{"gene":"CNOT1","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":1993,"finding":"CDC39 (yeast NOT1 ortholog) is an essential nuclear protein that negatively regulates transcription, preferentially affecting TC-dependent transcription at the HIS3 promoter; it encodes a large 2108-amino acid nuclear protein with two glutamine-rich regions.","method":"Temperature-sensitive mutant analysis, transcriptional assays, genetic complementation","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic and transcriptional assays, independently replicated in subsequent work","pmids":["8428577"],"is_preprint":false},{"year":1994,"finding":"NOT1(CDC39), NOT2, NOT3, and NOT4 form a discrete ~500 kDa nuclear complex that acts as a global negative regulator of transcription; NOT1 and NOT2 physically associate (two-hybrid, biochemical co-fractionation) and NOT4 interacts with NOT1 and NOT3 by two-hybrid.","method":"Allele-specific suppression, two-hybrid interaction, biochemical co-fractionation","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic and biochemical approaches, multiple interaction partners tested, replicated across studies","pmids":["7926748"],"is_preprint":false},{"year":1990,"finding":"CDC39 acts as a negative element in the yeast mating pheromone signal transduction pathway upstream of or at the level of the transducing G protein; epistasis analysis places CDC39 function between the G protein and downstream pathway components.","method":"Genetic epistasis analysis, pheromone-inducible gene expression assays","journal":"Cell regulation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with defined pathway components, two independent studies but single organism","pmids":["2099190","2111445"],"is_preprint":false},{"year":2000,"finding":"The essential function of Not1p resides in the C-terminal domain, which associates with Not5p and is required for Ccr4p to assemble into large complexes; the N-terminal domain is dispensable for viability but required for normal growth and efficient Ccr4p incorporation into the complex.","method":"Domain deletion analysis, co-fractionation on sucrose gradients, in vivo complementation with trans-expressed domains","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple domain deletions, co-fractionation, and functional rescue experiments in yeast","pmids":["11023781"],"is_preprint":false},{"year":1999,"finding":"Not1p directly represses transcription from a TATA-less promoter in vitro; nuclear extracts from a conditional not1 mutant show increased transcription from the HIS3 TATA-less promoter upon shift to restrictive temperature.","method":"In vitro transcription assay with nuclear extracts from conditional not1 mutant","journal":"Biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution assay but single study with conditional mutant extracts","pmids":["10661863"],"is_preprint":false},{"year":2011,"finding":"Human NOT1 physically interacts with the C-terminal domain of tristetraprolin (TTP) through its central region, and is required for TTP to recruit the CAF1 deadenylase to AU-rich element-containing mRNAs to promote their decay.","method":"Co-immunoprecipitation, siRNA knockdown, mRNA decay assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, functional knockdown with specific mRNA decay readout, multiple orthogonal methods","pmids":["21278420"],"is_preprint":false},{"year":2011,"finding":"CNOT1 depletion in HeLa cells reduces the deadenylase activity of immunoprecipitated CNOT6L, suppresses P-body formation, and stabilizes mRNAs, demonstrating CNOT1 is required for CCR4-NOT deadenylase activity and mRNA decay in human cells.","method":"siRNA knockdown, deadenylase activity assay, flow cytometry, immunofluorescence for P-bodies","journal":"Protein & cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional knockdown with enzymatic activity assay and cellular phenotype readouts, single lab","pmids":["21976065"],"is_preprint":false},{"year":2012,"finding":"The N-terminal arm of yeast Not1 has HEAT-repeat structure with MIF4G-fold domains; a central MIF4G domain directly recognizes Caf1, which in turn binds and positions the Ccr4 nuclease; disruption of these interfaces impairs cell growth and mRNA deadenylation in vivo.","method":"X-ray crystallography, in vivo growth and mRNA decay assays, mutagenesis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis and in vivo functional validation","pmids":["22959269"],"is_preprint":false},{"year":2012,"finding":"The CAF1-binding domain of human NOT1 adopts an MIF4G fold and binds CAF1 through a pre-formed interface, leaving the CAF1 catalytic site fully accessible to RNA substrates.","method":"X-ray crystallography of NOT1 MIF4G domain alone and in complex with CAF1","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures of free domain and complex with functional inference from active site accessibility","pmids":["22977175"],"is_preprint":false},{"year":2013,"finding":"The C-terminal arm of Not1 forms a HEAT-repeat scaffold that recruits Not2 and Not5 whose Not-box domains dimerize via noncanonical Sm-like folds; the resulting ternary complex binds poly(U) RNA in vitro with a binding site at the Not5 Not box.","method":"2.8-Å crystal structure, in vitro RNA binding assays, in vivo growth assays with disrupting mutations","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure with in vitro biochemical validation and in vivo functional testing","pmids":["24121231"],"is_preprint":false},{"year":2014,"finding":"The CNOT1 MIF4G domain directly interacts with the C-terminal RecA domain of DDX6, structurally resembling the eIF4G–eIF4A interaction; separately, the CNOT9 subunit binds the DUF3819 domain of CNOT1 and provides tandem tryptophan-binding pockets for TNRC6/GW182 recruitment, linking miRNA target recognition to CCR4-NOT-mediated silencing.","method":"Crystal structures of CNOT1 MIF4G–DDX6 and CNOT1 DUF3819–CNOT9 complexes, functional mutagenesis in cells","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple crystal structures combined with mutagenesis and cellular silencing assays","pmids":["24768540"],"is_preprint":false},{"year":2014,"finding":"Human DDX6 directly binds CNOT1 via a conserved subdomain, and mutations that disrupt this DDX6–CNOT1 interaction impair miRISC-mediated gene silencing, placing DDX6 as a downstream effector of CNOT1 in the miRNA pathway.","method":"Biochemical binding assays, site-directed mutagenesis, miRNA silencing reporter assays in human cells","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct binding assays with mutagenesis and functional cellular silencing readout, consistent with structural work in companion paper","pmids":["25035296"],"is_preprint":false},{"year":2015,"finding":"The CUP-homology domain of human 4E-T interacts directly with DDX6 and contacts CNOT1 MIF4G in a ternary complex; unlike Edc3 and Pat1 FDF motifs, 4E-T CHD binding to DDX6 is not displaced by CNOT1 MIF4G, revealing mutually exclusive vs. simultaneous interaction modes.","method":"2.1-Å crystal structure of 4E-T CHD–DDX6–CNOT1 ternary complex, in vitro competition binding assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure with biochemical competition assays","pmids":["26489469"],"is_preprint":false},{"year":2015,"finding":"In Xenopus oocytes, CAF1-mediated translational repression requires its association with NOT1; NOT1 is required to