| 1994 |
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. |
Allele-specific suppressor screens, yeast two-hybrid, biochemical co-fractionation, transcriptional reporter assays |
Genes & development |
High |
7926748
|
| 1990 |
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. |
Genetic epistasis analysis, pheromone-inducible reporter (FUS1) assays, temperature-sensitive mutant analysis |
Cell regulation |
High |
2099190 2111445
|
| 1999 |
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. |
Biochemical co-fractionation, co-immunoprecipitation, deletion mapping, genetic interaction analysis |
Molecular and cellular biology |
High |
10490603
|
| 2002 |
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. |
Biochemical co-fractionation, co-immunoprecipitation, site-directed mutagenesis, yeast genetic analysis |
Journal of molecular biology |
High |
12215412
|
| 2004 |
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. |
Promoter-targeting (Gal4-fusion) transient transfection reporter assays in human cells, Not-Box deletion and mutagenesis |
The Journal of biological chemistry |
Medium |
14707134
|
| 2006 |
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. |
Co-immunoprecipitation, reporter gene repression assays, co-expression of SMRT/NCoR-HDAC3 subunits, Not-Box deletion analysis |
The Biochemical journal |
Medium |
16712523
|
| 2013 |
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. |
X-ray crystallography (2.8 Å), in vitro RNA-binding assay, structure-guided mutagenesis with in vivo growth assays |
Nature structural & molecular biology |
High |
24121231
|
| 2011 |
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. |
siRNA knockdown, sucrose gradient fractionation, deadenylase activity assay, fluorescence microscopy (P-bodies), RT-qPCR, flow cytometry (apoptosis) |
Genes to cells |
High |
21299754
|
| 2005 |
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. |
Yeast two-hybrid screening, in vitro binding assay, co-immunoprecipitation in human cells, co-localization by fluorescence microscopy |
Biochemical and biophysical research communications |
Medium |
16039607
|
| 2012 |
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). |
siRNA/shRNA knockdown in mouse and human ESCs, gene expression analysis, genetic epistasis, immunofluorescence, co-IP to establish complex |
Stem cells |
Medium |
22367759
|
| 2015 |
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. |
siRNA knockdown, co-immunoprecipitation, immunofluorescence co-localization, Western blot, Oil Red O staining |
Cellular physiology and biochemistry |
Medium |
26584287
|
| 2017 |
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. |
siRNA/shRNA knockdown and overexpression, ATG5 knockout MEF cells, ubiquitination assay, co-immunoprecipitation, LC3 puncta imaging, autophagic flux assay |
Oncotarget |
Medium |
28537904
|
| 2019 |
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. |
RNAi screen, siRNA knockdown, qRT-PCR for mRNA stability, co-immunoprecipitation (CNOT2 with deadenylase subunits), viral replication assays |
Biochemical and biophysical research communications |
Medium |
31155293
|
| 2021 |
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. |
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 |
RNA biology |
High |
35129087
|
| 2026 |
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. |
Co-immunoprecipitation, siRNA knockdown, overexpression rescue, Western blot for glycolytic markers and apoptotic markers |
Scientific reports |
Low |
41629418
|
| 2026 |
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. |
Co-immunoprecipitation, siRNA knockdown, overexpression, VEGF luciferase reporter assay, cycloheximide chase assay, tube formation assay, CAM assay |
Scientific reports |
Low |
42045595
|