PMID:39032490]. In mice, UBE3D interacts with and can de-ubiquitinate CPSF3, and its deficiency reduces CPSF3 and downregulates Homeobox gene expression, while dominant-negative CPSF3 reproduces this loss [PMID:40075082]. UBE3D additionally functions in DNA double-strand break repair: it is recruited to damage sites via a PIP-box interaction with PCNA, binds KAP1 through residues R377/R378, and its depletion impairs chromatin relaxation and KAP1 recruitment, implicating it in homologous recombination via heterochromatin de-condensation; the AMD-associated V379M mutation abolishes KAP1 binding upon oxidation [PMID:36094642]. Its yeast ortholog Ipa1 belongs to the same HECT_2 family, requires conserved cysteines for activity, interacts with multiple E2 enzymes, and modulates proteasome activity [PMID:29519818].","teleology":[{"year":2005,"claim":"Established that UBE3D (H10BH) is an E3-like ubiquitin ligase by showing it pairs with a specific E2 enzyme and can ubiquitinylate a substrate, defining its biochemical activity.","evidence":"Yeast two-hybrid, in vitro ubiquitinylation, and deletion mutagenesis mapping UbcH10 binding and HECT-dependent activity","pmids":["15749827"],"confidence":"Medium","gaps":["Single lab without independent replication","Physiological substrates beyond cyclin B not identified","In vitro activity not validated in cells"]},{"year":2018,"claim":"Showed the yeast ortholog Ipa1 defines a conserved HECT_2 family with essential active-site cysteines and links the family to proteasome regulation, framing UBE3D within proteostasis.","evidence":"Genetic mutant analysis, in vivo E2 interaction assays, Rpn4 reporter and substrate turnover in yeast","pmids":["29519818"],"confidence":"Medium","gaps":["Findings are in yeast ortholog, not human UBE3D","Direct proteasome substrates not defined","Mechanism linking Ipa1 to Rpn4 regulon unresolved"]},{"year":2022,"claim":"Identified the primary cellular role of UBE3D as a stabilizer of CPSF73, connecting it to pre-mRNA 3'-end processing rather than substrate destruction.","evidence":"siRNA depletion with CPSF73 western blot, pre-mRNA cleavage assays, gene expression analysis","pmids":["35992060"],"confidence":"Medium","gaps":["Mechanism by which UBE3D blocks CPSF73 degradation not resolved","Direct ubiquitin chain editing on CPSF73 not demonstrated","Single lab"]},{"year":2022,"claim":"Confirmed via genetic knockout and rescue that UBE3D acts upstream of CPSF73 and that this axis is required for a physiological output, adipogenic differentiation.","evidence":"CRISPR/Cas9 knockout in 3T3-L1, overexpression rescue, RNA-seq, western blot, differentiation assay","pmids":["36519931"],"confidence":"Medium","gaps":["Direct enzymatic mechanism on CPSF73 not shown","Generality beyond adipocytes untested in this work","Single lab"]},{"year":2022,"claim":"Revealed a second, distinct function in DNA double-strand break repair, showing UBE3D is recruited via PCNA and engages KAP1 to drive heterochromatin de-condensation.","evidence":"I-SceI GFP reporter, IP-MS, Co-IP, pulldown, MNase assays, site-directed mutagenesis","pmids":["36094642"],"confidence":"Medium","gaps":["Whether repair role depends on ligase activity unknown","Relationship between repair function and CPSF73 stabilization unclear","AMD V379M mechanism shown only at the binding level"]},{"year":2024,"claim":"Provided a structural rationale for CPSF73 stabilization, proposing UBE3D uses a conserved cysteine to coordinate CPSF73 active-site metals analogous to BRAT1-INTS11.","evidence":"Structural analysis using a paralogous cryo-EM template, binding partner identification, functional complementation analogy","pmids":["39032490"],"confidence":"Medium","gaps":["Direct structure of the UBE3D-CPSF73 complex not reported","Cysteine-metal coordination not biochemically validated","Reconciliation with ligase/de-ubiquitination activities pending"]},{"year":2025,"claim":"Extended the CPSF3 axis in vivo and reframed UBE3D activity as de-ubiquitination of CPSF3, linking the interaction to developmental Homeobox gene expression.","evidence":"CRISPR/Cas9 knockout mice, Co-IP, de-ubiquitination