{"gene":"CSTF2","run_date":"2026-04-28T17:28:53","timeline":{"discoveries":[{"year":1996,"finding":"CstF-64 is limiting for formation of intact CstF complex, CstF has higher affinity for the IgM μm poly(A) site than the μs site, and overexpression of CstF-64 is sufficient to switch IgM heavy chain expression from membrane-bound to secreted form in a reconstituted in vitro processing reaction.","method":"Reconstituted in vitro polyadenylation/processing assay, overexpression in B cells, gel-shift/affinity assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro, multiple orthogonal methods, foundational study with 355 citations","pmids":["8945520"],"is_preprint":false},{"year":1996,"finding":"CstF-64 and CPSF-100 are concentrated in discrete nuclear foci ('cleavage bodies') closely associated with coiled bodies; transcription inhibition causes complete co-localization of cleavage bodies with coiled bodies, indicating a transcription-dependent dynamic interaction.","method":"Immunofluorescence, monoclonal antibody labeling, electron microscopy immunogold double-labeling, transcription inhibition with α-amanitin/DRB","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — direct localization with functional consequence (transcription-dependence), electron microscopy confirmation","pmids":["8654386"],"is_preprint":false},{"year":1998,"finding":"Reducing CstF-64 concentration 10-fold specifically and dramatically reduces IgM heavy chain mRNA accumulation; further reduction causes reversible G0/G1 cell cycle arrest, and depletion causes apoptotic cell death, demonstrating CstF-64 plays roles in regulating gene expression and cell growth in B cells.","method":"Gene disruption and regulatable transgene replacement in DT40 B cell line; cell growth and cell cycle assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — clean genetic KO with defined cellular phenotypes, multiple orthogonal readouts","pmids":["9885564"],"is_preprint":false},{"year":2003,"finding":"The N-terminal RRM of CstF-64 recognizes GU-rich downstream elements; the C-terminal helix of the RRM unfolds upon RNA binding and extends into the hinge domain where interactions with other polyadenylation complex factors occur, suggesting this conformational change initiates polyadenylation complex assembly. UU dinucleotides are specifically recognized within an RRM pocket.","method":"NMR structure determination of CstF-64 RRM domain, RNA-binding assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — NMR structure with functional validation and mechanistic interpretation, 126 citations","pmids":["12773396"],"is_preprint":false},{"year":2005,"finding":"The protein-RNA interface of the CstF-64 RRM acquires significant mobility on the micro-to-millisecond timescale upon binding GU-rich RNA, while the free protein is uniformly rigid; this dynamic binding is proposed as the mechanism enabling discrimination between GU-rich and non-GU-rich RNAs.","method":"NMR relaxation dynamics experiments of free and RNA-bound CstF-64 RRM","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — NMR with multiple RNA substrates, mechanistic interpretation supported by structural data","pmids":["15769465"],"is_preprint":false},{"year":2006,"finding":"The C-terminal domain of CstF-64 (and yeast ortholog Rna15) folds into a three-helix bundle with an uncommon topological arrangement; a cluster of conserved exposed residues is essential for interaction with Pcf11 (yeast), and this interaction is critical for 3'-end processing but dispensable for transcription termination.","method":"NMR structure determination, mutagenesis, yeast functional assays (3'-end processing and transcription termination)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — NMR structure plus mutagenesis with functional validation in two distinct assays","pmids":["17116658"],"is_preprint":false},{"year":2009,"finding":"The hinge domain of CstF-64 is essential for interaction with CstF-77; this interaction is required for nuclear localization of CstF-64, suggesting that nuclear import of a preformed CstF complex is an essential step in polyadenylation.","method":"SLAP (stem-loop luciferase assay for polyadenylation) in vivo assay, domain mutagenesis, nuclear/cytoplasmic fractionation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — in vivo functional assay plus localization, multiple domain mutants tested","pmids":["19887456"],"is_preprint":false},{"year":2009,"finding":"Enterovirus 71 3Cpro cleaves CstF-64 at position 251 in the P/G-rich domain and at multiple sites near position 500 in the C-terminus; this cleavage inhibits host cell 3'-end pre-mRNA processing and polyadenylation, and this impairment is rescued by adding purified recombinant CstF-64.","method":"In vitro cleavage assay with wild-type and catalytic mutant 3Cpro, serial mutagenesis of CstF-64, in vitro polyadenylation assay with nuclear extracts, rescue with purified CstF-64","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 — in vitro cleavage reconstitution, mutagenesis, and rescue experiment with orthogonal methods","pmids":["19779565"],"is_preprint":false},{"year":2009,"finding":"CstF-64 interaction with CstF-77 is required for nuclear accumulation of CstF-64, whereas interaction with symplekin is limiting for histone RNA 3' processing but relatively unimportant for cleavage/polyadenylation; CstF-64 and symplekin bind mutually exclusively to the hinge domain.","method":"Identification of CstF-64 and symplekin mutants that distinguish these interactions; nuclear localization assays; histone 3' processing assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal mutant analysis with multiple functional readouts distinguishing two interactions","pmids":["21119002"],"is_preprint":false},{"year":2000,"finding":"A variant form of CstF-64 (tauCstF-64, encoded by autosomal Cstf2t on chromosome 19) is expressed specifically in meiotic and postmeiotic male germ cells; it contains a Pro→Ser substitution in the RNA-binding domain and significant changes in the CstF-77 interaction region, suggesting altered polyadenylation specificities.","method":"cDNA cloning, chromosomal mapping, immunoblot with antibody reactivity and proteolytic digest pattern comparison","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — cloning and characterization with functional domain implications but no direct polyadenylation assay in this paper","pmids":["11113135"],"is_preprint":false},{"year":2007,"finding":"CstF-64 and tauCstF-64 RNA-binding domains show differential affinities for RNA polymers: CstF-64 has higher affinity for poly(U) while tauCstF-64 has higher affinity for poly(GU); the region C-terminal to the RRM contributes to RNA sequence recognition.","method":"RNA cross-linking assay with Kd quantification, site-directed mutagenesis of the RRM","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 — biochemical Kd measurements with mutagenesis, single lab","pmids":["17029590"],"is_preprint":false},{"year":2001,"finding":"In C. elegans, CstF-64 forms a complex with the SL2 snRNP (but not SL1 or other U snRNAs); SL2 RNA stem/loop III is required for both SL2 identity and complex formation with CstF-64, providing a molecular framework for coupling of 3' end formation and trans-splicing in polycistronic pre-mRNA processing.","method":"Immunoprecipitation with anti-CstF-64 antibody, SL2 RNA mutational analysis in vivo and in vitro","journal":"Genes & development","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal IP combined with mutational analysis, replicated in vivo and in vitro","pmids":["11581161"],"is_preprint":false},{"year":2001,"finding":"Elevated levels of CstF-64 in male germ cells enhance selection of the proximal poly(A) site on TB-RBP pre-mRNA, increasing the 1 kb mRNA isoform; CstF-64 preferentially binds to a distal site that produces the 3 kb mRNA, and overexpression shifts poly(A) site selection toward the 1 kb form.","method":"RNA cross-linking/binding assay, overexpression experiment with isoform quantification","journal":"Molecular reproduction and development","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding assay plus functional overexpression result with defined molecular readout","pmids":["11241784"],"is_preprint":false},{"year":2014,"finding":"CstF-64 is required for correct histone mRNA 3' end processing in mouse embryonic stem cells; loss of CstF-64 results in increased polyadenylation of histone mRNAs, slower growth, loss of