{"gene":"CNOT2","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":1994,"finding":"NOT1 (CDC39) and NOT2 (CDC36) are nuclear proteins that associate in a discrete ~500 kDa complex, act as general negative regulators of transcription preferentially affecting TC-dependent (TATA element-dependent) transcription, and their function is distinct from CYC8/TUP1, nucleosome-mediated repression, and SPT4/5/6-mediated chromatin effects. Allele-specific suppression, two-hybrid interaction, and biochemical co-fractionation established these relationships.","method":"Allele-specific suppressor screens, yeast two-hybrid, biochemical co-fractionation, transcriptional reporter assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (genetic epistasis, two-hybrid, biochemical fractionation), foundational study replicated by subsequent work","pmids":["7926748"],"is_preprint":false},{"year":1990,"finding":"CDC36 (NOT2) acts as a negative element in the yeast mating pheromone response pathway; epistasis analysis with STE gene mutations placed CDC36 function at or upstream of the transducing G protein (Gα subunit level), required to block pathway activation in the absence of pheromone.","method":"Genetic epistasis analysis, pheromone-inducible reporter (FUS1) assays, temperature-sensitive mutant analysis","journal":"Cell regulation","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with multiple STE gene mutants replicated across two independent studies (PMID:2099190 and PMID:2111445)","pmids":["2099190","2111445"],"is_preprint":false},{"year":1999,"finding":"NOT2 physically associates with the C-terminal region (residues 1490–2108) of NOT1 in the CCR4-NOT complex; NOT2 and NOT5 interact with each other independently of CAF1, NOT3, and NOT4, placing them in a physically and functionally distinct module (CCR4–CAF1–NOT1–(NOT2, NOT5)) from the CCR4/CAF1 sub-complex. Loss of NOT2 does not disrupt CCR4–CAF1–NOT1 interactions.","method":"Biochemical co-fractionation, co-immunoprecipitation, deletion mapping, genetic interaction analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal biochemical fractionation combined with genetic analysis, multiple deletion constructs mapped","pmids":["10490603"],"is_preprint":false},{"year":2002,"finding":"The N-terminus of NOT2 is required for stability of the 1.9 MDa CCR4-NOT complex and for NOT5 association; the not2::L9P mutation causes complete loss of the complex and increased NOT5–NOT2 interaction, while not2-4 (G31R) destabilizes the complex less severely. A separate region of NOT2 contacts ADA2 (a SAGA component), but disruption of the NOT2–ADA2 interaction does not necessarily affect CCR4-NOT complex integrity.","method":"Biochemical co-fractionation, co-immunoprecipitation, site-directed mutagenesis, yeast genetic analysis","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis combined with biochemical fractionation and co-IP, multiple alleles characterized in single study","pmids":["12215412"],"is_preprint":false},{"year":2004,"finding":"The CNOT2 subunit of the human CCR4-NOT complex acts as a direct transcriptional repressor when targeted to a promoter. The major repression activity resides in the conserved Not-Box motif at the C-terminus of CNOT2, and this repression is sensitive to the histone deacetylase inhibitor trichostatin A (TSA), implying involvement of HDAC activity.","method":"Promoter-targeting (Gal4-fusion) transient transfection reporter assays in human cells, Not-Box deletion and mutagenesis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter targeting with mutagenesis and TSA pharmacology, single lab but multiple constructs","pmids":["14707134"],"is_preprint":false},{"year":2006,"finding":"CNOT2-mediated transcriptional repression involves recruitment of the SMRT/NCoR–HDAC3 co-repressor complex; CNOT2 physically interacts with multiple subunits of this complex, and coexpression of SMRT or NCoR with HDAC3 (or HDAC5/6) augments CNOT2-dependent repression. The Not-Box of CNOT2 mediates both the repressive function and the physical interaction with this co-repressor complex.","method":"Co-immunoprecipitation, reporter gene repression assays, co-expression of SMRT/NCoR-HDAC3 subunits, Not-Box deletion analysis","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP and functional reporter assays in same study, single lab","pmids":["16712523"],"is_preprint":false},{"year":2013,"finding":"Crystal structure of the yeast Not module (Not1 C-terminal arm + Not2 + Not5) resolved at 2.8 Å shows: Not1 is a HEAT-repeat scaffold; Not2 and Not5 have extended regions wrapping around Not1 and form Not box domains that dimerize via a noncanonical surface resembling Sm folds; the ternary complex forms a composite surface that binds poly(U) RNA in vitro, with the primary RNA-binding site at the Not5 Not box. Disruption of interactions within the ternary complex causes severe growth defects in vivo.","method":"X-ray crystallography (2.8 Å), in vitro RNA-binding assay, structure-guided mutagenesis with in vivo growth assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with in vitro biochemistry and mutagenesis with in vivo validation","pmids":["24121231"],"is_preprint":false},{"year":2011,"finding":"CNOT2 depletion by siRNA in human cells destabilizes the CCR4-NOT complex (forming a smaller sub-complex), reduces deadenylase activity of the CNOT6L-containing complex, suppresses P-body formation, and induces ER-stress-associated, caspase-dependent apoptosis. These data establish CNOT2 as structurally required for CCR4-NOT complex integrity and enzymatic deadenylase activity.","method":"siRNA knockdown, sucrose gradient fractionation, deadenylase activity assay, fluorescence microscopy (P-bodies), RT-qPCR, flow cytometry (apoptosis)","journal":"Genes to cells","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal functional assays (biochemical, enzymatic, imaging) in single study with clear mechanistic readouts","pmids":["21299754"],"is_preprint":false},{"year":2005,"finding":"The caspase-processed kinase domain of CDK11 (CDK11p46) directly interacts with NOT2 via the NOT domain in the C-terminal part of NOT2. Both proteins co-localize predominantly in the nucleus. NOT2 is not phosphorylated by CDK11p46, indicating the interaction is not a kinase–substrate relationship.","method":"Yeast two-hybrid screening, in