{"gene":"CPSF4","run_date":"2026-06-09T22:57:19","timeline":{"discoveries":[{"year":2014,"finding":"CPSF30 (CPSF4) directly contacts the AAUAAA polyadenylation signal in mammalian mRNA 3' processing; this interaction is primarily mediated by zinc fingers 2 and 3 (ZF2-ZF3) and is essential for mRNA 3' end processing.","method":"In vitro RNA binding assays and in vivo crosslinking/immunoprecipitation (iCLIP); mutagenesis of zinc finger domains","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro and in vivo binding assays with domain mutagenesis, replicated across multiple orthogonal methods in one rigorous study","pmids":["25301780"],"is_preprint":false},{"year":2006,"finding":"CPSF30 binding to the influenza A NS1A protein is mediated specifically by zinc fingers 2 and 3 (F2F3) of CPSF30, and this interaction inhibits 3'-end processing of cellular pre-mRNAs including IFN-β. Constitutive nuclear expression of F2F3 inhibits influenza A virus replication by competing with endogenous CPSF30 for NS1A binding.","method":"Mutagenesis, stable cell line expression of epitope-tagged F2F3 fragment, viral replication assay, IFN-β mRNA induction assay","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutagenesis defining binding domain, functional rescue/competition experiments, multiple orthogonal readouts","pmids":["16571812"],"is_preprint":false},{"year":2008,"finding":"The influenza A virus polymerase complex (specifically PA protein and NP, but not PB1/PB2) is an integral component of the CPSF30–NS1A complex in infected cells, stabilizing CPSF30 binding to NS1A even when NS1A contains suboptimal hydrophobic residues at positions 103/106.","method":"Co-immunoprecipitation in infected cells, reverse genetics with cognate/non-cognate polymerase swaps, mutant NS1A viruses","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP in infected cells, multiple polymerase subunit knockouts, cognate/non-cognate swap experiments establishing specific subunit requirements","pmids":["19052083"],"is_preprint":false},{"year":2014,"finding":"An I106M substitution in H7N9 influenza NS1 restores CPSF30 binding and the ability to block host gene expression, and a recombinant virus expressing NS1-I106M replicates to higher titers in vivo, demonstrating that CPSF30 binding by NS1 is a virulence determinant.","method":"Site-directed mutagenesis, reverse genetics, in vivo infection, Co-immunoprecipitation","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutagenesis + reverse genetics + in vivo virulence assay, multiple orthogonal methods in one study","pmids":["25078692"],"is_preprint":false},{"year":2020,"finding":"The crystal structure of human CPSF30 ZF4-ZF5 in complex with hFip1 residues 161–200 (1.9 Å resolution) reveals that one hFip1 molecule binds each of ZF4 and ZF5 with a conserved interaction mode (1:2 stoichiometry). ZF4 has higher affinity for hFip1 (Kd = 1.8 nM). The CPSF30–hFip1 complex recruits two copies of poly(A) polymerase (PAP) and both hFip1 binding sites in CPSF30 support polyadenylation.","method":"X-ray crystallography (1.9 Å), mutagenesis of CPSF30 binding sites, fluorescence polarization binding assays, in vitro polyadenylation assay","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional mutagenesis and in vitro reconstitution of polyadenylation, multiple orthogonal methods in one study","pmids":["33122294"],"is_preprint":false},{"year":2020,"finding":"Full-length CPSF30 contains one 2Fe-2S iron-sulfur cluster in addition to five zinc ions. Full-length CPSF30 binds both AAUAAA and polyU pre-mRNA motifs with high affinity; AAUAAA binding requires all five CCCH domains whereas polyU binding requires full-length CPSF30 (implicating the CCHC zinc knuckle). Truncated forms (ZF2-ZF3 alone or CCHC alone) do not exhibit RNA binding.","method":"ICP-MS, UV-Vis spectroscopy, X-ray absorption spectroscopy (metal content), fluorescence anisotropy RNA binding assays with truncation variants","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical reconstitution with multiple spectroscopic methods and domain deletion analysis in one study","pmids":["32027124"],"is_preprint":false},{"year":2021,"finding":"Bases at positions 1, 2, 4, and 5 within the AAUAAA hexamer are important for CPSF30 binding; flanking A/U residues promote higher-affinity binding than G/C. The CCHC zinc knuckle restores binding to AU hexamer variants that are not recognized by the five CCCH domains alone, indicating the two RNA-binding modules (CCCH cluster and zinc knuckle) act cooperatively.","method":"Fluorescence anisotropy binding assays with systematic AAUAAA sequence variants; comparison of CPSF30-5F (five CCCH domains) vs full-length CPSF30","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — rigorous in vitro binding assays with systematic mutagenesis, single lab, no independent replication reported","pmids":["33615774"],"is_preprint":false},{"year":2019,"finding":"CPSF4 (CPSF30), independently and through its interaction with influenza NS1, modulates alternative splicing of TP53 transcripts, altering expression of p53 isoforms and affecting type I interferon secretion and viral replication.","method":"TP53 minigene splicing assay, siRNA knockdown of CPSF4, NS1–CPSF4 interaction in infected cells, IFN secretion measurement","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional minigene assay + siRNA knockdown + interaction studies, multiple readouts in one study, single lab","pmids":["30651364"],"is_preprint":false},{"year":2015,"finding":"CBP (CREB-binding protein) co-localizes and physically interacts with CPSF4 in lung cancer cells. Knockdown of CPSF4 inhibits hTERT transcription and cell growth induced by CBP, and vice versa, demonstrating a synergistic relationship between CBP and CPSF4 in regulating lung cancer cell growth.","method":"Co-immunoprecipitation, immunofluorescence co-localization, siRNA knockdown of CPSF4 and CBP, hTERT transcription assay, cell proliferation assay","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and co-localization plus functional siRNA rescue, two orthogonal methods, single lab","pmids":["26628108"],"is_preprint":false},{"year":2016,"finding":"CPSF4 binds to the hTERT promoter in colorectal cancer cells, as demonstrated by pulldown assays, luciferase reporter assays, and chromatin immunoprecipitation (ChIP), identifying CPSF4 as an hTERT