pluripotency, and lengthened G1 phase.","method":"CstF-64 knockout mouse ESCs, histone mRNA polyadenylation assay, cell cycle analysis, pluripotency marker assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 — clean genetic KO with multiple orthogonal phenotypic readouts including direct molecular assay of histone mRNA polyadenylation","pmids":["24957598"],"is_preprint":false},{"year":2014,"finding":"CstF-64 is essential for endoderm differentiation in mouse ESCs; loss of CstF-64 abolishes endodermal lineage differentiation and prevents cardiomyocyte formation, which can be rescued by conditioned medium from extraembryonic endodermal stem cells.","method":"CstF-64 knockout mouse ESCs, lineage marker analysis, cardiomyocyte differentiation assay, XEN cell conditioned medium rescue experiment","journal":"Stem cell research","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO with rescue experiment; mechanism downstream of endoderm specification not fully resolved","pmids":["25460602"],"is_preprint":false},{"year":2018,"finding":"The carboxy-terminus of CstF-77 (last 30 amino acids) enhances cleavage/polyadenylation by increasing the stability of the CstF-64 RRM, thereby altering the affinity of the complex for RNA; excess CstF-64 not bound to CstF-77 localizes to the cytoplasm, potentially via interaction with cytoplasmic RNAs.","method":"Reverse genetics, NMR studies of recombinant CstF-64 RRM-Hinge and CstF-77 domains, nuclear/cytoplasmic localization assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — NMR structural analysis plus functional genetics, multiple orthogonal methods","pmids":["30257008"],"is_preprint":false},{"year":2018,"finding":"CSTF2 induces 3'UTR shortening of RAC1 by cotranscriptional recruitment to the GUAAU motif at the proximal polyadenylation site, which attenuates recruitment of transcription elongation factors AFF1 and AFF4, causing defects in transcriptional elongation and promoting use of the proximal poly(A) site.","method":"RNA-seq, ChIP, RIP, CSTF2 overexpression/knockdown, polyadenylation site usage assays in UCB cells","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP and RIP combined with functional polyadenylation assays, but mechanism partly inferred from correlation","pmids":["30143523"],"is_preprint":false},{"year":2020,"finding":"A missense mutation in the CstF-64 RRM (p.D50A) reduces C/P efficiency by altering amino acid side chain positions, changing the electrostatic potential of the RRM and resulting in greater affinity for RNA; in mice, this mutation alters polyadenylation sites in over 1300 genes critical for brain development.","method":"Reporter gene C/P assay, NMR structural analysis of mutant RRM, mouse model with D50A knock-in, genome-wide poly(A) site analysis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — NMR structure, mutagenesis, in vivo mouse model, genome-wide functional readout","pmids":["32816001"],"is_preprint":false},{"year":2022,"finding":"Electrostatic attraction is the dominant factor in CstF-64 RRM binding to U-rich RNA; binding involves enthalpy-entropy compensation supported by changes in picosecond-to-nanosecond timescale dynamics; competition between fast, high-affinity RNA binding and efficient correct C/P exists in vivo.","method":"NMR spectroscopy, mutagenesis, biophysical assays (ITC/SPR), in vivo C/P assays","journal":"Biophysical journal","confidence":"High","confidence_rationale":"Tier 1 — NMR with mutagenesis and in vivo validation, multiple orthogonal methods","pmids":["35090899"],"is_preprint":false},{"year":2023,"finding":"CSTF2 co-transcriptionally regulates m6A installation by slowing RNA Pol II elongation rate during gene transcription; CSTF2-regulated m6As are recognized by IGF2BP2, an m6A reader that stabilizes mRNAs.","method":"Transcriptomic m6A profiling (MeRIP-seq) in PDAC tissues, CSTF2 manipulation with RNA Pol II elongation rate assays, IGF2BP2 RIP","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 — multiple molecular methods but m6A regulatory mechanism via transcription elongation partially correlative","pmids":["37816727"],"is_preprint":false},{"year":2024,"finding":"The CSTF2 RRM domain binds U-rich RNA through a multistep binding process involving differences in picosecond-to-nanosecond timescale dynamics and structural changes in the C-terminal α-helix.","method":"NMR titration, spin relaxation experiments, paramagnetic relaxation enhancement measurements, rigid-body docking","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — multiple NMR methods with structural characterization","pmids":["39305233"],"is_preprint":false},{"year":2025,"finding":"CSTF2 shortens the 3'UTR of PGK1 pre-mRNA by binding near the proximal polyadenylation site, causing loss of m6A modification sites; this prevents YTHDF2-mediated mRNA degradation and increases PGK1 protein to enhance glycolysis under hypoxia. YTHDC1 recognizes hypoxia-induced m6A near the proximal poly(A) site and recruits CSTF2 to enhance 3'UTR shortening.","method":"RIP, APA site usage assays, m6A mapping, CSTF2 knockdown/overexpression, xenograft models, patient-derived organoids, small-molecule screen","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — multiple molecular and in vivo methods but mechanism involves several sequential steps each supported by single-method evidence","pmids":["39514400"],"is_preprint":false},{"year":2025,"finding":"CSTF2 promotes PolyA polymerase alpha (PAPα) binding to the 3'UTR of CXCL10 RNA, resulting in shortened poly(A) tails and reduced CXCL10 mRNA stability; this diminishes CXCL10-mediated recruitment of innate αβ T cells, suppressing anti-tumor immunity in PDAC.","method":"RIP, poly(A) tail length assay, CSTF2 knockdown, CXCL10 mRNA stability assay, tumor infiltration analysis","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods but mechanism partly inferred; single lab study","pmids":["39972059"],"is_preprint":false},{"year":2022,"finding":"CSTF2 promotes 3'UTR shortening and upregulation of FGF2 mRNA by inducing use of the proximal polyadenylation site, stabilizing FGF2 mRNA through miRNA evasion; FGF2 in turn enhances CSTF2 expression forming a positive feedback loop that drives epithelial-mesenchymal transition in tubular epithelial cells.","method":"CSTF2 knockdown/overexpression, APA site usage assays, mRNA stability assay, in vivo UUO mouse model, antisense oligonucleotide treatment","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo and in vitro evidence with APA mechanistic assays, but feedback loop mechanism relies on correlative FGF2 upregulation data","pmids":["36113752"],"is_preprint":false}],"current_model":"CSTF2 (CstF-64) is an RNA-binding protein whose N-terminal RRM domain recognizes GU-rich downstream sequence elements of pre-mRNAs through electrostatic and dynamic interactions, with its C-terminal helix undergoing conformational change upon RNA binding to initiate polyadenylation complex assembly; the hinge domain mediates mutually exclusive binding to CstF-77 (required for nuclear localization and polyadenylation) or symplekin (required for histone 3' processing); cellular levels of CstF-64 are rate-limiting for CstF complex formation and directly regulate poly(A) site selection during IgM switching and other alternative polyadenylation events, while also co-transcriptionally influencing RNA Pol II elongation rate to affect m6A modification and 3'UTR length of target mRNAs."