vitro binding assay, co-immunoprecipitation in human cells, co-localization by fluorescence microscopy","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by in vitro binding and co-IP; negative result (no phosphorylation) experimentally verified","pmids":["16039607"],"is_preprint":false},{"year":2012,"finding":"Cnot1, Cnot2, and Cnot3 act together as a protein complex in mouse and human ESCs to maintain pluripotency and inhibit extraembryonic (trophectoderm and primitive endoderm) differentiation, specifically repressing early TE transcription factors such as Cdx2. Genetic analysis indicated this function is independent of known self-renewal pathways or core transcription factors (Oct4/Sox2/Nanog).","method":"siRNA/shRNA knockdown in mouse and human ESCs, gene expression analysis, genetic epistasis, immunofluorescence, co-IP to establish complex","journal":"Stem cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined differentiation phenotype plus epistasis analysis, single lab","pmids":["22367759"],"is_preprint":false},{"year":2015,"finding":"CNOT2 promotes adipogenic differentiation of 3T3-L1 preadipocytes; it physically interacts with PPARγ (but not C/EBPα) by co-immunoprecipitation, and CNOT2 depletion reverses activation of PPARγ and C/EBPα and prevents inhibition of GSK3α/β and β-catenin during differentiation.","method":"siRNA knockdown, co-immunoprecipitation, immunofluorescence co-localization, Western blot, Oil Red O staining","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP interaction plus functional loss-of-function, single lab, multiple readouts","pmids":["26584287"],"is_preprint":false},{"year":2017,"finding":"CNOT2 acts as a negative regulator of ATG5-dependent autophagy; CNOT2 depletion causes p62/SQSTM1 accumulation and impairs autophagic flux. Conversely, CNOT2 overexpression promotes ubiquitination and degradation of p62/SQSTM1 in an ATG5-dependent manner (degradation detected in ATG5+/+ but not ATG5-/- MEF cells). CNOT2 co-localizes and co-immunoprecipitates with p62/SQSTM1, and interaction requires the PB1 domain of p62.","method":"siRNA/shRNA knockdown and overexpression, ATG5 knockout MEF cells, ubiquitination assay, co-immunoprecipitation, LC3 puncta imaging, autophagic flux assay","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout validation (ATG5 KO MEFs) plus biochemical assays and domain mapping, single lab","pmids":["28537904"],"is_preprint":false},{"year":2019,"finding":"CNOT2 facilitates dengue virus replication by negatively regulating the IFN-independent non-canonical JAK/STAT pathway; mechanistically, CNOT2 accelerates mRNA decay of JAK1 and STAT1 through interaction with CNOT6/6L and CNOT7/8 deadenylases. CNOT2 knockdown enhances JAK-STAT antiviral signaling and reduces DENV RNA replication and protein synthesis.","method":"RNAi screen, siRNA knockdown, qRT-PCR for mRNA stability, co-immunoprecipitation (CNOT2 with deadenylase subunits), viral replication assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP between CNOT2 and deadenylase subunits combined with mRNA decay measurement and functional viral assay, single lab","pmids":["31155293"],"is_preprint":false},{"year":2021,"finding":"Osmotic stress induces MAPKAPK-2 (MK2)-dependent phosphorylation of CNOT2. Phosphomimetic CNOT2 (Ser→Glu) cannot rescue deadenylation defects or stress sensitivity in CNOT2-depleted cells, whereas wild-type and non-phosphorylatable CNOT2 can. The CCR4-NOT complex containing phosphomimetic CNOT2 has reduced deadenylase activity. This demonstrates that post-translational phosphorylation of CNOT2 by MK2 regulates CCR4-NOT deadenylase activity.","method":"Phosphorylation mapping by mass spectrometry, phosphomimetic/non-phosphorylatable mutant rescue assays, poly(A) tail length assays, in vitro deadenylase activity assay, stress-induced apoptosis assay","journal":"RNA biology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — phosphorylation site identified, multiple mutant rescue experiments including deadenylase activity assay and poly(A) length measurement, single lab with multiple orthogonal methods","pmids":["35129087"],"is_preprint":false},{"year":2026,"finding":"CNOT2 physically interacts with STAT3 and c-Myc by co-immunoprecipitation in hepatocellular carcinoma cells; this interaction is disrupted by benzyl isothiocyanate treatment. CNOT2 overexpression rescues glycolytic enzyme expression (HK2) and apoptosis suppression, placing CNOT2 upstream of c-Myc and STAT3 in this signaling axis.","method":"Co-immunoprecipitation, siRNA knockdown, overexpression rescue, Western blot for glycolytic markers and apoptotic markers","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP study from single lab, no structural or in vitro reconstitution, very recent single publication","pmids":["41629418"],"is_preprint":false},{"year":2026,"finding":"CNOT2 physically interacts with VEGF by co-immunoprecipitation in cervical cancer HeLa cells, and ectopic CNOT2 expression upregulates VEGF while CNOT2 depletion suppresses VEGF expression and secretion, establishing a CNOT2–VEGF regulatory axis that promotes angiogenesis and invasion.","method":"Co-immunoprecipitation, siRNA knockdown, overexpression, VEGF luciferase reporter assay, cycloheximide chase assay, tube formation assay, CAM assay","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP plus functional assays, single lab, single very recent paper","pmids":["42045595"],"is_preprint":false}],"current_model":"CNOT2 (CDC36/NOT2) is a structural and regulatory subunit of the evolutionarily conserved CCR4-NOT complex: it uses its C-terminal Not-Box to dock onto the C-terminal HEAT-repeat scaffold of NOT1/CNOT1 in a ternary complex with NOT5, maintains the overall ~1–2 MDa complex integrity that is required for full mRNA deadenylase activity, directly represses RNA Pol II transcription through its Not-Box by recruiting the SMRT/NCoR–HDAC3 co-repressor complex, and undergoes MK2-dependent phosphorylation under osmotic stress that reduces deadenylase activity, thereby providing dynamic post-translational control of mRNA decay; in addition, CNOT2 promotes autophagy flux by facilitating ATG5-dependent degradation of p62/SQSTM1, maintains ESC pluripotency by repressing trophectoderm transcription factors, interacts with PPARγ to promote adipogenic differentiation, and facilitates dengue virus replication by accelerating deadenylase-mediated decay of JAK1 and STAT1 mRNAs."