| 2012 |
Crystal structure of the CAF1-binding (MIF4G) domain of human NOT1 alone and in complex with CAF1 was solved, revealing that NOT1 bridges the interaction between the catalytic module (CAF1/CCR4) and the NOT module, acting as a scaffold for CCR4-NOT complex assembly. The NOT1 MIF4G domain binds CAF1 through a pre-formed interface while leaving the CAF1 catalytic site fully accessible to RNA substrates. |
X-ray crystallography with functional validation of interface residues |
Nucleic acids research |
High |
22977175
|
| 2019 |
Reconstitution of a complete recombinant human CCR4-NOT complex showed that CCR4a (CNOT6) and CAF1 have distinct deadenylation profiles in vitro, and that the complex is more active and selective for poly(A) than the isolated exonucleases alone. Non-enzymatic modules (CAF40 and NOT10:NOT11) stimulate deadenylation in a partially redundant manner. |
In vitro reconstitution of recombinant human CCR4-NOT complex; biochemical deadenylation assay with purified component variants |
Nature communications |
High |
31320642
|
| 2015 |
Using a purified minimal human BTG2-Caf1-Ccr4 (CNOT6) nuclease sub-complex reconstituted from bacteria, chemical inhibition and inactivating amino acid substitutions demonstrated that both the Caf1 and Ccr4 (CNOT6) enzyme activities are required for deadenylation in vitro, indicating they cooperate and may regulate each other allosterically within the nuclease module. |
In vitro deadenylation assay with purified recombinant sub-complex; active-site mutagenesis; chemical inhibition |
The Biochemical journal |
High |
25944446
|
| 2011 |
Knockdown of Ccr4a (CNOT6) and Ccr4b (CNOT6L) in human cells plays a role in cell survival and prevention of senescence, distinct from knockdown of Caf1a/Caf1b or non-catalytic subunits. CNOT6/6L knockdown differentially affects processing-body formation. CNOT6/6L regulate IGFBP5 mRNA, mediating cell cycle arrest and senescence via a p53-dependent pathway. The LRR domain of Ccr4b influences subcellular localization but is not required for deadenylase activity. |
siRNA knockdown with cell viability, senescence (SA-β-gal), cell cycle, and P-body formation readouts; gene expression profiling; subcellular localization studies |
Molecular biology of the cell |
Medium |
21233283
|
| 2020 |
In mouse embryonic fibroblasts (MEFs), CNOT6/6L double knockout (dKO) cells remain viable whereas CNOT7/8 dKO cells undergo cell death, demonstrating that CNOT7/8 are the essential deadenylase subunits for cell viability. In Cnot7/8-dKO MEFs, CNOT6/6L are also absent from the CCR4-NOT complex, whereas in Cnot6/6l-dKO MEFs, CNOT7/8 still interacts with other subunits. Bulk poly(A) tail analysis showed more mRNAs with longer poly(A) tails in Cnot7/8-dKO than Cnot6/6l-dKO MEFs. Cnot6/6l-dKO mice are viable and grow normally. |
CRISPR/genetic double knockout of Cnot6/6l in MEFs and mice; co-immunoprecipitation; bulk poly(A) tail analysis; mRNA stability measurement |
RNA biology |
High |
31924127
|
| 2008 |
CCR4 (CNOT6) potentiates nuclear hormone receptor transcriptional activity and mediates its effect through the ligand binding domain of nuclear receptors. siRNA knockdown of CCR4 decreased nuclear receptor activation and attenuated stimulation of RARα target genes Sox9 and HoxA1. CCR4 associates in vivo with NIF-1, and the CCR4-enhanced transcriptional activation is dependent on NIF-1. |
siRNA knockdown; co-immunoprecipitation (in vivo and in vitro); quantitative PCR of target genes; reporter assays |
The Journal of biological chemistry |
Medium |
18180299
|
| 2008 |
Expression of a GSE fragment of hCCR4/CNOT6 or siRNA knockdown of CNOT6 decreased sensitivity of mammalian cells to DNA-damaging agents (cisplatin). Overexpression of hCCR4 targeted Chk2 following cisplatin exposure without interfering with the upstream ATM/ATR pathway, while strongly increasing γH2AX phosphorylation. |
Genetic suppressor element (GSE) selection; siRNA knockdown; Western blot for DNA damage response proteins (Chk2, γH2AX) |
Cancer letters |
Medium |
18818012
|
| 2021 |
ENO1 acts as an RNA-binding protein that recruits CNOT6 to accelerate mRNA decay of IRP1 in hepatocellular carcinoma cells, thereby suppressing mitochondrial iron-induced ferroptosis. This establishes CNOT6 as a downstream effector of an ENO1-dependent mRNA decay pathway. |
Co-immunoprecipitation; RNA immunoprecipitation; siRNA knockdown; in vitro and in vivo functional assays |
Nature cancer |
Medium |
35121990
|
| 2018 |
CNOT6 is present in cortical foci of mouse oocytes and regulates a novel pattern of mRNA deadenylation during oocyte meiotic maturation, specifically targeting a subset of mRNAs that are deadenylated in growing oocytes, polyadenylated during early maturation, and then re-deadenylated during late maturation. PUF-binding elements (PBEs) regulate this deadenylation in mature oocytes. |
Immunofluorescence localization; poly(A) tail length assay; functional perturbation experiments in oocytes |
Scientific reports |
Medium |
29717177
|
| 2021 |