},"narrative":{"teleology":[{"year":1996,"claim":"Establishing that CstF-64 is the rate-limiting factor for CstF assembly and APA-mediated immunoglobulin class switching resolved how a stoichiometric change in a single polyadenylation factor could redirect gene expression.","evidence":"Reconstituted in vitro polyadenylation with overexpression in B cells and gel-shift affinity measurements","pmids":["8945520"],"confidence":"High","gaps":["Mechanism by which CstF-64 levels are themselves regulated during B-cell activation","Whether other APA targets respond similarly to CstF-64 dosage"]},{"year":1996,"claim":"Showing that CstF-64 concentrates in transcription-dependent nuclear 'cleavage bodies' linked its function to active transcription sites rather than a diffuse nuclear pool.","evidence":"Immunofluorescence, immunogold EM, and transcription inhibition with α-amanitin/DRB","pmids":["8654386"],"confidence":"High","gaps":["Identity of signals that recruit CstF-64 to cleavage bodies","Whether cleavage body localization is required for processing efficiency"]},{"year":1998,"claim":"Genetic depletion of CstF-64 in DT40 cells demonstrated that it is essential for viability, with graded reduction causing IgM mRNA loss, G0/G1 arrest, and apoptosis — proving CstF-64 is not merely a polyadenylation accessory but a cell-growth regulator.","evidence":"Regulatable transgene replacement in DT40 chicken B cells with cell-cycle and viability assays","pmids":["9885564"],"confidence":"High","gaps":["Which specific mRNA targets mediate the cell-cycle arrest phenotype","Whether the growth arrest is solely APA-dependent or involves additional functions"]},{"year":2003,"claim":"Solving the NMR structure of the CstF-64 RRM revealed that UU dinucleotide recognition occurs within a defined pocket and that the C-terminal α-helix unfolds upon RNA binding to expose the hinge domain, providing the first structural model for how RNA recognition triggers polyadenylation complex assembly.","evidence":"NMR structure determination of CstF-64 RRM with RNA-binding assays","pmids":["12773396"],"confidence":"High","gaps":["Structure of the full-length protein or CstF holocomplex","How the conformational change is transmitted to downstream factors"]},{"year":2005,"claim":"NMR relaxation dynamics showed that the RNA-binding surface of the RRM becomes mobile on the μs–ms timescale upon binding GU-rich RNA, establishing that sequence discrimination relies on dynamic rather than static complementarity.","evidence":"NMR relaxation experiments comparing free and RNA-bound CstF-64 RRM with multiple RNA substrates","pmids":["15769465"],"confidence":"High","gaps":["Whether dynamics-based discrimination operates identically in the context of the full CstF complex","Thermodynamic decomposition of the selectivity mechanism"]},{"year":2006,"claim":"Determination of the C-terminal domain structure as a three-helix bundle and identification of its interaction with Pcf11 distinguished CstF-64's role in 3′-end cleavage from its dispensability in transcription termination.","evidence":"NMR structure of the CTD, mutagenesis, and yeast functional assays for processing vs. termination","pmids":["17116658"],"confidence":"High","gaps":["Whether the human ortholog uses the same surface for Pcf11/PCF11 interaction","Whether additional partners bind this domain"]},{"year":2009,"claim":"Mapping the hinge domain as the site of mutually exclusive binding to CstF-77 (for nuclear import and polyadenylation) or symplekin (for histone 3′ processing) defined how a single protein partitions between two distinct 3′-end processing pathways.","evidence":"Reciprocal separation-of-function mutants analyzed by SLAP assay, histone processing assay, and nuclear/cytoplasmic fractionation","pmids":["19887456","21119002"],"confidence":"High","gaps":["Whether switching between CstF-77 and symplekin is regulated by post-translational modification","Structural basis of mutual exclusivity"]},{"year":2009,"claim":"Demonstration that enterovirus 71 3Cpro cleaves CstF-64 at specific sites to shut down host polyadenylation — rescuable by recombinant CstF-64 — established CstF-64 as a viral target for host gene expression shutoff.","evidence":"In vitro cleavage with wild-type/mutant 3Cpro, serial mutagenesis, polyadenylation rescue assay","pmids":["19779565"],"confidence":"High","gaps":["Whether other viruses target CstF-64 by analogous mechanisms","In vivo relevance during natural infection"]},{"year":2014,"claim":"CstF-64 knockout in mouse ESCs revealed its requirement for proper histone mRNA 3′ processing, pluripotency maintenance, and endoderm differentiation, extending its biological role from constitutive RNA processing to developmental competence.","evidence":"CstF-64 KO ESCs with histone mRNA polyadenylation assay, cell-cycle analysis, lineage differentiation, and conditioned-medium rescue","pmids":["24957598","25460602"],"confidence":"High","gaps":["Which specific histone mRNA or APA changes drive the pluripotency loss","Whether tauCstF-64 can partially compensate in vivo"]},{"year":2020,"claim":"A D50A missense mutation in the RRM altered its electrostatic potential and RNA affinity, changing poly(A) site usage in >1300 genes critical for brain development in mice, directly linking RRM biophysics to genome-wide APA and organismal phenotype.","evidence":"NMR of mutant RRM, reporter C/P assay, D50A knock-in mouse with genome-wide poly(A) site profiling","pmids":["32816001"],"confidence":"High","gaps":["Precise neurological phenotype and behavioral consequences in the mouse model","Whether similar mutations occur in human neurodevelopmental disease"]},{"year":2022,"claim":"Biophysical dissection showed that electrostatic attraction dominates CstF-64 RRM–RNA binding with enthalpy–entropy compensation, and that a trade-off exists between high-affinity RNA binding and efficient cleavage/polyadenylation in vivo.","evidence":"NMR, ITC/SPR thermodynamics, mutagenesis, in vivo C/P assays","pmids":["35090899"],"confidence":"High","gaps":["How the affinity–efficiency trade-off is tuned under different physiological conditions","Role of post-translational modifications in modulating RRM electrostatics"]},{"year":2023,"claim":"CSTF2 was shown to co-transcriptionally influence m6A deposition by slowing RNA Pol II elongation, revealing a previously unrecognized link between polyadenylation factor binding and epitranscriptomic modification.","evidence":"MeRIP-seq in PDAC tissues, Pol II elongation rate assays, CSTF2 manipulation, IGF2BP2 RIP","pmids":["37816727"],"confidence":"Medium","gaps":["Whether elongation-rate modulation is a general property of CSTF2 or context-specific","Direct measurement of Pol II speed changes at single-gene resolution","Independent replication in non-cancer cells"]},{"year":2025,"claim":"Studies in pancreatic cancer and renal fibrosis showed CSTF2-driven 3′UTR shortening of specific mRNAs (PGK1, CXCL10, FGF2) alters their stability, m6A landscape, and downstream signaling, illustrating how CSTF2-mediated APA reprograms gene expression in disease contexts.","evidence":"RIP, APA assays, poly(A) tail length assays, knockdown/overexpression, xenografts, organoids, and UUO mouse model","pmids":["39514400","39972059","36113752"],"confidence":"Medium","gaps":["Whether CSTF2 is a direct therapeutic target or a downstream effector","Genome-wide specificity of CSTF2-mediated APA changes in these disease settings","Independent validation of the YTHDC1–CSTF2 recruitment axis"]},{"year":null,"claim":"No high-resolution structure of the full CstF complex or of CstF-64 bound simultaneously to RNA and a protein partner exists; the mechanisms that regulate CstF-64 protein levels, its post-translational modifications, and the logic by which it selects specific poly(A) sites genome-wide remain incompletely defined.","evidence":"","pmids":[],"confidence":"High","gaps":["Full CstF complex structure","Regulation of CstF-64 protein turnover","Genome-wide rules for poly(A) site selectivity by CstF-64 in vivo"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,3,4,10,17,18,20]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,12,16,19,21,22]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,8,13]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,6,8]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[15]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,3,6,7,8,13,17]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[16,19]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[2,13]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[14,17]}],"complexes":["CstF (cleavage stimulation factor)","symplekin–CPSF–CstF histone processing complex"],"partners":["CSTF3","SYMPK","PCF11","CPSF2","AFF1","AFF4","IGF2BP2","YTHDC1"],"other_free_text":[]},"mechanistic_narrative":"CSTF2 (CstF-64) is the RNA-binding subunit of the cleavage stimulation factor (CstF) complex and a central regulator of pre-mRNA 3′-end processing, alternative polyadenylation (APA), and replication-dependent histone mRNA 3′ maturation. Its N-terminal RRM domain recognizes GU/U-rich downstream sequence elements through an electrostatically driven, multistep binding mechanism in which the C-terminal α-helix of the RRM unfolds upon RNA engagement, propagating a conformational change into the hinge domain that nucleates polyadenylation complex assembly [PMID:12773396, PMID:35090899, PMID:39305233]. The hinge domain mediates mutually exclusive interactions with CstF-77 — required for nuclear import and canonical cleavage/polyadenylation — and with symplekin, which is essential for histone RNA 3′ processing; CstF-64 protein levels are rate-limiting for CstF complex formation, and altering its concentration directly shifts poly(A) site choice, as demonstrated by IgM heavy-chain switching, genome-wide APA changes in a mouse D50A knock-in, and 3′UTR shortening of specific transcripts that affects mRNA stability and m6A deposition [PMID:8945520, PMID:21119002, PMID:32816001, PMID:37816727]. Loss of CstF-64 causes aberrant polyadenylation of histone mRNAs, cell-cycle arrest, loss of pluripotency in embryonic stem cells, and failure of endoderm differentiation, establishing it as essential for both housekeeping RNA processing and developmental gene regulation [PMID:9885564, PMID:24957598, PMID:25460602]."