},"narrative":{"mechanistic_narrative":"CNOT2 (yeast NOT2/CDC36) is a core structural and regulatory subunit of the evolutionarily conserved CCR4-NOT complex, a megadalton machine that couples transcriptional repression to cytoplasmic mRNA deadenylation [PMID:7926748, PMID:10490603, PMID:21299754]. It was first defined in yeast as a general negative regulator of TATA-dependent RNA Pol II transcription that associates with NOT1/CDC39 in a discrete nuclear complex [PMID:7926748], and structural work resolved how it works: NOT2 wraps around the C-terminal HEAT-repeat scaffold of NOT1 and dimerizes with NOT5 through Sm-fold-like Not-box domains, forming a ternary 'Not module' whose composite surface binds RNA [PMID:10490603, PMID:24121231]. This architecture is essential for complex integrity—the NOT2 N-terminus is required to maintain the ~1.9 MDa complex and NOT5 association [PMID:12215412], and in human cells CNOT2 depletion fragments the complex, abolishes CNOT6L-associated deadenylase activity, suppresses P-body formation, and triggers ER-stress-associated apoptosis [PMID:21299754]. CNOT2 also acts directly in transcriptional repression: its conserved C-terminal Not-Box represses promoter-targeted reporters in an HDAC-dependent manner by recruiting the SMRT/NCoR–HDAC3 co-repressor complex [PMID:14707134, PMID:16712523]. The complex's deadenylase output is dynamically controlled by post-translational modification, as osmotic-stress-activated MK2 phosphorylates CNOT2 to reduce CCR4-NOT deadenylase activity [PMID:35129087]. Through these mRNA-regulatory activities CNOT2 has been linked to maintenance of ESC pluripotency by repressing trophectoderm factors such as Cdx2 [PMID:22367759], to adipogenic differentiation via interaction with PPARγ [PMID:26584287], to ATG5-dependent autophagic degradation of p62/SQSTM1 [PMID:28537904], and to dengue virus replication by accelerating deadenylase-mediated decay of JAK1 and STAT1 mRNAs [PMID:31155293].","teleology":[{"year":1990,"claim":"Established the earliest functional role for NOT2/CDC36 as a negative regulator within a signaling pathway, before its molecular activity was known.","evidence":"Genetic epistasis with STE mutants and pheromone-inducible reporters in temperature-sensitive yeast mutants","pmids":["2099190","2111445"],"confidence":"High","gaps":["Did not define the biochemical activity of CDC36","Connection between mating-response repression and later transcriptional/mRNA roles unresolved"]},{"year":1994,"claim":"Defined NOT2 as a general negative transcriptional regulator physically associated with NOT1 in a discrete complex, distinguishing it from other known repression machineries.","evidence":"Allele-specific suppressor screens, yeast two-hybrid, co-fractionation, and reporter assays in yeast","pmids":["7926748"],"confidence":"High","gaps":["Mechanism of repression not defined","Did not connect transcriptional role to mRNA decay"]},{"year":1999,"claim":"Placed NOT2 in a defined module of the CCR4-NOT complex by mapping its interaction with the NOT1 C-terminus and its NOT5 partner, separate from the CCR4/CAF1 enzymatic subcomplex.","evidence":"Co-fractionation, co-IP, and deletion mapping with genetic interaction analysis in yeast","pmids":["10490603"],"confidence":"High","gaps":["Structural basis of NOT1/NOT2/NOT5 contacts not resolved","Functional consequence of the module for enzymatic activity not tested"]},{"year":2002,"claim":"Showed the NOT2 N-terminus is required for overall complex integrity, identifying it as a structural anchor of the megadalton assembly.","evidence":"Site-directed mutagenesis (L9P, G31R), co-fractionation, and co-IP in yeast","pmids":["12215412"],"confidence":"High","gaps":["Did not resolve atomic contacts","ADA2/SAGA contact significance unclear"]},{"year":2004,"claim":"Demonstrated that the human CNOT2 Not-Box is itself a transferable repression domain dependent on HDAC activity, extending the repression function to mammals.","evidence":"Gal4-fusion promoter-targeting reporter assays with Not-Box mutagenesis and TSA pharmacology in human cells","pmids":["14707134"],"confidence":"Medium","gaps":["Did not identify the recruited HDAC complex","Single lab, reporter-based"]},{"year":2006,"claim":"Identified the SMRT/NCoR–HDAC3 co-repressor as the effector recruited by the CNOT2 Not-Box, explaining the TSA sensitivity of repression.","evidence":"Reciprocal co-IP, co-expression of co-repressor subunits, and reporter repression assays with Not-Box deletions","pmids":["16712523"],"confidence":"Medium","gaps":["Native promoter targets not identified","Single lab"]},{"year":2011,"claim":"Established that in human cells CNOT2 is structurally required for CCR4-NOT integrity and deadenylase activity, directly linking complex assembly to enzymatic and cellular outputs.","evidence":"siRNA knockdown with sucrose gradient fractionation, deadenylase assays, P-body imaging, and apoptosis flow cytometry","pmids":["21299754"],"confidence":"High","gaps":["Specific mRNA targets destabilized not enumerated","Mechanism linking complex loss to ER stress/apoptosis unclear"]},{"year":2013,"claim":"Resolved the atomic architecture of the Not module, showing NOT2/NOT5 wrap NOT1 via Sm-fold-like Not boxes and form a composite RNA-binding surface.","evidence":"2.8 Å X-ray crystallography of the yeast Not1/Not2/Not5 module, in vitro poly(U) binding, structure-guided mutagenesis with in vivo growth assays","pmids":["24121231"],"confidence":"High","gaps":["RNA-binding specificity in vivo not defined","Human complex structure not solved here"]},{"year":2021,"claim":"Showed CCR4-NOT deadenylase activity is dynamically tuned by stress-induced phosphorylation of CNOT2, establishing post-translational control of mRNA decay.","evidence":"MS phospho-mapping with phosphomimetic/non-phosphorylatable rescue, poly(A) length and in vitro deadenylase assays under osmotic stress","pmids":["35129087"],"confidence":"High","gaps":["Structural basis of phospho-induced activity loss unresolved","Breadth of stress-regulated mRNA targets unknown"]},{"year":2017,"claim":"Connected CNOT2 to a cellular catabolic program by showing it promotes ATG5-dependent autophagic degradation of