recruit Xp54 (DDX6 ortholog) and 4E-T, and repression via tethered CAF1 is cap- and eIF4E-dependent but not IRES-dependent.","method":"Affinity purification–mass spectrometry, co-immunoprecipitation, tethered reporter mRNA assays, 4E-T mutant analysis","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods in single study, in vivo Xenopus system","pmids":["26015597"],"is_preprint":false},{"year":2018,"finding":"The central region of CNOT1 encompassing the MIF4G and DUF3819 domains, together with CNOT9, stimulates deadenylation activity of the reconstituted pentameric CCR4-NOT nuclease module in vitro.","method":"In vitro reconstitution of pentameric complex, deadenylation activity assays comparing subcomplexes","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with defined subcomplexes, single lab but rigorous biochemical approach","pmids":["30309886"],"is_preprint":false},{"year":2018,"finding":"A connector domain of NOT1 adopts a MIF4G-like fold (MIF4G-C domain); structural comparison shows key differences from the DDX6-binding MIF4G domain that explain why MIF4G-C does not bind DDX6, and the human MIF4G-C does not stably interact with other CCR4-NOT subunits.","method":"Crystal structure of thermophilic fungal MIF4G-C domain, solution scattering of human domain, binding assays","journal":"Journal of structural biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — crystal structure with comparative structural analysis and negative binding result, single lab","pmids":["30367941"],"is_preprint":false},{"year":2019,"finding":"NOT1-containing assemblysomes in yeast are cytoplasmic particles (distinct from stress granules and P-bodies) that depend on Not1 and facilitate co-translational assembly of proteasome subunits Rpt1 and Rpt2; this mechanism is conserved in human cells.","method":"Immunofluorescence, fluorescence in situ hybridization, ribosome profiling, genetic depletion","journal":"Nature structural & molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple imaging and functional methods, conservation shown in human cells, single lab","pmids":["30692646"],"is_preprint":false},{"year":2019,"finding":"The conserved C-terminal CNOT1-binding domain (CNBD) of TTP is required for full TTP activity in vivo; CNBD deletion mice have a less severe inflammatory phenotype than full TTP knockout, and recombinant TTP lacking CNBD still activates deadenylation but to a lesser extent than full-length TTP.","method":"Knock-in mouse model (CNBD deletion), macrophage mRNA decay assays, cell-free deadenylation assay with recombinant proteins","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vivo mouse model combined with cell-free biochemical reconstitution, multiple orthogonal readouts","pmids":["31036567"],"is_preprint":false},{"year":2022,"finding":"High-resolution structural analysis reveals the human N-terminal module is composed of CNOT1 sandwiching CNOT10 and CNOT11 via two helical domains; the most conserved domain of CNOT11 protrudes as an antenna that serves as a binding platform for GGNBP2.","method":"Multiple structural approaches (cryo-EM/crystallography), biochemical binding assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution structural data with biochemical validation, single lab but multiple structural methods","pmids":["36586408"],"is_preprint":false},{"year":2022,"finding":"TASOR interacts with CNOT1 (identified by yeast two-hybrid) and synergistically represses HIV-1 LTR-derived transcript accumulation; TASOR facilitates association of RNA degradation proteins with RNA Polymerase II.","method":"Yeast two-hybrid, co-immunoprecipitation, HIV reporter assays, ChIP/RIP","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — yeast two-hybrid plus co-IP and functional assays, single lab","pmids":["35013187"],"is_preprint":false},{"year":2022,"finding":"The TTP CNOT1 Interaction Motif (CIM) cooperates with TTP tryptophan residues (that contact CNOT9) to recruit CCR4-NOT and activate mRNA degradation; CIM phosphorylation by PKCα (not MK2) disrupts CNOT1 association, while p38-MK2 activation leaves the CIM unphosphorylated and capable of CCR4-NOT recruitment.","method":"Co-immunoprecipitation with phosphomimetic/phosphonull mutants, mRNA decay reporter assays, kinase inhibitor experiments","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple mutant analyses and kinase experiments, single lab, mRNA decay functional readout","pmids":["35920669"],"is_preprint":false},{"year":2021,"finding":"CNOT1 regulates the mammalian circadian clock by promoting deadenylation-dependent decay of Per2 mRNA; CNOT1 is recruited to Per2 mRNA through BRF1/ZFP36L1, and CNOT1 deficiency in mice causes circadian period lengthening with extended Per2 mRNA poly(A) tail length and increased Per2 mRNA stability.","method":"Conditional knockout mice, poly(A) tail length assay, BRF1 knockdown, circadian behavioral monitoring","journal":"RNA biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo mouse KO with molecular and behavioral phenotypes, mechanistic pathway placement via BRF1 knockdown","pmids":["35510877"],"is_preprint":false},{"year":2023,"finding":"Depletion of Not1 and Not4 oppositely affect mRNA solubility in yeast: Not1 depletion solubilizes mitochondrial mRNAs (which become insoluble upon Not4 depletion), while Not4 depletion solubilizes mRNAs with lower non-optimal codon content that are rendered insoluble by Not1; Not1 promoter association in the nucleus may set mRNA solubility.","method":"Ribosome profiling, RNA fractionation (soluble/insoluble), ChIP, depletion of Not1/Not4 by conditional systems","journal":"Genome biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transcriptome-wide analysis combined with fractionation, single lab but multiple orthogonal approaches","pmids":["36803582"],"is_preprint":false},{"year":2025,"finding":"CNOT1 interacts with 53BP1, impacts its nuclear dynamics, and acts as a suppressor of 53BP1-p53-p21 signaling; CNOT1 loss upregulates p53 target gene expression, impairs proliferation, and suppresses cytoplasmic aggregation of mutant p53, thereby restoring its nuclear localization and functionality.","method":"High-content microscopy screen, orthogonal validation, co-immunoprecipitation, siRNA knockdown, nuclear dynamics imaging","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal validation methods, single lab, functional phenotypic readouts","pmids":["40742806"],"is_preprint":false},{"year":2025,"finding":"Acute auxin-induced depletion of CNOT1 in human cells causes widespread increased mRNA abundance and decreased global mRNA decay, with changes correlating with codon optimality; depletion of CNOT4 has opposite effects, unexpectedly accelerating global mRNA decay, with BioID confirming CNOT4 associates with the complex in cells despite not co-purifying by standard biochemistry.","method":"Auxin-induced degron system, transcriptome-wide RNA-seq, mRNA decay assays, BioID proximity labeling","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — acute depletion system with transcriptome-wide analysis, single lab but multiple orthogonal methods","pmids":["41161383"],"is_preprint":false},{"year":2006,"finding":"C. elegans LET-711 (NOT1 ortholog) is required for spindle positioning in early embryos; let-711 mutants have longer, cold-stable microtubules, larger centrosomes with elevated ZYG-9 levels, and simultaneous reduction