},"prefetch_data":{"uniprot":{"accession":"P33240","full_name":"Cleavage stimulation factor subunit 2","aliases":["CF-1 64 kDa subunit","Cleavage stimulation factor 64 kDa subunit","CSTF 64 kDa subunit","CstF-64"],"length_aa":577,"mass_kda":61.0,"function":"One of the multiple factors required for polyadenylation and 3'-end cleavage of mammalian pre-mRNAs. This subunit is directly involved in the binding to pre-mRNAs","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P33240/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CSTF2","classification":"Not Classified","n_dependent_lines":70,"n_total_lines":1208,"dependency_fraction":0.057947019867549666},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CPSF6","stoichiometry":0.2},{"gene":"RBM14","stoichiometry":0.2},{"gene":"SNRPA","stoichiometry":0.2},{"gene":"SNRPB","stoichiometry":0.2},{"gene":"SNRPC","stoichiometry":0.2},{"gene":"SSRP1","stoichiometry":0.2},{"gene":"TOP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CSTF2","total_profiled":1310},"omim":[{"mim_id":"611968","title":"CLEAVAGE STIMULATION FACTOR, 3-PRIME PRE-RNA, SUBUNIT 2, 64-KD, TAU VARIANT; CSTF2T","url":"https://www.omim.org/entry/611968"},{"mim_id":"602388","title":"SYMPLEKIN; SYMPK","url":"https://www.omim.org/entry/602388"},{"mim_id":"301116","title":"INTELLECTUAL DEVELOPMENTAL DISORDER, X-LINKED 113; XLID113","url":"https://www.omim.org/entry/301116"},{"mim_id":"300907","title":"CLEAVAGE STIMULATION FACTOR, 3-PRIME PRE-RNA, SUBUNIT 2, 64-KD; CSTF2","url":"https://www.omim.org/entry/300907"},{"mim_id":"194355","title":"X BOX-BINDING PROTEIN 1; XBP1","url":"https://www.omim.org/entry/194355"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"},{"location":"Nuclear bodies","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CSTF2"},"hgnc":{"alias_symbol":["CstF-64"],"prev_symbol":[]},"alphafold":{"accession":"P33240","domains":[{"cath_id":"3.30.70.330","chopping":"8-103","consensus_level":"high","plddt":88.6825,"start":8,"end":103},{"cath_id":"-","chopping":"122-182","consensus_level":"high","plddt":87.4725,"start":122,"end":182},{"cath_id":"-","chopping":"535-573","consensus_level":"high","plddt":79.089,"start":535,"end":573}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P33240","model_url":"https://alphafold.ebi.ac.uk/files/AF-P33240-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P33240-F1-predicted_aligned_error_v6.png","plddt_mean":59.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CSTF2","jax_strain_url":"https://www.jax.org/strain/search?query=CSTF2"},"sequence":{"accession":"P33240","fasta_url":"https://rest.uniprot.org/uniprotkb/P33240.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P33240/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P33240"}},"corpus_meta":[{"pmid":"8945520","id":"PMC_8945520","title":"The polyadenylation factor CstF-64 regulates alternative processing of IgM heavy chain pre-mRNA during B cell differentiation.","date":"1996","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/8945520","citation_count":355,"is_preprint":false},{"pmid":"9885564","id":"PMC_9885564","title":"Levels of polyadenylation factor CstF-64 control IgM heavy chain mRNA accumulation and other events associated with B cell differentiation.","date":"1998","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/9885564","citation_count":193,"is_preprint":false},{"pmid":"12773396","id":"PMC_12773396","title":"Recognition of GU-rich polyadenylation regulatory elements by human CstF-64 protein.","date":"2003","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/12773396","citation_count":126,"is_preprint":false},{"pmid":"19779565","id":"PMC_19779565","title":"Enterovirus 71 3C protease cleaves a novel target CstF-64 and inhibits cellular polyadenylation.","date":"2009","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/19779565","citation_count":124,"is_preprint":false},{"pmid":"8654386","id":"PMC_8654386","title":"The RNA 3' cleavage factors CstF 64 kDa and CPSF 100 kDa are concentrated in nuclear domains closely associated with coiled bodies and newly synthesized RNA.","date":"1996","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/8654386","citation_count":84,"is_preprint":false},{"pmid":"15767428","id":"PMC_15767428","title":"A 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hFip1, CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein.","date":"2005","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/15767428","citation_count":62,"is_preprint":false},{"pmid":"30143523","id":"PMC_30143523","title":"CSTF2-Induced Shortening of the RAC1 3'UTR Promotes the Pathogenesis of Urothelial Carcinoma of the Bladder.","date":"2018","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/30143523","citation_count":60,"is_preprint":false},{"pmid":"15769465","id":"PMC_15769465","title":"Protein and RNA dynamics play key roles in determining the specific recognition of GU-rich polyadenylation regulatory elements by human Cstf-64 protein.","date":"2005","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/15769465","citation_count":57,"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":51,"is_preprint":false},{"pmid":"17116658","id":"PMC_17116658","title":"The C-terminal domains of vertebrate CstF-64 and its yeast orthologue Rna15 form a new structure critical for mRNA 3'-end processing.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17116658","citation_count":49,"is_preprint":false},{"pmid":"16542450","id":"PMC_16542450","title":"A multispecies comparison of the metazoan 3'-processing downstream elements and the CstF-64 RNA recognition motif.","date":"2006","source":"BMC genomics","url":"https://pubmed.ncbi.nlm.nih.gov/16542450","citation_count":42,"is_preprint":false},{"pmid":"11113135","id":"PMC_11113135","title":"The gene for a variant form of the polyadenylation protein CstF-64 is on chromosome 19 and is expressed in pachytene spermatocytes in mice.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11113135","citation_count":39,"is_preprint":false},{"pmid":"21813631","id":"PMC_21813631","title":"Characterization of a cleavage stimulation factor, 3' pre-RNA, subunit 2, 64 kDa (CSTF2) as a therapeutic target for lung cancer.","date":"2011","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/21813631","citation_count":37,"is_preprint":false},{"pmid":"11581161","id":"PMC_11581161","title":"A complex containing CstF-64 and the SL2 snRNP connects mRNA 3' end formation and trans-splicing in C. elegans operons.","date":"2001","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/11581161","citation_count":34,"is_preprint":false},{"pmid":"11369601","id":"PMC_11369601","title":"Overexpression of the CstF-64 and CPSF-160 polyadenylation protein messenger RNAs in mouse male germ cells.","date":"2001","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/11369601","citation_count":28,"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":"14681198","id":"PMC_14681198","title":"Developmental distribution of the polyadenylation protein CstF-64 and the variant tauCstF-64 in mouse and rat testis.","date":"2003","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/14681198","citation_count":26,"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":"19284619","id":"PMC_19284619","title":"A family of splice variants of CstF-64 expressed in vertebrate nervous systems.","date":"2009","source":"BMC molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/19284619","citation_count":22,"is_preprint":false},{"pmid":"12408968","id":"PMC_12408968","title":"The gene CSTF2T, encoding the human variant CstF-64 polyadenylation protein