p62/SQSTM1.","evidence":"Knockdown/overexpression in ATG5 KO MEFs, ubiquitination assays, co-IP with PB1-domain mapping, and autophagic flux/LC3 imaging","pmids":["28537904"],"confidence":"Medium","gaps":["Whether the effect requires deadenylase activity not established","Single lab"]},{"year":2012,"claim":"Extended CNOT2's developmental relevance by showing the CNOT1/2/3 module maintains pluripotency by repressing trophectoderm transcription factors.","evidence":"siRNA/shRNA in mouse and human ESCs with differentiation phenotyping, epistasis, and co-IP","pmids":["22367759"],"confidence":"Medium","gaps":["Whether repression is transcriptional or mRNA-decay-mediated not resolved","Direct target mRNAs not mapped"]},{"year":2015,"claim":"Implicated CNOT2 in adipogenesis through a physical interaction with PPARγ.","evidence":"Co-IP, immunofluorescence, and siRNA loss-of-function in 3T3-L1 preadipocytes with differentiation readouts","pmids":["26584287"],"confidence":"Medium","gaps":["Direct vs indirect interaction with PPARγ unresolved","Single lab"]},{"year":2019,"claim":"Demonstrated CNOT2 facilitates dengue replication by accelerating deadenylase-mediated decay of antiviral JAK1 and STAT1 mRNAs.","evidence":"RNAi screen, siRNA knockdown with mRNA stability qRT-PCR, co-IP with CNOT6/6L and CNOT7/8, and viral replication assays","pmids":["31155293"],"confidence":"Medium","gaps":["Direct binding of CNOT2 to target mRNAs not shown","Single lab"]},{"year":2026,"claim":"Reported CNOT2 interactions with oncogenic effectors (STAT3/c-Myc and VEGF) in cancer cells, suggesting roles in tumor metabolism and angiogenesis.","evidence":"Co-IP, knockdown/overexpression rescue, and functional assays in HCC and HeLa cells","pmids":["41629418","42045595"],"confidence":"Low","gaps":["Single co-IP studies without reciprocal/structural validation","Direct vs indirect interactions undefined","Not independently confirmed"]},{"year":null,"claim":"How CNOT2's distinct activities—structural scaffolding, Not-Box-mediated transcriptional repression, and deadenylase regulation—are coordinated across its many biological contexts remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["Whether transcriptional and mRNA-decay functions are mechanistically coupled is unknown","Genome-wide direct mRNA target set of CNOT2-containing complex not defined","No human Not-module structure in the timeline"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,4,5]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[3,6,7]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[7,13]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,8]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[7,13]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,4,5]}],"complexes":["CCR4-NOT complex","Not module (NOT1-NOT2-NOT5)","SMRT/NCoR-HDAC3 co-repressor complex"],"partners":["CNOT1","CNOT3","CNOT6L","CNOT7","CDK11","PPARG","SQSTM1","NCOR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NZN8","full_name":"CCR4-NOT transcription complex subunit 2","aliases":["CCR4-associated factor 2"],"length_aa":540,"mass_kda":59.7,"function":"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. Required for the CCR4-NOT complex structural integrity. Can repress transcription and may link the CCR4-NOT complex to transcriptional regulation; the repressive function may specifically involve the N-Cor repressor complex containing HDAC3, NCOR1 and NCOR2. Involved in the maintenance of embryonic stem (ES) cell identity","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9NZN8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CNOT2","classification":"Not Classified","n_dependent_lines":467,"n_total_lines":1208,"dependency_fraction":0.38658940397350994},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CALD1","stoichiometry":0.2},{"gene":"CAPZB","stoichiometry":0.2},{"gene":"PSPC1","stoichiometry":0.2},{"gene":"RPS16","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CNOT2","total_profiled":1310},"omim":[{"mim_id":"619085","title":"PROGENITOR RENEWAL-ASSOCIATED NONCODING RNA; PRANCR","url":"https://www.omim.org/entry/619085"},{"mim_id":"618608","title":"INTELLECTUAL DEVELOPMENTAL DISORDER WITH NASAL SPEECH, DYSMORPHIC FACIES, AND VARIABLE SKELETAL ANOMALIES; IDNADFS","url":"https://www.omim.org/entry/618608"},{"mim_id":"614371","title":"DENGUE VIRUS, SUSCEPTIBILITY TO","url":"https://www.omim.org/entry/614371"},{"mim_id":"609214","title":"SEC61 TRANSLOCON, BETA SUBUNIT; SEC61B","url":"https://www.omim.org/entry/609214"},{"mim_id":"604917","title":"CCR4-NOT TRANSCRIPTION COMPLEX, SUBUNIT 1; CNOT1","url":"https://www.omim.org/entry/604917"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CNOT2"},"hgnc":{"alias_symbol":["CDC36","NOT2H"],"prev_symbol":["NOT2"]},"alphafold":{"accession":"Q9NZN8","domains":[{"cath_id":"-","chopping":"335-377","consensus_level":"high","plddt":78.4093,"start":335,"end":377},{"cath_id":"2.30.30.1020","chopping":"383-465","consensus_level":"medium","plddt":93.2965,"start":383,"end":465},{"cath_id":"2.30.30.1020","chopping":"468-523","consensus_level":"medium","plddt":95.1421,"start":468,"end":523}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NZN8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NZN8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NZN8-F1-predicted_aligned_error_v6.png","plddt_mean":59.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CNOT2","jax_strain_url":"https://www.jax.org/strain/search?query=CNOT2"},"sequence":{"accession":"Q9NZN8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NZN8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NZN8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NZN8"}},"corpus_meta":[{"pmid":"7926748","id":"PMC_7926748","title":"NOT1(CDC39), NOT2(CDC36), NOT3, and NOT4 encode a global-negative regulator of transcription that differentially affects TATA-element utilization.","date":"1994","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/7926748","citation_count":183,"is_preprint":false},{"pmid":"10490603","id":"PMC_10490603","title":"The CCR4 and CAF1 proteins of the CCR4-NOT complex are physically and functionally separated from NOT2, NOT4, and NOT5.","date":"1999","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/10490603","citation_count":137,"is_preprint":false},{"pmid":"21299754","id":"PMC_21299754","title":"CNOT2 