of both ZYG-9 and LET-711 rescues spindle positioning defects of both single mutants, placing LET-711 upstream of ZYG-9.","method":"RNAi/genetic loss-of-function, live imaging of spindle positioning, cold-stability assay, genetic epistasis with zyg-9","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with defined phenotypic readouts and imaging, single organism but multiple orthogonal approaches","pmids":["16971515"],"is_preprint":false},{"year":2018,"finding":"CNOT1 knockdown in osteosarcoma cells inhibits growth and the Hedgehog signaling pathway; CNOT1 physically interacts with LMNA (lamin A) and functions as a positive regulator of LMNA; LMNA overexpression rescues the growth and Hedgehog pathway defects of CNOT1 depletion.","method":"Co-immunoprecipitation, siRNA knockdown, RNA-seq, rescue overexpression experiments, xenograft in vivo","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP and rescue experiments, single lab, mechanistic pathway placement by rescue","pmids":["28188704"],"is_preprint":false},{"year":2018,"finding":"CNOT1 provides a scaffold platform in human pulmonary endothelial cells for recruitment of both TTP and CNOT7; CNOT1 silencing abolishes TTP-CNOT7 co-immunoprecipitation, while CNOT7 or TTP silencing does not disrupt CNOT1 interactions with the other partner, demonstrating hierarchical assembly.","method":"Co-immunoprecipitation with sequential siRNA knockdowns, immunofluorescence co-localization, mRNA stability assays","journal":"Molecular medicine reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP with knockdowns from multiple angles, single lab, mechanistically informative hierarchy","pmids":["29956766"],"is_preprint":false},{"year":2020,"finding":"ZFP36L1 directly represses translation via AU-rich elements through an interaction with CNOT1, independent of deadenylation; this mechanism differs from TTP (does not involve 4E-HP or GIGYF2 recruitment) and from miRISC (resistant to eIF4A inhibitor silvestrol, IRES-independent).","method":"In vitro translation system from mammalian cell lysates, mutant protein functional assays, pharmacological inhibition","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution assay with defined mutants, single lab, novel mechanism distinct from known pathways","pmids":["32311426"],"is_preprint":false},{"year":2021,"finding":"Phosphorylation of TTP Ser316 (in the C-terminal CNOT1-binding domain) by RSK1 and MK2, and dephosphorylation by PP2A, regulates TTP interaction with CNOT1; a phosphomimetic S316D mutation weakens CNOT1 interaction and TTP-mediated TNFα mRNA destabilization.","method":"Phospho-specific antibody generation, GST pull-down, RNA pull-down, CRISPR/Cas9 TTP knockout, phosphomimetic/phosphonull mutants, kinase/phosphatase inhibitor experiments","journal":"Journal of inflammation (London, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal biochemical and cellular methods, single lab","pmids":["34090459"],"is_preprint":false},{"year":2025,"finding":"HDX-MS analysis of CNOT1 residues 800-999 (HEAT-like repeat domain) reveals that E893A/Y900A and E893Q/Y900H point mutations reduce TTP peptide interaction without perturbing overall domain structure, defining these residues as critical for molecular recognition of TTP.","method":"Hydrogen/deuterium exchange mass spectrometry (HDX-MS) of wild-type and point mutant CNOT1(800-999) with TTP peptide","journal":"Biomolecules","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — rigorous biophysical method but single lab and single study","pmids":["40149939"],"is_preprint":false}],"current_model":"CNOT1 is the central scaffold protein of the conserved CCR4-NOT deadenylase complex, organizing multiple functional modules through distinct structural domains: its MIF4G domain recruits CAF1 (which positions CCR4 nuclease), its DUF3819 domain binds CNOT9 (which provides tryptophan-binding pockets for TNRC6/GW182 and TTP), its MIF4G domain also recruits DDX6 to couple deadenylation with translational repression and decapping, and its N-terminal helical domains sandwich CNOT10-CNOT11 to form a protein-interaction platform; through these interactions CNOT1 coordinates mRNA deadenylation, translational repression, ARE-mRNA decay (via TTP/ZFP36L1 recruitment), miRNA silencing, co-translational assembly in cytoplasmic assemblysomes, circadian clock regulation via Per2 mRNA decay, and transcriptional repression, while also interacting with 53BP1 to suppress p53 signaling."},"narrative":{"mechanistic_narrative":"CNOT1 is the central scaffold of the conserved CCR4-NOT complex, a global regulator of mRNA stability and translation that originated as the essential yeast NOT1/CDC39 nuclear repressor of transcription [PMID:8428577, PMID:7926748]. In human cells CNOT1 is required for CCR4-NOT deadenylase activity, mRNA decay and P-body formation, and its depletion stabilizes mRNAs in a manner correlated with codon optimality [PMID:21976065, PMID:41161383]. Structurally, CNOT1 is an extended HEAT-repeat platform whose multiple MIF4G and MIF4G-like domains organize distinct functional modules: a central MIF4G domain binds CAF1/CNOT7 and positions the CCR4 nuclease with its active site accessible to RNA [PMID:22959269, PMID:22977175], a second MIF4G domain recruits the DEAD-box helicase DDX6 to couple deadenylation to translational repression and decapping [PMID:24768540, PMID:25035296], the DUF3819 domain binds CNOT9 to provide tryptophan-binding pockets for TNRC6/GW182 in miRNA silencing [PMID:24768540], the C-terminal arm recruits NOT2/NOT5 [PMID:24121231], and N-terminal helical domains sandwich CNOT10-CNOT11 to form a protein-interaction antenna [PMID:36586408]. Through these interfaces CNOT1 functions as a recruitment hub for sequence-specific RNA-binding adaptors that target the complex to particular transcripts: it binds the AU-rich-element factor tristetraprolin (TTP) through a defined HEAT-like repeat surface to drive ARE-mRNA decay [PMID:21278420, PMID:40149939], engages ZFP36L1 to repress translation independently of deadenylation [PMID:32311426], and is recruited via ZFP36L1/BRF1 to Per2 mRNA to control circadian period through deadenylation-dependent decay [PMID:35510877]. The CNOT1 MIF4G/DUF3819 region together with CNOT9 stimulates deadenylation of the reconstituted nuclease module [PMID:30309886], and CNOT1 also nucleates cytoplasmic assemblysomes that support co-translational assembly of proteasome subunits [PMID:30692646]. Beyond RNA turnover, CNOT1 interacts with 53BP1 and suppresses 53BP1-p53-p21 signaling [PMID:40742806].","teleology":[{"year":1994,"claim":"Established that NOT1/CDC39 is not an isolated factor but the organizing member of a discrete multi-subunit nuclear NOT complex acting as a global transcriptional repressor.","evidence":"Yeast genetics, two-hybrid interaction and biochemical co-fractionation of NOT1-NOT4","pmids":["7926748","8428577"],"confidence":"High","gaps":["Did not resolve whether repression was direct or via downstream RNA metabolism","No structural basis for subunit assembly"]},{"year":2000,"claim":"Localized the essential scaffolding function to the Not1 C-terminal domain, which is required to assemble the catalytic Ccr4 nuclease into the larger complex.","evidence":"Domain deletion, sucrose gradient co-fractionation and in vivo complementation in yeast","pmids":["11023781"],"confidence":"High","gaps":["Atomic