assay, RNA-seq, dominant-negative CPSF3, in situ hybridization","pmids":["40075082"],"confidence":"Medium","gaps":["Apparent de-ubiquitinase activity not reconciled with reported E3 ligase activity","Catalytic residues for CPSF3 de-ubiquitination not mapped","Single lab"]},{"year":null,"claim":"Whether UBE3D's enzymatic activity is fundamentally that of a ubiquitin ligase, a de-ubiquitinase, or a non-catalytic protective binder of CPSF73/CPSF3 remains unresolved, as does how its 3'-end processing and DSB-repair roles are coordinated.","evidence":"No single study reconciles the ligase, de-ubiquitination, and stabilization activities","pmids":[],"confidence":"Medium","gaps":["No direct structure of any UBE3D-substrate complex","Catalytic mechanism toward CPSF73/CPSF3 undefined","Functional link between RNA processing and DNA repair roles unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,5,6]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[2,3,5]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,5]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,4]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[2,3,6]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[4]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1]}],"complexes":[],"partners":["CPSF3","UBE2C","PCNA","KAP1","TRIM28"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q7Z6J8","full_name":"E3 ubiquitin-protein ligase E3D","aliases":["HECT-type E3 ubiquitin transferase E3D","UbcH10-binding protein with a HECT-like domain","Ubiquitin-conjugating enzyme E2C-binding protein"],"length_aa":389,"mass_kda":43.7,"function":"E3 ubiquitin-protein ligase which accepts ubiquitin from specific E2 ubiquitin-conjugating enzymes, and transfers it to substrates, generally promoting their degradation by the proteasome (PubMed:15749827). Independently of its E3 ubiquitin-protein ligase activity, acts as an inhibitor of CPSF3 endonuclease activity by blocking CPSF3 active site (PubMed:39032490)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q7Z6J8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UBE3D","classification":"Not Classified","n_dependent_lines":181,"n_total_lines":1208,"dependency_fraction":0.1498344370860927},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CAPZB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/UBE3D","total_profiled":1310},"omim":[{"mim_id":"612495","title":"UBIQUITIN-PROTEIN LIGASE E3D; UBE3D","url":"https://www.omim.org/entry/612495"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Rods & Rings","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/UBE3D"},"hgnc":{"alias_symbol":["DKFZp434A1520","H10BH","YJR141W"],"prev_symbol":["C6orf157","UBE2CBP"]},"alphafold":{"accession":"Q7Z6J8","domains":[{"cath_id":"2.60.40,2.60.40","chopping":"11-88","consensus_level":"high","plddt":87.2542,"start":11,"end":88},{"cath_id":"-","chopping":"99-143_155-176_205-244","consensus_level":"medium","plddt":83.2908,"start":99,"end":244},{"cath_id":"-","chopping":"247-299_307-389","consensus_level":"high","plddt":87.3569,"start":247,"end":389}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7Z6J8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q7Z6J8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q7Z6J8-F1-predicted_aligned_error_v6.png","plddt_mean":79.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=UBE3D","jax_strain_url":"https://www.jax.org/strain/search?query=UBE3D"},"sequence":{"accession":"Q7Z6J8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q7Z6J8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q7Z6J8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7Z6J8"}},"corpus_meta":[{"pmid":"25872646","id":"PMC_25872646","title":"Whole-exome sequencing implicates UBE3D in age-related macular degeneration in East Asian populations.","date":"2015","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/25872646","citation_count":36,"is_preprint":false},{"pmid":"35992060","id":"PMC_35992060","title":"Targeting the mRNA endonuclease CPSF73 inhibits breast cancer cell migration, invasion, and self-renewal.","date":"2022","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/35992060","citation_count":19,"is_preprint":false},{"pmid":"15749827","id":"PMC_15749827","title":"A