{"gene":"CNOT6","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2012,"finding":"Crystal structure of the CAF1-binding (MIF4G) domain of human NOT1 alone and in complex with CAF1 was solved, revealing that NOT1 bridges the interaction between the catalytic module (CAF1/CCR4) and the NOT module, acting as a scaffold for CCR4-NOT complex assembly. The NOT1 MIF4G domain binds CAF1 through a pre-formed interface while leaving the CAF1 catalytic site fully accessible to RNA substrates.","method":"X-ray crystallography with functional validation of interface residues","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures of apo and complex forms with functional interface analysis, mechanistically defines how CNOT6/CAF1 module is scaffolded within CCR4-NOT","pmids":["22977175"],"is_preprint":false},{"year":2019,"finding":"Reconstitution of a complete recombinant human CCR4-NOT complex showed that CCR4a (CNOT6) and CAF1 have distinct deadenylation profiles in vitro, and that the complex is more active and selective for poly(A) than the isolated exonucleases alone. Non-enzymatic modules (CAF40 and NOT10:NOT11) stimulate deadenylation in a partially redundant manner.","method":"In vitro reconstitution of recombinant human CCR4-NOT complex; biochemical deadenylation assay with purified component variants","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — full reconstitution of human complex with strict compositional control and multiple orthogonal in vitro assays in a single rigorous study","pmids":["31320642"],"is_preprint":false},{"year":2015,"finding":"Using a purified minimal human BTG2-Caf1-Ccr4 (CNOT6) nuclease sub-complex reconstituted from bacteria, chemical inhibition and inactivating amino acid substitutions demonstrated that both the Caf1 and Ccr4 (CNOT6) enzyme activities are required for deadenylation in vitro, indicating they cooperate and may regulate each other allosterically within the nuclease module.","method":"In vitro deadenylation assay with purified recombinant sub-complex; active-site mutagenesis; chemical inhibition","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution plus mutagenesis in a single study with multiple orthogonal approaches","pmids":["25944446"],"is_preprint":false},{"year":2011,"finding":"Knockdown of Ccr4a (CNOT6) and Ccr4b (CNOT6L) in human cells plays a role in cell survival and prevention of senescence, distinct from knockdown of Caf1a/Caf1b or non-catalytic subunits. CNOT6/6L knockdown differentially affects processing-body formation. CNOT6/6L regulate IGFBP5 mRNA, mediating cell cycle arrest and senescence via a p53-dependent pathway. The LRR domain of Ccr4b influences subcellular localization but is not required for deadenylase activity.","method":"siRNA knockdown with cell viability, senescence (SA-β-gal), cell cycle, and P-body formation readouts; gene expression profiling; subcellular localization studies","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with multiple defined cellular phenotypes and gene expression profiling, single lab","pmids":["21233283"],"is_preprint":false},{"year":2020,"finding":"In mouse embryonic fibroblasts (MEFs), CNOT6/6L double knockout (dKO) cells remain viable whereas CNOT7/8 dKO cells undergo cell death, demonstrating that CNOT7/8 are the essential deadenylase subunits for cell viability. In Cnot7/8-dKO MEFs, CNOT6/6L are also absent from the CCR4-NOT complex, whereas in Cnot6/6l-dKO MEFs, CNOT7/8 still interacts with other subunits. Bulk poly(A) tail analysis showed more mRNAs with longer poly(A) tails in Cnot7/8-dKO than Cnot6/6l-dKO MEFs. Cnot6/6l-dKO mice are viable and grow normally.","method":"CRISPR/genetic double knockout of Cnot6/6l in MEFs and mice; co-immunoprecipitation; bulk poly(A) tail analysis; mRNA stability measurement","journal":"RNA biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — clean genetic KO with multiple orthogonal readouts (viability, Co-IP, poly(A) profiling, transcriptomics), confirmed in vivo","pmids":["31924127"],"is_preprint":false},{"year":2008,"finding":"CCR4 (CNOT6) potentiates nuclear hormone receptor transcriptional activity and mediates its effect through the ligand binding domain of nuclear receptors. siRNA knockdown of CCR4 decreased nuclear receptor activation and attenuated stimulation of RARα target genes Sox9 and HoxA1. CCR4 associates in vivo with NIF-1, and the CCR4-enhanced transcriptional activation is dependent on NIF-1.","method":"siRNA knockdown; co-immunoprecipitation (in vivo and in vitro); quantitative PCR of target genes; reporter assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus functional knockdown with defined gene expression readouts, single lab","pmids":["18180299"],"is_preprint":false},{"year":2008,"finding":"Expression of a GSE fragment of hCCR4/CNOT6 or siRNA knockdown of CNOT6 decreased sensitivity of mammalian cells to DNA-damaging agents (cisplatin). Overexpression of hCCR4 targeted Chk2 following cisplatin exposure without interfering with the upstream ATM/ATR pathway, while strongly increasing γH2AX phosphorylation.","method":"Genetic suppressor element (GSE) selection; siRNA knockdown; Western blot for DNA damage response proteins (Chk2, γH2AX)","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — functional knockdown and overexpression with defined pathway readouts, single lab, single study","pmids":["18818012"],"is_preprint":false},{"year":2021,"finding":"ENO1 acts as an RNA-binding protein that recruits CNOT6 to accelerate mRNA decay of IRP1 in hepatocellular carcinoma cells, thereby suppressing mitochondrial iron-induced ferroptosis. This establishes CNOT6 as a downstream effector of an ENO1-dependent mRNA decay pathway.","method":"Co-immunoprecipitation; RNA immunoprecipitation; siRNA knockdown; in vitro and in vivo functional assays","journal":"Nature cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and RIP showing ENO1-CNOT6 interaction with mRNA decay functional readout, in vitro and in vivo validation","pmids":["35121990"],"is_preprint":false},{"year":2018,"finding":"CNOT6 is present in cortical foci of mouse oocytes and regulates a novel pattern of mRNA deadenylation during oocyte meiotic maturation, specifically targeting a subset of mRNAs that are deadenylated in growing oocytes, polyadenylated during early maturation, and then re-deadenylated during late maturation. PUF-binding elements (PBEs) regulate this deadenylation in mature oocytes.","method":"Immunofluorescence localization; poly(A) tail length assay; functional perturbation experiments in oocytes","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization tied to functional deadenylation readouts in oocytes, single lab","pmids":["29717177"],"is_preprint":false},{"year":2021,"finding":"FSH stimulates transcription and translation of CNOT6 and CNOT6L in ovarian granulosa