promoter-binding protein.","method":"Pulldown assay, luciferase reporter assay, ChIP assay","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — three orthogonal methods (pulldown, reporter, ChIP) establishing promoter binding, single lab","pmids":["27997899"],"is_preprint":false},{"year":2021,"finding":"CPSF4 reduces levels of circRNAs that contain a polyadenylation signal sequence, thereby decreasing miRNA accumulation and disrupting miRNA-mediated gene silencing in hepatocellular carcinoma. CPSF4 promotes HCC cell proliferation and tumorigenicity through this circRNA inhibition mechanism.","method":"Knockdown/overexpression of CPSF4 in cell culture and xenograft mouse models, circRNA profiling, miRNA accumulation measurement","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo functional assays with mechanistic circRNA/miRNA measurements, single lab","pmids":["34103682"],"is_preprint":false},{"year":2009,"finding":"In trypanosomes, TcCPSF30 physically interacts with TcFIP1-like (a polyadenylation factor); specific amino acids in each protein mediating this interaction were mapped, showing differences from the human CPSF30–FIP1 interaction surface.","method":"Yeast two-hybrid / biochemical interaction mapping, identification of interacting residues by mutagenesis","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single interaction mapping study in a divergent organism (trypanosome), limited direct applicability to human CPSF4 mechanism; single lab, single method","pmids":["19338765"],"is_preprint":false},{"year":2022,"finding":"WTAP-catalyzed m6A modification of the CPSF4 transcript reduces its stability through YTHDF2, thereby decreasing CPSF4 protein expression in oesophageal squamous cell carcinoma cells.","method":"m6A epitranscriptomic microarray, WTAP knockdown, YTHDF2 involvement assay, m6A quantification","journal":"Medical oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, indirect mechanistic evidence via m6A array and knockdown without direct reconstitution of the YTHDF2-mediated CPSF4 mRNA degradation","pmids":["36175708"],"is_preprint":false},{"year":2022,"finding":"CPSF4 binds to the promoters of VEGF and NRP2, activating their transcription; CPSF4/VEGF/NRP2 signaling drives tumor-initiating phenotype and chemoresistance through TAZ induction in lung cancer cells.","method":"Chromatin immunoprecipitation (ChIP) for VEGF and NRP2 promoters, siRNA knockdown, overexpression, in vitro and in vivo functional assays","journal":"Medical oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — ChIP supports promoter binding, but downstream pathway placement relies primarily on knockdown/overexpression with indirect readouts; single lab","pmids":["36567417"],"is_preprint":false},{"year":2024,"finding":"CPSF4 regulates alternative splicing of HMG20B by inhibiting alternative 3' splice site events, thereby promoting TNBC cell proliferation, migration, and invasion. RIP-seq identified CPSF4-interacting transcripts globally.","method":"RIP-seq (RNA immunoprecipitation sequencing), RNA-seq, qRT-PCR validation of splicing events, siRNA knockdown functional assays","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RIP-seq plus functional validation with orthogonal methods, single lab; mechanistic link between CPSF4 binding and specific splice site choice established","pmids":["39731153"],"is_preprint":false},{"year":2024,"finding":"CPSF4 negatively regulates NRF1 protein expression; knockdown of CPSF4 upregulates NRF1, and the additional knockdown of NRF1 partially reverses the growth-inhibitory effects of CPSF4 knockdown in bladder cancer cells and xenograft models.","method":"siRNA knockdown, Western blot, cell proliferation/migration/spheroid assays, in vivo xenograft","journal":"Biochemical genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — genetic epistasis via sequential knockdown, single lab, no direct biochemical mechanism for CPSF4-mediated NRF1 regulation established","pmids":["39039322"],"is_preprint":false},{"year":2013,"finding":"The CPSF30 binding function of influenza NS1 (from A/Texas/36/91) is essential for counteracting innate immune events (type I IFN and proinflammatory cytokine production) in human primary dendritic cells infected with influenza virus.","method":"Recombinant viruses encoding NS1 with mutant CPSF30-binding domain, infection of human primary dendritic cells, IFN and cytokine measurement","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — recombinant virus approach with loss-of-function NS1 mutations and primary cell readouts, single lab","pmids":["23255794"],"is_preprint":false}],"current_model":"CPSF4/CPSF30 is a zinc finger protein (five CCCH domains and one CCHC zinc knuckle, plus a 2Fe-2S cluster) that is a core subunit of the mammalian CPSF complex: its ZF2-ZF3 directly recognize the AAUAAA polyadenylation signal on pre-mRNA (essential for 3' cleavage and polyadenylation), while ZF4-ZF5 recruit two copies of hFip1 (which in turn recruit poly(A) polymerase) to support polyadenylation; the CCHC zinc knuckle mediates auxiliary polyU binding and can compensate for suboptimal AAUAAA sequences; influenza A NS1 hijacks ZF2-ZF3 to globally suppress host pre-mRNA 3' processing and innate immune responses, and CPSF4 additionally modulates TP53 alternative splicing and—in cancer cells—directly binds promoters of hTERT, VEGF, and NRP2 to activate transcription, while being subject to m6A-dependent post-transcriptional regulation via WTAP/YTHDF2."