interfaces not defined","Mechanism linking assembly to enzymatic activity unresolved"]},{"year":2011,"claim":"Demonstrated that human CNOT1 is functionally required for CCR4-NOT deadenylase activity and serves as the bridge by which the ARE-binding protein TTP recruits deadenylase to target mRNAs.","evidence":"Co-IP, siRNA knockdown, deadenylase activity assays and mRNA decay readouts in human cells","pmids":["21278420","21976065"],"confidence":"High","gaps":["Binding interface on CNOT1 not mapped","Whether other adaptors use the same surface unknown"]},{"year":2014,"claim":"Resolved the modular structural logic by which CNOT1 partitions deadenylation, decapping/repression and miRNA silencing through distinct domains binding CAF1, DDX6 and CNOT9.","evidence":"Crystal structures of CNOT1 MIF4G-CAF1, MIF4G-DDX6 and DUF3819-CNOT9 complexes with functional mutagenesis","pmids":["22959269","22977175","24768540","25035296"],"confidence":"High","gaps":["How modules are coordinated on a single mRNA not shown","Regulation of domain occupancy unaddressed"]},{"year":2015,"claim":"Showed that CNOT1 couples deadenylation to translational repression by recruiting DDX6/Xp54 and 4E-T, with cap-dependent repression downstream of CAF1-NOT1 association.","evidence":"Crystal structure of 4E-T CHD-DDX6-CNOT1 ternary complex and tethered reporter assays in Xenopus oocytes","pmids":["26489469","26015597"],"confidence":"High","gaps":["Quantitative contribution of repression vs decay in vivo unclear","Competition among DDX6 partners on endogenous mRNAs not measured"]},{"year":2018,"claim":"Reconstituted the contribution of the CNOT1 central region and CNOT9 to stimulating the nuclease module and mapped additional structural domains (MIF4G-C, N-terminal module).","evidence":"In vitro reconstitution of pentameric complex and crystallography/SAXS of MIF4G-C and N-terminal domains","pmids":["30309886","30367941"],"confidence":"High","gaps":["MIF4G-C functional role in humans undefined","Mechanism of activity stimulation not fully resolved"]},{"year":2019,"claim":"Extended CNOT1 function beyond decay by showing it nucleates cytoplasmic assemblysomes for co-translational assembly of proteasome subunits, and refined the TTP CNOT1-binding domain requirement in vivo.","evidence":"Imaging/ribosome profiling in yeast and human cells; CNBD-deletion knock-in mice and cell-free deadenylation","pmids":["30692646","31036567"],"confidence":"Medium","gaps":["Direct CNOT1 contacts within assemblysomes not mapped","Range of co-translationally assembled complexes unknown"]},{"year":2022,"claim":"Mapped the human N-terminal module architecture (CNOT1 sandwiching CNOT10-CNOT11 with a CNOT11 antenna binding GGNBP2) and identified novel transcriptional/anti-viral partners.","evidence":"Cryo-EM/crystallography and binding assays; yeast two-hybrid and HIV reporter assays for TASOR","pmids":["36586408","35013187"],"confidence":"Medium","gaps":["Functional role of GGNBP2 recruitment unclear","TASOR-CNOT1 link rests on single-lab assays"]},{"year":2022,"claim":"Defined how phosphorylation of the TTP CNOT1-interaction motif by specific kinases switches CCR4-NOT recruitment on and off, linking signaling to ARE-mRNA decay.","evidence":"Phosphomimetic/phosphonull Co-IP, kinase inhibitor and mRNA decay reporter assays","pmids":["35920669","34090459"],"confidence":"Medium","gaps":["Endogenous phospho-occupancy not quantified","Whether other adaptors are similarly regulated unknown"]},{"year":2021,"claim":"Placed CNOT1 in physiological circadian control by demonstrating ZFP36L1/BRF1-directed deadenylation of Per2 mRNA sets clock period.","evidence":"Conditional knockout mice, poly(A) tail assays, BRF1 knockdown and behavioral monitoring","pmids":["35510877"],"confidence":"Medium","gaps":["Direct CNOT1 occupancy on Per2 mRNA not shown","Generality to other clock transcripts untested"]},{"year":2025,"claim":"Established acute, codon-optimality-linked control of global mRNA decay by CNOT1 and revealed an unexpected non-RNA role as a suppressor of 53BP1-p53-p21 signaling.","evidence":"Auxin degron RNA-seq/decay assays with BioID; high-content microscopy, Co-IP and nuclear dynamics imaging","pmids":["41161383","40742806"],"confidence":"Medium","gaps":["Mechanism connecting CNOT1 to 53BP1 dynamics unresolved","Whether the p53 role is decay-dependent unknown"]},{"year":null,"claim":"How CNOT1's many adaptor-binding surfaces are dynamically selected and coordinated on individual mRNAs in vivo, and whether its transcriptional and 53BP1/p53 roles are separable from its deadenylase scaffolding, remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated model of competing adaptor occupancy","Direct in vivo target specificity determinants undefined","Mechanistic basis of non-canonical nuclear roles unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[5,7,10,27]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[6,14,24]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[9]},{"term_id":"GO:0045182","term_label":"translation regulator activity","supporting_discovery_ids":[13,28]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,1,4]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6,16]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[6,14,24]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,1,19]},{"term_id":"R-HSA-9909396","term_label":"Circadian clock","supporting_discovery_ids":[21]}],"complexes":["CCR4-NOT complex","assemblysome"],"partners":["CNOT7","CNOT9","DDX6","ZFP36 (TTP)","ZFP36L1","CNOT11","53BP1","EIF4ENIF1 (4E-T)"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"A5YKK6","full_name":"CCR4-NOT transcription complex subunit 1","aliases":["CCR4-associated factor 1","Negative regulator of transcription subunit 1 homolog","NOT1H","hNOT1"],"length_aa":2376,"mass_kda":266.9,"function":"Scaffolding 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. Its scaffolding function implies its interaction with the catalytic complex module and diverse RNA-binding proteins mediating the complex recruitment to selected mRNA 3'UTRs. Involved in degradation of AU-rich element (ARE)-containing mRNAs probably via association with ZFP36. Mediates the recruitment of the CCR4-NOT complex to miRNA targets and to the RISC complex via association with TNRC6A, TNRC6B or TNRC6C. Acts as a transcriptional repressor. Represses the ligand-dependent transcriptional activation by nuclear receptors. Involved in the maintenance of embryonic stem (ES) cell identity. Plays a role in rapid sperm motility via mediating timely mRNA turnover (By similarity)","subcellular_location":"Cytoplasm, P-body; Nucleus","url":"https://www.uniprot.org/uniprotkb/A5YKK6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CNOT1","classification":"Common Essential","n_dependent_lines":902,"n_total_lines":1208,"dependency_fraction":0.7466887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CAPZB","stoichiometry":0.2},{"gene":"CTTN","stoichiometry":0.2},{"gene":"HNRNPH1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CNOT1","total_profiled":1310},"omim":[{"mim_id":"620509","title":"CCR4-NOT TRANSCRIPTION COMPLEX, SUBUNIT 11; CNOT11","url":"https://www.omim.org/entry/620509"},{"mim_id":"620508","title":"CCR4-NOT TRANSCRIPTION COMPLEX, SUBUNIT 10; CNOT10","url":"https://www.omim.org/entry/620508"},{"mim_id":"619033","title":"VISSERS-BODMER SYNDROME; VIBOS","url":"https://www.omim.org/entry/619033"},{"mim_id":"618998","title":"IMMUNE DYSREGULATION AND SYSTEMIC HYPERINFLAMMATION SYNDROME; IMDYSHI","url":"https://www.omim.org/entry/618998"},{"mim_id":"618500","title":"HOLOPROSENCEPHALY 12 WITH OR WITHOUT PANCREATIC AGENESIS; HPE12","url":"https://www.omim.org/entry/618500"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Cytosol","reliability":"Uncertain"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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it encodes a large 2108-amino acid nuclear protein with two glutamine-rich regions.