cells. CNOT6/6L function as key effectors of FSH, triggering clearance of specific transcripts during preantral-to-antral follicle transition. Cnot6l-/- female mice are infertile with poor ovarian responses to gonadotropins. Cnot6/6l double knockout mice demonstrate that CNOT6/6L-mediated mRNA deadenylation is dispensable in most somatic cell types but essential for female reproductive endocrine regulation.","method":"Conditional and constitutive knockout mouse models; transcriptome analysis; gonadotropin stimulation assays; fertility phenotyping","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO with defined in vivo phenotype and transcriptome-level mechanistic characterization, multiple orthogonal approaches","pmids":["34788619"],"is_preprint":false},{"year":2014,"finding":"In sea urchin primordial germ cells, Nanos targets CNOT6 mRNA for degradation, creating a stable mRNA environment. Misexpression of CNOT6 in PGCs caused failure to retain Seawi transcripts and Vasa protein. Broad knockdown of CNOT6 expanded the domain of Seawi RNA and exogenous reporters, establishing spatially restricted CNOT6 expression as a mechanism for selective germline RNA localization.","method":"mRNA misexpression; morpholino-based knockdown; in situ hybridization; reporter assays in sea urchin embryos","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function and gain-of-function with defined molecular readouts in a developmental model, single lab","pmids":["25100654"],"is_preprint":false},{"year":2022,"finding":"Depletion of CNOT6 sensitizes human U2OS cells to the DNA-damaging agent MNNG, upregulates MMR pathway activity, decreases mutation frequency in MMR-proficient cells, and increases mRNA stability of MMR gene transcripts leading to increased MMR protein expression. This identifies CNOT6 as a novel regulator of DNA mismatch repair via mRNA decay of MMR gene transcripts.","method":"siRNA knockdown; cell survival assay; mRNA stability measurement; MMR reporter assay; Western blot","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown with multiple orthogonal readouts (survival, mRNA stability, mutation frequency, protein levels), single lab","pmids":["35159331"],"is_preprint":false},{"year":2021,"finding":"CNOT6/6L-mediated deadenylation targets p21 mRNA (but not Caf1a/b) in gastric cancer MKN28 cells, contributing to cell cycle regulation. Depletion of CNOT6/6L arrests cells at the G0/G1 phase and modulates CDK-cyclin inhibitor levels without affecting CDKs or cyclins directly. Depletion of CNOT6/6L also impairs processing-body formation.","method":"Stable shRNA knockdown; cell cycle analysis; Western blot; P-body imaging; mRNA stability assay","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined KD with multiple cellular and molecular readouts identifying a specific mRNA target, single lab","pmids":["33671234"],"is_preprint":false},{"year":2016,"finding":"Forced expression of a combination of Cnot6, Cnot6l, Cnot7, and Cnot8 increases the number of alkaline phosphatase-positive colonies after iPSC induction and decreases expression of Eomes and p21 mRNAs (which have longer poly(A) tails when Cnot1 is depleted), implicating CCR4-NOT deadenylase activity including CNOT6 in iPSC reprogramming via poly(A) tail regulation of specific mRNAs.","method":"Overexpression of deadenylase subunits in MEFs with iPSC induction; alkaline phosphatase assay; poly(A) tail length analysis; qRT-PCR","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — gain-of-function with defined readouts but CNOT6 contribution not separated from the other three subunits, single lab","pmids":["27037025"],"is_preprint":false},{"year":2019,"finding":"CNOT6 knockdown in human HepG2 cells led to significant alteration in stability of specific mRNAs; alterations in half-life were inversely associated with transcription rates, mostly not resulting in changes in mRNA abundance. This demonstrates a buffering mechanism whereby transcript stabilization upon CNOT6 depletion is compensated by decreased transcription.","method":"siRNA knockdown; mRNA stability measurement; transcription rate measurement in human cells","journal":"RNA biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KD with orthogonal stability and transcription rate measurements, single lab, single study","pmids":["31116665"],"is_preprint":false},{"year":2024,"finding":"The Legionella pneumophila effector PieF disrupts the association between CNOT6/6L EEP-type nucleases and CNOT7, demonstrating that PieF binding to CNOT7 is sufficient to displace CNOT6/6L from the complex. This mechanistically defines a protein-protein interaction dependency of CNOT6/6L on CNOT7 for complex incorporation.","method":"Co-immunoprecipitation; in vitro binding/inhibition assay; yeast genetic assay; mammalian cell overexpression","journal":"mSphere","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP showing CNOT6/6L displacement, complemented by in vitro and yeast orthogonal assays, single lab","pmids":["39699231"],"is_preprint":false},{"year":2024,"finding":"PCIF1 negatively regulates CNOT6 mRNA stability through m6A modification: siRNA-mediated suppression of PCIF1 upregulates CNOT6 at both mRNA and protein levels. MeRIP-qPCR showed that PCIF1 suppression significantly reduced m6A levels on CNOT6 mRNA, and rescue experiments confirmed the requirement for PCIF1's methyltransferase activity, establishing m6A modification as a mechanism controlling CNOT6 mRNA stability.","method":"siRNA knockdown; MeRIP-qPCR; Western blot; rescue with methyltransferase-dead mutant","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (MeRIP, knockdown, rescue with catalytic mutant), single lab","pmids":["39451207"],"is_preprint":false},{"year":2026,"finding":"CNOT6, but not its paralog CNOT6L, is an essential post-transcriptional regulator of neonatal growth: loss of Cnot6 causes severe growth retardation, multi-organ hypoplasia, and increased perinatal mortality. Mechanistically, Cnot6 deficiency elevates hepatic Fgf21 mRNA expression, suppresses the IGF1-IGFBP1 axis, and reprograms liver transcriptional networks, identifying Fgf21 mRNA as a direct CNOT6 decay target that limits anabolic metabolism during the neonatal period.","method":"Cnot6 knockout mouse model; hepatic transcriptome analysis; mRNA stability assay; metabolic phenotyping","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic KO with defined in vivo phenotype and mechanistic transcriptome data, preprint not yet peer-reviewed","pmids":["41757057"],"is_preprint":true},{"year":2025,"finding":"ENO1 lactylation at K71 (facilitated by the lactyltransferase P300) reduces binding of TRIM21 mRNA to ENO1, preventing CNOT6 recruitment and thereby stabilizing TRIM21 mRNA in endothelial cells during sepsis. This establishes that ENO1-CNOT6 interaction and mRNA decay depend on