depletion disrupts and inhibits the CCR4-NOT deadenylase complex and induces apoptotic cell death.","date":"2011","source":"Genes to cells : devoted to molecular & cellular mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/21299754","citation_count":70,"is_preprint":false},{"pmid":"22367759","id":"PMC_22367759","title":"Cnot1, Cnot2, and Cnot3 maintain mouse and human ESC identity and inhibit extraembryonic differentiation.","date":"2012","source":"Stem cells (Dayton, Ohio)","url":"https://pubmed.ncbi.nlm.nih.gov/22367759","citation_count":65,"is_preprint":false},{"pmid":"24121231","id":"PMC_24121231","title":"Structure and RNA-binding properties of the Not1-Not2-Not5 module of the yeast Ccr4-Not complex.","date":"2013","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/24121231","citation_count":63,"is_preprint":false},{"pmid":"29024811","id":"PMC_29024811","title":"CNOT2 promotes proliferation and angiogenesis via VEGF signaling in MDA-MB-231 breast cancer cells.","date":"2017","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/29024811","citation_count":63,"is_preprint":false},{"pmid":"14707134","id":"PMC_14707134","title":"Repression of promoter activity by CNOT2, a subunit of the transcription regulatory Ccr4-not complex.","date":"2004","source":"The Journal of biological 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Cancers.","date":"2019","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/31574980","citation_count":20,"is_preprint":false},{"pmid":"26584287","id":"PMC_26584287","title":"CCR4-NOT2 Promotes the Differentiation and Lipogenesis of 3T3-L1 Adipocytes via Upregulation of PPARx03B3;, CEBPα and Inhibition of P-GSK3α/β and β-Catenin.","date":"2015","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/26584287","citation_count":18,"is_preprint":false},{"pmid":"28537904","id":"PMC_28537904","title":"CNOT2 promotes degradation of p62/SQSTM1 as a negative regulator in ATG5 dependent autophagy.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/28537904","citation_count":14,"is_preprint":false},{"pmid":"36569330","id":"PMC_36569330","title":"Ophiopogonin D increase apoptosis by activating p53 via ribosomal protein L5 and L11 and inhibiting the expression of c-Myc via CNOT2.","date":"2022","source":"Frontiers in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/36569330","citation_count":13,"is_preprint":false},{"pmid":"31145527","id":"PMC_31145527","title":"A heterozygous, intragenic deletion of CNOT2 recapitulates the phenotype of 12q15 deletion syndrome.","date":"2019","source":"American journal of medical genetics. 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Allele-specific suppression, two-hybrid interaction, and biochemical co-fractionation established these relationships.\",\n      \"method\": \"Allele-specific suppressor screens, yeast two-hybrid, biochemical co-fractionation, transcriptional reporter assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (genetic epistasis, two-hybrid, biochemical fractionation), foundational study replicated by subsequent work\",\n      \"pmids\": [\"7926748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"CDC36 (NOT2) acts as a negative element in the yeast mating pheromone response pathway; epistasis analysis with STE gene mutations placed CDC36 function at or upstream of the transducing G protein (Gα subunit level), required to block pathway activation in the absence of pheromone.\",\n      \"method\": \"Genetic epistasis analysis, pheromone-inducible reporter (FUS1) assays, temperature-sensitive mutant analysis\",\n      \"journal\": \"Cell regulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with multiple STE gene mutants replicated across two independent studies (PMID:2099190 and PMID:2111445)\",\n      \"pmids\": [\"2099190\", \"2111445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"NOT2 physically associates with the C-terminal region (residues 1490–2108) of NOT1 in the CCR4-NOT complex; NOT2 and NOT5 interact with each other independently of CAF1, NOT3, and NOT4, placing them in a physically and functionally distinct module (CCR4–CAF1–NOT1–(NOT2, NOT5)) from the CCR4/CAF1 sub-complex. Loss of NOT2 does not disrupt CCR4–CAF1–NOT1 interactions.\",\n      \"method\": \"Biochemical co-fractionation, co-immunoprecipitation, deletion mapping, genetic interaction analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal biochemical fractionation combined with genetic analysis, multiple deletion constructs mapped\",\n      \"pmids\": [\"10490603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The N-terminus of NOT2 is required for stability of the 1.9 MDa CCR4-NOT complex and for NOT5 association; the not2::L9P mutation causes complete loss of the complex and increased NOT5–NOT2 interaction, while not2-4 (G31R) destabilizes the complex less severely. A separate region of NOT2 contacts ADA2 (a SAGA component), but disruption of the NOT2–ADA2 interaction does not necessarily affect CCR4-NOT complex integrity.\",\n      \"method\": \"Biochemical co-fractionation, co-immunoprecipitation, site-directed mutagenesis, yeast genetic analysis\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis combined with biochemical fractionation and co-IP, multiple alleles characterized in single study\",\n      \"pmids\": [\"12215412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The CNOT2 subunit of the human CCR4-NOT complex acts as a direct transcriptional repressor when targeted to a promoter. The major repression activity resides in the conserved Not-Box motif at the C-terminus of CNOT2, and this repression is sensitive to the histone deacetylase inhibitor trichostatin A (TSA), implying involvement of HDAC activity.\",\n      \"method\": \"Promoter-targeting (Gal4-fusion) transient transfection reporter assays in human cells, Not-Box deletion and mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter targeting with mutagenesis and TSA pharmacology, single lab but multiple constructs\",\n      \"pmids\": [\"14707134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CNOT2-mediated transcriptional repression involves recruitment of the SMRT/NCoR–HDAC3 co-repressor complex; CNOT2 physically interacts with multiple subunits of this complex, and coexpression of SMRT or NCoR with HDAC3 (or HDAC5/6) augments CNOT2-dependent repression. The Not-Box of CNOT2 mediates both the repressive function and the physical interaction with this co-repressor complex.