{"gene":"CSTF3","run_date":"2026-06-09T22:57:19","timeline":{"discoveries":[{"year":2007,"finding":"Crystal structure of the HAT (half a TPR) domain of murine CstF-77 revealed two subdomains (HAT-N and HAT-C) with drastically different helical orientations, forming a highly elongated homodimer spanning 165 Å mediated by the HAT-C domain. Light-scattering, yeast two-hybrid, and analytical ultracentrifugation confirmed this self-association, supporting a role for CstF dimerization in pre-mRNA 3' end processing.","method":"X-ray crystallography, light scattering, yeast two-hybrid, analytical ultracentrifugation","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure resolved with multiple orthogonal biochemical validations (light scattering, yeast two-hybrid, AUC) in a single rigorous study","pmids":["17386263"],"is_preprint":false},{"year":2007,"finding":"Crystal structure of CstF-77 from Encephalitozoon cuniculi at 2 Å resolution revealed 11 Half-a-TPR repeats defining two domains and a tight homodimer exposing phylogenetically conserved surface areas for interaction with protein partners. Mapping experiments identified the C-terminal region of Rna14p (yeast CstF-77 homologue) as the docking domain for Rna15p (yeast CstF-64 homologue).","method":"X-ray crystallography, domain mapping experiments","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure at 2 Å with biochemical domain mapping, independently replicating structural findings from PMID:17386263","pmids":["17584787"],"is_preprint":false},{"year":2010,"finding":"CstF-77 and symplekin bind mutually exclusively to the hinge domain of CstF-64. The nuclear accumulation of CstF-64 depends on its binding to CstF-77 (not symplekin), demonstrating that CstF-77 interaction is required for nuclear localization and maintenance of stoichiometric nuclear CstF complex levels.","method":"Mutant analysis (CstF-64 and symplekin mutants), nuclear localization assays, functional complementation","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal mutant analysis with two orthogonal functional readouts (localization and processing activity) in a single lab","pmids":["21119002"],"is_preprint":false},{"year":2009,"finding":"The hinge domain of CstF-64 is essential for interaction with CstF-77 and consequent nuclear localization of CstF-64, demonstrating that nuclear import of a preformed CstF complex is an essential step in polyadenylation.","method":"In vivo SLAP (stem-loop luciferase assay for polyadenylation), domain deletion/mutation analysis, nuclear localization assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — validated in vivo functional assay with structure-function analysis, replicated across multiple domain mutations, consistent with PMID:21119002","pmids":["19887456"],"is_preprint":false},{"year":2018,"finding":"The carboxy-terminus of CstF-77 (monkeytail-carboxy-terminal domain, last 30 amino acids) enhances cleavage/polyadenylation by increasing the stability of the RNA recognition motif (RRM) of CstF-64, thereby altering the affinity of the complex for RNA. CstF-64 relies on CstF-77 for nuclear transport; excess CstF-64 localizes to the cytoplasm, possibly via interaction with cytoplasmic RNAs.","method":"Reverse genetics, NMR spectroscopy of recombinant proteins (CstF-64 RRM-Hinge and CstF-77 monkeytail-CTD), nuclear localization assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structural data combined with reverse genetics and localization assays, single lab but multiple orthogonal methods","pmids":["30257008"],"is_preprint":false},{"year":2002,"finding":"Drosophila Su(f) (CstF-77 homologue) is required for pre-mRNA cleavage during mRNA 3' end formation in vivo. Chimeric human CstF-77/Su(f) proteins rescue lethality and cleavage defects in su(f) mutants, but a domain in human CstF-77 limiting for rescue is incapable of reproducing protein interactions with Drosophila CstF subunits. Chimeric proteins rescuing lethality cannot restore utilization of a regulated poly(A) site, indicating CstF-77 has an additional role in poly(A) site regulation.","method":"Genetic complementation in Drosophila su(f) mutants, chimeric protein rescue assays, mRNA 3' end processing analysis in vivo","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with in vivo functional rescue and multiple chimeric constructs tested, clear mechanistic dissection of domains","pmids":["12149458"],"is_preprint":false},{"year":2013,"finding":"The CstF-77 gene contains a conserved intronic polyadenylation site (In3 pA) whose usage is responsive to CstF-77 expression levels and several other C/P factors, establishing a negative feedback autoregulatory mechanism. U1 snRNP inhibition also regulates In3 pA usage. Perturbation of CstF-77 expression leads to widespread alternative cleavage and polyadenylation (APA) and disturbance of cell proliferation and differentiation.","method":"Molecular