{"gene":"CPSF3","run_date":"2026-06-09T22:57:19","timeline":{"discoveries":[{"year":2006,"finding":"CPSF-73 (CPSF3) is the pre-mRNA 3'-end-processing endonuclease. Crystal structure at 2.1 Å resolution revealed a metallo-β-lactamase domain and a novel β-CASP domain with a two-zinc-ion active site at their interface. Purified recombinant CPSF-73 possesses RNA endonuclease activity, and mutations disrupting zinc binding in the active site abolish this activity.","method":"X-ray crystallography (2.1 Å), in vitro RNA endonuclease assay with recombinant protein, active-site mutagenesis of zinc-coordinating residues","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus reconstituted in vitro endonuclease activity plus active-site mutagenesis in a single rigorous study","pmids":["17128255"],"is_preprint":false},{"year":2005,"finding":"CPSF-73 is both the endonuclease and the 5'-3' exonuclease in histone pre-mRNA 3'-end processing. UV-crosslinking with site-specifically labeled RNA substrates (at the cleavage site) identified an ~85 kDa protein that crosslinked in a U7 snRNP-dependent manner; immunoprecipitation identified this as CPSF-73.","method":"UV-crosslinking with site-specific radiolabeled/phosphorothioate RNA substrates, immunoprecipitation","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — UV-crosslinking with chemically defined substrates plus immunoprecipitation, replicated by multiple subsequent studies","pmids":["16213211"],"is_preprint":false},{"year":2004,"finding":"CPSF-73 contacts the pre-mRNA cleavage site in an AAUAAA-dependent manner, and mutation of key residues in the yeast CPSF-73 homolog (metallo-β-lactamase domain) causes lethality. The 3'-cleavage reaction is metal (Zn2+)-dependent, consistent with CPSF-73 being a metallo-β-lactamase-family endonuclease.","method":"UV-crosslinking with site-specific labeling in HeLa nuclear extract, yeast lethality assay with active-site mutants, metal-dependence assay (chelation/rescue)","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal approaches (UV-crosslinking, yeast genetics, metal-dependence), replicated by subsequent structural work","pmids":["15037765"],"is_preprint":false},{"year":2009,"finding":"CPSF73, CPSF100, and Symplekin form a stable core subcomplex that associates with histone-specific processing factors and is required for histone pre-mRNA 3'-end processing; chromatin immunoprecipitation showed that CPSF73 and Symplekin (but not CstF50) cotranscriptionally associate with histone genes. This core complex can function in two alternative cleavage machineries (poly(A) and histone mRNA).","method":"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), RNAi knockdown with functional pre-mRNA processing readout","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ChIP, and functional knockdown with multiple orthogonal methods in one study","pmids":["19450530"],"is_preprint":false},{"year":2008,"finding":"Conserved residues in the metallo-β-lactamase motifs of both CPSF73 and CPSF100 are required for the endonuclease activity that cleaves histone pre-mRNAs, indicating that CPSF73 and CPSF100 act together (analogous to homodimeric RNase Z/J family members) to form the active endonuclease.","method":"In vitro point mutagenesis of conserved MBL residues in mammalian CPSF73 and CPSF100, histone pre-mRNA cleavage assay","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution assay with systematic mutagenesis of both subunits, single lab","pmids":["18688255"],"is_preprint":false},{"year":2008,"finding":"CPSF-73 functions as a 5'-3' exonuclease that degrades the downstream cleavage product (DCP) of histone pre-mRNA in a U7 snRNP-dependent manner; degradation requires the U7 snRNP only for initiation and is processive. UV-crosslinking showed CPSF-73 directly contacts the DCP. RNAi showed that Xrn2 is not required for DCP degradation in vivo, implicating CPSF-73 as the in vivo 5'-3' exonuclease.","method":"UV-crosslinking with modified RNA substrates, in vitro degradation assay with U7 snRNP, RNAi knockdown of Xrn2 in HeLa cells","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — UV-crosslinking plus functional in vitro assays plus RNAi genetic dissection, multiple orthogonal methods, single lab","pmids":["18955505"],"is_preprint":false},{"year":2012,"finding":"The N-terminal regions of FLASH and Lsm11 form a platform that recruits a specific combination of polyadenylation factors—symplekin, CstF64, and all CPSF subunits including CPSF73—to the U7 snRNP. This interaction is abolished by point mutations in FLASH that are detrimental for processing. The same polyadenylation factors are associated with endogenous U7 snRNP and are recruited to histone pre-mRNA in a U7-dependent manner.","method":"Co-immunoprecipitation, pulldown assays, mass spectrometry, point mutagenesis of FLASH/Lsm11, chromatin association assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, MS, mutagenesis, and chromatin assays; independently corroborated by reconstitution studies","pmids":["23071092"],"is_preprint":false},{"year":2018,"finding":"CPSF73 endonuclease activity is required for efficient transcriptional termination of protein-coding genes by RNA Pol II. Catalytically inactive CPSF73 cannot restore termination to cells lacking functional CPSF73, placing RNA cleavage by CPSF73 upstream of Xrn2-mediated cotranscriptional degradation and termination. CPSF73 loss causes more extensive readthrough transcription than Xrn2 elimination, indicating CPSF73 has a more foundational role.","method":"Conditional depletion via gene editing (auxin-inducible degron), catalytic mutant rescue experiments, mammalian native elongating transcript sequencing (mNET-seq) genome-wide","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — engineered conditional depletion plus catalytic mutant rescue plus genome-wide mNET-seq, multiple orthogonal approaches","pmids":["29432121"],"is_preprint":false},{"year":2008,"finding":"CSR1 (a tumor suppressor) binds the C-terminus of CPSF3 (amino acids 440–543) via yeast two-hybrid and further direct binding assays. This interaction causes CPSF3 translocation from the nucleus to the cytoplasm, inhibiting polyadenylation activity both in vitro and in vivo. A CSR1 mutant unable to bind CPSF3 neither re-localizes it nor inhibits polyadenylation. CPSF3 knockdown by siRNA mimics CSR1-induced cell death.","method":"Yeast two-hybrid, co-immunoprecipitation, subcellular fractionation/localization, in vitro and in vivo polyadenylation assay, siRNA knockdown","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple assays (Y2H, Co-IP, localization, functional polyadenylation assay) in single lab","pmids":["18806823"],"is_preprint":false},{"year":2006,"finding":"The C-terminal domain of yeast Brr5/Ysh1 (CPSF3 ortholog) lying outside β-lactamase homology is essential for cell viability and mRNA 3'-end processing. A homologous protein Syc1, by mimicking the essential Brr5 C-terminus, acts as a negative regulator of mRNA 3'-end formation.","method":"Yeast genetics (deletion analysis), in vitro 3'-end processing assay with yeast extracts, genetic rescue experiments","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast genetic complementation plus in vitro processing assay, single lab","pmids":["16431986"],"is_preprint":false},{"year":2019,"finding":"CPSF3 is the direct molecular target of JTE-607 (a prodrug converted by ester hydrolysis to its active form), which binds CPSF3 as identified by compound-immobilized affinity chromatography. JTE-607 treatment causes accumulation of pre-mRNAs, and CPSF3 knockdown similarly causes pre-mRNA accumulation and suppresses inflammatory cytokine expression.","method":"Compound-immobilized affinity chromatography, CPSF3 knockdown, pre-mRNA accumulation assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — affinity chromatography plus genetic knockdown with functional readout, single lab","pmids":["31399191"],"is_preprint":false},{"year":2019,"finding":"CPSF3 is the target of JTE-607 in AML and Ewing's sarcoma. Chemical genetics/phenotypic screening identified CPSF3. Inhibition of CPSF3 prevents release of newly synthesized pre-mRNAs and causes transcriptional readthrough and formation of DNA-RNA R-loop structures.","method":"Phenotypic screening, chemical genetics, gene editing resistance mutations, pre-mRNA accumulation assays, R-loop detection","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — forward genetics resistance mapping, functional pre-mRNA assays, R-loop detection; corroborated by independent study (PMID:31399191)","pmids":["31819276"],"is_preprint":false},{"year":2019,"finding":"Crystal structures of Cryptosporidium CPSF3 