},"narrative":{"mechanistic_narrative":"CPSF4 (CPSF30) is a multi-zinc-finger RNA-binding protein that serves as a sequence-recognition core of the mammalian pre-mRNA 3'-end cleavage and polyadenylation machinery [PMID:25301780, PMID:32027124]. Its zinc fingers 2 and 3 directly contact the AAUAAA polyadenylation signal, an interaction essential for mRNA 3' processing, while AAUAAA recognition requires all five CCCH domains acting in concert [PMID:25301780, PMID:32027124]. Beyond signal recognition, CPSF4 carries a 2Fe-2S cluster in addition to five zinc ions, and its CCHC zinc knuckle provides a second RNA-binding mode that binds auxiliary polyU motifs and cooperatively rescues binding to suboptimal AU-rich hexamer variants not recognized by the CCCH cluster alone [PMID:32027124, PMID:33615774]. On the polyadenylation side, ZF4 and ZF5 each bind one molecule of hFip1 in a 1:2 stoichiometry — ZF4 with higher affinity — and this CPSF30–hFip1 assembly recruits two copies of poly(A) polymerase, with both binding sites supporting polyadenylation [PMID:33122294]. The ZF2-ZF3 module is the target of influenza A NS1, which binds CPSF30 to globally suppress host pre-mRNA 3' processing and innate immune signaling; this binding is a virulence determinant that blocks type I interferon and proinflammatory cytokine induction [PMID:16571812, PMID:25078692, PMID:23255794]. Independently of its core processing role, CPSF4 modulates alternative splicing of TP53 and HMG20B transcripts [PMID:30651364, PMID:39731153], and in cancer cells binds promoters of hTERT, VEGF, and NRP2 to activate their transcription and drive proliferative and tumor-initiating programs [PMID:27997899, PMID:36567417].","teleology":[{"year":2006,"claim":"Established that influenza NS1 hijacks a defined CPSF30 module to shut down host gene expression, framing CPSF30 as both a 3'-processing factor and an antiviral interface.","evidence":"Mutagenesis mapping NS1 binding to F2F3 of CPSF30, stable F2F3 fragment expression, viral replication and IFN-β induction assays","pmids":["16571812"],"confidence":"High","gaps":["Did not resolve atomic basis of the F2F3-NS1 interface","Endogenous CPSF30 3'-processing role not directly assayed here"]},{"year":2008,"claim":"Showed the CPSF30–NS1 interaction is reinforced within an infection-specific complex, defining which viral partners stabilize host-factor sequestration.","evidence":"Co-IP in infected cells, reverse genetics with polymerase subunit swaps, mutant NS1A viruses","pmids":["19052083"],"confidence":"High","gaps":["Mechanism by which PA/NP stabilize binding not structurally defined","Consequences for specific host transcripts not mapped"]},{"year":2013,"claim":"Linked CPSF30 sequestration by NS1 to suppression of innate immunity in physiologically relevant primary cells, establishing functional consequence of the interaction.","evidence":"Recombinant viruses with mutant CPSF30-binding NS1, infection of human primary dendritic cells, IFN/cytokine measurement","pmids":["23255794"],"confidence":"Medium","gaps":["Single virus strain background","Did not separate 3'-processing block from other NS1 functions"]},{"year":2014,"claim":"Pinpointed ZF2-ZF3 as the direct AAUAAA-recognition module, answering how the polyadenylation signal is read by CPSF.","evidence":"In vitro RNA binding, iCLIP, and zinc-finger domain mutagenesis","pmids":["25301780"],"confidence":"High","gaps":["Atomic-resolution structure of the AAUAAA-bound state not provided","Contribution of other CPSF subunits to specificity not isolated"]},{"year":2014,"claim":"Demonstrated that CPSF30 binding by NS1 is a determinant of viral virulence in vivo, elevating the interaction from molecular curiosity to pathogenesis driver.","evidence":"Site-directed I106M mutagenesis, reverse genetics, in vivo infection, Co-IP","pmids":["25078692"],"confidence":"High","gaps":["Host 3'-processing changes underlying virulence not directly profiled"]},{"year":2020,"claim":"Resolved how CPSF30 couples signal recognition to polyadenylation by showing ZF4/ZF5 each recruit hFip1, which in turn brings in poly(A) polymerase.","evidence":"1.9 Å crystal structure of CPSF30 ZF4-ZF5–hFip1, fluorescence polarization, mutagenesis, in vitro polyadenylation","pmids":["33122294"],"confidence":"High","gaps":["Stoichiometry within the full CPSF holocomplex in cells not addressed","Functional necessity of dual hFip1 sites in vivo untested"]},{"year":2020,"claim":"Revealed a 2Fe-2S cluster and a second RNA-binding mode (CCHC zinc knuckle for polyU), expanding CPSF30 beyond a simple AAUAAA reader.","evidence":"ICP-MS, UV-Vis and X-ray absorption spectroscopy, fluorescence anisotropy with truncation variants","pmids":["32027124"],"confidence":"High","gaps":["Functional role of the 2Fe-2S cluster unknown","polyU-binding contribution to processing in cells not established"]},{"year":2021,"claim":"Defined how the CCCH cluster and CCHC knuckle cooperate, explaining tolerance of suboptimal AAUAAA variants.","evidence":"Fluorescence anisotropy with systematic AAUAAA variants comparing CPSF30-5F vs full-length","pmids":["33615774"],"confidence":"Medium","gaps":["No independent replication","Cooperativity not validated in a cellular processing context"]},{"year":2019,"claim":"Extended CPSF4 function to alternative splicing of TP53, connecting it to p53 isoform output and interferon responses.","evidence":"TP53 minigene splicing assay, siRNA knockdown, NS1–CPSF4 interaction, IFN secretion","pmids":["30651364"],"confidence":"Medium","gaps":["Direct binding of CPSF4 to TP53 pre-mRNA not shown","Mechanism distinguishing splicing from 3'-processing role unclear"]},{"year":2024,"claim":"Generalized the splicing-regulatory role by globally mapping CPSF4-bound transcripts and showing control of HMG20B 3' splice-site choice.","evidence":"RIP-seq, RNA-seq, qRT-PCR splicing validation, siRNA functional assays in TNBC cells","pmids":["39731153"],"confidence":"Medium","gaps":["Direct mechanism linking binding to splice-site suppression not biochemically reconstituted","Single cancer context"]},{"year":2016,"claim":"Identified CPSF4 as a direct promoter-binding transcriptional activator of hTERT, a role distinct from RNA processing.","evidence":"Pulldown, luciferase reporter, and ChIP assays in colorectal cancer cells","pmids":["27997899"],"confidence":"Medium","gaps":["Does not define how an RNA-binding factor engages DNA promoters","No structural basis for promoter binding"]},{"year":2015,"claim":"Placed CPSF4 in a cancer growth circuit with CBP driving hTERT transcription, linking it to proliferative control.","evidence":"Co-IP, immunofluorescence co-localization, reciprocal siRNA knockdown, hTERT and proliferation assays in lung cancer","pmids":["26628108"],"confidence":"Medium","gaps":["Direct vs indirect nature of CBP–CPSF4 functional synergy unresolved"]},{"year":2021,"claim":"Connected CPSF4's polyadenylation-signal recognition to circRNA/miRNA regulation in driving hepatocellular carcinoma.","evidence":"Knockdown/overexpression in culture and xenografts, circRNA profiling, miRNA accumulation measurement","pmids":["34103682"],"confidence":"Medium","gaps":["Direct CPSF4 action on individual circRNAs not biochemically dissected"]},{"year":2022,"claim":"Proposed