biology validation of intronic poly(A) site, expression manipulation (overexpression/knockdown), U1 snRNP inhibition, global APA analysis","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple molecular methods in single lab; feedback mechanism established by expression perturbation and correlative analysis","pmids":["23874216"],"is_preprint":false},{"year":2006,"finding":"In Xenopus oocytes, CstF-77 (X77K) localizes mainly to the nucleus but also to punctate cytoplasmic foci, where it resides in a cytoplasmic complex with eIF4E, CPEB, CPSF-100, and XGLD2. X77K is not required for cytoplasmic polyadenylation per se, but impairment of X77K function accelerates the G2/M transition with premature synthesis of Mos and AuroraA proteins. X77K represses mRNA translation in vitro, suggesting a role in mRNA masking prior to polyadenylation.","method":"Co-immunoprecipitation, subcellular fractionation, in vivo function impairment (dominant negative/antibody injection), in vitro translation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP identifies complex, functional localization and cell cycle phenotype established, but single lab and some mechanistic aspects remain indirect","pmids":["16882666"],"is_preprint":false},{"year":2024,"finding":"CSTF3 directly binds downstream of the NEAT1 proximal polyadenylation site to promote usage of the proximal PAS, generating the short NEAT1_1 isoform. CSTF3 knockdown reduces proximal PAS usage, shifting expression toward the longer NEAT1_2 isoform and increasing platinum sensitivity in ovarian cancer cells. NEAT1_1 overexpression reverses platinum resistance after CSTF3 knockdown, and CSTF3/NEAT1_1 activity is linked to activation of the PI3K/AKT/mTOR pathway.","method":"CSTF3 knockdown in cell lines, RNA isoform analysis, lncRNA isoform-specific overexpression/knockdown, pathway activity measurement","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional knockdown with specific isoform rescue experiment; single lab, mechanistic link to PI3K/AKT/mTOR is correlative","pmids":["38898019"],"is_preprint":false},{"year":2026,"finding":"α3β1 integrin-MEK/ERK signaling induces CSTF3 expression in keratinocytes to promote proximal polyadenylation site usage in the Mmp9 gene, generating a short, more stable Mmp9 mRNA. CSTF3 knockdown shifts Mmp9 toward distal PAS usage. α3 deletion reduced Cstf3 gene expression and altered APA genome-wide in vivo.","method":"Inducible epidermis-specific α3 knockout mice, RNA in situ hybridization, CSTF3 knockdown, DaPars2 genome-wide APA analysis","journal":"Matrix biology : journal of the International Society for Matrix Biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic model with genome-wide APA analysis and targeted knockdown; single lab, pathway placement supported by multiple methods","pmids":["41628695"],"is_preprint":false},{"year":2005,"finding":"Human and mouse CstF-77 genes contain an intronic polyadenylation site that produces short CstF-77 transcripts lacking sequences encoding domains involved in CstF-77 functions, analogous to the Drosophila su(f) intronic poly(A) site. The intronic poly(A) site is utilized across a wide range of tissues based on SAGE data validation.","method":"Bioinformatic identification with molecular biology experimental validation (RT-PCR, poly(A) site analysis), SAGE data analysis","journal":"Gene","confidence":"Low","confidence_rationale":"Tier 3 / Moderate — molecular validation of intronic pA site but limited direct mechanistic functional data; primarily identification study","pmids":["16316725"],"is_preprint":false}],"current_model":"CstF-77 (CSTF3) is the central scaffold subunit of the heterotrimeric CstF complex essential for pre-mRNA 3' cleavage and polyadenylation: its HAT domain forms an elongated homodimer that bridges CstF-64 and CstF-50 subunits, its C-terminal domain directly stabilizes the CstF-64 RRM to enhance RNA binding and cleavage/polyadenylation activity, and it is required for nuclear import of the CstF complex (CstF-64 nuclear localization depends on its hinge-domain interaction with CstF-77); additionally, CstF-77 expression is subject to negative feedback autoregulation through a conserved intronic polyadenylation site, and upstream signals such as integrin α3β1-MEK/ERK can regulate CSTF3 expression to orchestrate alternative polyadenylation of target genes including Mmp9."