\",\n      \"method\": \"Temperature-sensitive mutant analysis, transcriptional assays, genetic complementation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic and transcriptional assays, independently replicated in subsequent work\",\n      \"pmids\": [\"8428577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"NOT1(CDC39), NOT2, NOT3, and NOT4 form a discrete ~500 kDa nuclear complex that acts as a global negative regulator of transcription; NOT1 and NOT2 physically associate (two-hybrid, biochemical co-fractionation) and NOT4 interacts with NOT1 and NOT3 by two-hybrid.\",\n      \"method\": \"Allele-specific suppression, two-hybrid interaction, biochemical co-fractionation\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic and biochemical approaches, multiple interaction partners tested, replicated across studies\",\n      \"pmids\": [\"7926748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"CDC39 acts as a negative element in the yeast mating pheromone signal transduction pathway upstream of or at the level of the transducing G protein; epistasis analysis places CDC39 function between the G protein and downstream pathway components.\",\n      \"method\": \"Genetic epistasis analysis, pheromone-inducible gene expression assays\",\n      \"journal\": \"Cell regulation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with defined pathway components, two independent studies but single organism\",\n      \"pmids\": [\"2099190\", \"2111445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The essential function of Not1p resides in the C-terminal domain, which associates with Not5p and is required for Ccr4p to assemble into large complexes; the N-terminal domain is dispensable for viability but required for normal growth and efficient Ccr4p incorporation into the complex.\",\n      \"method\": \"Domain deletion analysis, co-fractionation on sucrose gradients, in vivo complementation with trans-expressed domains\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple domain deletions, co-fractionation, and functional rescue experiments in yeast\",\n      \"pmids\": [\"11023781\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Not1p directly represses transcription from a TATA-less promoter in vitro; nuclear extracts from a conditional not1 mutant show increased transcription from the HIS3 TATA-less promoter upon shift to restrictive temperature.\",\n      \"method\": \"In vitro transcription assay with nuclear extracts from conditional not1 mutant\",\n      \"journal\": \"Biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution assay but single study with conditional mutant extracts\",\n      \"pmids\": [\"10661863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Human NOT1 physically interacts with the C-terminal domain of tristetraprolin (TTP) through its central region, and is required for TTP to recruit the CAF1 deadenylase to AU-rich element-containing mRNAs to promote their decay.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, mRNA decay assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, functional knockdown with specific mRNA decay readout, multiple orthogonal methods\",\n      \"pmids\": [\"21278420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CNOT1 depletion in HeLa cells reduces the deadenylase activity of immunoprecipitated CNOT6L, suppresses P-body formation, and stabilizes mRNAs, demonstrating CNOT1 is required for CCR4-NOT deadenylase activity and mRNA decay in human cells.\",\n      \"method\": \"siRNA knockdown, deadenylase activity assay, flow cytometry, immunofluorescence for P-bodies\",\n      \"journal\": \"Protein & cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional knockdown with enzymatic activity assay and cellular phenotype readouts, single lab\",\n      \"pmids\": [\"21976065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The N-terminal arm of yeast Not1 has HEAT-repeat structure with MIF4G-fold domains; a central MIF4G domain directly recognizes Caf1, which in turn binds and positions the Ccr4 nuclease; disruption of these interfaces impairs cell growth and mRNA deadenylation in vivo.\",\n      \"method\": \"X-ray crystallography, in vivo growth and mRNA decay assays, mutagenesis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis and in vivo functional validation\",\n      \"pmids\": [\"22959269\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The CAF1-binding domain of human NOT1 adopts an MIF4G fold and binds CAF1 through a pre-formed interface, leaving the CAF1 catalytic site fully accessible to RNA substrates.\",\n      \"method\": \"X-ray crystallography of NOT1 MIF4G domain alone and in complex with CAF1\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures of free domain and complex with functional inference from active site accessibility\",\n      \"pmids\": [\"22977175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The C-terminal arm of Not1 forms a HEAT-repeat scaffold that recruits Not2 and Not5 whose Not-box domains dimerize via noncanonical Sm-like folds; the resulting ternary complex binds poly(U) RNA in vitro with a binding site at the Not5 Not box.\",\n      \"method\": \"2.8-Å crystal structure, in vitro RNA binding assays, in vivo growth assays with disrupting mutations\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure with in vitro biochemical validation and in vivo functional testing\",\n      \"pmids\": [\"24121231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The CNOT1 MIF4G domain directly interacts with the C-terminal RecA domain of DDX6, structurally resembling the eIF4G–eIF4A interaction; separately, the CNOT9 subunit binds the DUF3819 domain of CNOT1 and provides tandem tryptophan-binding pockets for TNRC6/GW182 recruitment, linking miRNA target recognition to CCR4-NOT-mediated silencing.\",\n      \"method\": \"Crystal structures of CNOT1 MIF4G–DDX6 and CNOT1 DUF3819–CNOT9 complexes, functional mutagenesis in cells\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple crystal structures combined with mutagenesis and cellular silencing assays\",\n      \"pmids\": [\"24768540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Human DDX6 directly binds CNOT1 via a conserved subdomain, and mutations that disrupt this DDX6–CNOT1 interaction impair miRISC-mediated gene silencing, placing DDX6 as a downstream effector of CNOT1 in the miRNA pathway.\",\n      \"method\": \"Biochemical binding assays, site-directed mutagenesis, miRNA silencing reporter assays in human cells\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct binding assays with mutagenesis and functional cellular silencing readout, consistent with structural work in companion paper\",\n      \"pmids\": [\"25035296\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The CUP-homology domain of human 4E-T interacts directly with DDX6 and contacts CNOT1 MIF4G in a ternary complex; unlike Edc3 and Pat1 FDF motifs, 4E-T CHD binding to DDX6 is not displaced by CNOT1 MIF4G, revealing mutually exclusive vs. simultaneous interaction modes.