FSH stimulates transcription and translation of CNOT6 and CNOT6L in ovarian granulosa cells. CNOT6/6L function as key effectors of FSH, triggering clearance of specific transcripts during preantral-to-antral follicle transition. Cnot6l-/- female mice are infertile with poor ovarian responses to gonadotropins. Cnot6/6l double knockout mice demonstrate that CNOT6/6L-mediated mRNA deadenylation is dispensable in most somatic cell types but essential for female reproductive endocrine regulation. |
Conditional and constitutive knockout mouse models; transcriptome analysis; gonadotropin stimulation assays; fertility phenotyping |
Cell reports |
High |
34788619
|
| 2014 |
In sea urchin primordial germ cells, Nanos targets CNOT6 mRNA for degradation, creating a stable mRNA environment. Misexpression of CNOT6 in PGCs caused failure to retain Seawi transcripts and Vasa protein. Broad knockdown of CNOT6 expanded the domain of Seawi RNA and exogenous reporters, establishing spatially restricted CNOT6 expression as a mechanism for selective germline RNA localization. |
mRNA misexpression; morpholino-based knockdown; in situ hybridization; reporter assays in sea urchin embryos |
Development (Cambridge, England) |
Medium |
25100654
|
| 2022 |
Depletion of CNOT6 sensitizes human U2OS cells to the DNA-damaging agent MNNG, upregulates MMR pathway activity, decreases mutation frequency in MMR-proficient cells, and increases mRNA stability of MMR gene transcripts leading to increased MMR protein expression. This identifies CNOT6 as a novel regulator of DNA mismatch repair via mRNA decay of MMR gene transcripts. |
siRNA knockdown; cell survival assay; mRNA stability measurement; MMR reporter assay; Western blot |
Cells |
Medium |
35159331
|
| 2021 |
CNOT6/6L-mediated deadenylation targets p21 mRNA (but not Caf1a/b) in gastric cancer MKN28 cells, contributing to cell cycle regulation. Depletion of CNOT6/6L arrests cells at the G0/G1 phase and modulates CDK-cyclin inhibitor levels without affecting CDKs or cyclins directly. Depletion of CNOT6/6L also impairs processing-body formation. |
Stable shRNA knockdown; cell cycle analysis; Western blot; P-body imaging; mRNA stability assay |
Cancers |
Medium |
33671234
|
| 2016 |
Forced expression of a combination of Cnot6, Cnot6l, Cnot7, and Cnot8 increases the number of alkaline phosphatase-positive colonies after iPSC induction and decreases expression of Eomes and p21 mRNAs (which have longer poly(A) tails when Cnot1 is depleted), implicating CCR4-NOT deadenylase activity including CNOT6 in iPSC reprogramming via poly(A) tail regulation of specific mRNAs. |
Overexpression of deadenylase subunits in MEFs with iPSC induction; alkaline phosphatase assay; poly(A) tail length analysis; qRT-PCR |
Biochemical and biophysical research communications |
Low |
27037025
|
| 2019 |
CNOT6 knockdown in human HepG2 cells led to significant alteration in stability of specific mRNAs; alterations in half-life were inversely associated with transcription rates, mostly not resulting in changes in mRNA abundance. This demonstrates a buffering mechanism whereby transcript stabilization upon CNOT6 depletion is compensated by decreased transcription. |
siRNA knockdown; mRNA stability measurement; transcription rate measurement in human cells |
RNA biology |
Medium |
31116665
|
| 2024 |
The Legionella pneumophila effector PieF disrupts the association between CNOT6/6L EEP-type nucleases and CNOT7, demonstrating that PieF binding to CNOT7 is sufficient to displace CNOT6/6L from the complex. This mechanistically defines a protein-protein interaction dependency of CNOT6/6L on CNOT7 for complex incorporation. |
Co-immunoprecipitation; in vitro binding/inhibition assay; yeast genetic assay; mammalian cell overexpression |
mSphere |
Medium |
39699231
|
| 2024 |
PCIF1 negatively regulates CNOT6 mRNA stability through m6A modification: siRNA-mediated suppression of PCIF1 upregulates CNOT6 at both mRNA and protein levels. MeRIP-qPCR showed that PCIF1 suppression significantly reduced m6A levels on CNOT6 mRNA, and rescue experiments confirmed the requirement for PCIF1's methyltransferase activity, establishing m6A modification as a mechanism controlling CNOT6 mRNA stability. |
siRNA knockdown; MeRIP-qPCR; Western blot; rescue with methyltransferase-dead mutant |
Cells |
Medium |
39451207
|
| 2026 |
CNOT6, but not its paralog CNOT6L, is an essential post-transcriptional regulator of neonatal growth: loss of Cnot6 causes severe growth retardation, multi-organ hypoplasia, and increased perinatal mortality. Mechanistically, Cnot6 deficiency elevates hepatic Fgf21 mRNA expression, suppresses the IGF1-IGFBP1 axis, and reprograms liver transcriptional networks, identifying Fgf21 mRNA as a direct CNOT6 decay target that limits anabolic metabolism during the neonatal period. |
Cnot6 knockout mouse model; hepatic transcriptome analysis; mRNA stability assay; metabolic phenotyping |
bioRxivpreprint |
Medium |
41757057
|
| 2025 |
ENO1 lactylation at K71 (facilitated by the lactyltransferase P300) reduces binding of TRIM21 mRNA to ENO1, preventing CNOT6 recruitment and thereby stabilizing TRIM21 mRNA in endothelial cells during sepsis. This establishes that ENO1-CNOT6 interaction and mRNA decay depend on the lactylation state of ENO1. |
Co-immunoprecipitation; RNA immunoprecipitation (RIP); RT-qPCR; Western blot; post-translational modification mass spectrometry |
Clinical and translational medicine |
Medium |
41532698
|