},"narrative":{"mechanistic_narrative":"CSTF3 (CstF-77) is the central scaffold subunit of the heterotrimeric cleavage stimulation factor (CstF) complex that drives pre-mRNA 3' end cleavage and polyadenylation [PMID:17386263, PMID:12149458]. Its HAT (half-a-TPR) domain folds into two subdomains (HAT-N and HAT-C) that assemble a highly elongated homodimer, with HAT-C mediating self-association and the conserved C-terminal region docking the CstF-64 subunit [PMID:17386263, PMID:17584787]. CstF-77 binds the hinge domain of CstF-64 mutually exclusively with symplekin, and this interaction is required for nuclear import of CstF-64 and maintenance of stoichiometric nuclear CstF complex levels, establishing nuclear assembly of the complex as an essential step in polyadenylation [PMID:21119002, PMID:19887456]. The CstF-77 carboxy-terminal \"monkeytail\" further enhances cleavage/polyadenylation by stabilizing the CstF-64 RNA recognition motif, thereby tuning the complex's affinity for RNA [PMID:30257008]. CSTF3 levels govern alternative polyadenylation outcomes genome-wide and are themselves autoregulated by a conserved intronic poly(A) site that creates a negative feedback loop [PMID:23874216]. Upstream integrin α3β1–MEK/ERK signaling induces CSTF3 expression to shift target genes toward proximal poly(A) site usage, generating short, stable isoforms of genes such as Mmp9 [PMID:41628695].","teleology":[{"year":2002,"claim":"Establishing that CstF-77 is genuinely required for pre-mRNA cleavage in vivo and carries a separable role in regulated poly(A) site choice, beyond complex assembly.","evidence":"genetic complementation and chimeric human/Drosophila rescue assays in su(f) mutants","pmids":["12149458"],"confidence":"High","gaps":["Domain limiting cross-species rescue not molecularly defined","Mechanism distinguishing constitutive cleavage from regulated poly(A) site choice unresolved"]},{"year":2006,"claim":"Asking whether CstF-77 has functions outside nuclear 3' processing revealed a cytoplasmic complex and translational repression role linked to cell cycle timing.","evidence":"Co-IP, subcellular fractionation, and in vitro translation assays in Xenopus oocytes","pmids":["16882666"],"confidence":"Medium","gaps":["Cytoplasmic complex partners (eIF4E, CPEB, CPSF-100, XGLD2) not validated reciprocally","Direct molecular basis of translational repression unknown","Relevance to somatic cells untested"]},{"year":2007,"claim":"Resolving how CstF-77 organizes the complex: structures showed a HAT-domain homodimer that physically bridges partner subunits.","evidence":"X-ray crystallography of murine and E. cuniculi CstF-77 with light scattering, yeast two-hybrid, AUC, and domain mapping","pmids":["17386263","17584787"],"confidence":"High","gaps":["Full trimeric CstF architecture not visualized","Functional consequence of dimerization for processing kinetics not quantified"]},{"year":2009,"claim":"Determining how the complex enters the nucleus: the CstF-64 hinge–CstF-77 interaction was shown to be essential for nuclear import of a preformed CstF complex.","evidence":"in vivo SLAP polyadenylation assay with domain deletion and nuclear localization analysis","pmids":["19887456"],"confidence":"High","gaps":["Nuclear import machinery recognizing the complex not identified","Order of complex assembly relative to import not fully resolved"]},{"year":2010,"claim":"Clarifying competitive binding at the CstF-64 hinge: CstF-77 and symplekin bind mutually exclusively, with CstF-77 specifically required for nuclear accumulation.","evidence":"reciprocal mutant analysis with localization and processing readouts","pmids":["21119002"],"confidence":"High","gaps":["Functional role of the symplekin-bound pool not defined","Regulation of the binding switch unknown"]},{"year":2013,"claim":"Identifying how CSTF3 levels are buffered: a conserved intronic poly(A) site mediates negative feedback autoregulation that influences global APA, proliferation, and differentiation.","evidence":"intronic poly(A) site validation, expression perturbation, U1 snRNP inhibition, and global APA analysis","pmids":["23874216"],"confidence":"Medium","gaps":["Quantitative contribution of feedback to steady-state CSTF3 not measured","Coupling between feedback and downstream phenotypes correlative"]},{"year":2018,"claim":"Defining the biochemical basis of CstF-77's stimulatory effect: its C-terminal monkeytail stabilizes the CstF-64 RRM to enhance RNA affinity and processing.","evidence":"NMR spectroscopy of recombinant CstF-64 RRM-Hinge and CstF-77 CTD with reverse genetics and localization assays","pmids":["30257008"],"confidence":"High","gaps":["Structural model of the CTD–RRM contact not fully resolved","Effect on specific RNA sequence preferences not mapped"]},{"year":2024,"claim":"Connecting CSTF3 to disease-relevant isoform choice: it directly promotes proximal PAS usage on NEAT1, controlling lncRNA isoform output and platinum response.","evidence":"CSTF3 knockdown, isoform-specific manipulation, and pathway analysis in ovarian cancer cells","pmids":["38898019"],"confidence":"Medium","gaps":["Direct CSTF3–NEAT1 binding site not structurally defined","Link to PI3K/AKT/mTOR is correlative"]},{"year":2026,"claim":"Placing CSTF3 downstream of a signaling pathway: integrin α3β1–MEK/ERK induces CSTF3 to drive proximal PAS usage and APA of Mmp9 in vivo.","evidence":"epidermis-specific