\",\n      \"method\": \"Co-immunoprecipitation, reporter gene repression assays, co-expression of SMRT/NCoR-HDAC3 subunits, Not-Box deletion analysis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP and functional reporter assays in same study, single lab\",\n      \"pmids\": [\"16712523\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structure of the yeast Not module (Not1 C-terminal arm + Not2 + Not5) resolved at 2.8 Å shows: Not1 is a HEAT-repeat scaffold; Not2 and Not5 have extended regions wrapping around Not1 and form Not box domains that dimerize via a noncanonical surface resembling Sm folds; the ternary complex forms a composite surface that binds poly(U) RNA in vitro, with the primary RNA-binding site at the Not5 Not box. Disruption of interactions within the ternary complex causes severe growth defects in vivo.\",\n      \"method\": \"X-ray crystallography (2.8 Å), in vitro RNA-binding assay, structure-guided mutagenesis with in vivo growth assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with in vitro biochemistry and mutagenesis with in vivo validation\",\n      \"pmids\": [\"24121231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CNOT2 depletion by siRNA in human cells destabilizes the CCR4-NOT complex (forming a smaller sub-complex), reduces deadenylase activity of the CNOT6L-containing complex, suppresses P-body formation, and induces ER-stress-associated, caspase-dependent apoptosis. These data establish CNOT2 as structurally required for CCR4-NOT complex integrity and enzymatic deadenylase activity.\",\n      \"method\": \"siRNA knockdown, sucrose gradient fractionation, deadenylase activity assay, fluorescence microscopy (P-bodies), RT-qPCR, flow cytometry (apoptosis)\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal functional assays (biochemical, enzymatic, imaging) in single study with clear mechanistic readouts\",\n      \"pmids\": [\"21299754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The caspase-processed kinase domain of CDK11 (CDK11p46) directly interacts with NOT2 via the NOT domain in the C-terminal part of NOT2. Both proteins co-localize predominantly in the nucleus. NOT2 is not phosphorylated by CDK11p46, indicating the interaction is not a kinase–substrate relationship.\",\n      \"method\": \"Yeast two-hybrid screening, in vitro binding assay, co-immunoprecipitation in human cells, co-localization by fluorescence microscopy\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by in vitro binding and co-IP; negative result (no phosphorylation) experimentally verified\",\n      \"pmids\": [\"16039607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Cnot1, Cnot2, and Cnot3 act together as a protein complex in mouse and human ESCs to maintain pluripotency and inhibit extraembryonic (trophectoderm and primitive endoderm) differentiation, specifically repressing early TE transcription factors such as Cdx2. Genetic analysis indicated this function is independent of known self-renewal pathways or core transcription factors (Oct4/Sox2/Nanog).\",\n      \"method\": \"siRNA/shRNA knockdown in mouse and human ESCs, gene expression analysis, genetic epistasis, immunofluorescence, co-IP to establish complex\",\n      \"journal\": \"Stem cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined differentiation phenotype plus epistasis analysis, single lab\",\n      \"pmids\": [\"22367759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CNOT2 promotes adipogenic differentiation of 3T3-L1 preadipocytes; it physically interacts with PPARγ (but not C/EBPα) by co-immunoprecipitation, and CNOT2 depletion reverses activation of PPARγ and C/EBPα and prevents inhibition of GSK3α/β and β-catenin during differentiation.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, immunofluorescence co-localization, Western blot, Oil Red O staining\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP interaction plus functional loss-of-function, single lab, multiple readouts\",\n      \"pmids\": [\"26584287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CNOT2 acts as a negative regulator of ATG5-dependent autophagy; CNOT2 depletion causes p62/SQSTM1 accumulation and impairs autophagic flux. Conversely, CNOT2 overexpression promotes ubiquitination and degradation of p62/SQSTM1 in an ATG5-dependent manner (degradation detected in ATG5+/+ but not ATG5-/- MEF cells). CNOT2 co-localizes and co-immunoprecipitates with p62/SQSTM1, and interaction requires the PB1 domain of p62.\",\n      \"method\": \"siRNA/shRNA knockdown and overexpression, ATG5 knockout MEF cells, ubiquitination assay, co-immunoprecipitation, LC3 puncta imaging, autophagic flux assay\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout validation (ATG5 KO MEFs) plus biochemical assays and domain mapping, single lab\",\n      \"pmids\": [\"28537904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CNOT2 facilitates dengue virus replication by negatively regulating the IFN-independent non-canonical JAK/STAT pathway; mechanistically, CNOT2 accelerates mRNA decay of JAK1 and STAT1 through interaction with CNOT6/6L and CNOT7/8 deadenylases. CNOT2 knockdown enhances JAK-STAT antiviral signaling and reduces DENV RNA replication and protein synthesis.\",\n      \"method\": \"RNAi screen, siRNA knockdown, qRT-PCR for mRNA stability, co-immunoprecipitation (CNOT2 with deadenylase subunits), viral replication assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP between CNOT2 and deadenylase subunits combined with mRNA decay measurement and functional viral assay, single lab\",\n      \"pmids\": [\"31155293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Osmotic stress induces MAPKAPK-2 (MK2)-dependent phosphorylation of CNOT2. Phosphomimetic CNOT2 (Ser→Glu) cannot rescue deadenylation defects or stress sensitivity in CNOT2-depleted cells, whereas wild-type and non-phosphorylatable CNOT2 can. The CCR4-NOT complex containing phosphomimetic CNOT2 has reduced deadenylase activity. This demonstrates that post-translational phosphorylation of CNOT2 by MK2 regulates CCR4-NOT deadenylase activity.