reveal that the oxaborole AN3661 blocks mRNA processing activity by binding at the metal-dependent catalytic center of CPSF3, with the oxaborole group directly occupying the active site.","method":"X-ray crystallography of recombinant Cryptosporidium CPSF3 with inhibitor bound, growth inhibition assay, in vivo efficacy in mouse models","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with inhibitor at active site plus functional antiparasitic validation in cell and animal models","pmids":["31694928"],"is_preprint":false},{"year":2020,"finding":"Reconstituted recombinant U7 snRNP (all 13 components) demonstrates that CPSF73 is both an endonuclease and a 5'-3' exonuclease: it cleaves histone pre-mRNAs endonucleolytically and degrades the downstream cleavage product 5'-to-3'. Both activities require base-pairing of U7 snRNA, presence of the symplekin N-terminal domain (NTD), and are abolished by active-site mutations in CPSF73. Binding of symplekin NTD to SSU72 phosphatase also abolishes activity.","method":"In vitro reconstitution of recombinant U7 snRNP from 13 components, endonuclease and exonuclease assays, active-site mutagenesis of CPSF73, substrate modification studies","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — full reconstitution from recombinant components plus active-site mutagenesis plus multiple substrate modifications; rigorous single-study multi-method approach","pmids":["32554553"],"is_preprint":false},{"year":2020,"finding":"In yeast, the Ysh1 (CPSF3 ortholog) endonuclease is regulated by ubiquitin-mediated proteasomal degradation. Ubiquitination of Ysh1 is mediated by the Ubc4 ubiquitin-conjugating enzyme and the RING ubiquitin ligase Mpe1. Ipa1 mutation causes striking depletion of Ysh1 protein (but not mRNA), rescued by proteasome inactivation or YSH1 overexpression.","method":"Yeast genetics (ipa1-1 mutant), proteasome inhibition, ubiquitination assays, YSH1 overexpression rescue, mRNA level analysis","journal":"RNA biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic and biochemical approaches in yeast ortholog, single lab","pmids":["32009536"],"is_preprint":false},{"year":2019,"finding":"Yeast Ipa1 interacts with the Ysh1 (CPSF3 ortholog) endonuclease and promotes proper transcription termination and poly(A) site selection globally. Ipa1 mutation leads to decreased Ysh1 levels and poor recruitment of the cleavage/polyadenylation complex to transcribed genes. Ysh1 overexpression in the Ipa1 mutant rescues the termination defect.","method":"Chromatin immunoprecipitation, yeast genetic suppression (Ysh1 overexpression rescue), Pol II elongation kinetics assay","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP, genetic rescue, Pol II kinetics, single lab; yeast ortholog","pmids":["30759400"],"is_preprint":false},{"year":2021,"finding":"UBE3D, a HECT-like E3 ligase, 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.","method":"UBE3D depletion, ubiquitination assay, pre-mRNA cleavage assay, protein stability assay","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — depletion with functional pre-mRNA cleavage readout plus ubiquitination assay, single lab","pmids":["35992060"],"is_preprint":false},{"year":2023,"finding":"X-ray crystallography demonstrated that benzoxaborole compounds bind to the active site of CPSF3 in a manner distinct from other known CPSF3 inhibitors. Forward genetics resistance mapping identified CPSF3 mutations that reduce benzoxaborole binding and confer resistance. Benzoxaboroles inhibit CPSF73 endonuclease activity in vitro and curb transcriptional termination in cells.","method":"X-ray crystallography of CPSF3-benzoxaborole complex, forward genetics resistance screen, in vitro endonuclease activity assay, transcription termination assay in cells","journal":"Cell chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of inhibitor complex plus forward genetics resistance mapping plus in vitro enzymatic assay","pmids":["37967558"],"is_preprint":false},{"year":2023,"finding":"The C-terminal domain 3 (CTD3) of CPSF73 is required for binding to Symplekin, as demonstrated by biochemical assays; the CPSF73-CPSF100 C-terminal heterodimer structure was determined by NMR for the Encephalitozoon cuniculi minimal construct, revealing extensive inter-protein contacts and TBP-like CTD2 folds.","method":"NMR solution structure of minimal CPSF73-CPSF100 C-terminal heterodimer, biochemical binding assays (CTD3–Symplekin interaction)","journal":"Open biology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — NMR structure plus biochemical binding assay for one domain interaction, single lab, parasite ortholog","pmids":["37989222"],"is_preprint":false},{"year":2023,"finding":"The active site of CPSF73 contains a mixture of metal ions (Fe, Zn, Mn) rather than exclusively zinc ions. The abundance of the metal ions varies with the expression host, yet the enzyme retains endonuclease activity with less than 20% zinc occupancy, indicating that iron and/or manganese can support catalysis.","method":"Inductively coupled plasma mass spectrometry (ICP-MS), X-ray diffraction metal anomalous scattering, in vitro pre-mRNA cleavage assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — ICP-MS plus X-ray diffraction plus in vitro activity assay, multiple orthogonal methods, single lab","pmids":["36822327"],"is_preprint":false},{"year":2024,"finding":"UBE3D is a binding partner and stabilizer of CPSF73 in the cytoplasm; structural studies showed that the conserved C-terminal cysteine of UBE3D likely coordinates the active-site metal ions of CPSF73, functioning as a cytoplasmic chaperone required for CPSF73's nuclear pre-mRNA processing function. Loss of UBE3D leads to CPSF73 downregulation.","method":"Structural studies (cryo-EM of INTS9-INTS11-BRAT1 complex providing mechanistic analogy), co-immunoprecipitation, depletion experiments with pre-mRNA processing readout","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — structural analogy (cryo-EM of paralog complex) plus Co-IP plus depletion functional assay; direct CPSF73 structure not solved, inference from paralog","pmids":["39032490"],"is_preprint":false},{"year":2024,"finding":"RBBP6 E3 ligase mediates K63-linked ubiquitination of CPSF3, which stabilizes CPSF3 and regulates alternative polyadenylation events. RBBP6 depletion induces shortening of 3'UTRs of MYC competing-endogenous RNAs, releasing miR-590-3p to decrease MYC expression in glioblastoma stem cells.","method":"CRISPR/Cas9 knockout screens, Co-immunoprecipitation, ubiquitination assay (K63-linkage), 3'UTR sequencing, alternative polyadenylation profiling, CPSF3 inhibitor (JTE-607) functional assay","journal":"Cell discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screens, Co-IP ubiquitination assay, APA profiling, single lab","pmids":["38503731"],"is_preprint":false},{"year":2025,"finding":"UBE3D interacts with CPSF3 and can de-ubiquitinate CPSF3; deficiency of UBE3D leads to reduced CPSF3 protein levels in both mouse and human cells. Overexpression of dominant negative CPSF3 mutants partially reduces mRNA levels of Homeobox genes, linking CPSF3-mediated pre-mRNA 3'-end processing to Homeobox gene expression during early embryogenesis.","method":"Co-immunoprecipitation, CRISPR/Cas9 knockout mice, in situ hybridization, immunofluorescence, RNA-seq, dominant-negative CPSF3 overexpression","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, KO mouse, dominant-negative functional assay, multiple methods; single lab","pmids":["40075082"],"is_preprint":false},{"year":2026,"finding":"A conserved helix in the Lsm11 N-terminal extension directly contacts the metallo-β-lactamase domain of CPSF73 and is required to hold CPSF73 in the correct position for the cleavage reaction in U7 snRNP. Cryo-EM also revealed that CPSF73 can achieve an open (active) conformation independent of RNA binding in its active site. Additionally, the C-terminal end of CstF77 contacts CPSF100 at the CPSF73-CPSF100 interface, with a small effect on cleavage activity.","method":"Cryo-EM structure determination of reconstituted U7 snRNP complexes, mutagenesis/deletion of Lsm11 helix, in vitro pre-mRNA cleavage assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure plus mutagenesis plus in vitro reconstituted cleavage assay, single study with multiple orthogonal methods","pmids":["41495886"],"is_preprint":false},{"year":2026,"finding":"Heat shock causes loss of CPSF73 endonucleolytic cleavage activity at thousands of genes despite CPSF73 remaining present. Overexpression of RBBP6, an activator of CPSF73, during heat shock rescues the loss of cleavage and dampens readthrough transcription. Heat shock also increases