CPSF4 as an upstream transcriptional activator of VEGF and NRP2 driving tumor-initiating phenotypes.","evidence":"ChIP for VEGF/NRP2 promoters, siRNA/overexpression, in vitro and in vivo assays in lung cancer","pmids":["36567417"],"confidence":"Low","gaps":["Downstream TAZ pathway placement rests on indirect readouts","No direct structural basis for promoter engagement"]},{"year":2022,"claim":"Suggested CPSF4 is itself epitranscriptomically controlled via WTAP-deposited m6A and YTHDF2-mediated decay.","evidence":"m6A microarray, WTAP knockdown, YTHDF2 involvement assays in oesophageal carcinoma","pmids":["36175708"],"confidence":"Low","gaps":["No direct reconstitution of YTHDF2-mediated CPSF4 mRNA degradation","Single lab, indirect evidence"]},{"year":2024,"claim":"Linked CPSF4 to negative regulation of NRF1 in bladder cancer growth via genetic epistasis.","evidence":"siRNA knockdown, Western blot, proliferation/spheroid assays, xenograft with sequential NRF1 knockdown","pmids":["39039322"],"confidence":"Low","gaps":["No direct biochemical mechanism for CPSF4 control of NRF1","Single lab"]},{"year":null,"claim":"How CPSF4's core 3'-processing activity is mechanistically repurposed for promoter-level transcriptional activation and splice-site selection in cancer remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking RNA-processing and DNA-binding/transcriptional functions","Role of the 2Fe-2S cluster in any function undefined","Structure of CPSF4 bound to promoter DNA absent"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,5,6,14]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[9,13]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[8,9,13]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,8]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,4,5]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[8,9,13]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,16]}],"complexes":["CPSF"],"partners":["FIP1L1","NS1","CREBBP","PAP"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O95639","full_name":"Cleavage and polyadenylation specificity factor subunit 4","aliases":["Cleavage and polyadenylation specificity factor 30 kDa subunit","CPSF 30 kDa subunit","NS1 effector domain-binding protein 1","Neb-1","No arches homolog"],"length_aa":269,"mass_kda":30.3,"function":"Component of the cleavage and polyadenylation specificity factor (CPSF) complex that play a key role in pre-mRNA 3'-end formation, recognizing the AAUAAA signal sequence and interacting with poly(A) polymerase and other factors to bring about cleavage and poly(A) addition. CPSF4 binds RNA polymers with a preference for poly(U)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/O95639/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CPSF4","classification":"Common Essential","n_dependent_lines":1205,"n_total_lines":1208,"dependency_fraction":0.9975165562913907},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CAPZB","stoichiometry":0.2},{"gene":"CPSF6","stoichiometry":0.2},{"gene":"POLR1C","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CPSF4","total_profiled":1310},"omim":[{"mim_id":"618082","title":"WD REPEAT-CONTAINING PROTEIN 33; WDR33","url":"https://www.omim.org/entry/618082"},{"mim_id":"603052","title":"CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR 4; CPSF4","url":"https://www.omim.org/entry/603052"},{"mim_id":"602388","title":"SYMPLEKIN; SYMPK","url":"https://www.omim.org/entry/602388"},{"mim_id":"118493","title":"CHOLINERGIC RECEPTOR, MUSCARINIC, 2; CHRM2","url":"https://www.omim.org/entry/118493"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CPSF4"},"hgnc":{"alias_symbol":["NAR","CPSF30"],"prev_symbol":[]},"alphafold":{"accession":"O95639","domains":[{"cath_id":"-","chopping":"121-171","consensus_level":"medium","plddt":86.2678,"start":121,"end":171}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O95639","model_url":"https://alphafold.ebi.ac.uk/files/AF-O95639-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O95639-F1-predicted_aligned_error_v6.png","plddt_mean":75.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CPSF4","jax_strain_url":"https://www.jax.org/strain/search?query=CPSF4"},"sequence":{"accession":"O95639","fasta_url":"https://rest.uniprot.org/uniprotkb/O95639.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O95639/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O95639"}},"corpus_meta":[{"pmid":"25301780","id":"PMC_25301780","title":"CPSF30 and Wdr33 directly bind to AAUAAA in mammalian mRNA 3' processing.","date":"2014","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/25301780","citation_count":186,"is_preprint":false},{"pmid":"9751746","id":"PMC_9751746","title":"Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins.","date":"1998","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/9751746","citation_count":182,"is_preprint":false},{"pmid":"2832370","id":"PMC_2832370","title":"Identification and expression of genes narL and narX of the nar (nitrate reductase) locus in Escherichia coli K-12.","date":"1988","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/2832370","citation_count":157,"is_preprint":false},{"pmid":"16571812","id":"PMC_16571812","title":"The CPSF30 binding site on the NS1A protein of influenza A virus is a potential antiviral 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that of mammalian Ig genes and to spontaneous mutations in evolution: the translesion synthesis model of somatic hypermutation.","date":"1999","source":"International immunology","url":"https://pubmed.ncbi.nlm.nih.gov/10330287","citation_count":89,"is_preprint":false},{"pmid":"16549799","id":"PMC_16549799","title":"An evolutionarily mobile antigen receptor variable region gene: doubly rearranging NAR-TcR genes in sharks.","date":"2006","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/16549799","citation_count":83,"is_preprint":false},{"pmid":"12244263","id":"PMC_12244263","title":"Nar-1 and Nar-2, Two Loci Required for Mla12-Specified Race-Specific Resistance to Powdery Mildew in Barley.","date":"1994","source":"The Plant cell","url":"https://pubmed.ncbi.nlm.nih.gov/12244263","citation_count":81,"is_preprint":false},{"pmid":"2649492","id":"PMC_2649492","title":"Structure of genes narL and narX of the nar 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MT-1.","date":"2015","source":"The