tauCstF-64, lacks introns and may be associated with male sterility.","date":"2002","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/12408968","citation_count":22,"is_preprint":false},{"pmid":"37816727","id":"PMC_37816727","title":"CSTF2 mediated mRNA N6-methyladenosine modification drives pancreatic ductal adenocarcinoma m6A subtypes.","date":"2023","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/37816727","citation_count":20,"is_preprint":false},{"pmid":"11241784","id":"PMC_11241784","title":"Elevated levels of the polyadenylation factor CstF 64 enhance formation of the 1kB Testis brain RNA-binding protein (TB-RBP) mRNA in male germ cells.","date":"2001","source":"Molecular reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/11241784","citation_count":19,"is_preprint":false},{"pmid":"24957598","id":"PMC_24957598","title":"CstF-64 supports pluripotency and regulates cell cycle progression in embryonic stem cells through histone 3' end processing.","date":"2014","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/24957598","citation_count":18,"is_preprint":false},{"pmid":"17029590","id":"PMC_17029590","title":"Polyadenylation proteins CstF-64 and tauCstF-64 exhibit differential binding affinities for RNA polymers.","date":"2007","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/17029590","citation_count":18,"is_preprint":false},{"pmid":"32816001","id":"PMC_32816001","title":"A missense mutation in the CSTF2 gene that impairs the function of the RNA recognition motif and causes defects in 3' end processing is associated with intellectual disability in humans.","date":"2020","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/32816001","citation_count":17,"is_preprint":false},{"pmid":"35875122","id":"PMC_35875122","title":"CSTF2 Promotes Hepatocarcinogenesis and Hepatocellular Carcinoma Progression via Aerobic Glycolysis.","date":"2022","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/35875122","citation_count":13,"is_preprint":false},{"pmid":"25460602","id":"PMC_25460602","title":"CstF-64 is necessary for endoderm differentiation resulting in cardiomyocyte defects.","date":"2014","source":"Stem cell research","url":"https://pubmed.ncbi.nlm.nih.gov/25460602","citation_count":13,"is_preprint":false},{"pmid":"24590791","id":"PMC_24590791","title":"High-throughput sequencing of RNA isolated by cross-linking and immunoprecipitation (HITS-CLIP) to determine sites of binding of CstF-64 on nascent RNAs.","date":"2014","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/24590791","citation_count":9,"is_preprint":false},{"pmid":"39514400","id":"PMC_39514400","title":"CSTF2 Supports Hypoxia Tolerance in Hepatocellular Carcinoma by Enabling m6A Modification Evasion of PGK1 to Enhance Glycolysis.","date":"2025","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/39514400","citation_count":8,"is_preprint":false},{"pmid":"36521710","id":"PMC_36521710","title":"The RNA-binding protein CSTF2 regulates BAD to inhibit apoptosis in glioblastoma.","date":"2022","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/36521710","citation_count":7,"is_preprint":false},{"pmid":"36113752","id":"PMC_36113752","title":"Alternative polyadenylation writer CSTF2 forms a positive loop with FGF2 to promote tubular epithelial-mesenchymal transition and renal fibrosis.","date":"2022","source":"Biochimica et biophysica acta. Molecular basis of disease","url":"https://pubmed.ncbi.nlm.nih.gov/36113752","citation_count":7,"is_preprint":false},{"pmid":"35090899","id":"PMC_35090899","title":"Electrostatic Interactions between CSTF2 and pre-mRNA Drive Cleavage and Polyadenylation.","date":"2022","source":"Biophysical journal","url":"https://pubmed.ncbi.nlm.nih.gov/35090899","citation_count":4,"is_preprint":false},{"pmid":"24590783","id":"PMC_24590783","title":"The stem-loop luciferase assay for polyadenylation (SLAP) method for determining CstF-64-dependent polyadenylation activity.","date":"2014","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/24590783","citation_count":3,"is_preprint":false},{"pmid":"38166492","id":"PMC_38166492","title":"The role of CSTF2 in cancer: from technology to clinical application.","date":"2024","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/38166492","citation_count":2,"is_preprint":false},{"pmid":"39972059","id":"PMC_39972059","title":"CSTF2-impeded innate αβ T cell infiltration and activation exacerbate immune evasion of pancreatic cancer.","date":"2025","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/39972059","citation_count":1,"is_preprint":false},{"pmid":"39722572","id":"PMC_39722572","title":"The host gene CSTF2 regulates HBV replication via HBV PRE-induced nuclear export.","date":"2024","source":"Acta biochimica et biophysica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/39722572","citation_count":0,"is_preprint":false},{"pmid":"39305233","id":"PMC_39305233","title":"Human CSTF2 RNA Recognition Motif Domain Binds to a U-Rich RNA Sequence through a Multistep Binding Process.","date":"2024","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/39305233","citation_count":0,"is_preprint":false},{"pmid":"41310899","id":"PMC_41310899","title":"The role of CSTF2 in gastric cancer: implications for therapy.","date":"2025","source":"European journal of medical research","url":"https://pubmed.ncbi.nlm.nih.gov/41310899","citation_count":0,"is_preprint":false},{"pmid":"38058835","id":"PMC_38058835","title":"Detection of CSTF2 by nano fluorescent probe and its correlation with malignant biological characteristics in liver cancer.","date":"2023","source":"American journal of cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/38058835","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":19762,"output_tokens":5454,"usd":0.070548},"stage2":{"model":"claude-opus-4-6","input_tokens":9043,"output_tokens":3451,"usd":0.197235},"total_usd":0.267783,"stage1_batch_id":"msgbatch_011kfMfE81o6CTcyhVUqumuW","stage2_batch_id":"msgbatch_01DTputS6WNkH745x1nVSoBo","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"CstF-64 is limiting for formation of intact CstF complex, CstF has higher affinity for the IgM μm poly(A) site than the μs site, and overexpression of CstF-64 is sufficient to switch IgM heavy chain expression from membrane-bound to secreted form in a reconstituted in vitro processing reaction.\",\n      \"method\": \"Reconstituted in vitro polyadenylation/processing assay, overexpression in B cells, gel-shift/affinity assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro, multiple orthogonal methods, foundational study with 355 citations\",\n      \"pmids\": [\"8945520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"CstF-64 and CPSF-100 are concentrated in discrete nuclear foci ('cleavage bodies') closely associated with coiled bodies; transcription inhibition causes complete co-localization of cleavage bodies with coiled bodies, indicating a transcription-dependent dynamic interaction.\",\n      \"method\": \"Immunofluorescence, monoclonal antibody labeling, electron microscopy immunogold double-labeling, transcription inhibition with α-amanitin/DRB\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization with functional consequence (transcription-dependence), electron microscopy confirmation\",\n      \"pmids\": [\"8654386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Reducing CstF-64 concentration 10-fold specifically and dramatically reduces IgM heavy chain mRNA accumulation; further reduction causes reversible G0/G1 cell cycle arrest, and depletion causes apoptotic cell death, demonstrating CstF-64 plays roles in regulating gene expression and cell growth in B cells.\",\n      \"method\": \"Gene disruption and regulatable transgene replacement in DT40 B cell line; cell growth and cell cycle assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO with defined cellular phenotypes, multiple orthogonal readouts\",\n      \"pmids\": [\"9885564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The N-terminal RRM of CstF-64 recognizes GU-rich downstream elements; the C-terminal helix of the RRM unfolds upon RNA binding and extends into the hinge domain where interactions with other polyadenylation complex factors occur, suggesting this conformational change initiates polyadenylation complex assembly. UU dinucleotides are specifically recognized within an RRM pocket.