the lactylation state of ENO1.","method":"Co-immunoprecipitation; RNA immunoprecipitation (RIP); RT-qPCR; Western blot; post-translational modification mass spectrometry","journal":"Clinical and translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and RIP showing direct mechanistic link between ENO1 PTM status and CNOT6 recruitment, single lab","pmids":["41532698"],"is_preprint":false}],"current_model":"CNOT6 (CCR4a) is an EEP-domain deadenylase subunit of the conserved CCR4-NOT complex that cooperates with the CAF1 (CNOT7/8) nuclease to shorten poly(A) tails of target mRNAs, is scaffolded into the complex via a NOT1 MIF4G domain that binds CAF1, requires CNOT7/8 for its own incorporation into the complex, and executes substrate-selective mRNA decay in contexts including oocyte maturation, granulosa cell gonadotropin signaling, neonatal hepatic Fgf21 regulation, ferroptosis suppression via IRP1 mRNA decay, and modulation of DNA mismatch repair, while also potentiating nuclear hormone receptor transcription through an NIF-1-dependent mechanism."},"narrative":{"mechanistic_narrative":"CNOT6 (CCR4a) is an EEP-domain exonuclease subunit of the conserved CCR4-NOT deadenylase complex that shortens mRNA poly(A) tails to drive substrate-selective decay [PMID:31320642, PMID:31924127]. It is scaffolded into the complex through the NOT1 MIF4G domain, which bridges the catalytic module while leaving the nuclease active sites accessible to RNA [PMID:22977175], and its incorporation depends on CNOT7/8 (CAF1) — loss of CNOT7/8 removes CNOT6/6L from the complex, whereas loss of CNOT6/6L leaves CNOT7/8 bound to other subunits [PMID:31924127], a dependency that can be exploited by the Legionella effector PieF, which displaces CNOT6/6L by binding CNOT7 [PMID:39699231]. Within the reconstituted complex CNOT6 and CAF1 have distinct, cooperative deadenylation profiles, with both nuclease activities required and non-catalytic modules (CAF40, NOT10:NOT11) stimulating activity [PMID:31320642, PMID:25944446]. Functionally CNOT6 (acting with its paralog CNOT6L) executes targeted decay of specific transcripts: it regulates IGFBP5 and p21 mRNAs to control cell-cycle arrest and senescence [PMID:21233283, PMID:33671234], is recruited by the RNA-binding protein ENO1 to degrade IRP1 mRNA and suppress ferroptosis, an interaction governed by ENO1 lactylation state [PMID:35121990, PMID:41532698], regulates DNA mismatch-repair gene transcripts to modulate genome stability and drug sensitivity [PMID:35159331, PMID:18818012], and directs developmental and endocrine programs including oocyte meiotic maturation, gonadotropin-driven granulosa-cell transcript clearance and female fertility [PMID:29717177, PMID:34788619]. Its own expression is controlled post-transcriptionally by PCIF1-dependent m6A modification of CNOT6 mRNA [PMID:39451207]. CNOT6 also potentiates nuclear hormone receptor transcription through an NIF-1-dependent mechanism acting on the receptor ligand-binding domain [PMID:18180299].","teleology":[{"year":2008,"claim":"Established a non-deadenylase role: CNOT6 was shown to potentiate nuclear hormone receptor transcription, indicating functions beyond mRNA turnover.","evidence":"siRNA knockdown, reciprocal Co-IP and reporter/qPCR of RARα targets in human cells","pmids":["18180299"],"confidence":"Medium","gaps":["Mechanism linking a cytoplasmic deadenylase to nuclear receptor transcription unresolved","NIF-1 dependency not structurally defined"]},{"year":2008,"claim":"Linked CNOT6 to the DNA-damage response, showing its expression level modulates cellular sensitivity to genotoxic agents.","evidence":"GSE selection, siRNA/overexpression with Chk2 and γH2AX Western blots after cisplatin","pmids":["18818012"],"confidence":"Medium","gaps":["Direct mRNA targets mediating Chk2 effect not identified","Single study, single cell context"]},{"year":2011,"claim":"Distinguished CNOT6/6L from the CAF1 nucleases functionally, tying them to IGFBP5 mRNA decay and a p53-dependent senescence program.","evidence":"siRNA knockdown with senescence, cell-cycle, P-body and expression-profiling readouts in human cells","pmids":["21233283"],"confidence":"Medium","gaps":["Direct vs indirect targeting of IGFBP5 not fully resolved","LRR-localization link is for the paralog CNOT6L"]},{"year":2012,"claim":"Defined how the CNOT6/CAF1 catalytic module is assembled into CCR4-NOT, answering how the nuclease is positioned within the complex.","evidence":"X-ray crystallography of NOT1 MIF4G apo and CAF1-bound forms with interface mutagenesis","pmids":["22977175"],"confidence":"High","gaps":["Structure does not capture CNOT6 directly bound","Full-complex architecture not resolved"]},{"year":2015,"claim":"Demonstrated catalytic cooperation: both CNOT6 (Ccr4) and CAF1 enzymatic activities are required for deadenylation within a minimal module.","evidence":"In vitro deadenylation with purified BTG2-CAF1-CCR4 sub-complex, active-site mutagenesis and chemical inhibition","pmids":["25944446"],"confidence":"High","gaps":["Allosteric regulation mechanism inferred, not structurally shown","Substrate-specific division of labor unclear"]},{"year":2019,"claim":"Resolved distinct enzymatic profiles of CNOT6 versus CAF1 and the stimulatory role of non-catalytic modules in a fully reconstituted human complex.","evidence":"In vitro reconstitution of recombinant human CCR4-NOT with compositional control and deadenylation assays","pmids":["31320642"],"confidence":"High","gaps":["In-cell substrate selectivity not addressed","Regulatory inputs determining CNOT6 vs CAF1 usage unknown"]},{"year":2019,"claim":"Showed that CNOT6-driven changes in mRNA stability are buffered by compensatory transcription, explaining why decay activity does not always alter steady-state abundance.","evidence":"siRNA knockdown with paired mRNA stability and transcription-rate measurements in HepG2 cells","pmids":["31116665"],"confidence":"Medium","gaps":["Sensing mechanism coupling decay to transcription not defined","Single cell line"]},{"year":2020,"claim":"Established the hierarchy of subunit essentiality, showing CNOT6/6L are dispensable for viability while their complex incorporation requires CNOT7/8.","evidence":"CRISPR double knockouts in MEFs and mice, Co-IP, and bulk poly(A) profiling","pmids":["31924127"],"confidence":"High","gaps":["Specific transcripts uniquely dependent on CNOT6/6L not enumerated","Tissue-specific essential roles not probed here"]},{"year":2021,"claim":"Defined an in vivo physiological requirement: CNOT6/6L act as FSH effectors essential for female reproductive endocrine regulation.","evidence":"Constitutive/conditional knockout mice, gonadotropin stimulation, transcriptome analysis and fertility phenotyping","pmids":["34788619"],"confidence":"High","gaps":["Direct FSH-induced decay targets not fully