Affinage

CNOT6L

CCR4-NOT transcription complex subunit 6-like · UniProt Q96LI5

Length
555 aa
Mass
63.0 kDa
Annotated
2026-06-09
13 papers in source corpus 10 papers cited in narrative 10 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 5/5 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

CNOT6L is a cytoplasmic EEP-family deadenylase subunit of the CCR4-NOT complex that controls the stability of specific mRNAs by shortening their poly(A) tails (PMID:17452450, PMID:20628353). Its nuclease domain adopts an alpha/beta sandwich fold with conserved active-site residues and carries out Mg2+-dependent catalysis with strict poly(A) RNA substrate specificity, while its N-terminal leucine-rich repeat governs subcellular localization without contributing to catalysis (PMID:20628353, PMID:21233283). Through this deadenylase activity CNOT6L sets the turnover of defined transcripts—p27Kip1 to permit cell cycle progression, IL-8 during myogenesis, Zeb1 downstream of a miR-146a axis in epithelial-mesenchymal transition, and tenascin-C in cardiac fibroblasts—coupling targeted mRNA decay to growth control, differentiation, and tissue remodeling (PMID:17452450, PMID:26608607, PMID:23591815, PMID:40023604). Substrate selection is achieved through RNA-binding adaptors and 3'-UTR cis-elements: in oocytes ZFP36L2 recruits CNOT6L (distinct from the BTG4–CNOT7/CNOT8 arm) to degrade a subset of maternal mRNAs required for meiotic spindle organization, and a tenascin-C 3'-UTR cis-element directs CNOT6L-mediated decay in the heart (PMID:30478191, PMID:40023604). CNOT6L abundance is itself regulated post-translationally by the E3 ubiquitin ligase RNF219, which ubiquitinates CNOT6L and promotes its proteasomal degradation (PMID:40598799).

Mechanistic history

Synthesis pass · year-by-year structured walk · 9 steps
  1. 2007 High

    Established that the previously uncharacterized CCR4b protein is a functional cytoplasmic deadenylase within a CCR4-NOT-like complex that controls a specific mRNA, answering whether it has catalytic and physiological relevance.

    Evidence in vitro/in vivo deadenylase assays, RNAi with catalytic-mutant rescue, and poly(A) tail analysis of p27Kip1 in NIH 3T3 cells

    PMID:17452450

    Open questions at the time
    • Structural basis of catalysis and substrate specificity unresolved
    • Mechanism of recruitment to p27Kip1 mRNA unknown
  2. 2010 High

    Defined the structural and chemical basis of CNOT6L activity, explaining how it achieves Mg2+-dependent catalysis and strict poly(A) specificity.

    Evidence X-ray crystallography of the nuclease domain, co-crystals with AMP and poly(A), in vitro deadenylase assays with active-site mutagenesis

    PMID:20628353

    Open questions at the time
    • Structure of full-length protein with LRR not determined
    • No structure within the assembled CCR4-NOT complex
  3. 2011 Medium

    Distinguished CNOT6L from the CAF1 (CNOT7/CNOT8) catalytic arm functionally and mapped the LRR to localization rather than catalysis, refining how the two deadenylase modules are deployed.

    Evidence RNAi knockdown, domain-deletion overexpression, senescence/viability assays, and P-body microscopy

    PMID:21233283

    Open questions at the time
    • No structural confirmation of LRR localization role
    • Molecular basis of differential P-body effects unresolved
  4. 2013 Medium

    Placed CNOT6L within a regulatory circuit by showing it is itself a miRNA target whose deadenylase output controls an EMT effector mRNA.

    Evidence Git2 loss-of-function, miR-146a target validation, and Zeb1 mRNA stability/poly(A) assays

    PMID:23591815

    Open questions at the time
    • Direct biochemical demonstration of CNOT6L acting on Zeb1 limited
    • Single study, single system
  5. 2015 Medium

    Extended CNOT6L target repertoire to inflammatory signaling in differentiation by identifying IL-8 mRNA as a direct substrate controlling myogenesis.

    Evidence siRNA knockdown, poly(A) tail length assays, and IL-8 gain/loss-of-function in human myoblasts

    PMID:26608607

    Open questions at the time
    • Adaptor mediating IL-8 mRNA recruitment unknown
    • Single lab, single study
  6. 2018 High

    Demonstrated in vivo that CNOT6L drives stage-specific maternal mRNA decay and revealed adaptor-specific recruitment, answering how distinct CCR4-NOT catalytic subunits are targeted to different transcripts.