Tyr1 and Ser2 phosphorylation of the Pol II CTD at 3'-ends, which is attenuated at readthrough genes.","method":"mNET-seq or equivalent nascent RNA sequencing, overexpression rescue of RBBP6, cleavage assay, ChIP for Pol II CTD phosphorylation marks","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide sequencing plus RBBP6 overexpression rescue plus CTD phosphorylation analysis, single lab","pmids":["41758649"],"is_preprint":false}],"current_model":"CPSF3 (CPSF-73) is a metallo-β-lactamase/β-CASP family endoribonuclease that catalyzes the endonucleolytic cleavage of pre-mRNAs at poly(A) sites using a two-metal-ion (Fe/Zn/Mn mixture) active site; it also functions as a 5'-3' exonuclease to degrade downstream cleavage products, particularly in the U7 snRNP machinery for histone pre-mRNA 3'-end processing, where its recruitment depends on a FLASH/Lsm11 platform and a symplekin N-terminal domain. CPSF3 cleavage activity is essential for RNA Pol II transcription termination on protein-coding genes upstream of Xrn2-mediated cotranscriptional degradation. CPSF3 protein stability is regulated by ubiquitin-mediated proteasomal degradation, with UBE3D acting as a stabilizing cytoplasmic chaperone and RBBP6 mediating K63-linked ubiquitination to stabilize the protein. The CTD3 domain of CPSF3 mediates interaction with Symplekin to form a core cleavage complex with CPSF100, and active-site mutations abolish both endonuclease and exonuclease activities."},"narrative":{"mechanistic_narrative":"CPSF3 (CPSF-73) is the catalytic endoribonuclease of the eukaryotic pre-mRNA 3'-end processing machinery, executing the endonucleolytic cleavage at poly(A) sites that defines mRNA 3'-ends [PMID:17128255, PMID:15037765]. Its catalytic core is a metallo-β-lactamase domain joined to a β-CASP domain, with a metal-ion active site at their interface; the enzyme uses a mixture of Fe, Zn and Mn ions rather than zinc exclusively, and active-site mutations that disrupt metal coordination abolish activity [PMID:17128255, PMID:36822327]. CPSF3 acts together with CPSF100, whose conserved metallo-β-lactamase residues are also required for cleavage, the two subunits forming the active endonuclease, and its C-terminal CTD3 domain binds Symplekin to assemble the core cleavage complex [PMID:18688255, PMID:37989222]. Beyond polyadenylation, CPSF3 is the dual endonuclease/5'-3' exonuclease of histone pre-mRNA 3'-end processing within the U7 snRNP, where it is recruited via a FLASH/Lsm11 platform and a symplekin N-terminal domain and is positioned for catalysis by direct Lsm11 contacts to its metallo-β-lactamase domain [PMID:16213211, PMID:18955505, PMID:23071092, PMID:32554553, PMID:41495886]. CPSF3-mediated cleavage is foundational for RNA polymerase II transcription termination, acting upstream of Xrn2-dependent cotranscriptional degradation, and its loss produces extensive readthrough transcription and R-loop accumulation [PMID:29432121, PMID:31819276]. CPSF3 protein levels are set by ubiquitin-dependent control: UBE3D acts as a cytoplasmic chaperone/stabilizer and RBBP6 mediates stabilizing K63-linked ubiquitination that also tunes alternative polyadenylation [PMID:35992060, PMID:39032490, PMID:38503731]. CPSF3 is the direct molecular target of the inhibitors JTE-607 and active-site-binding benzoxaboroles/oxaboroles, which block its endonuclease activity [PMID:31399191, PMID:31819276, PMID:31694928, PMID:37967558].","teleology":[{"year":2004,"claim":"Established which factor contacts the pre-mRNA cleavage site and that cleavage is metal-dependent, pointing to a metallo-β-lactamase endonuclease.","evidence":"Site-specific UV-crosslinking in HeLa nuclear extract, yeast active-site mutant lethality, and metal-dependence assays","pmids":["15037765"],"confidence":"High","gaps":["Did not directly demonstrate purified recombinant nuclease activity","Active-site architecture not yet resolved structurally"]},{"year":2005,"claim":"Identified CPSF-73 as the U7 snRNP-dependent factor contacting the histone pre-mRNA cleavage site, implicating one enzyme in both endo- and exonucleolytic steps.","evidence":"UV-crosslinking with site-specific RNA substrates plus immunoprecipitation","pmids":["16213211"],"confidence":"High","gaps":["Crosslinking does not prove catalysis","Exonuclease role still inferred, not reconstituted"]},{"year":2006,"claim":"Resolved the catalytic basis of CPSF3 by showing a metallo-β-lactamase/β-CASP fold with a two-metal active site and reconstituting endonuclease activity, settling that CPSF3 is the 3'-processing nuclease.","evidence":"2.1 Å crystal structure, recombinant in vitro endonuclease assay, active-site mutagenesis","pmids":["17128255"],"confidence":"High","gaps":["Did not address how the enzyme is positioned within larger processing complexes","Exonuclease activity not structurally explained"]},{"year":2006,"claim":"Showed that a non-catalytic CPSF3 C-terminal region (via the yeast ortholog) is essential for processing and is a target of negative regulation, revealing functional roles beyond the catalytic domain.","evidence":"Yeast deletion genetics and in vitro 3'-end processing with extracts","pmids":["16431986"],"confidence":"Medium","gaps":["Yeast ortholog; human C-terminal regulation not directly tested here","Molecular function of the C-terminus left undefined"]},{"year":2008,"claim":"Demonstrated that CPSF73 and CPSF100 metallo-β-lactamase residues both contribute, defining the endonuclease as a two-subunit unit rather than CPSF73 alone.","evidence":"Systematic MBL-residue mutagenesis of both subunits with histone pre-mRNA cleavage assays","pmids":["18688255"],"confidence":"High","gaps":["Did not resolve the heterodimer interface structurally","Relative catalytic contribution of each subunit unquantified"]},{"year":2008,"claim":"Established CPSF-73 as the in vivo processive 5'-3' exonuclease degrading the downstream cleavage product, distinguishing it from Xrn2 for this step.","evidence":"UV-crosslinking, in vitro U7 snRNP degradation assays, Xrn2 RNAi","pmids":["18955505"],"confidence":"High","gaps":["Structural basis of switching between endo- and exonuclease modes unresolved"]},{"year":2008,"claim":"Identified a regulatory partner (CSR1) that re-localizes CPSF3 to the cytoplasm and inhibits polyadenylation, linking CPSF3 activity to subcellular sequestration and cell death.","evidence":"Yeast two-hybrid, Co-IP, fractionation, in vitro/in vivo polyadenylation assays, siRNA","pmids":["18806823"],"confidence":"Medium","gaps":["Single-lab finding without reciprocal structural mapping","Physiological context of CSR1 regulation unclear"]},{"year":2009,"claim":"Defined the CPSF73-CPSF100-Symplekin core subcomplex shared between poly(A) and histone machineries and showed cotranscriptional association with target genes.","evidence":"Reciprocal Co-IP, ChIP, RNAi with processing readouts","pmids":["19450530"],"confidence":"High","gaps":["Stoichiometry and architecture of the core complex not resolved","Mechanism of switching between two machineries unaddressed"]},{"year":2012,"claim":"Showed that FLASH/Lsm11 N-terminal regions form the platform recruiting CPSF subunits including CPSF73 to U7 snRNP, defining the recruitment route for histone processing.","evidence":"Co-IP, pulldowns, mass spectrometry, FLASH/Lsm11 mutagenesis, chromatin assays","pmids":["23071092"],"confidence":"High","gaps":["Did not place CPSF73 catalytic geometry within the assembled complex"]},{"year":2018,"claim":"Established that CPSF73 catalytic cleavage is required for and upstream of Pol II transcription termination, making it more foundational than Xrn2.","evidence":"Auxin-inducible degron depletion, catalytic-mutant rescue, genome-wide mNET-seq","pmids":["29432121"],"confidence":"High","gaps":["Did not define how readthrough relates to R-loop formation","Termination defect mechanism downstream of cleavage incomplete"]},{"year":2019,"claim":"Identified CPSF3 as the direct target of small-molecule inhibitors (JTE-607, oxaboroles) that block processing, validating it as a druggable node and confirming functional consequences of inhibition.","evidence":"Affinity chromatography, phenotypic/chemical-genetic screening, resistance mutation mapping, pre-mRNA accumulation and R-loop assays, inhibitor crystal structure (Cryptosporidium ortholog)","pmids":["31399191","31819276","31694928"],"confidence":"High","gaps":["Selectivity over related β-CASP nucleases not fully defined","Human inhibitor co-structure inferred partly from parasite ortholog"]},{"year":2019,"claim":"Showed that CPSF3 protein abundance is actively regulated via partner interactions and recruitment in yeast, connecting Ysh1 levels to