mapped","Relative contributions of CNOT6 vs CNOT6L not separated"]},{"year":2021,"claim":"Identified CNOT6 as a recruited effector of sequence-specific decay, linking the RNA-binding protein ENO1 to IRP1 mRNA degradation and ferroptosis suppression.","evidence":"Co-IP, RIP, knockdown and in vitro/in vivo functional assays in hepatocellular carcinoma","pmids":["35121990"],"confidence":"Medium","gaps":["Whether ENO1 recruits the full complex or CNOT6 alone unclear","Generality of ENO1-CNOT6 axis beyond HCC unknown"]},{"year":2021,"claim":"Extended target-specific decay to cell-cycle control, identifying p21 mRNA as a CNOT6/6L (but not CAF1) substrate.","evidence":"shRNA knockdown with cell-cycle analysis, P-body imaging and mRNA stability assays in gastric cancer cells","pmids":["33671234"],"confidence":"Medium","gaps":["Mechanism of target selectivity over CAF1 unknown","Single cancer cell line"]},{"year":2022,"claim":"Connected CNOT6 to genome maintenance, showing it limits mismatch-repair capacity by destabilizing MMR gene mRNAs.","evidence":"siRNA knockdown with survival, mRNA stability, mutation frequency and MMR reporter assays in U2OS cells","pmids":["35159331"],"confidence":"Medium","gaps":["Direct vs indirect targeting of MMR transcripts not established","Single cell context"]},{"year":2024,"claim":"Mechanistically defined CNOT6's dependency on CNOT7 through a bacterial effector that displaces it from the complex.","evidence":"Co-IP, in vitro binding/inhibition, yeast genetics and mammalian overexpression with Legionella PieF","pmids":["39699231"],"confidence":"Medium","gaps":["Physiological consequences of PieF-mediated displacement during infection not detailed"]},{"year":2024,"claim":"Revealed upstream control of CNOT6 itself, showing PCIF1-dependent m6A modification destabilizes CNOT6 mRNA.","evidence":"siRNA knockdown, MeRIP-qPCR and rescue with a methyltransferase-dead PCIF1 mutant","pmids":["39451207"],"confidence":"Medium","gaps":["m6A reader mediating CNOT6 decay not identified","Functional output of altered CNOT6 dosage not measured"]},{"year":2025,"claim":"Showed the ENO1-CNOT6 decay axis is switchable by ENO1 lactylation, providing a metabolic control point for transcript stability.","evidence":"Co-IP, RIP, PTM mass spectrometry and RT-qPCR in sepsis endothelial cells (TRIM21 mRNA)","pmids":["41532698"],"confidence":"Medium","gaps":["Generality of lactylation-controlled CNOT6 recruitment unknown","Quantitative contribution to TRIM21 stability not isolated"]},{"year":2026,"claim":"Distinguished CNOT6 from its paralog as a non-redundant regulator of neonatal growth via hepatic Fgf21 mRNA decay.","evidence":"Cnot6 knockout mouse, hepatic transcriptomics, mRNA stability and metabolic phenotyping (preprint)","pmids":["41757057"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","Direct binding of CNOT6 to Fgf21 mRNA not shown","Basis of CNOT6 vs CNOT6L specificity unresolved"]},{"year":null,"claim":"The molecular basis by which CNOT6 selects specific substrates — versus the paralog CNOT6L or the CAF1 nucleases — and how diverse RNA-binding adaptors (ENO1, PUF/PBE factors) recruit it to particular transcripts remains undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of CNOT6 substrate engagement","Determinants of CNOT6 vs CNOT6L target specificity unknown","Rules governing adaptor-mediated recruitment not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[1,2,4]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1,2]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,8]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,12]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[1,4]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[3,12]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[8,9]}],"complexes":["CCR4-NOT complex"],"partners":["CNOT1","CNOT7","CNOT8","CNOT6L","ENO1","NIF-1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9ULM6","full_name":"CCR4-NOT transcription complex subunit 6","aliases":["CCR4 carbon catabolite repression 4-like","Carbon catabolite repressor protein 4 homolog","Cytoplasmic deadenylase"],"length_aa":557,"mass_kda":63.3,"function":"Poly(A) nuclease with 3'-5' RNase activity. Catalytic component of the CCR4-NOT complex which is one of the major cellular mRNA deadenylases and is linked to various cellular processes including bulk mRNA degradation, miRNA-mediated repression, translational repression during translational initiation and general transcription regulation. Additional complex functions may be a consequence of its influence on mRNA expression. Involved in mRNA decay mediated by the major-protein-coding determinant of instability (mCRD) of the FOS gene in the cytoplasm. In the presence of ZNF335, enhances ligand-dependent transcriptional activity of nuclear hormone receptors, including RARA. The increase of ligand-dependent ESR1-mediated transcription is much smaller, if any. Mediates cell proliferation and cell survival and prevents cellular senescence","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9ULM6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CNOT6","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CAPZB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CNOT6","total_profiled":1310},"omim":[{"mim_id":"618069","title":"CCR4-NOT TRANSCRIPTION COMPLEX, SUBUNIT 6-LIKE; CNOT6L","url":"https://www.omim.org/entry/618069"},{"mim_id":"612054","title":"CCR4-NOT TRANSCRIPTION COMPLEX, SUBUNIT 9; CNOT9","url":"https://www.omim.org/entry/612054"},{"mim_id":"608951","title":"CCR4-NOT TRANSCRIPTION COMPLEX, SUBUNIT 6; CNOT6","url":"https://www.omim.org/entry/608951"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CNOT6"},"hgnc":{"alias_symbol":["CCR4","KIAA1194","Ccr4a"],"prev_symbol":[]},"alphafold":{"accession":"Q9ULM6","domains":[{"cath_id":"3.80.10.10","chopping":"30-158","consensus_level":"high","plddt":95.6254,"start":30,"end":158},{"cath_id":"3.60.10.10","chopping":"174-338_350-428_465-540","consensus_level":"high","plddt":94.6653,"start":174,"end":540}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9ULM6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9ULM6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9ULM6-F1-predicted_aligned_error_v6.png","plddt_mean":89.