\",\n      \"method\": \"NMR structure determination of CstF-64 RRM domain, RNA-binding assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with functional validation and mechanistic interpretation, 126 citations\",\n      \"pmids\": [\"12773396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The protein-RNA interface of the CstF-64 RRM acquires significant mobility on the micro-to-millisecond timescale upon binding GU-rich RNA, while the free protein is uniformly rigid; this dynamic binding is proposed as the mechanism enabling discrimination between GU-rich and non-GU-rich RNAs.\",\n      \"method\": \"NMR relaxation dynamics experiments of free and RNA-bound CstF-64 RRM\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR with multiple RNA substrates, mechanistic interpretation supported by structural data\",\n      \"pmids\": [\"15769465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The C-terminal domain of CstF-64 (and yeast ortholog Rna15) folds into a three-helix bundle with an uncommon topological arrangement; a cluster of conserved exposed residues is essential for interaction with Pcf11 (yeast), and this interaction is critical for 3'-end processing but dispensable for transcription termination.\",\n      \"method\": \"NMR structure determination, mutagenesis, yeast functional assays (3'-end processing and transcription termination)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure plus mutagenesis with functional validation in two distinct assays\",\n      \"pmids\": [\"17116658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The hinge domain of CstF-64 is essential for interaction with CstF-77; this interaction is required for nuclear localization of CstF-64, suggesting that nuclear import of a preformed CstF complex is an essential step in polyadenylation.\",\n      \"method\": \"SLAP (stem-loop luciferase assay for polyadenylation) in vivo assay, domain mutagenesis, nuclear/cytoplasmic fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo functional assay plus localization, multiple domain mutants tested\",\n      \"pmids\": [\"19887456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Enterovirus 71 3Cpro cleaves CstF-64 at position 251 in the P/G-rich domain and at multiple sites near position 500 in the C-terminus; this cleavage inhibits host cell 3'-end pre-mRNA processing and polyadenylation, and this impairment is rescued by adding purified recombinant CstF-64.\",\n      \"method\": \"In vitro cleavage assay with wild-type and catalytic mutant 3Cpro, serial mutagenesis of CstF-64, in vitro polyadenylation assay with nuclear extracts, rescue with purified CstF-64\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro cleavage reconstitution, mutagenesis, and rescue experiment with orthogonal methods\",\n      \"pmids\": [\"19779565\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CstF-64 interaction with CstF-77 is required for nuclear accumulation of CstF-64, whereas interaction with symplekin is limiting for histone RNA 3' processing but relatively unimportant for cleavage/polyadenylation; CstF-64 and symplekin bind mutually exclusively to the hinge domain.\",\n      \"method\": \"Identification of CstF-64 and symplekin mutants that distinguish these interactions; nuclear localization assays; histone 3' processing assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal mutant analysis with multiple functional readouts distinguishing two interactions\",\n      \"pmids\": [\"21119002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"A variant form of CstF-64 (tauCstF-64, encoded by autosomal Cstf2t on chromosome 19) is expressed specifically in meiotic and postmeiotic male germ cells; it contains a Pro→Ser substitution in the RNA-binding domain and significant changes in the CstF-77 interaction region, suggesting altered polyadenylation specificities.\",\n      \"method\": \"cDNA cloning, chromosomal mapping, immunoblot with antibody reactivity and proteolytic digest pattern comparison\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cloning and characterization with functional domain implications but no direct polyadenylation assay in this paper\",\n      \"pmids\": [\"11113135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CstF-64 and tauCstF-64 RNA-binding domains show differential affinities for RNA polymers: CstF-64 has higher affinity for poly(U) while tauCstF-64 has higher affinity for poly(GU); the region C-terminal to the RRM contributes to RNA sequence recognition.\",\n      \"method\": \"RNA cross-linking assay with Kd quantification, site-directed mutagenesis of the RRM\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — biochemical Kd measurements with mutagenesis, single lab\",\n      \"pmids\": [\"17029590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"In C. elegans, CstF-64 forms a complex with the SL2 snRNP (but not SL1 or other U snRNAs); SL2 RNA stem/loop III is required for both SL2 identity and complex formation with CstF-64, providing a molecular framework for coupling of 3' end formation and trans-splicing in polycistronic pre-mRNA processing.\",\n      \"method\": \"Immunoprecipitation with anti-CstF-64 antibody, SL2 RNA mutational analysis in vivo and in vitro\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal IP combined with mutational analysis, replicated in vivo and in vitro\",\n      \"pmids\": [\"11581161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Elevated levels of CstF-64 in male germ cells enhance selection of the proximal poly(A) site on TB-RBP pre-mRNA, increasing the 1 kb mRNA isoform; CstF-64 preferentially binds to a distal site that produces the 3 kb mRNA, and overexpression shifts poly(A) site selection toward the 1 kb form.\",\n      \"method\": \"RNA cross-linking/binding assay, overexpression experiment with isoform quantification\",\n      \"journal\": \"Molecular reproduction and development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding assay plus functional overexpression result with defined molecular readout\",\n      \"pmids\": [\"11241784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CstF-64 is required for correct histone mRNA 3' end processing in mouse embryonic stem cells; loss of CstF-64 results in increased polyadenylation of histone mRNAs, slower growth, loss of pluripotency, and lengthened G1 phase.\",\n      \"method\": \"CstF-64 knockout mouse ESCs, histone mRNA polyadenylation assay, cell cycle analysis, pluripotency marker assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO with multiple orthogonal phenotypic readouts including direct molecular assay of histone mRNA polyadenylation\",\n      \"pmids\": [\"24957598\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CstF-64 is essential for endoderm differentiation in mouse ESCs; loss of CstF-64 abolishes endodermal lineage differentiation and prevents cardiomyocyte formation, which can be rescued by conditioned medium from extraembryonic endodermal stem cells.\",\n      \"method\": \"CstF-64 knockout mouse ESCs, lineage marker analysis, cardiomyocyte differentiation assay, XEN cell conditioned medium rescue experiment\",\n      \"journal\": \"Stem cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with rescue experiment; mechanism downstream of endoderm specification not fully resolved\",\n      \"pmids\": [\"25460602\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The carboxy-terminus of CstF-77 (last 30 amino acids) enhances cleavage/polyadenylation by increasing the stability of the CstF-64 RRM, thereby altering the affinity of the complex for RNA; excess CstF-64 not bound to CstF-77 localizes to the cytoplasm, potentially via interaction with cytoplasmic RNAs.\",\n      \"method\": \"Reverse genetics, NMR studies of recombinant CstF-64 RRM-Hinge and CstF-77 domains, nuclear/cytoplasmic localization assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structural analysis plus functional genetics, multiple orthogonal methods\",\n      \"pmids\": [\"30257008\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CSTF2 induces 3'UTR shortening of RAC1 by cotranscriptional recruitment to the GUAAU motif at the proximal polyadenylation site, which attenuates recruitment of transcription elongation factors AFF1 and AFF4, causing defects in transcriptional elongation and promoting use of the proximal poly(A) site.