novel UbcH10-binding protein facilitates the ubiquitinylation of cyclin B in vitro.","date":"2005","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15749827","citation_count":12,"is_preprint":false},{"pmid":"32200270","id":"PMC_32200270","title":"ube3d, a New Gene Associated with Age-Related Macular Degeneration, Induces Functional Changes in Both In Vivo and In Vitro Studies.","date":"2020","source":"Molecular therapy. Nucleic acids","url":"https://pubmed.ncbi.nlm.nih.gov/32200270","citation_count":11,"is_preprint":false},{"pmid":"33050709","id":"PMC_33050709","title":"Genetic determinants of ammonia-induced acute lung injury in mice.","date":"2020","source":"American journal of physiology. Lung cellular and molecular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/33050709","citation_count":11,"is_preprint":false},{"pmid":"29519818","id":"PMC_29519818","title":"Proteasome Activity Is Influenced by the HECT_2 Protein Ipa1 in Budding Yeast.","date":"2018","source":"Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29519818","citation_count":11,"is_preprint":false},{"pmid":"35873486","id":"PMC_35873486","title":"Genomic Diversity and Selection Signatures for Weining Cattle on the Border of Yunnan-Guizhou.","date":"2022","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/35873486","citation_count":10,"is_preprint":false},{"pmid":"39032490","id":"PMC_39032490","title":"Cytoplasmic binding partners of the Integrator endonuclease INTS11 and its paralog CPSF73 are required for their nuclear function.","date":"2024","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/39032490","citation_count":8,"is_preprint":false},{"pmid":"36519931","id":"PMC_36519931","title":"UBE3D Regulates mRNA 3'-End Processing and Maintains Adipogenic Potential in 3T3-L1 Cells.","date":"2022","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/36519931","citation_count":7,"is_preprint":false},{"pmid":"36094642","id":"PMC_36094642","title":"UBE3D Is Involved in Blue Light-Induced Retinal Damage by Regulating Double-Strand Break Repair.","date":"2022","source":"Investigative ophthalmology & visual science","url":"https://pubmed.ncbi.nlm.nih.gov/36094642","citation_count":5,"is_preprint":false},{"pmid":"37772657","id":"PMC_37772657","title":"Conditional loss of Ube3d in the retinal pigment epithelium accelerates age-associated alterations in the retina of mice.","date":"2023","source":"The Journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/37772657","citation_count":5,"is_preprint":false},{"pmid":"39199913","id":"PMC_39199913","title":"Novel Candidate Genes Involved in an Initial Stage of White Striping Development in Broiler Chickens.","date":"2024","source":"Animals : an open access journal from MDPI","url":"https://pubmed.ncbi.nlm.nih.gov/39199913","citation_count":3,"is_preprint":false},{"pmid":"40075082","id":"PMC_40075082","title":"Deficiency of UBE3D in mice leads to severe embryonic abnormalities and disrupts the mRNA of Homeobox genes via CPSF3.","date":"2025","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/40075082","citation_count":0,"is_preprint":false},{"pmid":"40629317","id":"PMC_40629317","title":"The related SNPs and genes to body size using GWAS- latent variable modeling in dromedaries.","date":"2025","source":"BMC genomics","url":"https://pubmed.ncbi.nlm.nih.gov/40629317","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8646,"output_tokens":2127,"usd":0.028921,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9034,"output_tokens":3146,"usd":0.06191,"stage2_stop_reason":"end_turn"},"total_usd":0.090831,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"H10BH (UBE3D) binds UbcH10 (UBE2C) via amino acids 235–257, exhibits self-ubiquitinylation activity dependent on its HECT-like domain, and the C-terminal half (aa 188–389) can bind cyclin B and ubiquitinylate cyclin B in vitro, suggesting it functions as an E3 ubiquitin ligase using UbcH10 as its cognate E2.