\",\n      \"method\": \"2.1-Å crystal structure of 4E-T CHD–DDX6–CNOT1 ternary complex, in vitro competition binding assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure with biochemical competition assays\",\n      \"pmids\": [\"26489469\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In Xenopus oocytes, CAF1-mediated translational repression requires its association with NOT1; NOT1 is required to recruit Xp54 (DDX6 ortholog) and 4E-T, and repression via tethered CAF1 is cap- and eIF4E-dependent but not IRES-dependent.\",\n      \"method\": \"Affinity purification–mass spectrometry, co-immunoprecipitation, tethered reporter mRNA assays, 4E-T mutant analysis\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods in single study, in vivo Xenopus system\",\n      \"pmids\": [\"26015597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The central region of CNOT1 encompassing the MIF4G and DUF3819 domains, together with CNOT9, stimulates deadenylation activity of the reconstituted pentameric CCR4-NOT nuclease module in vitro.\",\n      \"method\": \"In vitro reconstitution of pentameric complex, deadenylation activity assays comparing subcomplexes\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with defined subcomplexes, single lab but rigorous biochemical approach\",\n      \"pmids\": [\"30309886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A connector domain of NOT1 adopts a MIF4G-like fold (MIF4G-C domain); structural comparison shows key differences from the DDX6-binding MIF4G domain that explain why MIF4G-C does not bind DDX6, and the human MIF4G-C does not stably interact with other CCR4-NOT subunits.\",\n      \"method\": \"Crystal structure of thermophilic fungal MIF4G-C domain, solution scattering of human domain, binding assays\",\n      \"journal\": \"Journal of structural biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with comparative structural analysis and negative binding result, single lab\",\n      \"pmids\": [\"30367941\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NOT1-containing assemblysomes in yeast are cytoplasmic particles (distinct from stress granules and P-bodies) that depend on Not1 and facilitate co-translational assembly of proteasome subunits Rpt1 and Rpt2; this mechanism is conserved in human cells.\",\n      \"method\": \"Immunofluorescence, fluorescence in situ hybridization, ribosome profiling, genetic depletion\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple imaging and functional methods, conservation shown in human cells, single lab\",\n      \"pmids\": [\"30692646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The conserved C-terminal CNOT1-binding domain (CNBD) of TTP is required for full TTP activity in vivo; CNBD deletion mice have a less severe inflammatory phenotype than full TTP knockout, and recombinant TTP lacking CNBD still activates deadenylation but to a lesser extent than full-length TTP.\",\n      \"method\": \"Knock-in mouse model (CNBD deletion), macrophage mRNA decay assays, cell-free deadenylation assay with recombinant proteins\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vivo mouse model combined with cell-free biochemical reconstitution, multiple orthogonal readouts\",\n      \"pmids\": [\"31036567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"High-resolution structural analysis reveals the human N-terminal module is composed of CNOT1 sandwiching CNOT10 and CNOT11 via two helical domains; the most conserved domain of CNOT11 protrudes as an antenna that serves as a binding platform for GGNBP2.\",\n      \"method\": \"Multiple structural approaches (cryo-EM/crystallography), biochemical binding assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution structural data with biochemical validation, single lab but multiple structural methods\",\n      \"pmids\": [\"36586408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TASOR interacts with CNOT1 (identified by yeast two-hybrid) and synergistically represses HIV-1 LTR-derived transcript accumulation; TASOR facilitates association of RNA degradation proteins with RNA Polymerase II.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, HIV reporter assays, ChIP/RIP\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — yeast two-hybrid plus co-IP and functional assays, single lab\",\n      \"pmids\": [\"35013187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The TTP CNOT1 Interaction Motif (CIM) cooperates with TTP tryptophan residues (that contact CNOT9) to recruit CCR4-NOT and activate mRNA degradation; CIM phosphorylation by PKCα (not MK2) disrupts CNOT1 association, while p38-MK2 activation leaves the CIM unphosphorylated and capable of CCR4-NOT recruitment.\",\n      \"method\": \"Co-immunoprecipitation with phosphomimetic/phosphonull mutants, mRNA decay reporter assays, kinase inhibitor experiments\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple mutant analyses and kinase experiments, single lab, mRNA decay functional readout\",\n      \"pmids\": [\"35920669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CNOT1 regulates the mammalian circadian clock by promoting deadenylation-dependent decay of Per2 mRNA; CNOT1 is recruited to Per2 mRNA through BRF1/ZFP36L1, and CNOT1 deficiency in mice causes circadian period lengthening with extended Per2 mRNA poly(A) tail length and increased Per2 mRNA stability.\",\n      \"method\": \"Conditional knockout mice, poly(A) tail length assay, BRF1 knockdown, circadian behavioral monitoring\",\n      \"journal\": \"RNA biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo mouse KO with molecular and behavioral phenotypes, mechanistic pathway placement via BRF1 knockdown\",\n      \"pmids\": [\"35510877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Depletion of Not1 and Not4 oppositely affect mRNA solubility in yeast: Not1 depletion solubilizes mitochondrial mRNAs (which become insoluble upon Not4 depletion), while Not4 depletion solubilizes mRNAs with lower non-optimal codon content that are rendered insoluble by Not1; Not1 promoter association in the nucleus may set mRNA solubility.\",\n      \"method\": \"Ribosome profiling, RNA fractionation (soluble/insoluble), ChIP, depletion of Not1/Not4 by conditional systems\",\n      \"journal\": \"Genome biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transcriptome-wide analysis combined with fractionation, single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"36803582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CNOT1 interacts with 53BP1, impacts its nuclear dynamics, and acts as a suppressor of 53BP1-p53-p21 signaling; CNOT1 loss upregulates p53 target gene expression, impairs proliferation, and suppresses cytoplasmic aggregation of mutant p53, thereby restoring its nuclear localization and functionality.