Journal of general and applied microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/25833675","citation_count":2,"is_preprint":false},{"pmid":"17616601","id":"PMC_17616601","title":"Artificial control of nitrate respiration through the lac promoter permits the assessment of oxygen-mediated posttranslational regulation of the nar operon in Pseudomonas aeruginosa.","date":"2007","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/17616601","citation_count":2,"is_preprint":false},{"pmid":"39039322","id":"PMC_39039322","title":"Knockdown of CPSF4 Inhibits Bladder Cancer Cell Growth by Upregulating NRF1.","date":"2024","source":"Biochemical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/39039322","citation_count":1,"is_preprint":false},{"pmid":"39590459","id":"PMC_39590459","title":"Functional Analysis of CPSF30 in Nilaparvata lugens Using RNA Interference Reveals Its Essential Role in Development and Survival.","date":"2024","source":"Insects","url":"https://pubmed.ncbi.nlm.nih.gov/39590459","citation_count":1,"is_preprint":false},{"pmid":"39938213","id":"PMC_39938213","title":"A coaxial electrospun PLLA/PPDO/NAR mesh for abdominal wall hernia repair.","date":"2025","source":"Biomedical materials (Bristol, England)","url":"https://pubmed.ncbi.nlm.nih.gov/39938213","citation_count":1,"is_preprint":false},{"pmid":"39337258","id":"PMC_39337258","title":"A New Real-Time Simple Method to Measure the Endogenous Nitrate Reductase Activity (Nar) in Paracoccus denitrificans and Other Denitrifying Bacteria.","date":"2024","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/39337258","citation_count":1,"is_preprint":false},{"pmid":"17343210","id":"PMC_17343210","title":"Tandem and single genes of three membrane-bound nitrate transporters in the nar gene cluster of the moderately halophilic denitrifier, Halomonas halodenitrificans.","date":"2006","source":"DNA sequence : the journal of DNA sequencing and mapping","url":"https://pubmed.ncbi.nlm.nih.gov/17343210","citation_count":1,"is_preprint":false},{"pmid":"41235944","id":"PMC_41235944","title":"Bioresponsive Nar-Zn@GelMA Hydrogel Reprograms the Diabetic Wound Microenvironment via Antioxidant and Immunoregulatory Synergy.","date":"2025","source":"Advanced healthcare materials","url":"https://pubmed.ncbi.nlm.nih.gov/41235944","citation_count":1,"is_preprint":false},{"pmid":"39758454","id":"PMC_39758454","title":"Does the Nutrient Adequacy Ratio (NAR) Predict Metabolic Profile and Glycemic Status Among the Obese Population?","date":"2025","source":"Nutrition and metabolic insights","url":"https://pubmed.ncbi.nlm.nih.gov/39758454","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":97570,"output_tokens":5314,"usd":0.291305,"retried_sync":true,"stage1_stop_reason":"refusal"},"stage2":{"model":"claude-opus-4-8","input_tokens":12092,"output_tokens":4476,"usd":0.08618,"stage2_stop_reason":"end_turn"},"total_usd":0.377485,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2014,\n      \"finding\": \"CPSF30 (CPSF4) directly contacts the AAUAAA polyadenylation signal in mammalian mRNA 3' processing; this interaction is primarily mediated by zinc fingers 2 and 3 (ZF2-ZF3) and is essential for mRNA 3' end processing.\",\n      \"method\": \"In vitro RNA binding assays and in vivo crosslinking/immunoprecipitation (iCLIP); mutagenesis of zinc finger domains\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro and in vivo binding assays with domain mutagenesis, replicated across multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"25301780\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CPSF30 binding to the influenza A NS1A protein is mediated specifically by zinc fingers 2 and 3 (F2F3) of CPSF30, and this interaction inhibits 3'-end processing of cellular pre-mRNAs including IFN-β. Constitutive nuclear expression of F2F3 inhibits influenza A virus replication by competing with endogenous CPSF30 for NS1A binding.\",\n      \"method\": \"Mutagenesis, stable cell line expression of epitope-tagged F2F3 fragment, viral replication assay, IFN-β mRNA induction assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutagenesis defining binding domain, functional rescue/competition experiments, multiple orthogonal readouts\",\n      \"pmids\": [\"16571812\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The influenza A virus polymerase complex (specifically PA protein and NP, but not PB1/PB2) is an integral component of the CPSF30–NS1A complex in infected cells, stabilizing CPSF30 binding to NS1A even when NS1A contains suboptimal hydrophobic residues at positions 103/106.\",\n      \"method\": \"Co-immunoprecipitation in infected cells, reverse genetics with cognate/non-cognate polymerase swaps, mutant NS1A viruses\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP in infected cells, multiple polymerase subunit knockouts, cognate/non-cognate swap experiments establishing specific subunit requirements\",\n      \"pmids\": [\"19052083\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"An I106M substitution in H7N9 influenza NS1 restores CPSF30 binding and the ability to block host gene expression, and a recombinant virus expressing NS1-I106M replicates to higher titers in vivo, demonstrating that CPSF30 binding by NS1 is a virulence determinant.\",\n      \"method\": \"Site-directed mutagenesis, reverse genetics, in vivo infection, Co-immunoprecipitation\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutagenesis + reverse genetics + in vivo virulence assay, multiple orthogonal methods in one study\",\n      \"pmids\": [\"25078692\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The crystal structure of human CPSF30 ZF4-ZF5 in complex with hFip1 residues 161–200 (1.9 Å resolution) reveals that one hFip1 molecule binds each of ZF4 and ZF5 with a conserved interaction mode (1:2 stoichiometry). ZF4 has higher affinity for hFip1 (Kd = 1.8 nM). The CPSF30–hFip1 complex recruits two copies of poly(A) polymerase (PAP) and both hFip1 binding sites in CPSF30 support polyadenylation.