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CNOT6","jax_strain_url":"https://www.jax.org/strain/search?query=CNOT6"},"sequence":{"accession":"Q9ULM6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9ULM6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9ULM6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9ULM6"}},"corpus_meta":[{"pmid":"35121990","id":"PMC_35121990","title":"ENO1 suppresses cancer cell ferroptosis by degrading the mRNA of iron regulatory protein 1.","date":"2021","source":"Nature cancer","url":"https://pubmed.ncbi.nlm.nih.gov/35121990","citation_count":172,"is_preprint":false},{"pmid":"22977175","id":"PMC_22977175","title":"The structural basis for the interaction between the CAF1 nuclease and the NOT1 scaffold of the human CCR4-NOT deadenylase complex.","date":"2012","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/22977175","citation_count":113,"is_preprint":false},{"pmid":"21233283","id":"PMC_21233283","title":"The Ccr4a (CNOT6) and Ccr4b (CNOT6L) deadenylase subunits of the human Ccr4-Not complex contribute to the prevention of cell death and senescence.","date":"2011","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/21233283","citation_count":99,"is_preprint":false},{"pmid":"31320642","id":"PMC_31320642","title":"Reconstitution of recombinant human CCR4-NOT reveals molecular insights 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facilitates dengue virus infection via negatively modulating IFN-Independent Non-Canonical JAK/STAT pathway.","date":"2019","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/31155293","citation_count":10,"is_preprint":false},{"pmid":"27805284","id":"PMC_27805284","title":"Susceptibility loci of CNOT6 in the general mRNA degradation pathway and lung cancer risk-A re-analysis of eight GWASs.","date":"2016","source":"Molecular carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/27805284","citation_count":9,"is_preprint":false},{"pmid":"35159331","id":"PMC_35159331","title":"CNOT6: A Novel Regulator of DNA Mismatch Repair.","date":"2022","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/35159331","citation_count":9,"is_preprint":false},{"pmid":"33671234","id":"PMC_33671234","title":"Human Ccr4 and Caf1 Deadenylases Regulate Proliferation and Tumorigenicity of Human Gastric Cancer Cells via Modulating Cell Cycle 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complex with CAF1 was solved, revealing that NOT1 bridges the interaction between the catalytic module (CAF1/CCR4) and the NOT module, acting as a scaffold for CCR4-NOT complex assembly. The NOT1 MIF4G domain binds CAF1 through a pre-formed interface while leaving the CAF1 catalytic site fully accessible to RNA substrates.\",\n      \"method\": \"X-ray crystallography with functional validation of interface residues\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures of apo and complex forms with functional interface analysis, mechanistically defines how CNOT6/CAF1 module is scaffolded within CCR4-NOT\",\n      \"pmids\": [\"22977175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Reconstitution of a complete recombinant human CCR4-NOT complex showed that CCR4a (CNOT6) and CAF1 have distinct deadenylation profiles in vitro, and that the complex is more active and selective for poly(A) than the isolated exonucleases alone. Non-enzymatic modules (CAF40 and NOT10:NOT11) stimulate deadenylation in a partially redundant manner.\",\n      \"method\": \"In vitro reconstitution of recombinant human CCR4-NOT complex; biochemical deadenylation assay with purified component variants\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — full reconstitution of human complex with strict compositional control and multiple orthogonal in vitro assays in a single rigorous study\",\n      \"pmids\": [\"31320642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Using a purified minimal human BTG2-Caf1-Ccr4 (CNOT6) nuclease sub-complex reconstituted from bacteria, chemical inhibition and inactivating amino acid substitutions demonstrated that both the Caf1 and Ccr4 (CNOT6) enzyme activities are required for deadenylation in vitro, indicating they cooperate and may regulate each other allosterically within the nuclease module.\",\n      \"method\": \"In vitro deadenylation assay with purified recombinant sub-complex; active-site mutagenesis; chemical inhibition\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution plus mutagenesis in a single study with multiple orthogonal approaches\",\n      \"pmids\": [\"25944446\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Knockdown of Ccr4a (CNOT6) and Ccr4b (CNOT6L) in human cells plays a role in cell survival and prevention of senescence, distinct from knockdown of Caf1a/Caf1b or non-catalytic subunits. CNOT6/6L knockdown differentially affects processing-body formation. CNOT6/6L regulate IGFBP5 mRNA, mediating cell cycle arrest and senescence via a p53-dependent pathway. The LRR domain of Ccr4b influences subcellular localization but is not required for deadenylase activity.\",\n      \"method\": \"siRNA knockdown with cell viability, senescence (SA-β-gal), cell cycle, and P-body formation readouts; gene expression profiling; subcellular localization studies\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with multiple defined cellular phenotypes and gene expression profiling, single lab\",\n      \"pmids\": [\"21233283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In mouse embryonic fibroblasts (MEFs), CNOT6/6L double knockout (dKO) cells remain viable whereas CNOT7/8 dKO cells undergo cell death, demonstrating that CNOT7/8 are the essential deadenylase subunits for cell viability. In Cnot7/8-dKO MEFs, CNOT6/6L are also absent from the CCR4-NOT complex, whereas in Cnot6/6l-dKO MEFs, CNOT7/8 still interacts with other subunits. Bulk poly(A) tail analysis showed more mRNAs with longer poly(A) tails in Cnot7/8-dKO than Cnot6/6l-dKO MEFs. Cnot6/6l-dKO mice are viable and grow normally.\",\n      \"method\": \"CRISPR/genetic double knockout of Cnot6/6l in MEFs and mice; co-immunoprecipitation; bulk poly(A) tail analysis; mRNA stability measurement\",\n      \"journal\": \"RNA biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — clean genetic KO with multiple orthogonal readouts (viability, Co-IP, poly(A) profiling, transcriptomics), confirmed in vivo\",\n      \"pmids\": [\"31924127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CCR4 (CNOT6) potentiates nuclear hormone receptor transcriptional activity and mediates its effect through the ligand binding domain of nuclear receptors. siRNA knockdown of CCR4 decreased nuclear receptor activation and attenuated stimulation of RARα target genes Sox9 and HoxA1. CCR4 associates in vivo with NIF-1, and the CCR4-enhanced transcriptional activation is dependent on NIF-1.\",\n      \"method\": \"siRNA knockdown; co-immunoprecipitation (in vivo and in vitro); quantitative PCR of target genes; reporter assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus functional knockdown with defined gene expression readouts, single lab\",\n      \"pmids\": [\"18180299\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Expression of a GSE fragment of hCCR4/CNOT6 or siRNA knockdown of CNOT6 decreased sensitivity of mammalian cells to DNA-damaging agents (cisplatin). Overexpression of hCCR4 targeted Chk2 following cisplatin exposure without interfering with the upstream ATM/ATR pathway, while strongly increasing γH2AX phosphorylation.