global termination and poly(A) site selection.","evidence":"Yeast ChIP, genetic suppression by Ysh1 overexpression, Pol II elongation kinetics","pmids":["30759400"],"confidence":"Medium","gaps":["Yeast ortholog; human Ipa1 equivalent not tested","Direct mechanism of Ysh1 destabilization not defined here"]},{"year":2020,"claim":"Achieved full recombinant reconstitution of U7 snRNP, proving CPSF73 carries both endo- and exonuclease activities and that symplekin NTD and SSU72 binding gate these activities.","evidence":"13-component recombinant U7 snRNP reconstitution, cleavage/degradation assays, CPSF73 active-site mutagenesis","pmids":["32554553"],"confidence":"High","gaps":["Structural snapshot of the active enzyme within the assembled complex not yet obtained"]},{"year":2020,"claim":"Demonstrated ubiquitin-proteasome control of CPSF3 stability in yeast, identifying the conjugating enzyme and ligase that target the nuclease.","evidence":"Yeast ipa1-1 mutant, proteasome inhibition, ubiquitination assays, overexpression rescue","pmids":["32009536"],"confidence":"Medium","gaps":["Mammalian conservation of Ubc4/Mpe1 axis not established here"]},{"year":2021,"claim":"Identified UBE3D as a stabilizer that protects CPSF73 from proteasomal degradation, linking E3-ligase machinery to processing competence in human cells.","evidence":"UBE3D depletion, ubiquitination and protein stability assays, pre-mRNA cleavage readout","pmids":["35992060"],"confidence":"Medium","gaps":["Direct enzymatic relationship of UBE3D to CPSF73 ubiquitin chains unresolved at this stage"]},{"year":2023,"claim":"Refined the metal identity and the C-terminal interaction module, showing the active site uses a Fe/Zn/Mn mixture and that CTD3 mediates Symplekin binding within a CPSF73-CPSF100 heterodimer.","evidence":"ICP-MS and anomalous X-ray scattering with activity assays; NMR of CPSF73-CPSF100 C-terminal heterodimer plus binding assays (parasite ortholog)","pmids":["36822327","37989222"],"confidence":"High","gaps":["Physiological metal occupancy in cells uncertain","Heterodimer structure from parasite minimal construct"]},{"year":2023,"claim":"Provided a distinct inhibitor-bound active-site structure and resistance map, advancing structure-guided inhibition of CPSF73 endonuclease activity.","evidence":"X-ray crystallography of CPSF3-benzoxaborole complex, forward-genetics resistance screen, in vitro endonuclease and cellular termination assays","pmids":["37967558"],"confidence":"High","gaps":["In vivo therapeutic window and selectivity not defined here"]},{"year":2024,"claim":"Showed two complementary ubiquitin-pathway mechanisms set CPSF3 levels: UBE3D acts as a cytoplasmic chaperone coordinating the active-site metals, while RBBP6 deposits stabilizing K63 chains that tune alternative polyadenylation.","evidence":"Co-IP, depletion with processing readout, structural analogy from INTS9-INTS11-BRAT1 cryo-EM (UBE3D); CRISPR screens, K63-ubiquitination assay, APA/3'UTR profiling (RBBP6)","pmids":["39032490","38503731"],"confidence":"Medium","gaps":["Direct CPSF73-UBE3D structure inferred from paralog complex","How RBBP6 stabilization integrates with UBE3D chaperoning unresolved"]},{"year":2025,"claim":"Connected CPSF3 stability control by UBE3D to a developmental output, linking CPSF3-dependent processing to Homeobox gene expression in embryogenesis.","evidence":"Co-IP, CRISPR knockout mice, in situ hybridization, RNA-seq, dominant-negative CPSF3 overexpression","pmids":["40075082"],"confidence":"Medium","gaps":["Whether UBE3D de-ubiquitinates or stabilizes CPSF3 mechanistically remains to be reconciled across studies","Direct gene-by-gene processing effects not mapped"]},{"year":2026,"claim":"Resolved how CPSF73 is held in catalytic position by Lsm11 contacts and showed it can adopt an active open conformation independent of RNA, plus revealed regulation of cleavage activity by RBBP6 and stress-responsive CTD phosphorylation.","evidence":"Cryo-EM of reconstituted U7 snRNP with mutagenesis and cleavage assays; nascent RNA sequencing with RBBP6 overexpression rescue and Pol II CTD ChIP under heat shock","pmids":["41495886","41758649"],"confidence":"High","gaps":["Mechanism by which heat shock inactivates CPSF73 cleavage despite its presence not fully defined","How RBBP6 acts as cleavage activator at the catalytic step unresolved"]},{"year":null,"claim":"It remains unresolved how CPSF3 dynamically switches between endonuclease and exonuclease modes and between poly(A) versus histone machineries, and how its ubiquitin-based stability control, metal loading, and conformational gating are integrated in cells.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of mode-switching within an assembled complex","Integration of UBE3D chaperoning, RBBP6 ubiquitination, and metal occupancy in vivo unknown","Determinants of activity loss under stress undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,1,4,5,13,19,23]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,19]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,1,2,5]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8,20]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[8,20]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,3,13]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[7,11,24]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[16,21,20]}],"complexes":["CPSF (cleavage and polyadenylation specificity factor)","CPSF73-CPSF100-Symplekin core cleavage complex","U7 snRNP"],"partners":["CPSF100","SYMPLEKIN","LSM11","FLASH","UBE3D","RBBP6","CSTF77","CSR1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UKF6","full_name":"Cleavage and polyadenylation specificity factor subunit 3","aliases":["Cleavage and polyadenylation specificity factor 73 kDa subunit","CPSF 73 kDa subunit","mRNA 3'-end-processing endonuclease CPSF-73"],"length_aa":684,"mass_kda":77.5,"function":"Component of the cleavage and polyadenylation specificity factor (CPSF) complex that plays 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. Has endonuclease activity, and functions as an mRNA 3'-end-processing endonuclease (PubMed:30507380). Also involved in the histone 3'-end pre-mRNA processing (PubMed:30507380). U7 snRNP-dependent protein that induces both the 3'-endoribonucleolytic cleavage of histone pre-mRNAs and acts as a 5' to 3' exonuclease for degrading the subsequent downstream cleavage product (DCP) of mature histone mRNAs. Cleavage occurs after the 5'-ACCCA-3' sequence in the histone pre-mRNA leaving a 3'hydroxyl group on the upstream fragment containing the stem loop (SL) and 5' phosphate on the downstream cleavage product (DCP) starting with CU nucleotides. The U7-dependent 5' to 3' exonuclease activity is processive and degrades the DCP RNA substrate even after complete removal of the U7-binding site. Binds to the downstream cleavage product (DCP) of histone pre-mRNAs and the cleaved DCP RNA substrate in a U7 snRNP dependent manner. Required for entering/progressing through S-phase of the cell cycle (PubMed:30507380). Required for the selective processing of microRNAs (miRNAs) during embryonic stem cell differentiation via its interaction with ISY1 (By similarity). Required for the biogenesis of all miRNAs from the pri-miR-17-92 primary transcript except miR-92a (By similarity). Only required for the biogenesis of miR-290 and miR-96 from the pri-miR-290-295 and pri-miR-96-183 primary transcripts, respectively (By similarity)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9UKF6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CPSF3","classification":"Common Essential","n_dependent_lines":1198,"n_total_lines":1208,"dependency_fraction":0.9917218543046358},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CPSF6","stoichiometry":0.2},{"gene":"RBM14","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CPSF3","total_profiled":1310},"omim":[{"mim_id":"619876","title":"NEURODEVELOPMENTAL DISORDER WITH MICROCEPHALY, HYPOTONIA, NYSTAGMUS, AND SEIZURES; NEDMHS","url":"https://www.omim.org/entry/619876"},{"mim_id":"606880","title":"CASPASE 8-ASSOCIATED PROTEIN 2; CASP8AP2","url":"https://www.omim.org/entry/606880"},{"mim_id":"606029","title":"CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR 3; CPSF3","url":"https://www.omim.org/entry/606029"},{"mim_id":"603824","title":"UDP-N-ACETYLGLUCOSAMINE 2-EPIMERASE/N-ACETYLMANNOSAMINE KINASE; GNE","url":"https://www.omim.org/entry/603824"},{"mim_id":"603371","title":"GLE1, RNA EXPORT MEDIATOR; GLE1","url":"https://www.omim.org/entry/603371"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CPSF3"},"hgnc":{"alias_symbol":["CPSF-73","CPSF73","YSH1"],"prev_symbol":[]},"alphafold":{"accession":"Q9UKF6","domains":[{"cath_id":"3.60.15.10","chopping":"7-209_397-458","consensus_level":"medium","plddt":95.1484,"start":7,"end":458},{"cath_id":"3.40.50.10890","chopping":"216-391","consensus_level":"high","plddt":92.624,"start":216,"end":391},{"cath_id":"-","chopping":"465-504","consensus_level":"high","plddt":84.6782,"start":465,"end":504},{"cath_id":"3.30.160,3.30.310","chopping":"513-598","consensus_level":"high","plddt":88.2283,"start":513,"end":598},{"cath_id":"-","chopping":"608-681","consensus_level":"high","plddt":82.7024,"start":608,"end":681}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UKF6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UKF6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UKF6-F1-predicted_aligned_error_v6.png","plddt_mean":90.