\",\n      \"method\": \"RNA-seq, ChIP, RIP, CSTF2 overexpression/knockdown, polyadenylation site usage assays in UCB cells\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP and RIP combined with functional polyadenylation assays, but mechanism partly inferred from correlation\",\n      \"pmids\": [\"30143523\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A missense mutation in the CstF-64 RRM (p.D50A) reduces C/P efficiency by altering amino acid side chain positions, changing the electrostatic potential of the RRM and resulting in greater affinity for RNA; in mice, this mutation alters polyadenylation sites in over 1300 genes critical for brain development.\",\n      \"method\": \"Reporter gene C/P assay, NMR structural analysis of mutant RRM, mouse model with D50A knock-in, genome-wide poly(A) site analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure, mutagenesis, in vivo mouse model, genome-wide functional readout\",\n      \"pmids\": [\"32816001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Electrostatic attraction is the dominant factor in CstF-64 RRM binding to U-rich RNA; binding involves enthalpy-entropy compensation supported by changes in picosecond-to-nanosecond timescale dynamics; competition between fast, high-affinity RNA binding and efficient correct C/P exists in vivo.\",\n      \"method\": \"NMR spectroscopy, mutagenesis, biophysical assays (ITC/SPR), in vivo C/P assays\",\n      \"journal\": \"Biophysical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR with mutagenesis and in vivo validation, multiple orthogonal methods\",\n      \"pmids\": [\"35090899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CSTF2 co-transcriptionally regulates m6A installation by slowing RNA Pol II elongation rate during gene transcription; CSTF2-regulated m6As are recognized by IGF2BP2, an m6A reader that stabilizes mRNAs.\",\n      \"method\": \"Transcriptomic m6A profiling (MeRIP-seq) in PDAC tissues, CSTF2 manipulation with RNA Pol II elongation rate assays, IGF2BP2 RIP\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple molecular methods but m6A regulatory mechanism via transcription elongation partially correlative\",\n      \"pmids\": [\"37816727\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The CSTF2 RRM domain binds U-rich RNA through a multistep binding process involving differences in picosecond-to-nanosecond timescale dynamics and structural changes in the C-terminal α-helix.\",\n      \"method\": \"NMR titration, spin relaxation experiments, paramagnetic relaxation enhancement measurements, rigid-body docking\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple NMR methods with structural characterization\",\n      \"pmids\": [\"39305233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CSTF2 shortens the 3'UTR of PGK1 pre-mRNA by binding near the proximal polyadenylation site, causing loss of m6A modification sites; this prevents YTHDF2-mediated mRNA degradation and increases PGK1 protein to enhance glycolysis under hypoxia. YTHDC1 recognizes hypoxia-induced m6A near the proximal poly(A) site and recruits CSTF2 to enhance 3'UTR shortening.\",\n      \"method\": \"RIP, APA site usage assays, m6A mapping, CSTF2 knockdown/overexpression, xenograft models, patient-derived organoids, small-molecule screen\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple molecular and in vivo methods but mechanism involves several sequential steps each supported by single-method evidence\",\n      \"pmids\": [\"39514400\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CSTF2 promotes PolyA polymerase alpha (PAPα) binding to the 3'UTR of CXCL10 RNA, resulting in shortened poly(A) tails and reduced CXCL10 mRNA stability; this diminishes CXCL10-mediated recruitment of innate αβ T cells, suppressing anti-tumor immunity in PDAC.\",\n      \"method\": \"RIP, poly(A) tail length assay, CSTF2 knockdown, CXCL10 mRNA stability assay, tumor infiltration analysis\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods but mechanism partly inferred; single lab study\",\n      \"pmids\": [\"39972059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CSTF2 promotes 3'UTR shortening and upregulation of FGF2 mRNA by inducing use of the proximal polyadenylation site, stabilizing FGF2 mRNA through miRNA evasion; FGF2 in turn enhances CSTF2 expression forming a positive feedback loop that drives epithelial-mesenchymal transition in tubular epithelial cells.\",\n      \"method\": \"CSTF2 knockdown/overexpression, APA site usage assays, mRNA stability assay, in vivo UUO mouse model, antisense oligonucleotide treatment\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo and in vitro evidence with APA mechanistic assays, but feedback loop mechanism relies on correlative FGF2 upregulation data\",\n      \"pmids\": [\"36113752\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CSTF2 (CstF-64) is an RNA-binding protein whose N-terminal RRM domain recognizes GU-rich downstream sequence elements of pre-mRNAs through electrostatic and dynamic interactions, with its C-terminal helix undergoing conformational change upon RNA binding to initiate polyadenylation complex assembly; the hinge domain mediates mutually exclusive binding to CstF-77 (required for nuclear localization and polyadenylation) or symplekin (required for histone 3' processing); cellular levels of CstF-64 are rate-limiting for CstF complex formation and directly regulate poly(A) site selection during IgM switching and other alternative polyadenylation events, while also co-transcriptionally influencing RNA Pol II elongation rate to affect m6A modification and 3'UTR length of target mRNAs.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CSTF2 (CstF-64) is the RNA-binding subunit of the cleavage stimulation factor (CstF) complex and a central regulator of pre-mRNA 3′-end processing, alternative polyadenylation (APA), and replication-dependent histone mRNA 3′ maturation. Its N-terminal RRM domain recognizes GU/U-rich downstream sequence elements through an electrostatically driven, multistep binding mechanism in which the C-terminal α-helix of the RRM unfolds upon RNA engagement, propagating a conformational change into the hinge domain that nucleates polyadenylation complex assembly [PMID:12773396, PMID:35090899, PMID:39305233]. The hinge domain mediates mutually exclusive interactions with CstF-77 — required for nuclear import and canonical cleavage/polyadenylation — and with symplekin, which is essential for histone RNA 3′ processing; CstF-64 protein levels are rate-limiting for CstF complex formation, and altering its concentration directly shifts poly(A) site choice, as demonstrated by IgM heavy-chain switching, genome-wide APA changes in a mouse D50A knock-in, and 3′UTR shortening of specific transcripts that affects mRNA stability and m6A deposition [PMID:8945520, PMID:21119002, PMID:32816001, PMID:37816727]. Loss of CstF-64 causes aberrant polyadenylation of histone mRNAs, cell-cycle arrest, loss of pluripotency in embryonic stem cells, and failure of endoderm differentiation, establishing it as essential for both housekeeping RNA processing and developmental gene regulation [PMID:9885564, PMID:24957598, PMID:25460602].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Establishing that CstF-64 is the rate-limiting factor for CstF assembly and APA-mediated immunoglobulin class switching resolved how a stoichiometric change in a single polyadenylation factor could redirect gene expression.\",\n      \"evidence\": \"Reconstituted in vitro polyadenylation with overexpression in B cells and gel-shift affinity measurements\",\n      \"pmids\": [\"8945520\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which CstF-64 levels are themselves regulated during B-cell activation\", \"Whether other APA targets respond similarly to CstF-64 dosage\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Showing that CstF-64 concentrates in transcription-dependent nuclear 'cleavage bodies' linked its function to active transcription sites rather than a diffuse nuclear pool.\",\n      \"evidence\": \"Immunofluorescence, immunogold EM, and transcription inhibition with α-amanitin/DRB\",\n      \"pmids\": [\"8654386\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of signals that recruit CstF-64 to cleavage bodies\", \"Whether cleavage body localization is required for processing efficiency\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Genetic depletion of CstF-64 in DT40 cells demonstrated that it is essential for viability, with graded reduction causing IgM mRNA loss, G0/G1 arrest, and apoptosis — proving CstF-64 is not merely a polyadenylation accessory but a cell-growth regulator.