α3 knockout mice, in situ hybridization, CSTF3 knockdown, and genome-wide DaPars2 APA analysis","pmids":["41628695"],"confidence":"Medium","gaps":["Transcriptional mechanism linking MEK/ERK to CSTF3 induction unknown","Direct vs indirect control of Mmp9 PAS choice not separated"]},{"year":null,"claim":"How CSTF3 expression levels are translated into selective, gene-specific alternative polyadenylation decisions across the transcriptome remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of the assembled trimeric or RNA-bound CstF complex","Determinants of target poly(A) site selectivity unknown","Integration of upstream signaling, autoregulation, and APA output not mechanistically unified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[5,4]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[8,4]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,3,4]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4,7]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,5,6]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[5,6,9]}],"complexes":["CstF complex"],"partners":["CSTF2","CSTF1","SYMPK"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q12996","full_name":"Cleavage stimulation factor subunit 3","aliases":["CF-1 77 kDa subunit","Cleavage stimulation factor 77 kDa subunit","CSTF 77 kDa subunit","CstF-77"],"length_aa":717,"mass_kda":82.9,"function":"One of the multiple factors required for polyadenylation and 3'-end cleavage of mammalian pre-mRNAs","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q12996/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CSTF3","classification":"Common Essential","n_dependent_lines":1192,"n_total_lines":1208,"dependency_fraction":0.9867549668874173},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CPSF6","stoichiometry":0.2},{"gene":"RBM14","stoichiometry":0.2},{"gene":"TOP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CSTF3","total_profiled":1310},"omim":[{"mim_id":"612297","title":"CHROMOSOME 11 OPEN READING FRAME 41; C11ORF41","url":"https://www.omim.org/entry/612297"},{"mim_id":"612294","title":"DEP DOMAIN-CONTAINING PROTEIN 7; DEPDC7","url":"https://www.omim.org/entry/612294"},{"mim_id":"606027","title":"CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR 1; CPSF1","url":"https://www.omim.org/entry/606027"},{"mim_id":"602388","title":"SYMPLEKIN; SYMPK","url":"https://www.omim.org/entry/602388"},{"mim_id":"600369","title":"CLEAVAGE STIMULATION FACTOR, 3-PRIME PRE-RNA, SUBUNIT 1, 50-KD; CSTF1","url":"https://www.omim.org/entry/600369"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CSTF3"},"hgnc":{"alias_symbol":["CstF-77"],"prev_symbol":[]},"alphafold":{"accession":"Q12996","domains":[{"cath_id":"1.25.40.10","chopping":"21-226","consensus_level":"medium","plddt":96.7687,"start":21,"end":226},{"cath_id":"1.25.40.10","chopping":"327-556","consensus_level":"medium","plddt":92.627,"start":327,"end":556},{"cath_id":"1.20.58","chopping":"245-326","consensus_level":"medium","plddt":97.9872,"start":245,"end":326}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q12996","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q12996-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q12996-F1-predicted_aligned_error_v6.png","plddt_mean":86.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CSTF3","jax_strain_url":"https://www.jax.org/strain/search?query=CSTF3"},"sequence":{"accession":"Q12996","fasta_url":"https://rest.uniprot.org/uniprotkb/Q12996.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q12996/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q12996"}},"corpus_meta":[{"pmid":"17386263","id":"PMC_17386263","title":"Crystal structure of murine CstF-77: dimeric association and implications for polyadenylation of mRNA precursors.","date":"2007","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/17386263","citation_count":76,"is_preprint":false},{"pmid":"21119002","id":"PMC_21119002","title":"Interactions of CstF-64, CstF-77, and symplekin: implications on localisation and function.","date":"2010","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/21119002","citation_count":52,"is_preprint":false},{"pmid":"23874216","id":"PMC_23874216","title":"The conserved intronic cleavage and polyadenylation site of CstF-77 gene imparts control of 3' end processing activity through feedback autoregulation and by U1 snRNP.","date":"2013","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/23874216","citation_count":47,"is_preprint":false},{"pmid":"17584787","id":"PMC_17584787","title":"The structure of the CstF-77 homodimer provides insights into CstF assembly.","date":"2007","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/17584787","citation_count":42,"is_preprint":false},{"pmid":"16316725","id":"PMC_16316725","title":"An intronic polyadenylation site in human and mouse CstF-77 genes suggests an evolutionarily conserved regulatory mechanism.","date":"2005","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/16316725","citation_count":35,"is_preprint":false},{"pmid":"30257008","id":"PMC_30257008","title":"The structural basis of CstF-77 modulation of cleavage and polyadenylation through stimulation of CstF-64 activity.","date":"2018","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/30257008","citation_count":27,"is_preprint":false},{"pmid":"19887456","id":"PMC_19887456","title":"The hinge domain of the cleavage stimulation factor protein CstF-64 is essential for CstF-77 interaction, nuclear localization, and polyadenylation.