\",\n      \"method\": \"Yeast two-hybrid screening, in vitro ubiquitinylation assay, deletion mutagenesis\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution of ubiquitinylation with mutagenesis, but single lab and single study without independent replication\",\n      \"pmids\": [\"15749827\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The yeast ortholog of UBE3D, Yjr141w/Ipa1, belongs to the HECT_2 family; five cysteine residues within the HECT_2 signature and the C-terminus are essential for its activity; Ipa1 interacts with multiple ubiquitin-conjugating enzymes in vivo, localizes to cytosol and nucleus, and influences proteasome activity as evidenced by activation of the Rpn4 regulon and decreased turnover of destabilized proteasome substrates in ipa1 mutants.\",\n      \"method\": \"Genetic mutant analysis, in vivo interaction assays, proteasome substrate turnover assays, transcriptional reporter (Rpn4 regulon), subcellular localization\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (mutagenesis, genetic epistasis, localization, substrate turnover) in single study in yeast ortholog\",\n      \"pmids\": [\"29519818\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBE3D stabilizes CPSF73 protein by preventing its ubiquitin-mediated proteasomal degradation; depletion of UBE3D leads to CPSF73 downregulation, a pre-mRNA cleavage defect, and dysregulated gene expression in cells.\",\n      \"method\": \"siRNA depletion of UBE3D, western blot for CPSF73 protein levels, pre-mRNA cleavage assays, gene expression analysis\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined molecular phenotype (CPSF73 degradation, cleavage defect) using multiple readouts, single lab\",\n      \"pmids\": [\"35992060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CRISPR/Cas9 knockout of Ube3d in 3T3-L1 cells decreases CPSF73 protein levels and causes global changes in cellular mRNAs consistent with loss of 3'-end processing capacity; overexpression of either UBE3D or CPSF73 rescues the adipogenic differentiation defect and partially restores preadipogenic marker expression, placing UBE3D upstream of CPSF73 in the mRNA 3'-end processing pathway.\",\n      \"method\": \"CRISPR/Cas9 knockout, overexpression rescue, RNA-seq/global mRNA analysis, western blot, adipocyte differentiation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with rescue experiment and multiple orthogonal readouts, single lab\",\n      \"pmids\": [\"36519931\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"GFP-UBE3D is recruited to DNA double-strand break damage sites via a PCNA-interacting protein (PIP) box interaction with PCNA; UBE3D interacts with KAP1 via residues R377/R378; the AMD-associated V379M mutation abolishes KAP1-UBE3D binding upon oxidation; UBE3D depletion reduces chromatin relaxation and KAP1 recruitment upon DNA damage, implicating UBE3D in homologous recombination repair via heterochromatin de-condensation.\",\n      \"method\": \"I-SceI-inducible GFP reporter system, immunoprecipitation-mass spectrometry, co-immunoprecipitation, pulldown assays, micrococcal nuclease (MNase) assays, site-directed mutagenesis\",\n      \"journal\": \"Investigative ophthalmology & visual science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (MS, CoIP, pulldown, MNase, mutagenesis) in single study, single lab\",\n      \"pmids\": [\"36094642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UBE3D is a cytoplasmic binding partner for CPSF73 and likely uses a conserved cysteine residue to directly coordinate the active-site metal ions of CPSF73, analogous to how BRAT1 engages INTS11; this interaction stabilizes CPSF73 and is required for its nuclear function in pre-mRNA 3'-end processing.\",\n      \"method\": \"Structural analysis (cryo-EM of paralogous complex as template), binding partner identification, functional complementation analogy with BRAT1-INTS11 complex structure\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — binding partner identification supported by structural analogy and functional evidence, but direct structural validation of UBE3D-CPSF73 complex not fully reported in abstract\",\n      \"pmids\": [\"39032490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UBE3D interacts with CPSF3 (the mouse homolog of CPSF73/Ysh1), can de-ubiquitinate CPSF3, and deficiency of UBE3D leads to reduced CPSF3 protein levels in both mouse and human cells; RNA-seq of Ube3d knockout embryos shows downregulation of multiple Homeobox genes, and overexpression of dominant-negative CPSF3 partially reduces mRNA levels of several Homeobox genes, placing UBE3D-CPSF3 interaction upstream of Homeobox gene expression.