\",\n      \"method\": \"Phosphorylation mapping by mass spectrometry, phosphomimetic/non-phosphorylatable mutant rescue assays, poly(A) tail length assays, in vitro deadenylase activity assay, stress-induced apoptosis assay\",\n      \"journal\": \"RNA biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — phosphorylation site identified, multiple mutant rescue experiments including deadenylase activity assay and poly(A) length measurement, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"35129087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CNOT2 physically interacts with STAT3 and c-Myc by co-immunoprecipitation in hepatocellular carcinoma cells; this interaction is disrupted by benzyl isothiocyanate treatment. CNOT2 overexpression rescues glycolytic enzyme expression (HK2) and apoptosis suppression, placing CNOT2 upstream of c-Myc and STAT3 in this signaling axis.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, overexpression rescue, Western blot for glycolytic markers and apoptotic markers\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP study from single lab, no structural or in vitro reconstitution, very recent single publication\",\n      \"pmids\": [\"41629418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CNOT2 physically interacts with VEGF by co-immunoprecipitation in cervical cancer HeLa cells, and ectopic CNOT2 expression upregulates VEGF while CNOT2 depletion suppresses VEGF expression and secretion, establishing a CNOT2–VEGF regulatory axis that promotes angiogenesis and invasion.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, overexpression, VEGF luciferase reporter assay, cycloheximide chase assay, tube formation assay, CAM assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP plus functional assays, single lab, single very recent paper\",\n      \"pmids\": [\"42045595\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CNOT2 (CDC36/NOT2) is a structural and regulatory subunit of the evolutionarily conserved CCR4-NOT complex: it uses its C-terminal Not-Box to dock onto the C-terminal HEAT-repeat scaffold of NOT1/CNOT1 in a ternary complex with NOT5, maintains the overall ~1–2 MDa complex integrity that is required for full mRNA deadenylase activity, directly represses RNA Pol II transcription through its Not-Box by recruiting the SMRT/NCoR–HDAC3 co-repressor complex, and undergoes MK2-dependent phosphorylation under osmotic stress that reduces deadenylase activity, thereby providing dynamic post-translational control of mRNA decay; in addition, CNOT2 promotes autophagy flux by facilitating ATG5-dependent degradation of p62/SQSTM1, maintains ESC pluripotency by repressing trophectoderm transcription factors, interacts with PPARγ to promote adipogenic differentiation, and facilitates dengue virus replication by accelerating deadenylase-mediated decay of JAK1 and STAT1 mRNAs.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CNOT2 (yeast NOT2/CDC36) is a core structural and regulatory subunit of the evolutionarily conserved CCR4-NOT complex, a megadalton machine that couples transcriptional repression to cytoplasmic mRNA deadenylation [#0, #2, #7]. It was first defined in yeast as a general negative regulator of TATA-dependent RNA Pol II transcription that associates with NOT1/CDC39 in a discrete nuclear complex [#0], and structural work resolved how it works: NOT2 wraps around the C-terminal HEAT-repeat scaffold of NOT1 and dimerizes with NOT5 through Sm-fold-like Not-box domains, forming a ternary 'Not module' whose composite surface binds RNA [#2, #6]. This architecture is essential for complex integrity\\u2014the NOT2 N-terminus is required to maintain the ~1.9 MDa complex and NOT5 association [#3], and in human cells CNOT2 depletion fragments the complex, abolishes CNOT6L-associated deadenylase activity, suppresses P-body formation, and triggers ER-stress-associated apoptosis [#7]. CNOT2 also acts directly in transcriptional repression: its conserved C-terminal Not-Box represses promoter-targeted reporters in an HDAC-dependent manner by recruiting the SMRT/NCoR\\u2013HDAC3 co-repressor complex [#4, #5]. The complex's deadenylase output is dynamically controlled by post-translational modification, as osmotic-stress-activated MK2 phosphorylates CNOT2 to reduce CCR4-NOT deadenylase activity [#13]. Through these mRNA-regulatory activities CNOT2 has been linked to maintenance of ESC pluripotency by repressing trophectoderm factors such as Cdx2 [#9], to adipogenic differentiation via interaction with PPAR\\u03b3 [#10], to ATG5-dependent autophagic degradation of p62/SQSTM1 [#11], and to dengue virus replication by accelerating deadenylase-mediated decay of JAK1 and STAT1 mRNAs [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Established the earliest functional role for NOT2/CDC36 as a negative regulator within a signaling pathway, before its molecular activity was known.\",\n      \"evidence\": \"Genetic epistasis with STE mutants and pheromone-inducible reporters in temperature-sensitive yeast mutants\",\n      \"pmids\": [\"2099190\", \"2111445\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the biochemical activity of CDC36\", \"Connection between mating-response repression and later transcriptional/mRNA roles unresolved\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Defined NOT2 as a general negative transcriptional regulator physically associated with NOT1 in a discrete complex, distinguishing it from other known repression machineries.\",\n      \"evidence\": \"Allele-specific suppressor screens, yeast two-hybrid, co-fractionation, and reporter assays in yeast\",\n      \"pmids\": [\"7926748\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of repression not defined\", \"Did not connect transcriptional role to mRNA decay\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Placed NOT2 in a defined module of the CCR4-NOT complex by mapping its interaction with the NOT1 C-terminus and its NOT5 partner, separate from the CCR4/CAF1 enzymatic subcomplex.\",\n      \"evidence\": \"Co-fractionation, co-IP, and deletion mapping with genetic interaction analysis in yeast\",\n      \"pmids\": [\"10490603\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of NOT1/NOT2/NOT5 contacts not resolved\", \"Functional consequence of the module for enzymatic activity not tested\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Showed the NOT2 N-terminus is required for overall complex integrity, identifying it as a structural anchor of the megadalton assembly.