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CPSF3","jax_strain_url":"https://www.jax.org/strain/search?query=CPSF3"},"sequence":{"accession":"Q9UKF6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UKF6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UKF6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UKF6"}},"corpus_meta":[{"pmid":"17128255","id":"PMC_17128255","title":"Polyadenylation 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3'-end-processing endonuclease. Crystal structure at 2.1 Å resolution revealed a metallo-β-lactamase domain and a novel β-CASP domain with a two-zinc-ion active site at their interface. Purified recombinant CPSF-73 possesses RNA endonuclease activity, and mutations disrupting zinc binding in the active site abolish this activity.\",\n      \"method\": \"X-ray crystallography (2.1 Å), in vitro RNA endonuclease assay with recombinant protein, active-site mutagenesis of zinc-coordinating residues\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus reconstituted in vitro endonuclease activity plus active-site mutagenesis in a single rigorous study\",\n      \"pmids\": [\"17128255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CPSF-73 is both the endonuclease and the 5'-3' exonuclease in histone pre-mRNA 3'-end processing. UV-crosslinking with site-specifically labeled RNA substrates (at the cleavage site) identified an ~85 kDa protein that crosslinked in a U7 snRNP-dependent manner; immunoprecipitation identified this as CPSF-73.\",\n      \"method\": \"UV-crosslinking with site-specific radiolabeled/phosphorothioate RNA substrates, immunoprecipitation\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — UV-crosslinking with chemically defined substrates plus immunoprecipitation, replicated by multiple subsequent studies\",\n      \"pmids\": [\"16213211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CPSF-73 contacts the pre-mRNA cleavage site in an AAUAAA-dependent manner, and mutation of key residues in the yeast CPSF-73 homolog (metallo-β-lactamase domain) causes lethality. The 3'-cleavage reaction is metal (Zn2+)-dependent, consistent with CPSF-73 being a metallo-β-lactamase-family endonuclease.\",\n      \"method\": \"UV-crosslinking with site-specific labeling in HeLa nuclear extract, yeast lethality assay with active-site mutants, metal-dependence assay (chelation/rescue)\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal approaches (UV-crosslinking, yeast genetics, metal-dependence), replicated by subsequent structural work\",\n      \"pmids\": [\"15037765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CPSF73, CPSF100, and Symplekin form a stable core subcomplex that associates with histone-specific processing factors and is required for histone pre-mRNA 3'-end processing; chromatin immunoprecipitation showed that CPSF73 and Symplekin (but not CstF50) cotranscriptionally associate with histone genes. This core complex can function in two alternative cleavage machineries (poly(A) and histone mRNA).\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), RNAi knockdown with functional pre-mRNA processing readout\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ChIP, and functional knockdown with multiple orthogonal methods in one study\",\n      \"pmids\": [\"19450530\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Conserved residues in the metallo-β-lactamase motifs of both CPSF73 and CPSF100 are required for the endonuclease activity that cleaves histone pre-mRNAs, indicating that CPSF73 and CPSF100 act together (analogous to homodimeric RNase Z/J family members) to form the active endonuclease.\",\n      \"method\": \"In vitro point mutagenesis of conserved MBL residues in mammalian CPSF73 and CPSF100, histone pre-mRNA cleavage assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution assay with systematic mutagenesis of both subunits, single lab\",\n      \"pmids\": [\"18688255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CPSF-73 functions as a 5'-3' exonuclease that degrades the downstream cleavage product (DCP) of histone pre-mRNA in a U7 snRNP-dependent manner; degradation requires the U7 snRNP only for initiation and is processive. UV-crosslinking showed CPSF-73 directly contacts the DCP. RNAi showed that Xrn2 is not required for DCP degradation in vivo, implicating CPSF-73 as the in vivo 5'-3' exonuclease.\",\n      \"method\": \"UV-crosslinking with modified RNA substrates, in vitro degradation assay with U7 snRNP, RNAi knockdown of Xrn2 in HeLa cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — UV-crosslinking plus functional in vitro assays plus RNAi genetic dissection, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"18955505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The N-terminal regions of FLASH and Lsm11 form a platform that recruits a specific combination of polyadenylation factors—symplekin, CstF64, and all CPSF subunits including CPSF73—to the U7 snRNP. This interaction is abolished by point mutations in FLASH that are detrimental for processing. The same polyadenylation factors are associated with endogenous U7 snRNP and are recruited to histone pre-mRNA in a U7-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, pulldown assays, mass spectrometry, point mutagenesis of FLASH/Lsm11, chromatin association assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, MS, mutagenesis, and chromatin assays; independently corroborated by reconstitution studies\",\n      \"pmids\": [\"23071092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CPSF73 endonuclease activity is required for efficient transcriptional termination of protein-coding genes by RNA Pol II. Catalytically inactive CPSF73 cannot restore termination to cells lacking functional CPSF73, placing RNA cleavage by CPSF73 upstream of Xrn2-mediated cotranscriptional degradation and termination. CPSF73 loss causes more extensive readthrough transcription than Xrn2 elimination, indicating CPSF73 has a more foundational role.\",\n      \"method\": \"Conditional depletion via gene editing (auxin-inducible degron), catalytic mutant rescue experiments, mammalian native elongating transcript sequencing (mNET-seq) genome-wide\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — engineered conditional depletion plus catalytic mutant rescue plus genome-wide mNET-seq, multiple orthogonal approaches\",\n      \"pmids\": [\"29432121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CSR1 (a tumor suppressor) binds the C-terminus of CPSF3 (amino acids 440–543) via yeast two-hybrid and further direct binding assays. This interaction causes CPSF3 translocation from the nucleus to the cytoplasm, inhibiting polyadenylation activity both in vitro and in vivo. A CSR1 mutant unable to bind CPSF3 neither re-localizes it nor inhibits polyadenylation. CPSF3 knockdown by siRNA mimics CSR1-induced cell death.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, subcellular fractionation/localization, in vitro and in vivo polyadenylation assay, siRNA knockdown\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple assays (Y2H, Co-IP, localization, functional polyadenylation assay) in single lab\",\n      \"pmids\": [\"18806823\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The C-terminal domain of yeast Brr5/Ysh1 (CPSF3 ortholog) lying outside β-lactamase homology is essential for cell viability and mRNA 3'-end processing. A homologous protein Syc1, by mimicking the essential Brr5 C-terminus, acts as a negative regulator of mRNA 3'-end formation.\",\n      \"method\": \"Yeast genetics (deletion analysis), in vitro 3'-end processing assay with yeast extracts, genetic rescue experiments\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast genetic complementation plus in vitro processing assay, single lab\",\n      \"pmids\": [\"16431986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CPSF3 is the direct molecular target of JTE-607 (a prodrug converted by ester hydrolysis to its active form), which binds CPSF3 as identified by compound-immobilized affinity chromatography. JTE-607 treatment causes accumulation of pre-mRNAs, and CPSF3 knockdown similarly causes pre-mRNA accumulation and suppresses inflammatory cytokine expression.