\",\n      \"method\": \"CRISPR/Cas9 knockout mice, co-immunoprecipitation, de-ubiquitination assay, RNA-seq, dominant-negative overexpression, in situ hybridization, immunofluorescence\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (CoIP, deubiquitination assay, RNA-seq, genetic rescue) in single study, single lab\",\n      \"pmids\": [\"40075082\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UBE3D is a HECT_2-family E3-like ubiquitin ligase that binds the E2 enzyme UbcH10 and can ubiquitinylate substrates (e.g., cyclin B) in vitro; its primary established cellular role is to stabilize the mRNA 3'-end processing endonuclease CPSF73/CPSF3 by preventing its ubiquitin-mediated proteasomal degradation, thereby maintaining pre-mRNA 3'-end cleavage and polyadenylation capacity—a function conserved from its yeast ortholog Ipa1 (which also modulates proteasome activity)—while additionally participating in DNA double-strand break repair through PCNA-directed recruitment and KAP1-mediated heterochromatin de-condensation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UBE3D is a HECT_2-family E3-like ubiquitin ligase whose principal characterized role is to stabilize the pre-mRNA 3'-end processing endonuclease CPSF73/CPSF3, thereby maintaining cleavage and polyadenylation capacity and the downstream gene expression programs it controls [#2, #3, #6]. It binds the E2 ubiquitin-conjugating enzyme UbcH10/UBE2C through residues 235–257, displays HECT-dependent self-ubiquitinylation, and ubiquitinylates cyclin B in vitro [#0]. Mechanistically, UBE3D is a cytoplasmic binding partner of CPSF73 that uses a conserved cysteine to coordinate the enzyme's active-site metal ions, and its loss—by siRNA depletion or CRISPR knockout—lowers CPSF73/CPSF3 protein, produces pre-mRNA cleavage defects, and globally dysregulates mRNAs; overexpression of UBE3D or CPSF73 rescues the resulting phenotypes, placing UBE3D genetically upstream of CPSF73 [#2, #3, #5]. In mice, UBE3D interacts with and can de-ubiquitinate CPSF3, and its deficiency reduces CPSF3 and downregulates Homeobox gene expression, while dominant-negative CPSF3 reproduces this loss [#6]. UBE3D additionally functions in DNA double-strand break repair: it is recruited to damage sites via a PIP-box interaction with PCNA, binds KAP1 through residues R377/R378, and its depletion impairs chromatin relaxation and KAP1 recruitment, implicating it in homologous recombination via heterochromatin de-condensation; the AMD-associated V379M mutation abolishes KAP1 binding upon oxidation [#4]. Its yeast ortholog Ipa1 belongs to the same HECT_2 family, requires conserved cysteines for activity, interacts with multiple E2 enzymes, and modulates proteasome activity [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established that UBE3D (H10BH) is an E3-like ubiquitin ligase by showing it pairs with a specific E2 enzyme and can ubiquitinylate a substrate, defining its biochemical activity.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro ubiquitinylation, and deletion mutagenesis mapping UbcH10 binding and HECT-dependent activity\",\n      \"pmids\": [\"15749827\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single lab without independent replication\",\n        \"Physiological substrates beyond cyclin B not identified\",\n        \"In vitro activity not validated in cells\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed the yeast ortholog Ipa1 defines a conserved HECT_2 family with essential active-site cysteines and links the family to proteasome regulation, framing UBE3D within proteostasis.