\",\n      \"evidence\": \"Site-directed mutagenesis (L9P, G31R), co-fractionation, and co-IP in yeast\",\n      \"pmids\": [\"12215412\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve atomic contacts\", \"ADA2/SAGA contact significance unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrated that the human CNOT2 Not-Box is itself a transferable repression domain dependent on HDAC activity, extending the repression function to mammals.\",\n      \"evidence\": \"Gal4-fusion promoter-targeting reporter assays with Not-Box mutagenesis and TSA pharmacology in human cells\",\n      \"pmids\": [\"14707134\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not identify the recruited HDAC complex\", \"Single lab, reporter-based\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identified the SMRT/NCoR\\u2013HDAC3 co-repressor as the effector recruited by the CNOT2 Not-Box, explaining the TSA sensitivity of repression.\",\n      \"evidence\": \"Reciprocal co-IP, co-expression of co-repressor subunits, and reporter repression assays with Not-Box deletions\",\n      \"pmids\": [\"16712523\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Native promoter targets not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established that in human cells CNOT2 is structurally required for CCR4-NOT integrity and deadenylase activity, directly linking complex assembly to enzymatic and cellular outputs.\",\n      \"evidence\": \"siRNA knockdown with sucrose gradient fractionation, deadenylase assays, P-body imaging, and apoptosis flow cytometry\",\n      \"pmids\": [\"21299754\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific mRNA targets destabilized not enumerated\", \"Mechanism linking complex loss to ER stress/apoptosis unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved the atomic architecture of the Not module, showing NOT2/NOT5 wrap NOT1 via Sm-fold-like Not boxes and form a composite RNA-binding surface.\",\n      \"evidence\": \"2.8 \\u00c5 X-ray crystallography of the yeast Not1/Not2/Not5 module, in vitro poly(U) binding, structure-guided mutagenesis with in vivo growth assays\",\n      \"pmids\": [\"24121231\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RNA-binding specificity in vivo not defined\", \"Human complex structure not solved here\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed CCR4-NOT deadenylase activity is dynamically tuned by stress-induced phosphorylation of CNOT2, establishing post-translational control of mRNA decay.\",\n      \"evidence\": \"MS phospho-mapping with phosphomimetic/non-phosphorylatable rescue, poly(A) length and in vitro deadenylase assays under osmotic stress\",\n      \"pmids\": [\"35129087\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of phospho-induced activity loss unresolved\", \"Breadth of stress-regulated mRNA targets unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected CNOT2 to a cellular catabolic program by showing it promotes ATG5-dependent autophagic degradation of p62/SQSTM1.\",\n      \"evidence\": \"Knockdown/overexpression in ATG5 KO MEFs, ubiquitination assays, co-IP with PB1-domain mapping, and autophagic flux/LC3 imaging\",\n      \"pmids\": [\"28537904\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the effect requires deadenylase activity not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended CNOT2's developmental relevance by showing the CNOT1/2/3 module maintains pluripotency by repressing trophectoderm transcription factors.\",\n      \"evidence\": \"siRNA/shRNA in mouse and human ESCs with differentiation phenotyping, epistasis, and co-IP\",\n      \"pmids\": [\"22367759\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether repression is transcriptional or mRNA-decay-mediated not resolved\", \"Direct target mRNAs not mapped\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Implicated CNOT2 in adipogenesis through a physical interaction with PPAR\\u03b3.\",\n      \"evidence\": \"Co-IP, immunofluorescence, and siRNA loss-of-function in 3T3-L1 preadipocytes with differentiation readouts\",\n      \"pmids\": [\"26584287\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect interaction with PPAR\\u03b3 unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated CNOT2 facilitates dengue replication by accelerating deadenylase-mediated decay of antiviral JAK1 and STAT1 mRNAs.\",\n      \"evidence\": \"RNAi screen, siRNA knockdown with mRNA stability qRT-PCR, co-IP with CNOT6/6L and CNOT7/8, and viral replication assays\",\n      \"pmids\": [\"31155293\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding of CNOT2 to target mRNAs not shown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Reported CNOT2 interactions with oncogenic effectors (STAT3/c-Myc and VEGF) in cancer cells, suggesting roles in tumor metabolism and angiogenesis.\",\n      \"evidence\": \"Co-IP, knockdown/overexpression rescue, and functional assays in HCC and HeLa cells\",\n      \"pmids\": [\"41629418\", \"42045595\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single co-IP studies without reciprocal/structural validation\", \"Direct vs indirect interactions undefined\", \"Not independently confirmed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CNOT2's distinct activities\\u2014structural scaffolding, Not-Box-mediated transcriptional repression, and deadenylase regulation\\u2014are coordinated across its many biological contexts remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Whether transcriptional and mRNA-decay functions are mechanistically coupled is unknown\", \"Genome-wide direct mRNA target set of CNOT2-containing complex not defined\", \"No human Not-module structure in the timeline\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 4, 5]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [3, 6, 7]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [7, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [7, 13]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 4, 5]}\n    ],\n    \"complexes\": [\"CCR4-NOT complex\", \"Not module (NOT1-NOT2-NOT5)\", \"SMRT/NCoR-HDAC3 co-repressor complex\"],\n    \"partners\": [\"CNOT1\", \"CNOT3\", \"CNOT6L\", \"CNOT7\", \"CDK11\", \"PPARG\", \"SQSTM1\", \"NCOR\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}