\",\n      \"method\": \"Compound-immobilized affinity chromatography, CPSF3 knockdown, pre-mRNA accumulation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — affinity chromatography plus genetic knockdown with functional readout, single lab\",\n      \"pmids\": [\"31399191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CPSF3 is the target of JTE-607 in AML and Ewing's sarcoma. Chemical genetics/phenotypic screening identified CPSF3. Inhibition of CPSF3 prevents release of newly synthesized pre-mRNAs and causes transcriptional readthrough and formation of DNA-RNA R-loop structures.\",\n      \"method\": \"Phenotypic screening, chemical genetics, gene editing resistance mutations, pre-mRNA accumulation assays, R-loop detection\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — forward genetics resistance mapping, functional pre-mRNA assays, R-loop detection; corroborated by independent study (PMID:31399191)\",\n      \"pmids\": [\"31819276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Crystal structures of Cryptosporidium CPSF3 reveal that the oxaborole AN3661 blocks mRNA processing activity by binding at the metal-dependent catalytic center of CPSF3, with the oxaborole group directly occupying the active site.\",\n      \"method\": \"X-ray crystallography of recombinant Cryptosporidium CPSF3 with inhibitor bound, growth inhibition assay, in vivo efficacy in mouse models\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with inhibitor at active site plus functional antiparasitic validation in cell and animal models\",\n      \"pmids\": [\"31694928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Reconstituted recombinant U7 snRNP (all 13 components) demonstrates that CPSF73 is both an endonuclease and a 5'-3' exonuclease: it cleaves histone pre-mRNAs endonucleolytically and degrades the downstream cleavage product 5'-to-3'. Both activities require base-pairing of U7 snRNA, presence of the symplekin N-terminal domain (NTD), and are abolished by active-site mutations in CPSF73. Binding of symplekin NTD to SSU72 phosphatase also abolishes activity.\",\n      \"method\": \"In vitro reconstitution of recombinant U7 snRNP from 13 components, endonuclease and exonuclease assays, active-site mutagenesis of CPSF73, substrate modification studies\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — full reconstitution from recombinant components plus active-site mutagenesis plus multiple substrate modifications; rigorous single-study multi-method approach\",\n      \"pmids\": [\"32554553\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In yeast, the Ysh1 (CPSF3 ortholog) endonuclease is regulated by ubiquitin-mediated proteasomal degradation. Ubiquitination of Ysh1 is mediated by the Ubc4 ubiquitin-conjugating enzyme and the RING ubiquitin ligase Mpe1. Ipa1 mutation causes striking depletion of Ysh1 protein (but not mRNA), rescued by proteasome inactivation or YSH1 overexpression.\",\n      \"method\": \"Yeast genetics (ipa1-1 mutant), proteasome inhibition, ubiquitination assays, YSH1 overexpression rescue, mRNA level analysis\",\n      \"journal\": \"RNA biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic and biochemical approaches in yeast ortholog, single lab\",\n      \"pmids\": [\"32009536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Yeast Ipa1 interacts with the Ysh1 (CPSF3 ortholog) endonuclease and promotes proper transcription termination and poly(A) site selection globally. Ipa1 mutation leads to decreased Ysh1 levels and poor recruitment of the cleavage/polyadenylation complex to transcribed genes. Ysh1 overexpression in the Ipa1 mutant rescues the termination defect.\",\n      \"method\": \"Chromatin immunoprecipitation, yeast genetic suppression (Ysh1 overexpression rescue), Pol II elongation kinetics assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP, genetic rescue, Pol II kinetics, single lab; yeast ortholog\",\n      \"pmids\": [\"30759400\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"UBE3D, a HECT-like E3 ligase, 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.\",\n      \"method\": \"UBE3D depletion, ubiquitination assay, pre-mRNA cleavage assay, protein stability assay\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — depletion with functional pre-mRNA cleavage readout plus ubiquitination assay, single lab\",\n      \"pmids\": [\"35992060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"X-ray crystallography demonstrated that benzoxaborole compounds bind to the active site of CPSF3 in a manner distinct from other known CPSF3 inhibitors. Forward genetics resistance mapping identified CPSF3 mutations that reduce benzoxaborole binding and confer resistance. Benzoxaboroles inhibit CPSF73 endonuclease activity in vitro and curb transcriptional termination in cells.\",\n      \"method\": \"X-ray crystallography of CPSF3-benzoxaborole complex, forward genetics resistance screen, in vitro endonuclease activity assay, transcription termination assay in cells\",\n      \"journal\": \"Cell chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of inhibitor complex plus forward genetics resistance mapping plus in vitro enzymatic assay\",\n      \"pmids\": [\"37967558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The C-terminal domain 3 (CTD3) of CPSF73 is required for binding to Symplekin, as demonstrated by biochemical assays; the CPSF73-CPSF100 C-terminal heterodimer structure was determined by NMR for the Encephalitozoon cuniculi minimal construct, revealing extensive inter-protein contacts and TBP-like CTD2 folds.\",\n      \"method\": \"NMR solution structure of minimal CPSF73-CPSF100 C-terminal heterodimer, biochemical binding assays (CTD3–Symplekin interaction)\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — NMR structure plus biochemical binding assay for one domain interaction, single lab, parasite ortholog\",\n      \"pmids\": [\"37989222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The active site of CPSF73 contains a mixture of metal ions (Fe, Zn, Mn) rather than exclusively zinc ions. The abundance of the metal ions varies with the expression host, yet the enzyme retains endonuclease activity with less than 20% zinc occupancy, indicating that iron and/or manganese can support catalysis.\",\n      \"method\": \"Inductively coupled plasma mass spectrometry (ICP-MS), X-ray diffraction metal anomalous scattering, in vitro pre-mRNA cleavage assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — ICP-MS plus X-ray diffraction plus in vitro activity assay, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"36822327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UBE3D is a binding partner and stabilizer of CPSF73 in the cytoplasm; structural studies showed that the conserved C-terminal cysteine of UBE3D likely coordinates the active-site metal ions of CPSF73, functioning as a cytoplasmic chaperone required for CPSF73's nuclear pre-mRNA processing function. Loss of UBE3D leads to CPSF73 downregulation.\",\n      \"method\": \"Structural studies (cryo-EM of INTS9-INTS11-BRAT1 complex providing mechanistic analogy), co-immunoprecipitation, depletion experiments with pre-mRNA processing readout\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — structural analogy (cryo-EM of paralog complex) plus Co-IP plus depletion functional assay; direct CPSF73 structure not solved, inference from paralog\",\n      \"pmids\": [\"39032490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RBBP6 E3 ligase mediates K63-linked ubiquitination of CPSF3, which stabilizes CPSF3 and regulates alternative polyadenylation events. RBBP6 depletion induces shortening of 3'UTRs of MYC competing-endogenous RNAs, releasing miR-590-3p to decrease MYC expression in glioblastoma stem cells.\",\n      \"method\": \"CRISPR/Cas9 knockout screens, Co-immunoprecipitation, ubiquitination assay (K63-linkage), 3'UTR sequencing, alternative polyadenylation profiling, CPSF3 inhibitor (JTE-607) functional assay\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screens, Co-IP ubiquitination assay, APA profiling, single lab\",\n      \"pmids\": [\"38503731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UBE3D interacts with CPSF3 and can de-ubiquitinate CPSF3; deficiency of UBE3D leads to reduced CPSF3 protein levels in both mouse and human cells. Overexpression of dominant negative CPSF3 mutants partially reduces mRNA levels of Homeobox genes, linking CPSF3-mediated pre-mRNA 3'-end processing to Homeobox gene expression during early embryogenesis.