\",\n      \"method\": \"X-ray crystallography (1.9 Å), mutagenesis of CPSF30 binding sites, fluorescence polarization binding assays, in vitro polyadenylation assay\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional mutagenesis and in vitro reconstitution of polyadenylation, multiple orthogonal methods in one study\",\n      \"pmids\": [\"33122294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Full-length CPSF30 contains one 2Fe-2S iron-sulfur cluster in addition to five zinc ions. Full-length CPSF30 binds both AAUAAA and polyU pre-mRNA motifs with high affinity; AAUAAA binding requires all five CCCH domains whereas polyU binding requires full-length CPSF30 (implicating the CCHC zinc knuckle). Truncated forms (ZF2-ZF3 alone or CCHC alone) do not exhibit RNA binding.\",\n      \"method\": \"ICP-MS, UV-Vis spectroscopy, X-ray absorption spectroscopy (metal content), fluorescence anisotropy RNA binding assays with truncation variants\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical reconstitution with multiple spectroscopic methods and domain deletion analysis in one study\",\n      \"pmids\": [\"32027124\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Bases at positions 1, 2, 4, and 5 within the AAUAAA hexamer are important for CPSF30 binding; flanking A/U residues promote higher-affinity binding than G/C. The CCHC zinc knuckle restores binding to AU hexamer variants that are not recognized by the five CCCH domains alone, indicating the two RNA-binding modules (CCCH cluster and zinc knuckle) act cooperatively.\",\n      \"method\": \"Fluorescence anisotropy binding assays with systematic AAUAAA sequence variants; comparison of CPSF30-5F (five CCCH domains) vs full-length CPSF30\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous in vitro binding assays with systematic mutagenesis, single lab, no independent replication reported\",\n      \"pmids\": [\"33615774\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CPSF4 (CPSF30), independently and through its interaction with influenza NS1, modulates alternative splicing of TP53 transcripts, altering expression of p53 isoforms and affecting type I interferon secretion and viral replication.\",\n      \"method\": \"TP53 minigene splicing assay, siRNA knockdown of CPSF4, NS1–CPSF4 interaction in infected cells, IFN secretion measurement\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional minigene assay + siRNA knockdown + interaction studies, multiple readouts in one study, single lab\",\n      \"pmids\": [\"30651364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CBP (CREB-binding protein) co-localizes and physically interacts with CPSF4 in lung cancer cells. Knockdown of CPSF4 inhibits hTERT transcription and cell growth induced by CBP, and vice versa, demonstrating a synergistic relationship between CBP and CPSF4 in regulating lung cancer cell growth.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization, siRNA knockdown of CPSF4 and CBP, hTERT transcription assay, cell proliferation assay\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and co-localization plus functional siRNA rescue, two orthogonal methods, single lab\",\n      \"pmids\": [\"26628108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CPSF4 binds to the hTERT promoter in colorectal cancer cells, as demonstrated by pulldown assays, luciferase reporter assays, and chromatin immunoprecipitation (ChIP), identifying CPSF4 as an hTERT promoter-binding protein.\",\n      \"method\": \"Pulldown assay, luciferase reporter assay, ChIP assay\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — three orthogonal methods (pulldown, reporter, ChIP) establishing promoter binding, single lab\",\n      \"pmids\": [\"27997899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CPSF4 reduces levels of circRNAs that contain a polyadenylation signal sequence, thereby decreasing miRNA accumulation and disrupting miRNA-mediated gene silencing in hepatocellular carcinoma. CPSF4 promotes HCC cell proliferation and tumorigenicity through this circRNA inhibition mechanism.\",\n      \"method\": \"Knockdown/overexpression of CPSF4 in cell culture and xenograft mouse models, circRNA profiling, miRNA accumulation measurement\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo functional assays with mechanistic circRNA/miRNA measurements, single lab\",\n      \"pmids\": [\"34103682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In trypanosomes, TcCPSF30 physically interacts with TcFIP1-like (a polyadenylation factor); specific amino acids in each protein mediating this interaction were mapped, showing differences from the human CPSF30–FIP1 interaction surface.\",\n      \"method\": \"Yeast two-hybrid / biochemical interaction mapping, identification of interacting residues by mutagenesis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single interaction mapping study in a divergent organism (trypanosome), limited direct applicability to human CPSF4 mechanism; single lab, single method\",\n      \"pmids\": [\"19338765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"WTAP-catalyzed m6A modification of the CPSF4 transcript reduces its stability through YTHDF2, thereby decreasing CPSF4 protein expression in oesophageal squamous cell carcinoma cells.\",\n      \"method\": \"m6A epitranscriptomic microarray, WTAP knockdown, YTHDF2 involvement assay, m6A quantification\",\n      \"journal\": \"Medical oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, indirect mechanistic evidence via m6A array and knockdown without direct reconstitution of the YTHDF2-mediated CPSF4 mRNA degradation\",\n      \"pmids\": [\"36175708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CPSF4 binds to the promoters of VEGF and NRP2, activating their transcription; CPSF4/VEGF/NRP2 signaling drives tumor-initiating phenotype and chemoresistance through TAZ induction in lung cancer cells.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP) for VEGF and NRP2 promoters, siRNA knockdown, overexpression, in vitro and in vivo functional assays\",\n      \"journal\": \"Medical oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — ChIP supports promoter binding, but downstream pathway placement relies primarily on knockdown/overexpression with indirect readouts; single lab\",\n      \"pmids\": [\"36567417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CPSF4 regulates alternative splicing of HMG20B by inhibiting alternative 3' splice site events, thereby promoting TNBC cell proliferation, migration, and invasion. RIP-seq identified CPSF4-interacting transcripts globally.