Affinage

CSTF3

Cleavage stimulation factor subunit 3 · UniProt Q12996

Length
717 aa
Mass
82.9 kDa
Annotated
2026-06-09
13 papers in source corpus 11 papers cited in narrative 11 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 9 steps
  1. 2002 High

    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

    PMID:12149458

    Open questions at the time
    • Domain limiting cross-species rescue not molecularly defined
    • Mechanism distinguishing constitutive cleavage from regulated poly(A) site choice unresolved
  2. 2006 Medium

    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

    PMID:16882666

    Open questions at the time
    • Cytoplasmic complex partners (eIF4E, CPEB, CPSF-100, XGLD2) not validated reciprocally
    • Direct molecular basis of translational repression unknown
    • Relevance to somatic cells untested
  3. 2007 High

    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

    PMID:17386263 PMID:17584787

    Open questions at the time
    • Full trimeric CstF architecture not visualized
    • Functional consequence of dimerization for processing kinetics not quantified
  4. 2009 High

    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

    PMID:19887456

    Open questions at the time
    • Nuclear import machinery recognizing the complex not identified
    • Order of complex assembly relative to import not fully resolved
  5. 2010 High

    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

    PMID:21119002

    Open questions at the time
    • Functional role of the symplekin-bound pool not defined
    • Regulation of the binding switch unknown
  6. 2013 Medium

    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

    PMID:23874216

    Open questions at the time
    • Quantitative contribution of feedback to steady-state CSTF3 not measured
    • Coupling between feedback and downstream phenotypes correlative
  7. 2018 High

    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

    PMID:30257008

    Open questions at the time
    • Structural model of the CTD–RRM contact not fully resolved
    • Effect on specific RNA sequence preferences not mapped
  8. 2024 Medium

    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

    PMID:38898019

    Open questions at the time
    • Direct CSTF3–NEAT1 binding site not structurally defined
    • Link to PI3K/AKT/mTOR is correlative
  9. 2026 Medium

    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

    PMID:41628695

    Open questions at the time
    • Transcriptional mechanism linking MEK/ERK to CSTF3 induction unknown
    • Direct vs indirect control of Mmp9 PAS choice not separated

Open questions

Synthesis pass · forward-looking unresolved questions
  • How CSTF3 expression levels are translated into selective, gene-specific alternative polyadenylation decisions across the transcriptome remains unresolved.
  • 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

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0003723 RNA binding 2 GO:0005198 structural molecule activity 2 GO:0060090 molecular adaptor activity 2 GO:0140098 catalytic activity, acting on RNA 2
Localization
GO:0005634 nucleus 3 GO:0005829 cytosol 2
Pathway
R-HSA-74160 Gene expression (Transcription) 3 R-HSA-8953854 Metabolism of RNA 3
Partners
Complex memberships
CstF complex

Evidence

Reading pass · 11 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2007 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. X-ray crystallography, light scattering, yeast two-hybrid, analytical ultracentrifugation Molecular cell High 17386263
2007 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). X-ray crystallography, domain mapping experiments Nucleic acids research High 17584787
2010 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. Mutant analysis (CstF-64 and symplekin mutants), nuclear localization assays, functional complementation Molecular biology of the cell High 21119002
2009 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. In vivo SLAP (stem-loop luciferase assay for polyadenylation), domain deletion/mutation analysis, nuclear localization assays The Journal of biological chemistry High 19887456
2018 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. Reverse genetics, NMR spectroscopy of recombinant proteins (CstF-64 RRM-Hinge and CstF-77 monkeytail-CTD), nuclear localization assays Nucleic acids research High 30257008
2002 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. Genetic complementation in Drosophila su(f) mutants, chimeric protein rescue assays, mRNA 3' end processing analysis in vivo Proceedings of the National Academy of Sciences of the United States of America High 12149458
2013 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. Molecular biology validation of intronic poly(A) site, expression manipulation (overexpression/knockdown), U1 snRNP inhibition, global APA analysis PLoS genetics Medium 23874216
2006 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. Co-immunoprecipitation, subcellular fractionation, in vivo function impairment (dominant negative/antibody injection), in vitro translation assay The Journal of biological chemistry Medium 16882666
2024 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. CSTF3 knockdown in cell lines, RNA isoform analysis, lncRNA isoform-specific overexpression/knockdown, pathway activity measurement Cell death & disease Medium 38898019
2026 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. Inducible epidermis-specific α3 knockout mice, RNA in situ hybridization, CSTF3 knockdown, DaPars2 genome-wide APA analysis Matrix biology : journal of the International Society for Matrix Biology Medium 41628695
2005 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. Bioinformatic identification with molecular biology experimental validation (RT-PCR, poly(A) site analysis), SAGE data analysis Gene Low 16316725

Source papers

Stage 0 corpus · 13 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2007 Crystal structure of murine CstF-77: dimeric association and implications for polyadenylation of mRNA precursors. Molecular cell 76 17386263
2010 Interactions of CstF-64, CstF-77, and symplekin: implications on localisation and function. Molecular biology of the cell 52 21119002
2013 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. PLoS genetics 47 23874216
2007 The structure of the CstF-77 homodimer provides insights into CstF assembly. Nucleic acids research 42 17584787
2005 An intronic polyadenylation site in human and mouse CstF-77 genes suggests an evolutionarily conserved regulatory mechanism. Gene 35 16316725
2018 The structural basis of CstF-77 modulation of cleavage and polyadenylation through stimulation of CstF-64 activity. Nucleic acids research 27 30257008
2009 The hinge domain of the cleavage stimulation factor protein CstF-64 is essential for CstF-77 interaction, nuclear localization, and polyadenylation. The Journal of biological chemistry 24 19887456
2024 CSTF3 contributes to platinum resistance in ovarian cancer through alternative polyadenylation of lncRNA NEAT1 and generating the short isoform NEAT1_1. Cell death & disease 19 38898019
2002 Chimeric human CstF-77/Drosophila Suppressor of forked proteins rescue suppressor of forked mutant lethality and mRNA 3' end processing in Drosophila. Proceedings of the National Academy of Sciences of the United States of America 17 12149458
2007 The use of in situ proteolysis in the crystallization of murine CstF-77. Acta crystallographica. Section F, Structural biology and crystallization communications 16 17277459
2006 Cytoplasmic CstF-77 protein belongs to a masking complex with cytoplasmic polyadenylation element-binding protein in Xenopus oocytes. The Journal of biological chemistry 12 16882666
2026 An integrin α3β1-CSTF3 signaling axis regulates alternative polyadenylation of Mmp9 mRNA. Matrix biology : journal of the International Society for Matrix Biology 0 41628695
2026 Inflammatory Myofibroblastic Tumor of the Breast Mimicking Complex Fibrocystic Changes, With a Novel CSTF3::ALK Fusion. International journal of surgical pathology 0 42108690

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