\",\n      \"evidence\": \"Regulatable transgene replacement in DT40 chicken B cells with cell-cycle and viability assays\",\n      \"pmids\": [\"9885564\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which specific mRNA targets mediate the cell-cycle arrest phenotype\", \"Whether the growth arrest is solely APA-dependent or involves additional functions\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Solving the NMR structure of the CstF-64 RRM revealed that UU dinucleotide recognition occurs within a defined pocket and that the C-terminal α-helix unfolds upon RNA binding to expose the hinge domain, providing the first structural model for how RNA recognition triggers polyadenylation complex assembly.\",\n      \"evidence\": \"NMR structure determination of CstF-64 RRM with RNA-binding assays\",\n      \"pmids\": [\"12773396\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the full-length protein or CstF holocomplex\", \"How the conformational change is transmitted to downstream factors\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"NMR relaxation dynamics showed that the RNA-binding surface of the RRM becomes mobile on the μs–ms timescale upon binding GU-rich RNA, establishing that sequence discrimination relies on dynamic rather than static complementarity.\",\n      \"evidence\": \"NMR relaxation experiments comparing free and RNA-bound CstF-64 RRM with multiple RNA substrates\",\n      \"pmids\": [\"15769465\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether dynamics-based discrimination operates identically in the context of the full CstF complex\", \"Thermodynamic decomposition of the selectivity mechanism\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Determination of the C-terminal domain structure as a three-helix bundle and identification of its interaction with Pcf11 distinguished CstF-64's role in 3′-end cleavage from its dispensability in transcription termination.\",\n      \"evidence\": \"NMR structure of the CTD, mutagenesis, and yeast functional assays for processing vs. termination\",\n      \"pmids\": [\"17116658\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the human ortholog uses the same surface for Pcf11/PCF11 interaction\", \"Whether additional partners bind this domain\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Mapping the hinge domain as the site of mutually exclusive binding to CstF-77 (for nuclear import and polyadenylation) or symplekin (for histone 3′ processing) defined how a single protein partitions between two distinct 3′-end processing pathways.\",\n      \"evidence\": \"Reciprocal separation-of-function mutants analyzed by SLAP assay, histone processing assay, and nuclear/cytoplasmic fractionation\",\n      \"pmids\": [\"19887456\", \"21119002\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether switching between CstF-77 and symplekin is regulated by post-translational modification\", \"Structural basis of mutual exclusivity\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstration that enterovirus 71 3Cpro cleaves CstF-64 at specific sites to shut down host polyadenylation — rescuable by recombinant CstF-64 — established CstF-64 as a viral target for host gene expression shutoff.\",\n      \"evidence\": \"In vitro cleavage with wild-type/mutant 3Cpro, serial mutagenesis, polyadenylation rescue assay\",\n      \"pmids\": [\"19779565\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other viruses target CstF-64 by analogous mechanisms\", \"In vivo relevance during natural infection\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"CstF-64 knockout in mouse ESCs revealed its requirement for proper histone mRNA 3′ processing, pluripotency maintenance, and endoderm differentiation, extending its biological role from constitutive RNA processing to developmental competence.\",\n      \"evidence\": \"CstF-64 KO ESCs with histone mRNA polyadenylation assay, cell-cycle analysis, lineage differentiation, and conditioned-medium rescue\",\n      \"pmids\": [\"24957598\", \"25460602\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which specific histone mRNA or APA changes drive the pluripotency loss\", \"Whether tauCstF-64 can partially compensate in vivo\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"A D50A missense mutation in the RRM altered its electrostatic potential and RNA affinity, changing poly(A) site usage in >1300 genes critical for brain development in mice, directly linking RRM biophysics to genome-wide APA and organismal phenotype.\",\n      \"evidence\": \"NMR of mutant RRM, reporter C/P assay, D50A knock-in mouse with genome-wide poly(A) site profiling\",\n      \"pmids\": [\"32816001\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise neurological phenotype and behavioral consequences in the mouse model\", \"Whether similar mutations occur in human neurodevelopmental disease\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Biophysical dissection showed that electrostatic attraction dominates CstF-64 RRM–RNA binding with enthalpy–entropy compensation, and that a trade-off exists between high-affinity RNA binding and efficient cleavage/polyadenylation in vivo.\",\n      \"evidence\": \"NMR, ITC/SPR thermodynamics, mutagenesis, in vivo C/P assays\",\n      \"pmids\": [\"35090899\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the affinity–efficiency trade-off is tuned under different physiological conditions\", \"Role of post-translational modifications in modulating RRM electrostatics\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"CSTF2 was shown to co-transcriptionally influence m6A deposition by slowing RNA Pol II elongation, revealing a previously unrecognized link between polyadenylation factor binding and epitranscriptomic modification.\",\n      \"evidence\": \"MeRIP-seq in PDAC tissues, Pol II elongation rate assays, CSTF2 manipulation, IGF2BP2 RIP\",\n      \"pmids\": [\"37816727\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether elongation-rate modulation is a general property of CSTF2 or context-specific\", \"Direct measurement of Pol II speed changes at single-gene resolution\", \"Independent replication in non-cancer cells\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Studies in pancreatic cancer and renal fibrosis showed CSTF2-driven 3′UTR shortening of specific mRNAs (PGK1, CXCL10, FGF2) alters their stability, m6A landscape, and downstream signaling, illustrating how CSTF2-mediated APA reprograms gene expression in disease contexts.\",\n      \"evidence\": \"RIP, APA assays, poly(A) tail length assays, knockdown/overexpression, xenografts, organoids, and UUO mouse model\",\n      \"pmids\": [\"39514400\", \"39972059\", \"36113752\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CSTF2 is a direct therapeutic target or a downstream effector\", \"Genome-wide specificity of CSTF2-mediated APA changes in these disease settings\", \"Independent validation of the YTHDC1–CSTF2 recruitment axis\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"No high-resolution structure of the full CstF complex or of CstF-64 bound simultaneously to RNA and a protein partner exists; the mechanisms that regulate CstF-64 protein levels, its post-translational modifications, and the logic by which it selects specific poly(A) sites genome-wide remain incompletely defined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full CstF complex structure\", \"Regulation of CstF-64 protein turnover\", \"Genome-wide rules for poly(A) site selectivity by CstF-64 in vivo\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 3, 4, 10, 17, 18, 20]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 12, 16, 19, 21, 22]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 8, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 6, 8]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 3, 6, 7, 8, 13, 17]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [16, 19]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 13]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [14, 17]}\n    ],\n    \"complexes\": [\n      \"CstF (cleavage stimulation factor)\",\n      \"symplekin–CPSF–CstF histone processing complex\"\n    ],\n    \"partners\": [\n      \"CSTF3\",\n      \"SYMPK\",\n      \"PCF11\",\n      \"CPSF2\",\n      \"AFF1\",\n      \"AFF4\",\n      \"IGF2BP2\",\n      \"YTHDC1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}