\",\n      \"method\": \"RIP-seq (RNA immunoprecipitation sequencing), RNA-seq, qRT-PCR validation of splicing events, siRNA knockdown functional assays\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RIP-seq plus functional validation with orthogonal methods, single lab; mechanistic link between CPSF4 binding and specific splice site choice established\",\n      \"pmids\": [\"39731153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CPSF4 negatively regulates NRF1 protein expression; knockdown of CPSF4 upregulates NRF1, and the additional knockdown of NRF1 partially reverses the growth-inhibitory effects of CPSF4 knockdown in bladder cancer cells and xenograft models.\",\n      \"method\": \"siRNA knockdown, Western blot, cell proliferation/migration/spheroid assays, in vivo xenograft\",\n      \"journal\": \"Biochemical genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — genetic epistasis via sequential knockdown, single lab, no direct biochemical mechanism for CPSF4-mediated NRF1 regulation established\",\n      \"pmids\": [\"39039322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The CPSF30 binding function of influenza NS1 (from A/Texas/36/91) is essential for counteracting innate immune events (type I IFN and proinflammatory cytokine production) in human primary dendritic cells infected with influenza virus.\",\n      \"method\": \"Recombinant viruses encoding NS1 with mutant CPSF30-binding domain, infection of human primary dendritic cells, IFN and cytokine measurement\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — recombinant virus approach with loss-of-function NS1 mutations and primary cell readouts, single lab\",\n      \"pmids\": [\"23255794\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CPSF4/CPSF30 is a zinc finger protein (five CCCH domains and one CCHC zinc knuckle, plus a 2Fe-2S cluster) that is a core subunit of the mammalian CPSF complex: its ZF2-ZF3 directly recognize the AAUAAA polyadenylation signal on pre-mRNA (essential for 3' cleavage and polyadenylation), while ZF4-ZF5 recruit two copies of hFip1 (which in turn recruit poly(A) polymerase) to support polyadenylation; the CCHC zinc knuckle mediates auxiliary polyU binding and can compensate for suboptimal AAUAAA sequences; influenza A NS1 hijacks ZF2-ZF3 to globally suppress host pre-mRNA 3' processing and innate immune responses, and CPSF4 additionally modulates TP53 alternative splicing and—in cancer cells—directly binds promoters of hTERT, VEGF, and NRP2 to activate transcription, while being subject to m6A-dependent post-transcriptional regulation via WTAP/YTHDF2.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CPSF4 (CPSF30) is a multi-zinc-finger RNA-binding protein that serves as a sequence-recognition core of the mammalian pre-mRNA 3'-end cleavage and polyadenylation machinery [#0, #5]. Its zinc fingers 2 and 3 directly contact the AAUAAA polyadenylation signal, an interaction essential for mRNA 3' processing, while AAUAAA recognition requires all five CCCH domains acting in concert [#0, #5]. Beyond signal recognition, CPSF4 carries a 2Fe-2S cluster in addition to five zinc ions, and its CCHC zinc knuckle provides a second RNA-binding mode that binds auxiliary polyU motifs and cooperatively rescues binding to suboptimal AU-rich hexamer variants not recognized by the CCCH cluster alone [#5, #6]. On the polyadenylation side, ZF4 and ZF5 each bind one molecule of hFip1 in a 1:2 stoichiometry — ZF4 with higher affinity — and this CPSF30–hFip1 assembly recruits two copies of poly(A) polymerase, with both binding sites supporting polyadenylation [#4]. The ZF2-ZF3 module is the target of influenza A NS1, which binds CPSF30 to globally suppress host pre-mRNA 3' processing and innate immune signaling; this binding is a virulence determinant that blocks type I interferon and proinflammatory cytokine induction [#1, #3, #16]. Independently of its core processing role, CPSF4 modulates alternative splicing of TP53 and HMG20B transcripts [#7, #14], and in cancer cells binds promoters of hTERT, VEGF, and NRP2 to activate their transcription and drive proliferative and tumor-initiating programs [#9, #13].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established that influenza NS1 hijacks a defined CPSF30 module to shut down host gene expression, framing CPSF30 as both a 3'-processing factor and an antiviral interface.\",\n      \"evidence\": \"Mutagenesis mapping NS1 binding to F2F3 of CPSF30, stable F2F3 fragment expression, viral replication and IFN-\\u03b2 induction assays\",\n      \"pmids\": [\"16571812\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve atomic basis of the F2F3-NS1 interface\", \"Endogenous CPSF30 3'-processing role not directly assayed here\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showed the CPSF30\\u2013NS1 interaction is reinforced within an infection-specific complex, defining which viral partners stabilize host-factor sequestration.\",\n      \"evidence\": \"Co-IP in infected cells, reverse genetics with polymerase subunit swaps, mutant NS1A viruses\",\n      \"pmids\": [\"19052083\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which PA/NP stabilize binding not structurally defined\", \"Consequences for specific host transcripts not mapped\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Linked CPSF30 sequestration by NS1 to suppression of innate immunity in physiologically relevant primary cells, establishing functional consequence of the interaction.\",\n      \"evidence\": \"Recombinant viruses with mutant CPSF30-binding NS1, infection of human primary dendritic cells, IFN/cytokine measurement\",\n      \"pmids\": [\"23255794\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single virus strain background\", \"Did not separate 3'-processing block from other NS1 functions\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Pinpointed ZF2-ZF3 as the direct AAUAAA-recognition module, answering how the polyadenylation signal is read by CPSF.\",\n      \"evidence\": \"In vitro RNA binding, iCLIP, and zinc-finger domain mutagenesis\",\n      \"pmids\": [\"25301780\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure of the AAUAAA-bound state not provided\", \"Contribution of other CPSF subunits to specificity not isolated\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated that CPSF30 binding by NS1 is a determinant of viral virulence in vivo, elevating the interaction from molecular curiosity to pathogenesis driver.