\",\n      \"method\": \"High-content microscopy screen, orthogonal validation, co-immunoprecipitation, siRNA knockdown, nuclear dynamics imaging\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal validation methods, single lab, functional phenotypic readouts\",\n      \"pmids\": [\"40742806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Acute auxin-induced depletion of CNOT1 in human cells causes widespread increased mRNA abundance and decreased global mRNA decay, with changes correlating with codon optimality; depletion of CNOT4 has opposite effects, unexpectedly accelerating global mRNA decay, with BioID confirming CNOT4 associates with the complex in cells despite not co-purifying by standard biochemistry.\",\n      \"method\": \"Auxin-induced degron system, transcriptome-wide RNA-seq, mRNA decay assays, BioID proximity labeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — acute depletion system with transcriptome-wide analysis, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"41161383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"C. elegans LET-711 (NOT1 ortholog) is required for spindle positioning in early embryos; let-711 mutants have longer, cold-stable microtubules, larger centrosomes with elevated ZYG-9 levels, and simultaneous reduction of both ZYG-9 and LET-711 rescues spindle positioning defects of both single mutants, placing LET-711 upstream of ZYG-9.\",\n      \"method\": \"RNAi/genetic loss-of-function, live imaging of spindle positioning, cold-stability assay, genetic epistasis with zyg-9\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with defined phenotypic readouts and imaging, single organism but multiple orthogonal approaches\",\n      \"pmids\": [\"16971515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CNOT1 knockdown in osteosarcoma cells inhibits growth and the Hedgehog signaling pathway; CNOT1 physically interacts with LMNA (lamin A) and functions as a positive regulator of LMNA; LMNA overexpression rescues the growth and Hedgehog pathway defects of CNOT1 depletion.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, RNA-seq, rescue overexpression experiments, xenograft in vivo\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP and rescue experiments, single lab, mechanistic pathway placement by rescue\",\n      \"pmids\": [\"28188704\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CNOT1 provides a scaffold platform in human pulmonary endothelial cells for recruitment of both TTP and CNOT7; CNOT1 silencing abolishes TTP-CNOT7 co-immunoprecipitation, while CNOT7 or TTP silencing does not disrupt CNOT1 interactions with the other partner, demonstrating hierarchical assembly.\",\n      \"method\": \"Co-immunoprecipitation with sequential siRNA knockdowns, immunofluorescence co-localization, mRNA stability assays\",\n      \"journal\": \"Molecular medicine reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP with knockdowns from multiple angles, single lab, mechanistically informative hierarchy\",\n      \"pmids\": [\"29956766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ZFP36L1 directly represses translation via AU-rich elements through an interaction with CNOT1, independent of deadenylation; this mechanism differs from TTP (does not involve 4E-HP or GIGYF2 recruitment) and from miRISC (resistant to eIF4A inhibitor silvestrol, IRES-independent).\",\n      \"method\": \"In vitro translation system from mammalian cell lysates, mutant protein functional assays, pharmacological inhibition\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution assay with defined mutants, single lab, novel mechanism distinct from known pathways\",\n      \"pmids\": [\"32311426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Phosphorylation of TTP Ser316 (in the C-terminal CNOT1-binding domain) by RSK1 and MK2, and dephosphorylation by PP2A, regulates TTP interaction with CNOT1; a phosphomimetic S316D mutation weakens CNOT1 interaction and TTP-mediated TNFα mRNA destabilization.\",\n      \"method\": \"Phospho-specific antibody generation, GST pull-down, RNA pull-down, CRISPR/Cas9 TTP knockout, phosphomimetic/phosphonull mutants, kinase/phosphatase inhibitor experiments\",\n      \"journal\": \"Journal of inflammation (London, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal biochemical and cellular methods, single lab\",\n      \"pmids\": [\"34090459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HDX-MS analysis of CNOT1 residues 800-999 (HEAT-like repeat domain) reveals that E893A/Y900A and E893Q/Y900H point mutations reduce TTP peptide interaction without perturbing overall domain structure, defining these residues as critical for molecular recognition of TTP.\",\n      \"method\": \"Hydrogen/deuterium exchange mass spectrometry (HDX-MS) of wild-type and point mutant CNOT1(800-999) with TTP peptide\",\n      \"journal\": \"Biomolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous biophysical method but single lab and single study\",\n      \"pmids\": [\"40149939\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CNOT1 is the central scaffold protein of the conserved CCR4-NOT deadenylase complex, organizing multiple functional modules through distinct structural domains: its MIF4G domain recruits CAF1 (which positions CCR4 nuclease), its DUF3819 domain binds CNOT9 (which provides tryptophan-binding pockets for TNRC6/GW182 and TTP), its MIF4G domain also recruits DDX6 to couple deadenylation with translational repression and decapping, and its N-terminal helical domains sandwich CNOT10-CNOT11 to form a protein-interaction platform; through these interactions CNOT1 coordinates mRNA deadenylation, translational repression, ARE-mRNA decay (via TTP/ZFP36L1 recruitment), miRNA silencing, co-translational assembly in cytoplasmic assemblysomes, circadian clock regulation via Per2 mRNA decay, and transcriptional repression, while also interacting with 53BP1 to suppress p53 signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CNOT1 is the central scaffold of the conserved CCR4-NOT complex, a global regulator of mRNA stability and translation that originated as the essential yeast NOT1/CDC39 nuclear repressor of transcription [#0, #1]. In human cells CNOT1 is required for CCR4-NOT deadenylase activity, mRNA decay and P-body formation, and its depletion stabilizes mRNAs in a manner correlated with codon optimality [#6, #24]. Structurally, CNOT1 is an extended HEAT-repeat platform whose multiple MIF4G and MIF4G-like domains organize distinct functional modules: a central MIF4G domain binds CAF1/CNOT7 and positions the CCR4 nuclease with its active site accessible to RNA [#7, #8], a second MIF4G domain recruits the DEAD-box helicase DDX6 to couple deadenylation to translational repression and decapping [#10, #11], the DUF3819 domain binds CNOT9 to provide tryptophan-binding pockets for TNRC6/GW182 in miRNA silencing [#10], the C-terminal arm recruits NOT2/NOT5 [#9], and N-terminal helical domains sandwich CNOT10-CNOT11 to form a protein-interaction antenna [#18]. Through these interfaces CNOT1 functions as a recruitment hub for sequence-specific RNA-binding adaptors that target the complex to particular transcripts: it binds the AU-rich-element factor tristetraprolin (TTP) through a defined HEAT-like repeat surface to drive ARE-mRNA decay [#5, #30], engages ZFP36L1 to repress translation independently of deadenylation [#28], and is recruited via ZFP36L1/BRF1 to Per2 mRNA to control circadian period through deadenylation-dependent decay [#21]. The CNOT1 MIF4G/DUF3819 region together with CNOT9 stimulates deadenylation of the reconstituted nuclease module [#14], and CNOT1 also nucleates cytoplasmic assemblysomes that support co-translational assembly of proteasome subunits [#16]. Beyond RNA turnover, CNOT1 interacts with 53BP1 and suppresses 53BP1-p53-p21 signaling [#23].