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19887456","citation_count":24,"is_preprint":false},{"pmid":"38898019","id":"PMC_38898019","title":"CSTF3 contributes to platinum resistance in ovarian cancer through alternative polyadenylation of lncRNA NEAT1 and generating the short isoform NEAT1_1.","date":"2024","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/38898019","citation_count":19,"is_preprint":false},{"pmid":"12149458","id":"PMC_12149458","title":"Chimeric human CstF-77/Drosophila Suppressor of forked proteins rescue suppressor of forked mutant lethality and mRNA 3' end processing in Drosophila.","date":"2002","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/12149458","citation_count":17,"is_preprint":false},{"pmid":"17277459","id":"PMC_17277459","title":"The use of in situ proteolysis in the crystallization of murine CstF-77.","date":"2007","source":"Acta crystallographica. Section F, Structural biology and crystallization communications","url":"https://pubmed.ncbi.nlm.nih.gov/17277459","citation_count":16,"is_preprint":false},{"pmid":"16882666","id":"PMC_16882666","title":"Cytoplasmic CstF-77 protein belongs to a masking complex with cytoplasmic polyadenylation element-binding protein in Xenopus oocytes.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16882666","citation_count":12,"is_preprint":false},{"pmid":"41628695","id":"PMC_41628695","title":"An integrin α3β1-CSTF3 signaling axis regulates alternative polyadenylation of Mmp9 mRNA.","date":"2026","source":"Matrix biology : journal of the International Society for Matrix Biology","url":"https://pubmed.ncbi.nlm.nih.gov/41628695","citation_count":0,"is_preprint":false},{"pmid":"42108690","id":"PMC_42108690","title":"Inflammatory Myofibroblastic Tumor of the Breast Mimicking Complex Fibrocystic Changes, With a Novel CSTF3::ALK Fusion.","date":"2026","source":"International journal of surgical pathology","url":"https://pubmed.ncbi.nlm.nih.gov/42108690","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7859,"output_tokens":3110,"usd":0.035113,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10502,"output_tokens":3113,"usd":0.065168,"stage2_stop_reason":"end_turn"},"total_usd":0.100281,"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\": 2007,\n      \"finding\": \"Crystal structure of the HAT (half a TPR) domain of murine CstF-77 revealed two subdomains (HAT-N and HAT-C) with drastically different helical orientations, forming a highly elongated homodimer spanning 165 Å mediated by the HAT-C domain. Light-scattering, yeast two-hybrid, and analytical ultracentrifugation confirmed this self-association, supporting a role for CstF dimerization in pre-mRNA 3' end processing.\",\n      \"method\": \"X-ray crystallography, light scattering, yeast two-hybrid, analytical ultracentrifugation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure resolved with multiple orthogonal biochemical validations (light scattering, yeast two-hybrid, AUC) in a single rigorous study\",\n      \"pmids\": [\"17386263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structure of CstF-77 from Encephalitozoon cuniculi at 2 Å resolution revealed 11 Half-a-TPR repeats defining two domains and a tight homodimer exposing phylogenetically conserved surface areas for interaction with protein partners. Mapping experiments identified the C-terminal region of Rna14p (yeast CstF-77 homologue) as the docking domain for Rna15p (yeast CstF-64 homologue).\",\n      \"method\": \"X-ray crystallography, domain mapping experiments\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure at 2 Å with biochemical domain mapping, independently replicating structural findings from PMID:17386263\",\n      \"pmids\": [\"17584787\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CstF-77 and symplekin bind mutually exclusively to the hinge domain of CstF-64. The nuclear accumulation of CstF-64 depends on its binding to CstF-77 (not symplekin), demonstrating that CstF-77 interaction is required for nuclear localization and maintenance of stoichiometric nuclear CstF complex levels.