\",\n      \"method\": \"Co-immunoprecipitation, CRISPR/Cas9 knockout mice, in situ hybridization, immunofluorescence, RNA-seq, dominant-negative CPSF3 overexpression\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, KO mouse, dominant-negative functional assay, multiple methods; single lab\",\n      \"pmids\": [\"40075082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"A conserved helix in the Lsm11 N-terminal extension directly contacts the metallo-β-lactamase domain of CPSF73 and is required to hold CPSF73 in the correct position for the cleavage reaction in U7 snRNP. Cryo-EM also revealed that CPSF73 can achieve an open (active) conformation independent of RNA binding in its active site. Additionally, the C-terminal end of CstF77 contacts CPSF100 at the CPSF73-CPSF100 interface, with a small effect on cleavage activity.\",\n      \"method\": \"Cryo-EM structure determination of reconstituted U7 snRNP complexes, mutagenesis/deletion of Lsm11 helix, in vitro pre-mRNA cleavage assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure plus mutagenesis plus in vitro reconstituted cleavage assay, single study with multiple orthogonal methods\",\n      \"pmids\": [\"41495886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Heat shock causes loss of CPSF73 endonucleolytic cleavage activity at thousands of genes despite CPSF73 remaining present. Overexpression of RBBP6, an activator of CPSF73, during heat shock rescues the loss of cleavage and dampens readthrough transcription. Heat shock also increases Tyr1 and Ser2 phosphorylation of the Pol II CTD at 3'-ends, which is attenuated at readthrough genes.\",\n      \"method\": \"mNET-seq or equivalent nascent RNA sequencing, overexpression rescue of RBBP6, cleavage assay, ChIP for Pol II CTD phosphorylation marks\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide sequencing plus RBBP6 overexpression rescue plus CTD phosphorylation analysis, single lab\",\n      \"pmids\": [\"41758649\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CPSF3 (CPSF-73) is a metallo-β-lactamase/β-CASP family endoribonuclease that catalyzes the endonucleolytic cleavage of pre-mRNAs at poly(A) sites using a two-metal-ion (Fe/Zn/Mn mixture) active site; it also functions as a 5'-3' exonuclease to degrade downstream cleavage products, particularly in the U7 snRNP machinery for histone pre-mRNA 3'-end processing, where its recruitment depends on a FLASH/Lsm11 platform and a symplekin N-terminal domain. CPSF3 cleavage activity is essential for RNA Pol II transcription termination on protein-coding genes upstream of Xrn2-mediated cotranscriptional degradation. CPSF3 protein stability is regulated by ubiquitin-mediated proteasomal degradation, with UBE3D acting as a stabilizing cytoplasmic chaperone and RBBP6 mediating K63-linked ubiquitination to stabilize the protein. The CTD3 domain of CPSF3 mediates interaction with Symplekin to form a core cleavage complex with CPSF100, and active-site mutations abolish both endonuclease and exonuclease activities.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CPSF3 (CPSF-73) is the catalytic endoribonuclease of the eukaryotic pre-mRNA 3'-end processing machinery, executing the endonucleolytic cleavage at poly(A) sites that defines mRNA 3'-ends [#0, #2]. Its catalytic core is a metallo-β-lactamase domain joined to a β-CASP domain, with a metal-ion active site at their interface; the enzyme uses a mixture of Fe, Zn and Mn ions rather than zinc exclusively, and active-site mutations that disrupt metal coordination abolish activity [#0, #19]. CPSF3 acts together with CPSF100, whose conserved metallo-β-lactamase residues are also required for cleavage, the two subunits forming the active endonuclease, and its C-terminal CTD3 domain binds Symplekin to assemble the core cleavage complex [#4, #18]. Beyond polyadenylation, CPSF3 is the dual endonuclease/5'-3' exonuclease of histone pre-mRNA 3'-end processing within the U7 snRNP, where it is recruited via a FLASH/Lsm11 platform and a symplekin N-terminal domain and is positioned for catalysis by direct Lsm11 contacts to its metallo-β-lactamase domain [#1, #5, #6, #13, #23]. CPSF3-mediated cleavage is foundational for RNA polymerase II transcription termination, acting upstream of Xrn2-dependent cotranscriptional degradation, and its loss produces extensive readthrough transcription and R-loop accumulation [#7, #11]. CPSF3 protein levels are set by ubiquitin-dependent control: UBE3D acts as a cytoplasmic chaperone/stabilizer and RBBP6 mediates stabilizing K63-linked ubiquitination that also tunes alternative polyadenylation [#16, #20, #21]. CPSF3 is the direct molecular target of the inhibitors JTE-607 and active-site-binding benzoxaboroles/oxaboroles, which block its endonuclease activity [#10, #11, #12, #17].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established which factor contacts the pre-mRNA cleavage site and that cleavage is metal-dependent, pointing to a metallo-β-lactamase endonuclease.\",\n      \"evidence\": \"Site-specific UV-crosslinking in HeLa nuclear extract, yeast active-site mutant lethality, and metal-dependence assays\",\n      \"pmids\": [\"15037765\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not directly demonstrate purified recombinant nuclease activity\", \"Active-site architecture not yet resolved structurally\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified CPSF-73 as the U7 snRNP-dependent factor contacting the histone pre-mRNA cleavage site, implicating one enzyme in both endo- and exonucleolytic steps.\",\n      \"evidence\": \"UV-crosslinking with site-specific RNA substrates plus immunoprecipitation\",\n      \"pmids\": [\"16213211\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crosslinking does not prove catalysis\", \"Exonuclease role still inferred, not reconstituted\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Resolved the catalytic basis of CPSF3 by showing a metallo-β-lactamase/β-CASP fold with a two-metal active site and reconstituting endonuclease activity, settling that CPSF3 is the 3'-processing nuclease.\",\n      \"evidence\": \"2.1 Å crystal structure, recombinant in vitro endonuclease assay, active-site mutagenesis\",\n      \"pmids\": [\"17128255\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address how the enzyme is positioned within larger processing complexes\", \"Exonuclease activity not structurally explained\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Showed that a non-catalytic CPSF3 C-terminal region (via the yeast ortholog) is essential for processing and is a target of negative regulation, revealing functional roles beyond the catalytic domain.\",\n      \"evidence\": \"Yeast deletion genetics and in vitro 3'-end processing with extracts\",\n      \"pmids\": [\"16431986\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Yeast ortholog; human C-terminal regulation not directly tested here\", \"Molecular function of the C-terminus left undefined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated that CPSF73 and CPSF100 metallo-β-lactamase residues both contribute, defining the endonuclease as a two-subunit unit rather than CPSF73 alone.\",\n      \"evidence\": \"Systematic MBL-residue mutagenesis of both subunits with histone pre-mRNA cleavage assays\",\n      \"pmids\": [\"18688255\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the heterodimer interface structurally\", \"Relative catalytic contribution of each subunit unquantified\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Established CPSF-73 as the in vivo processive 5'-3' exonuclease degrading the downstream cleavage product, distinguishing it from Xrn2 for this step.\",\n      \"evidence\": \"UV-crosslinking, in vitro U7 snRNP degradation assays, Xrn2 RNAi\",\n      \"pmids\": [\"18955505\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of switching between endo- and exonuclease modes unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified a regulatory partner (CSR1) that re-localizes CPSF3 to the cytoplasm and inhibits polyadenylation, linking CPSF3 activity to subcellular sequestration and cell death.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, fractionation, in vitro/in vivo polyadenylation assays, siRNA\",\n      \"pmids\": [\"18806823\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding without reciprocal structural mapping\", \"Physiological context of CSR1 regulation unclear\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined the CPSF73-CPSF100-Symplekin core subcomplex shared between poly(A) and histone machineries and showed cotranscriptional association with target genes.