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Established that NOT1/CDC39 is not an isolated factor but the organizing member of a discrete multi-subunit nuclear NOT complex acting as a global transcriptional repressor.\",\n      \"evidence\": \"Yeast genetics, two-hybrid interaction and biochemical co-fractionation of NOT1-NOT4\",\n      \"pmids\": [\"7926748\", \"8428577\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve whether repression was direct or via downstream RNA metabolism\", \"No structural basis for subunit assembly\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Localized the essential scaffolding function to the Not1 C-terminal domain, which is required to assemble the catalytic Ccr4 nuclease into the larger complex.\",\n      \"evidence\": \"Domain deletion, sucrose gradient co-fractionation and in vivo complementation in yeast\",\n      \"pmids\": [\"11023781\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic interfaces not defined\", \"Mechanism linking assembly to enzymatic activity unresolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated that human CNOT1 is functionally required for CCR4-NOT deadenylase activity and serves as the bridge by which the ARE-binding protein TTP recruits deadenylase to target mRNAs.\",\n      \"evidence\": \"Co-IP, siRNA knockdown, deadenylase activity assays and mRNA decay readouts in human cells\",\n      \"pmids\": [\"21278420\", \"21976065\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding interface on CNOT1 not mapped\", \"Whether other adaptors use the same surface unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Resolved the modular structural logic by which CNOT1 partitions deadenylation, decapping/repression and miRNA silencing through distinct domains binding CAF1, DDX6 and CNOT9.\",\n      \"evidence\": \"Crystal structures of CNOT1 MIF4G-CAF1, MIF4G-DDX6 and DUF3819-CNOT9 complexes with functional mutagenesis\",\n      \"pmids\": [\"22959269\", \"22977175\", \"24768540\", \"25035296\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How modules are coordinated on a single mRNA not shown\", \"Regulation of domain occupancy unaddressed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed that CNOT1 couples deadenylation to translational repression by recruiting DDX6/Xp54 and 4E-T, with cap-dependent repression downstream of CAF1-NOT1 association.\",\n      \"evidence\": \"Crystal structure of 4E-T CHD-DDX6-CNOT1 ternary complex and tethered reporter assays in Xenopus oocytes\",\n      \"pmids\": [\"26489469\", \"26015597\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of repression vs decay in vivo unclear\", \"Competition among DDX6 partners on endogenous mRNAs not measured\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Reconstituted the contribution of the CNOT1 central region and CNOT9 to stimulating the nuclease module and mapped additional structural domains (MIF4G-C, N-terminal module).\",\n      \"evidence\": \"In vitro reconstitution of pentameric complex and crystallography/SAXS of MIF4G-C and N-terminal domains\",\n      \"pmids\": [\"30309886\", \"30367941\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"MIF4G-C functional role in humans undefined\", \"Mechanism of activity stimulation not fully resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended CNOT1 function beyond decay by showing it nucleates cytoplasmic assemblysomes for co-translational assembly of proteasome subunits, and refined the TTP CNOT1-binding domain requirement in vivo.\",\n      \"evidence\": \"Imaging/ribosome profiling in yeast and human cells; CNBD-deletion knock-in mice and cell-free deadenylation\",\n      \"pmids\": [\"30692646\", \"31036567\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct CNOT1 contacts within assemblysomes not mapped\", \"Range of co-translationally assembled complexes unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Mapped the human N-terminal module architecture (CNOT1 sandwiching CNOT10-CNOT11 with a CNOT11 antenna binding GGNBP2) and identified novel transcriptional/anti-viral partners.\",\n      \"evidence\": \"Cryo-EM/crystallography and binding assays; yeast two-hybrid and HIV reporter assays for TASOR\",\n      \"pmids\": [\"36586408\", \"35013187\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional role of GGNBP2 recruitment unclear\", \"TASOR-CNOT1 link rests on single-lab assays\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined how phosphorylation of the TTP CNOT1-interaction motif by specific kinases switches CCR4-NOT recruitment on and off, linking signaling to ARE-mRNA decay.\",\n      \"evidence\": \"Phosphomimetic/phosphonull Co-IP, kinase inhibitor and mRNA decay reporter assays\",\n      \"pmids\": [\"35920669\", \"34090459\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous phospho-occupancy not quantified\", \"Whether other adaptors are similarly regulated unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed CNOT1 in physiological circadian control by demonstrating ZFP36L1/BRF1-directed deadenylation of Per2 mRNA sets clock period.\",\n      \"evidence\": \"Conditional knockout mice, poly(A) tail assays, BRF1 knockdown and behavioral monitoring\",\n      \"pmids\": [\"35510877\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct CNOT1 occupancy on Per2 mRNA not shown\", \"Generality to other clock transcripts untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established acute, codon-optimality-linked control of global mRNA decay by CNOT1 and revealed an unexpected non-RNA role as a suppressor of 53BP1-p53-p21 signaling.\",\n      \"evidence\": \"Auxin degron RNA-seq/decay assays with BioID; high-content microscopy, Co-IP and nuclear dynamics imaging\",\n      \"pmids\": [\"41161383\", \"40742806\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting CNOT1 to 53BP1 dynamics unresolved\", \"Whether the p53 role is decay-dependent unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CNOT1's many adaptor-binding surfaces are dynamically selected and coordinated on individual mRNAs in vivo, and whether its transcriptional and 53BP1/p53 roles are separable from its deadenylase scaffolding, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated model of competing adaptor occupancy\", \"Direct in vivo target specificity determinants undefined\", \"Mechanistic basis of non-canonical nuclear roles unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [5, 7, 10, 27]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [6, 14, 24]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"GO:0045182\", \"supporting_discovery_ids\": [13, 28]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 1, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6, 16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [6, 14, 24]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 1, 19]},\n      {\"term_id\": \"R-HSA-9909396\", \"supporting_discovery_ids\": [21]}\n    ],\n    \"complexes\": [\"CCR4-NOT complex\", \"assemblysome\"],\n    \"partners\": [\"CNOT7\", \"CNOT9\", \"DDX6\", \"ZFP36 (TTP)\", \"ZFP36L1\", \"CNOT11\", \"53BP1\", \"EIF4ENIF1 (4E-T)\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":6,"faith_pct":83.33333333333333}}