\",\n      \"evidence\": \"Genetic mutant analysis, in vivo E2 interaction assays, Rpn4 reporter and substrate turnover in yeast\",\n      \"pmids\": [\"29519818\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Findings are in yeast ortholog, not human UBE3D\",\n        \"Direct proteasome substrates not defined\",\n        \"Mechanism linking Ipa1 to Rpn4 regulon unresolved\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified the primary cellular role of UBE3D as a stabilizer of CPSF73, connecting it to pre-mRNA 3'-end processing rather than substrate destruction.\",\n      \"evidence\": \"siRNA depletion with CPSF73 western blot, pre-mRNA cleavage assays, gene expression analysis\",\n      \"pmids\": [\"35992060\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism by which UBE3D blocks CPSF73 degradation not resolved\",\n        \"Direct ubiquitin chain editing on CPSF73 not demonstrated\",\n        \"Single lab\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Confirmed via genetic knockout and rescue that UBE3D acts upstream of CPSF73 and that this axis is required for a physiological output, adipogenic differentiation.\",\n      \"evidence\": \"CRISPR/Cas9 knockout in 3T3-L1, overexpression rescue, RNA-seq, western blot, differentiation assay\",\n      \"pmids\": [\"36519931\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct enzymatic mechanism on CPSF73 not shown\",\n        \"Generality beyond adipocytes untested in this work\",\n        \"Single lab\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed a second, distinct function in DNA double-strand break repair, showing UBE3D is recruited via PCNA and engages KAP1 to drive heterochromatin de-condensation.\",\n      \"evidence\": \"I-SceI GFP reporter, IP-MS, Co-IP, pulldown, MNase assays, site-directed mutagenesis\",\n      \"pmids\": [\"36094642\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether repair role depends on ligase activity unknown\",\n        \"Relationship between repair function and CPSF73 stabilization unclear\",\n        \"AMD V379M mechanism shown only at the binding level\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided a structural rationale for CPSF73 stabilization, proposing UBE3D uses a conserved cysteine to coordinate CPSF73 active-site metals analogous to BRAT1-INTS11.\",\n      \"evidence\": \"Structural analysis using a paralogous cryo-EM template, binding partner identification, functional complementation analogy\",\n      \"pmids\": [\"39032490\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct structure of the UBE3D-CPSF73 complex not reported\",\n        \"Cysteine-metal coordination not biochemically validated\",\n        \"Reconciliation with ligase/de-ubiquitination activities pending\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended the CPSF3 axis in vivo and reframed UBE3D activity as de-ubiquitination of CPSF3, linking the interaction to developmental Homeobox gene expression.\",\n      \"evidence\": \"CRISPR/Cas9 knockout mice, Co-IP, de-ubiquitination assay, RNA-seq, dominant-negative CPSF3, in situ hybridization\",\n      \"pmids\": [\"40075082\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Apparent de-ubiquitinase activity not reconciled with reported E3 ligase activity\",\n        \"Catalytic residues for CPSF3 de-ubiquitination not mapped\",\n        \"Single lab\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether UBE3D's enzymatic activity is fundamentally that of a ubiquitin ligase, a de-ubiquitinase, or a non-catalytic protective binder of CPSF73/CPSF3 remains unresolved, as does how its 3'-end processing and DSB-repair roles are coordinated.\",\n      \"evidence\": \"No single study reconciles the ligase, de-ubiquitination, and stabilization activities\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No direct structure of any UBE3D-substrate complex\",\n        \"Catalytic mechanism toward CPSF73/CPSF3 undefined\",\n        \"Functional link between RNA processing and DNA repair roles unknown\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 5, 6]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [2, 3, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [2, 3, 6]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CPSF3\", \"UBE2C\", \"PCNA\", \"KAP1\", \"TRIM28\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}