\",\n      \"method\": \"Genetic suppressor element (GSE) selection; siRNA knockdown; Western blot for DNA damage response proteins (Chk2, γH2AX)\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — functional knockdown and overexpression with defined pathway readouts, single lab, single study\",\n      \"pmids\": [\"18818012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ENO1 acts as an RNA-binding protein that recruits CNOT6 to accelerate mRNA decay of IRP1 in hepatocellular carcinoma cells, thereby suppressing mitochondrial iron-induced ferroptosis. This establishes CNOT6 as a downstream effector of an ENO1-dependent mRNA decay pathway.\",\n      \"method\": \"Co-immunoprecipitation; RNA immunoprecipitation; siRNA knockdown; in vitro and in vivo functional assays\",\n      \"journal\": \"Nature cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and RIP showing ENO1-CNOT6 interaction with mRNA decay functional readout, in vitro and in vivo validation\",\n      \"pmids\": [\"35121990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CNOT6 is present in cortical foci of mouse oocytes and regulates a novel pattern of mRNA deadenylation during oocyte meiotic maturation, specifically targeting a subset of mRNAs that are deadenylated in growing oocytes, polyadenylated during early maturation, and then re-deadenylated during late maturation. PUF-binding elements (PBEs) regulate this deadenylation in mature oocytes.\",\n      \"method\": \"Immunofluorescence localization; poly(A) tail length assay; functional perturbation experiments in oocytes\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization tied to functional deadenylation readouts in oocytes, single lab\",\n      \"pmids\": [\"29717177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FSH stimulates transcription and translation of CNOT6 and CNOT6L in ovarian granulosa cells. CNOT6/6L function as key effectors of FSH, triggering clearance of specific transcripts during preantral-to-antral follicle transition. Cnot6l-/- female mice are infertile with poor ovarian responses to gonadotropins. Cnot6/6l double knockout mice demonstrate that CNOT6/6L-mediated mRNA deadenylation is dispensable in most somatic cell types but essential for female reproductive endocrine regulation.\",\n      \"method\": \"Conditional and constitutive knockout mouse models; transcriptome analysis; gonadotropin stimulation assays; fertility phenotyping\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO with defined in vivo phenotype and transcriptome-level mechanistic characterization, multiple orthogonal approaches\",\n      \"pmids\": [\"34788619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In sea urchin primordial germ cells, Nanos targets CNOT6 mRNA for degradation, creating a stable mRNA environment. Misexpression of CNOT6 in PGCs caused failure to retain Seawi transcripts and Vasa protein. Broad knockdown of CNOT6 expanded the domain of Seawi RNA and exogenous reporters, establishing spatially restricted CNOT6 expression as a mechanism for selective germline RNA localization.\",\n      \"method\": \"mRNA misexpression; morpholino-based knockdown; in situ hybridization; reporter assays in sea urchin embryos\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function and gain-of-function with defined molecular readouts in a developmental model, single lab\",\n      \"pmids\": [\"25100654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Depletion of CNOT6 sensitizes human U2OS cells to the DNA-damaging agent MNNG, upregulates MMR pathway activity, decreases mutation frequency in MMR-proficient cells, and increases mRNA stability of MMR gene transcripts leading to increased MMR protein expression. This identifies CNOT6 as a novel regulator of DNA mismatch repair via mRNA decay of MMR gene transcripts.\",\n      \"method\": \"siRNA knockdown; cell survival assay; mRNA stability measurement; MMR reporter assay; Western blot\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown with multiple orthogonal readouts (survival, mRNA stability, mutation frequency, protein levels), single lab\",\n      \"pmids\": [\"35159331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CNOT6/6L-mediated deadenylation targets p21 mRNA (but not Caf1a/b) in gastric cancer MKN28 cells, contributing to cell cycle regulation. Depletion of CNOT6/6L arrests cells at the G0/G1 phase and modulates CDK-cyclin inhibitor levels without affecting CDKs or cyclins directly. Depletion of CNOT6/6L also impairs processing-body formation.\",\n      \"method\": \"Stable shRNA knockdown; cell cycle analysis; Western blot; P-body imaging; mRNA stability assay\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined KD with multiple cellular and molecular readouts identifying a specific mRNA target, single lab\",\n      \"pmids\": [\"33671234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Forced expression of a combination of Cnot6, Cnot6l, Cnot7, and Cnot8 increases the number of alkaline phosphatase-positive colonies after iPSC induction and decreases expression of Eomes and p21 mRNAs (which have longer poly(A) tails when Cnot1 is depleted), implicating CCR4-NOT deadenylase activity including CNOT6 in iPSC reprogramming via poly(A) tail regulation of specific mRNAs.\",\n      \"method\": \"Overexpression of deadenylase subunits in MEFs with iPSC induction; alkaline phosphatase assay; poly(A) tail length analysis; qRT-PCR\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — gain-of-function with defined readouts but CNOT6 contribution not separated from the other three subunits, single lab\",\n      \"pmids\": [\"27037025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CNOT6 knockdown in human HepG2 cells led to significant alteration in stability of specific mRNAs; alterations in half-life were inversely associated with transcription rates, mostly not resulting in changes in mRNA abundance. This demonstrates a buffering mechanism whereby transcript stabilization upon CNOT6 depletion is compensated by decreased transcription.\",\n      \"method\": \"siRNA knockdown; mRNA stability measurement; transcription rate measurement in human cells\",\n      \"journal\": \"RNA biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KD with orthogonal stability and transcription rate measurements, single lab, single study\",\n      \"pmids\": [\"31116665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The Legionella pneumophila effector PieF disrupts the association between CNOT6/6L EEP-type nucleases and CNOT7, demonstrating that PieF binding to CNOT7 is sufficient to displace CNOT6/6L from the complex. This mechanistically defines a protein-protein interaction dependency of CNOT6/6L on CNOT7 for complex incorporation.