\",\n      \"evidence\": \"Reciprocal Co-IP, ChIP, RNAi with processing readouts\",\n      \"pmids\": [\"19450530\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and architecture of the core complex not resolved\", \"Mechanism of switching between two machineries unaddressed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed that FLASH/Lsm11 N-terminal regions form the platform recruiting CPSF subunits including CPSF73 to U7 snRNP, defining the recruitment route for histone processing.\",\n      \"evidence\": \"Co-IP, pulldowns, mass spectrometry, FLASH/Lsm11 mutagenesis, chromatin assays\",\n      \"pmids\": [\"23071092\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not place CPSF73 catalytic geometry within the assembled complex\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established that CPSF73 catalytic cleavage is required for and upstream of Pol II transcription termination, making it more foundational than Xrn2.\",\n      \"evidence\": \"Auxin-inducible degron depletion, catalytic-mutant rescue, genome-wide mNET-seq\",\n      \"pmids\": [\"29432121\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how readthrough relates to R-loop formation\", \"Termination defect mechanism downstream of cleavage incomplete\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified CPSF3 as the direct target of small-molecule inhibitors (JTE-607, oxaboroles) that block processing, validating it as a druggable node and confirming functional consequences of inhibition.\",\n      \"evidence\": \"Affinity chromatography, phenotypic/chemical-genetic screening, resistance mutation mapping, pre-mRNA accumulation and R-loop assays, inhibitor crystal structure (Cryptosporidium ortholog)\",\n      \"pmids\": [\"31399191\", \"31819276\", \"31694928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Selectivity over related β-CASP nucleases not fully defined\", \"Human inhibitor co-structure inferred partly from parasite ortholog\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed that CPSF3 protein abundance is actively regulated via partner interactions and recruitment in yeast, connecting Ysh1 levels to global termination and poly(A) site selection.\",\n      \"evidence\": \"Yeast ChIP, genetic suppression by Ysh1 overexpression, Pol II elongation kinetics\",\n      \"pmids\": [\"30759400\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Yeast ortholog; human Ipa1 equivalent not tested\", \"Direct mechanism of Ysh1 destabilization not defined here\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Achieved full recombinant reconstitution of U7 snRNP, proving CPSF73 carries both endo- and exonuclease activities and that symplekin NTD and SSU72 binding gate these activities.\",\n      \"evidence\": \"13-component recombinant U7 snRNP reconstitution, cleavage/degradation assays, CPSF73 active-site mutagenesis\",\n      \"pmids\": [\"32554553\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural snapshot of the active enzyme within the assembled complex not yet obtained\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated ubiquitin-proteasome control of CPSF3 stability in yeast, identifying the conjugating enzyme and ligase that target the nuclease.\",\n      \"evidence\": \"Yeast ipa1-1 mutant, proteasome inhibition, ubiquitination assays, overexpression rescue\",\n      \"pmids\": [\"32009536\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mammalian conservation of Ubc4/Mpe1 axis not established here\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified UBE3D as a stabilizer that protects CPSF73 from proteasomal degradation, linking E3-ligase machinery to processing competence in human cells.\",\n      \"evidence\": \"UBE3D depletion, ubiquitination and protein stability assays, pre-mRNA cleavage readout\",\n      \"pmids\": [\"35992060\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct enzymatic relationship of UBE3D to CPSF73 ubiquitin chains unresolved at this stage\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Refined the metal identity and the C-terminal interaction module, showing the active site uses a Fe/Zn/Mn mixture and that CTD3 mediates Symplekin binding within a CPSF73-CPSF100 heterodimer.\",\n      \"evidence\": \"ICP-MS and anomalous X-ray scattering with activity assays; NMR of CPSF73-CPSF100 C-terminal heterodimer plus binding assays (parasite ortholog)\",\n      \"pmids\": [\"36822327\", \"37989222\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological metal occupancy in cells uncertain\", \"Heterodimer structure from parasite minimal construct\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Provided a distinct inhibitor-bound active-site structure and resistance map, advancing structure-guided inhibition of CPSF73 endonuclease activity.\",\n      \"evidence\": \"X-ray crystallography of CPSF3-benzoxaborole complex, forward-genetics resistance screen, in vitro endonuclease and cellular termination assays\",\n      \"pmids\": [\"37967558\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo therapeutic window and selectivity not defined here\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed two complementary ubiquitin-pathway mechanisms set CPSF3 levels: UBE3D acts as a cytoplasmic chaperone coordinating the active-site metals, while RBBP6 deposits stabilizing K63 chains that tune alternative polyadenylation.\",\n      \"evidence\": \"Co-IP, depletion with processing readout, structural analogy from INTS9-INTS11-BRAT1 cryo-EM (UBE3D); CRISPR screens, K63-ubiquitination assay, APA/3'UTR profiling (RBBP6)\",\n      \"pmids\": [\"39032490\", \"38503731\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct CPSF73-UBE3D structure inferred from paralog complex\", \"How RBBP6 stabilization integrates with UBE3D chaperoning unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected CPSF3 stability control by UBE3D to a developmental output, linking CPSF3-dependent processing to Homeobox gene expression in embryogenesis.\",\n      \"evidence\": \"Co-IP, CRISPR knockout mice, in situ hybridization, RNA-seq, dominant-negative CPSF3 overexpression\",\n      \"pmids\": [\"40075082\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether UBE3D de-ubiquitinates or stabilizes CPSF3 mechanistically remains to be reconciled across studies\", \"Direct gene-by-gene processing effects not mapped\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Resolved how CPSF73 is held in catalytic position by Lsm11 contacts and showed it can adopt an active open conformation independent of RNA, plus revealed regulation of cleavage activity by RBBP6 and stress-responsive CTD phosphorylation.\",\n      \"evidence\": \"Cryo-EM of reconstituted U7 snRNP with mutagenesis and cleavage assays; nascent RNA sequencing with RBBP6 overexpression rescue and Pol II CTD ChIP under heat shock\",\n      \"pmids\": [\"41495886\", \"41758649\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which heat shock inactivates CPSF73 cleavage despite its presence not fully defined\", \"How RBBP6 acts as cleavage activator at the catalytic step unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how CPSF3 dynamically switches between endonuclease and exonuclease modes and between poly(A) versus histone machineries, and how its ubiquitin-based stability control, metal loading, and conformational gating are integrated in cells.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of mode-switching within an assembled complex\", \"Integration of UBE3D chaperoning, RBBP6 ubiquitination, and metal occupancy in vivo unknown\", \"Determinants of activity loss under stress undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 1, 4, 5, 13, 19, 23]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 19]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 1, 2, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8, 20]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [8, 20]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 3, 13]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [7, 11, 24]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [16, 21, 20]}\n    ],\n    \"complexes\": [\n      \"CPSF (cleavage and polyadenylation specificity factor)\",\n      \"CPSF73-CPSF100-Symplekin core cleavage complex\",\n      \"U7 snRNP\"\n    ],\n    \"partners\": [\n      \"CPSF100\",\n      \"Symplekin\",\n      \"Lsm11\",\n      \"FLASH\",\n      \"UBE3D\",\n      \"RBBP6\",\n      \"CstF77\",\n      \"CSR1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}