\",\n      \"method\": \"Mutant analysis (CstF-64 and symplekin mutants), nuclear localization assays, functional complementation\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal mutant analysis with two orthogonal functional readouts (localization and processing activity) in a single lab\",\n      \"pmids\": [\"21119002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The hinge domain of CstF-64 is essential for interaction with CstF-77 and consequent nuclear localization of CstF-64, demonstrating that nuclear import of a preformed CstF complex is an essential step in polyadenylation.\",\n      \"method\": \"In vivo SLAP (stem-loop luciferase assay for polyadenylation), domain deletion/mutation analysis, nuclear localization assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — validated in vivo functional assay with structure-function analysis, replicated across multiple domain mutations, consistent with PMID:21119002\",\n      \"pmids\": [\"19887456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The carboxy-terminus of CstF-77 (monkeytail-carboxy-terminal domain, last 30 amino acids) enhances cleavage/polyadenylation by increasing the stability of the RNA recognition motif (RRM) of CstF-64, thereby altering the affinity of the complex for RNA. CstF-64 relies on CstF-77 for nuclear transport; excess CstF-64 localizes to the cytoplasm, possibly via interaction with cytoplasmic RNAs.\",\n      \"method\": \"Reverse genetics, NMR spectroscopy of recombinant proteins (CstF-64 RRM-Hinge and CstF-77 monkeytail-CTD), nuclear localization assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structural data combined with reverse genetics and localization assays, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"30257008\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Drosophila Su(f) (CstF-77 homologue) is required for pre-mRNA cleavage during mRNA 3' end formation in vivo. Chimeric human CstF-77/Su(f) proteins rescue lethality and cleavage defects in su(f) mutants, but a domain in human CstF-77 limiting for rescue is incapable of reproducing protein interactions with Drosophila CstF subunits. Chimeric proteins rescuing lethality cannot restore utilization of a regulated poly(A) site, indicating CstF-77 has an additional role in poly(A) site regulation.\",\n      \"method\": \"Genetic complementation in Drosophila su(f) mutants, chimeric protein rescue assays, mRNA 3' end processing analysis in vivo\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with in vivo functional rescue and multiple chimeric constructs tested, clear mechanistic dissection of domains\",\n      \"pmids\": [\"12149458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The CstF-77 gene contains a conserved intronic polyadenylation site (In3 pA) whose usage is responsive to CstF-77 expression levels and several other C/P factors, establishing a negative feedback autoregulatory mechanism. U1 snRNP inhibition also regulates In3 pA usage. Perturbation of CstF-77 expression leads to widespread alternative cleavage and polyadenylation (APA) and disturbance of cell proliferation and differentiation.\",\n      \"method\": \"Molecular biology validation of intronic poly(A) site, expression manipulation (overexpression/knockdown), U1 snRNP inhibition, global APA analysis\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple molecular methods in single lab; feedback mechanism established by expression perturbation and correlative analysis\",\n      \"pmids\": [\"23874216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"In Xenopus oocytes, CstF-77 (X77K) localizes mainly to the nucleus but also to punctate cytoplasmic foci, where it resides in a cytoplasmic complex with eIF4E, CPEB, CPSF-100, and XGLD2. X77K is not required for cytoplasmic polyadenylation per se, but impairment of X77K function accelerates the G2/M transition with premature synthesis of Mos and AuroraA proteins. X77K represses mRNA translation in vitro, suggesting a role in mRNA masking prior to polyadenylation.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, in vivo function impairment (dominant negative/antibody injection), in vitro translation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP identifies complex, functional localization and cell cycle phenotype established, but single lab and some mechanistic aspects remain indirect\",\n      \"pmids\": [\"16882666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CSTF3 directly binds downstream of the NEAT1 proximal polyadenylation site to promote usage of the proximal PAS, generating the short NEAT1_1 isoform. CSTF3 knockdown reduces proximal PAS usage, shifting expression toward the longer NEAT1_2 isoform and increasing platinum sensitivity in ovarian cancer cells. NEAT1_1 overexpression reverses platinum resistance after CSTF3 knockdown, and CSTF3/NEAT1_1 activity is linked to activation of the PI3K/AKT/mTOR pathway.