\",\n      \"evidence\": \"Site-directed I106M mutagenesis, reverse genetics, in vivo infection, Co-IP\",\n      \"pmids\": [\"25078692\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Host 3'-processing changes underlying virulence not directly profiled\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved how CPSF30 couples signal recognition to polyadenylation by showing ZF4/ZF5 each recruit hFip1, which in turn brings in poly(A) polymerase.\",\n      \"evidence\": \"1.9 \\u00c5 crystal structure of CPSF30 ZF4-ZF5\\u2013hFip1, fluorescence polarization, mutagenesis, in vitro polyadenylation\",\n      \"pmids\": [\"33122294\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry within the full CPSF holocomplex in cells not addressed\", \"Functional necessity of dual hFip1 sites in vivo untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed a 2Fe-2S cluster and a second RNA-binding mode (CCHC zinc knuckle for polyU), expanding CPSF30 beyond a simple AAUAAA reader.\",\n      \"evidence\": \"ICP-MS, UV-Vis and X-ray absorption spectroscopy, fluorescence anisotropy with truncation variants\",\n      \"pmids\": [\"32027124\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of the 2Fe-2S cluster unknown\", \"polyU-binding contribution to processing in cells not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined how the CCCH cluster and CCHC knuckle cooperate, explaining tolerance of suboptimal AAUAAA variants.\",\n      \"evidence\": \"Fluorescence anisotropy with systematic AAUAAA variants comparing CPSF30-5F vs full-length\",\n      \"pmids\": [\"33615774\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No independent replication\", \"Cooperativity not validated in a cellular processing context\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended CPSF4 function to alternative splicing of TP53, connecting it to p53 isoform output and interferon responses.\",\n      \"evidence\": \"TP53 minigene splicing assay, siRNA knockdown, NS1\\u2013CPSF4 interaction, IFN secretion\",\n      \"pmids\": [\"30651364\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding of CPSF4 to TP53 pre-mRNA not shown\", \"Mechanism distinguishing splicing from 3'-processing role unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Generalized the splicing-regulatory role by globally mapping CPSF4-bound transcripts and showing control of HMG20B 3' splice-site choice.\",\n      \"evidence\": \"RIP-seq, RNA-seq, qRT-PCR splicing validation, siRNA functional assays in TNBC cells\",\n      \"pmids\": [\"39731153\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mechanism linking binding to splice-site suppression not biochemically reconstituted\", \"Single cancer context\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified CPSF4 as a direct promoter-binding transcriptional activator of hTERT, a role distinct from RNA processing.\",\n      \"evidence\": \"Pulldown, luciferase reporter, and ChIP assays in colorectal cancer cells\",\n      \"pmids\": [\"27997899\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not define how an RNA-binding factor engages DNA promoters\", \"No structural basis for promoter binding\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Placed CPSF4 in a cancer growth circuit with CBP driving hTERT transcription, linking it to proliferative control.\",\n      \"evidence\": \"Co-IP, immunofluorescence co-localization, reciprocal siRNA knockdown, hTERT and proliferation assays in lung cancer\",\n      \"pmids\": [\"26628108\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect nature of CBP\\u2013CPSF4 functional synergy unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected CPSF4's polyadenylation-signal recognition to circRNA/miRNA regulation in driving hepatocellular carcinoma.\",\n      \"evidence\": \"Knockdown/overexpression in culture and xenografts, circRNA profiling, miRNA accumulation measurement\",\n      \"pmids\": [\"34103682\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct CPSF4 action on individual circRNAs not biochemically dissected\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Proposed CPSF4 as an upstream transcriptional activator of VEGF and NRP2 driving tumor-initiating phenotypes.\",\n      \"evidence\": \"ChIP for VEGF/NRP2 promoters, siRNA/overexpression, in vitro and in vivo assays in lung cancer\",\n      \"pmids\": [\"36567417\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Downstream TAZ pathway placement rests on indirect readouts\", \"No direct structural basis for promoter engagement\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Suggested CPSF4 is itself epitranscriptomically controlled via WTAP-deposited m6A and YTHDF2-mediated decay.\",\n      \"evidence\": \"m6A microarray, WTAP knockdown, YTHDF2 involvement assays in oesophageal carcinoma\",\n      \"pmids\": [\"36175708\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct reconstitution of YTHDF2-mediated CPSF4 mRNA degradation\", \"Single lab, indirect evidence\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked CPSF4 to negative regulation of NRF1 in bladder cancer growth via genetic epistasis.\",\n      \"evidence\": \"siRNA knockdown, Western blot, proliferation/spheroid assays, xenograft with sequential NRF1 knockdown\",\n      \"pmids\": [\"39039322\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct biochemical mechanism for CPSF4 control of NRF1\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CPSF4's core 3'-processing activity is mechanistically repurposed for promoter-level transcriptional activation and splice-site selection in cancer remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking RNA-processing and DNA-binding/transcriptional functions\", \"Role of the 2Fe-2S cluster in any function undefined\", \"Structure of CPSF4 bound to promoter DNA absent\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 5, 6, 14]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [9, 13]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [8, 9, 13]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 4, 5]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [8, 9, 13]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 16]}\n    ],\n    \"complexes\": [\"CPSF\"],\n    \"partners\": [\"FIP1L1\", \"NS1\", \"CREBBP\", \"PAP\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}