\",\n      \"method\": \"Co-immunoprecipitation; in vitro binding/inhibition assay; yeast genetic assay; mammalian cell overexpression\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP showing CNOT6/6L displacement, complemented by in vitro and yeast orthogonal assays, single lab\",\n      \"pmids\": [\"39699231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PCIF1 negatively regulates CNOT6 mRNA stability through m6A modification: siRNA-mediated suppression of PCIF1 upregulates CNOT6 at both mRNA and protein levels. MeRIP-qPCR showed that PCIF1 suppression significantly reduced m6A levels on CNOT6 mRNA, and rescue experiments confirmed the requirement for PCIF1's methyltransferase activity, establishing m6A modification as a mechanism controlling CNOT6 mRNA stability.\",\n      \"method\": \"siRNA knockdown; MeRIP-qPCR; Western blot; rescue with methyltransferase-dead mutant\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (MeRIP, knockdown, rescue with catalytic mutant), single lab\",\n      \"pmids\": [\"39451207\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CNOT6, but not its paralog CNOT6L, is an essential post-transcriptional regulator of neonatal growth: loss of Cnot6 causes severe growth retardation, multi-organ hypoplasia, and increased perinatal mortality. Mechanistically, Cnot6 deficiency elevates hepatic Fgf21 mRNA expression, suppresses the IGF1-IGFBP1 axis, and reprograms liver transcriptional networks, identifying Fgf21 mRNA as a direct CNOT6 decay target that limits anabolic metabolism during the neonatal period.\",\n      \"method\": \"Cnot6 knockout mouse model; hepatic transcriptome analysis; mRNA stability assay; metabolic phenotyping\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic KO with defined in vivo phenotype and mechanistic transcriptome data, preprint not yet peer-reviewed\",\n      \"pmids\": [\"41757057\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ENO1 lactylation at K71 (facilitated by the lactyltransferase P300) reduces binding of TRIM21 mRNA to ENO1, preventing CNOT6 recruitment and thereby stabilizing TRIM21 mRNA in endothelial cells during sepsis. This establishes that ENO1-CNOT6 interaction and mRNA decay depend on the lactylation state of ENO1.\",\n      \"method\": \"Co-immunoprecipitation; RNA immunoprecipitation (RIP); RT-qPCR; Western blot; post-translational modification mass spectrometry\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and RIP showing direct mechanistic link between ENO1 PTM status and CNOT6 recruitment, single lab\",\n      \"pmids\": [\"41532698\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CNOT6 (CCR4a) is an EEP-domain deadenylase subunit of the conserved CCR4-NOT complex that cooperates with the CAF1 (CNOT7/8) nuclease to shorten poly(A) tails of target mRNAs, is scaffolded into the complex via a NOT1 MIF4G domain that binds CAF1, requires CNOT7/8 for its own incorporation into the complex, and executes substrate-selective mRNA decay in contexts including oocyte maturation, granulosa cell gonadotropin signaling, neonatal hepatic Fgf21 regulation, ferroptosis suppression via IRP1 mRNA decay, and modulation of DNA mismatch repair, while also potentiating nuclear hormone receptor transcription through an NIF-1-dependent mechanism.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CNOT6 (CCR4a) is an EEP-domain exonuclease subunit of the conserved CCR4-NOT deadenylase complex that shortens mRNA poly(A) tails to drive substrate-selective decay [#1, #4]. It is scaffolded into the complex through the NOT1 MIF4G domain, which bridges the catalytic module while leaving the nuclease active sites accessible to RNA [#0], and its incorporation depends on CNOT7/8 (CAF1) — loss of CNOT7/8 removes CNOT6/6L from the complex, whereas loss of CNOT6/6L leaves CNOT7/8 bound to other subunits [#4], a dependency that can be exploited by the Legionella effector PieF, which displaces CNOT6/6L by binding CNOT7 [#15]. Within the reconstituted complex CNOT6 and CAF1 have distinct, cooperative deadenylation profiles, with both nuclease activities required and non-catalytic modules (CAF40, NOT10:NOT11) stimulating activity [#1, #2]. Functionally CNOT6 (acting with its paralog CNOT6L) executes targeted decay of specific transcripts: it regulates IGFBP5 and p21 mRNAs to control cell-cycle arrest and senescence [#3, #12], is recruited by the RNA-binding protein ENO1 to degrade IRP1 mRNA and suppress ferroptosis, an interaction governed by ENO1 lactylation state [#7, #18], regulates DNA mismatch-repair gene transcripts to modulate genome stability and drug sensitivity [#11, #6], and directs developmental and endocrine programs including oocyte meiotic maturation, gonadotropin-driven granulosa-cell transcript clearance and female fertility [#8, #9]. Its own expression is controlled post-transcriptionally by PCIF1-dependent m6A modification of CNOT6 mRNA [#16]. CNOT6 also potentiates nuclear hormone receptor transcription through an NIF-1-dependent mechanism acting on the receptor ligand-binding domain [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established a non-deadenylase role: CNOT6 was shown to potentiate nuclear hormone receptor transcription, indicating functions beyond mRNA turnover.\",\n      \"evidence\": \"siRNA knockdown, reciprocal Co-IP and reporter/qPCR of RARα targets in human cells\",\n      \"pmids\": [\"18180299\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking a cytoplasmic deadenylase to nuclear receptor transcription unresolved\", \"NIF-1 dependency not structurally defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Linked CNOT6 to the DNA-damage response, showing its expression level modulates cellular sensitivity to genotoxic agents.\",\n      \"evidence\": \"GSE selection, siRNA/overexpression with Chk2 and γH2AX Western blots after cisplatin\",\n      \"pmids\": [\"18818012\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mRNA targets mediating Chk2 effect not identified\", \"Single study, single cell context\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Distinguished CNOT6/6L from the CAF1 nucleases functionally, tying them to IGFBP5 mRNA decay and a p53-dependent senescence program.\",\n      \"evidence\": \"siRNA knockdown with senescence, cell-cycle, P-body and expression-profiling readouts in human cells\",\n      \"pmids\": [\"21233283\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect targeting of IGFBP5 not fully resolved\", \"LRR-localization link is for the paralog CNOT6L\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined how the CNOT6/CAF1 catalytic module is assembled into CCR4-NOT, answering how the nuclease is positioned within the complex.