\",\n      \"method\": \"CSTF3 knockdown in cell lines, RNA isoform analysis, lncRNA isoform-specific overexpression/knockdown, pathway activity measurement\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional knockdown with specific isoform rescue experiment; single lab, mechanistic link to PI3K/AKT/mTOR is correlative\",\n      \"pmids\": [\"38898019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"α3β1 integrin-MEK/ERK signaling induces CSTF3 expression in keratinocytes to promote proximal polyadenylation site usage in the Mmp9 gene, generating a short, more stable Mmp9 mRNA. CSTF3 knockdown shifts Mmp9 toward distal PAS usage. α3 deletion reduced Cstf3 gene expression and altered APA genome-wide in vivo.\",\n      \"method\": \"Inducible epidermis-specific α3 knockout mice, RNA in situ hybridization, CSTF3 knockdown, DaPars2 genome-wide APA analysis\",\n      \"journal\": \"Matrix biology : journal of the International Society for Matrix Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic model with genome-wide APA analysis and targeted knockdown; single lab, pathway placement supported by multiple methods\",\n      \"pmids\": [\"41628695\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Human and mouse CstF-77 genes contain an intronic polyadenylation site that produces short CstF-77 transcripts lacking sequences encoding domains involved in CstF-77 functions, analogous to the Drosophila su(f) intronic poly(A) site. The intronic poly(A) site is utilized across a wide range of tissues based on SAGE data validation.\",\n      \"method\": \"Bioinformatic identification with molecular biology experimental validation (RT-PCR, poly(A) site analysis), SAGE data analysis\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — molecular validation of intronic pA site but limited direct mechanistic functional data; primarily identification study\",\n      \"pmids\": [\"16316725\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CstF-77 (CSTF3) is the central scaffold subunit of the heterotrimeric CstF complex essential for pre-mRNA 3' cleavage and polyadenylation: its HAT domain forms an elongated homodimer that bridges CstF-64 and CstF-50 subunits, its C-terminal domain directly stabilizes the CstF-64 RRM to enhance RNA binding and cleavage/polyadenylation activity, and it is required for nuclear import of the CstF complex (CstF-64 nuclear localization depends on its hinge-domain interaction with CstF-77); additionally, CstF-77 expression is subject to negative feedback autoregulation through a conserved intronic polyadenylation site, and upstream signals such as integrin α3β1-MEK/ERK can regulate CSTF3 expression to orchestrate alternative polyadenylation of target genes including Mmp9.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CSTF3 (CstF-77) is the central scaffold subunit of the heterotrimeric cleavage stimulation factor (CstF) complex that drives pre-mRNA 3' end cleavage and polyadenylation [#0, #5]. Its HAT (half-a-TPR) domain folds into two subdomains (HAT-N and HAT-C) that assemble a highly elongated homodimer, with HAT-C mediating self-association and the conserved C-terminal region docking the CstF-64 subunit [#0, #1]. CstF-77 binds the hinge domain of CstF-64 mutually exclusively with symplekin, and this interaction is required for nuclear import of CstF-64 and maintenance of stoichiometric nuclear CstF complex levels, establishing nuclear assembly of the complex as an essential step in polyadenylation [#2, #3]. The CstF-77 carboxy-terminal \\\"monkeytail\\\" further enhances cleavage/polyadenylation by stabilizing the CstF-64 RNA recognition motif, thereby tuning the complex's affinity for RNA [#4]. CSTF3 levels govern alternative polyadenylation outcomes genome-wide and are themselves autoregulated by a conserved intronic poly(A) site that creates a negative feedback loop [#6]. Upstream integrin \\u03b13\\u03b21\\u2013MEK/ERK signaling induces CSTF3 expression to shift target genes toward proximal poly(A) site usage, generating short, stable isoforms of genes such as Mmp9 [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Establishing that CstF-77 is genuinely required for pre-mRNA cleavage in vivo and carries a separable role in regulated poly(A) site choice, beyond complex assembly.\",\n      \"evidence\": \"genetic complementation and chimeric human/Drosophila rescue assays in su(f) mutants\",\n      \"pmids\": [\"12149458\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Domain limiting cross-species rescue not molecularly defined\", \"Mechanism distinguishing constitutive cleavage from regulated poly(A) site choice unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Asking whether CstF-77 has functions outside nuclear 3' processing revealed a cytoplasmic complex and translational repression role linked to cell cycle timing.\",\n      \"evidence\": \"Co-IP, subcellular fractionation, and in vitro translation assays in Xenopus oocytes\",\n      \"pmids\": [\"16882666\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cytoplasmic complex partners (eIF4E, CPEB, CPSF-100, XGLD2) not validated reciprocally\", \"Direct molecular basis of translational repression unknown\", \"Relevance to somatic cells untested\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Resolving how CstF-77 organizes the complex: structures showed a HAT-domain homodimer that physically bridges partner subunits.\",\n      \"evidence\": \"X-ray crystallography of murine and E. cuniculi CstF-77 with light scattering, yeast two-hybrid, AUC, and domain mapping\",\n      \"pmids\": [\"17386263\", \"17584787\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full trimeric CstF architecture not visualized\", \"Functional consequence of dimerization for processing kinetics not quantified\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Determining how the complex enters the nucleus: the CstF-64 hinge\\u2013CstF-77 interaction was shown to be essential for nuclear import of a preformed CstF complex.\",\n      \"evidence\": \"in vivo SLAP polyadenylation assay with domain deletion and nuclear localization analysis\",\n      \"pmids\": [\"19887456\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear import machinery recognizing the complex not identified\", \"Order of complex assembly relative to import not fully resolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Clarifying competitive binding at the CstF-64 hinge: CstF-77 and symplekin bind mutually exclusively, with CstF-77 specifically required for nuclear accumulation.\",\n      \"evidence\": \"reciprocal mutant analysis with localization and processing readouts\",\n      \"pmids\": [\"21119002\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of the symplekin-bound pool not defined\", \"Regulation of the binding switch unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identifying how CSTF3 levels are buffered: a conserved intronic poly(A) site mediates negative feedback autoregulation that influences global APA, proliferation, and differentiation.\",\n      \"evidence\": \"intronic poly(A) site validation, expression perturbation, U1 snRNP inhibition, and global APA analysis\",\n      \"pmids\": [\"23874216\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Quantitative contribution of feedback to steady-state CSTF3 not measured\", \"Coupling between feedback and downstream phenotypes correlative\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defining the biochemical basis of CstF-77's stimulatory effect: its C-terminal monkeytail stabilizes the CstF-64 RRM to enhance RNA affinity and processing.\",\n      \"evidence\": \"NMR spectroscopy of recombinant CstF-64 RRM-Hinge and CstF-77 CTD with reverse genetics and localization assays\",\n      \"pmids\": [\"30257008\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural model of the CTD\\u2013RRM contact not fully resolved\", \"Effect on specific RNA sequence preferences not mapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connecting CSTF3 to disease-relevant isoform choice: it directly promotes proximal PAS usage on NEAT1, controlling lncRNA isoform output and platinum response.\",\n      \"evidence\": \"CSTF3 knockdown, isoform-specific manipulation, and pathway analysis in ovarian cancer cells\",\n      \"pmids\": [\"38898019\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct CSTF3\\u2013NEAT1 binding site not structurally defined\", \"Link to PI3K/AKT/mTOR is correlative\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Placing CSTF3 downstream of a signaling pathway: integrin \\u03b13\\u03b21\\u2013MEK/ERK induces CSTF3 to drive proximal PAS usage and APA of Mmp9 in vivo.\",\n      \"evidence\": \"epidermis-specific \\u03b13 knockout mice, in situ hybridization, CSTF3 knockdown, and genome-wide DaPars2 APA analysis\",\n      \"pmids\": [\"41628695\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcriptional mechanism linking MEK/ERK to CSTF3 induction unknown\", \"Direct vs indirect control of Mmp9 PAS choice not separated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CSTF3 expression levels are translated into selective, gene-specific alternative polyadenylation decisions across the transcriptome remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of the assembled trimeric or RNA-bound CstF complex\", \"Determinants of target poly(A) site selectivity unknown\", \"Integration of upstream signaling, autoregulation, and APA output not mechanistically unified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [5, 4]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [8, 4]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 3, 4]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 5, 6]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [5, 6, 9]}\n    ],\n    \"complexes\": [\"CstF complex\"],\n    \"partners\": [\"CSTF2\", \"CSTF1\", \"SYMPK\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}