\",\n      \"evidence\": \"X-ray crystallography of NOT1 MIF4G apo and CAF1-bound forms with interface mutagenesis\",\n      \"pmids\": [\"22977175\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure does not capture CNOT6 directly bound\", \"Full-complex architecture not resolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated catalytic cooperation: both CNOT6 (Ccr4) and CAF1 enzymatic activities are required for deadenylation within a minimal module.\",\n      \"evidence\": \"In vitro deadenylation with purified BTG2-CAF1-CCR4 sub-complex, active-site mutagenesis and chemical inhibition\",\n      \"pmids\": [\"25944446\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Allosteric regulation mechanism inferred, not structurally shown\", \"Substrate-specific division of labor unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Resolved distinct enzymatic profiles of CNOT6 versus CAF1 and the stimulatory role of non-catalytic modules in a fully reconstituted human complex.\",\n      \"evidence\": \"In vitro reconstitution of recombinant human CCR4-NOT with compositional control and deadenylation assays\",\n      \"pmids\": [\"31320642\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In-cell substrate selectivity not addressed\", \"Regulatory inputs determining CNOT6 vs CAF1 usage unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed that CNOT6-driven changes in mRNA stability are buffered by compensatory transcription, explaining why decay activity does not always alter steady-state abundance.\",\n      \"evidence\": \"siRNA knockdown with paired mRNA stability and transcription-rate measurements in HepG2 cells\",\n      \"pmids\": [\"31116665\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Sensing mechanism coupling decay to transcription not defined\", \"Single cell line\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established the hierarchy of subunit essentiality, showing CNOT6/6L are dispensable for viability while their complex incorporation requires CNOT7/8.\",\n      \"evidence\": \"CRISPR double knockouts in MEFs and mice, Co-IP, and bulk poly(A) profiling\",\n      \"pmids\": [\"31924127\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific transcripts uniquely dependent on CNOT6/6L not enumerated\", \"Tissue-specific essential roles not probed here\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined an in vivo physiological requirement: CNOT6/6L act as FSH effectors essential for female reproductive endocrine regulation.\",\n      \"evidence\": \"Constitutive/conditional knockout mice, gonadotropin stimulation, transcriptome analysis and fertility phenotyping\",\n      \"pmids\": [\"34788619\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct FSH-induced decay targets not fully mapped\", \"Relative contributions of CNOT6 vs CNOT6L not separated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified CNOT6 as a recruited effector of sequence-specific decay, linking the RNA-binding protein ENO1 to IRP1 mRNA degradation and ferroptosis suppression.\",\n      \"evidence\": \"Co-IP, RIP, knockdown and in vitro/in vivo functional assays in hepatocellular carcinoma\",\n      \"pmids\": [\"35121990\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether ENO1 recruits the full complex or CNOT6 alone unclear\", \"Generality of ENO1-CNOT6 axis beyond HCC unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended target-specific decay to cell-cycle control, identifying p21 mRNA as a CNOT6/6L (but not CAF1) substrate.\",\n      \"evidence\": \"shRNA knockdown with cell-cycle analysis, P-body imaging and mRNA stability assays in gastric cancer cells\",\n      \"pmids\": [\"33671234\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of target selectivity over CAF1 unknown\", \"Single cancer cell line\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected CNOT6 to genome maintenance, showing it limits mismatch-repair capacity by destabilizing MMR gene mRNAs.\",\n      \"evidence\": \"siRNA knockdown with survival, mRNA stability, mutation frequency and MMR reporter assays in U2OS cells\",\n      \"pmids\": [\"35159331\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect targeting of MMR transcripts not established\", \"Single cell context\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Mechanistically defined CNOT6's dependency on CNOT7 through a bacterial effector that displaces it from the complex.\",\n      \"evidence\": \"Co-IP, in vitro binding/inhibition, yeast genetics and mammalian overexpression with Legionella PieF\",\n      \"pmids\": [\"39699231\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological consequences of PieF-mediated displacement during infection not detailed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed upstream control of CNOT6 itself, showing PCIF1-dependent m6A modification destabilizes CNOT6 mRNA.\",\n      \"evidence\": \"siRNA knockdown, MeRIP-qPCR and rescue with a methyltransferase-dead PCIF1 mutant\",\n      \"pmids\": [\"39451207\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"m6A reader mediating CNOT6 decay not identified\", \"Functional output of altered CNOT6 dosage not measured\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed the ENO1-CNOT6 decay axis is switchable by ENO1 lactylation, providing a metabolic control point for transcript stability.\",\n      \"evidence\": \"Co-IP, RIP, PTM mass spectrometry and RT-qPCR in sepsis endothelial cells (TRIM21 mRNA)\",\n      \"pmids\": [\"41532698\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Generality of lactylation-controlled CNOT6 recruitment unknown\", \"Quantitative contribution to TRIM21 stability not isolated\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Distinguished CNOT6 from its paralog as a non-redundant regulator of neonatal growth via hepatic Fgf21 mRNA decay.\",\n      \"evidence\": \"Cnot6 knockout mouse, hepatic transcriptomics, mRNA stability and metabolic phenotyping (preprint)\",\n      \"pmids\": [\"41757057\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Direct binding of CNOT6 to Fgf21 mRNA not shown\", \"Basis of CNOT6 vs CNOT6L specificity unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular basis by which CNOT6 selects specific substrates — versus the paralog CNOT6L or the CAF1 nucleases — and how diverse RNA-binding adaptors (ENO1, PUF/PBE factors) recruit it to particular transcripts remains undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of CNOT6 substrate engagement\", \"Determinants of CNOT6 vs CNOT6L target specificity unknown\", \"Rules governing adaptor-mediated recruitment not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [1, 2, 4]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [3, 12]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [8, 9]}\n    ],\n    \"complexes\": [\"CCR4-NOT complex\"],\n    \"partners\": [\"CNOT1\", \"CNOT7\", \"CNOT8\", \"CNOT6L\", \"ENO1\", \"NIF-1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}