    Evidence Cnot6l conditional/constitutive knockout mice, poly(A) and transcriptome analysis, spindle imaging, and ZFP36L2 vs BTG4 epistasis; cross-species oocyte analysis

    PMID:30456367 PMID:30478191

    Open questions at the time
    • Structural basis of ZFP36L2-CNOT6L recruitment unresolved
    • Rules defining which transcripts each adaptor selects incomplete
  7. 2021 Low

    Linked CNOT6L loss to depletion of inosine-modified transcripts, raising the question of how deadenylation relates to clearance of modified mRNAs.

    Evidence computational inosine identification from RNA-seq and polysomal fractionation in Cnot6l-knockout oocytes

    PMID:33530472

    Open questions at the time
    • Correlative computational inference, no direct mechanistic link
    • Cannot distinguish direct from indirect effects on inosine RNAs
  8. 2024 High

    Established a tissue-level disease-relevant CNOT6L axis by showing it directly decays tenascin-C mRNA via a defined cis-element to restrain cardiac fibrosis.

    Evidence Cnot6l knockout under transverse aortic constriction, poly(A) and 3'-UTR luciferase reporter assays, and Cnot6l/tenascin-C double-knockout epistasis

    PMID:40023604

    Open questions at the time
    • Adaptor/RBP recognizing the tenascin-C cis-element not identified
    • Signaling that modulates CNOT6L activity during pressure overload unknown
  9. 2025 Medium

    Identified post-translational control of CNOT6L abundance, answering how its protein levels are regulated independently of transcription.

    Evidence MS of CCR4-NOT immunoprecipitates, in vitro ubiquitination, RNF219-CNOT1 pull-down, and RNF219 knockdown with proteasome inhibition in HEK293T

    PMID:40598799

    Open questions at the time
    • Ubiquitination site(s) on CNOT6L not mapped
    • Physiological contexts where RNF219 regulates CNOT6L unknown
    • Single lab

Open questions

Synthesis pass · forward-looking unresolved questions
  • How CNOT6L substrate selection is encoded—the full set of adaptor proteins, cis-elements, and structural determinants that route specific mRNAs to CNOT6L versus the CAF1 arm—remains unresolved.
  • No general code linking adaptors/cis-elements to CNOT6L target choice
  • No structure of CNOT6L engaged with an adaptor or within full CCR4-NOT
  • Regulation of CNOT6L activity by signaling poorly defined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140098 catalytic activity, acting on RNA 5 GO:0016787 hydrolase activity 2 GO:0003723 RNA binding 1
Localization
GO:0005829 cytosol 2
Pathway
R-HSA-8953854 Metabolism of RNA 3
Partners
CNOT1RNF219ZFP36L2
Complex memberships
CCR4-NOT

Evidence

Reading pass · 10 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2010 Crystal structure of the CNOT6L nuclease domain was determined by X-ray crystallography, revealing an alpha/beta sandwich fold typical of EEP-family hydrolases with conserved active-site residues similar to APE1. In vitro deadenylase assays confirmed critical active-site residues and demonstrated that the nuclease domain exhibits full Mg2+-dependent deadenylase activity with strict poly(A) RNA substrate specificity. Co-crystal structures with AMP and poly(A) DNA suggested a molecular mechanism involving a pentacovalent phosphate transition state. X-ray crystallography (SAD), in vitro deadenylase assay, active-site mutagenesis, co-crystal structures with substrates The EMBO journal High 20628353
2007 CNOT6L (CCR4b) is localized mainly in the cytoplasm, displays deadenylase activity both in vitro and in vivo, and forms a multisubunit complex analogous to the yeast CCR4-NOT complex. RNAi-mediated suppression of CCR4b in NIH 3T3 cells caused growth retardation, elevated p27Kip1 mRNA and protein, and preservation of the p27Kip1 poly(A) tail. Reintroduction of wild-type but not deadenylase-inactive CCR4b rescued cell growth, establishing that CNOT6L regulates p27Kip1 mRNA turnover through its deadenylase activity. In vitro and in vivo deadenylase assay, RNAi knockdown, rescue with wild-type vs. catalytic mutant, poly(A) tail length assay, subcellular fractionation/localization Molecular and cellular biology High 17452450
2011 CNOT6L (Ccr4b) plays a role in cell survival and prevention of senescence distinct from the CAF1 subunits (CNOT7/CNOT8) of the CCR4-NOT complex. The N-terminal leucine-rich repeat (LRR) domain of CNOT6L influences its subcellular localization but is not required for deadenylase activity. Overexpression of LRR-deleted CNOT6L interfered with cell cycle progression but not cell viability. Knockdown of Ccr4a/Ccr4b, but not Caf1a/Caf1b, differentially affected cytoplasmic processing-body (P-body) foci formation. RNAi knockdown, domain-deletion overexpression, cell viability/senescence assays, subcellular localization analysis, P-body microscopy, gene expression profiling Molecular biology of the cell Medium 21233283
2018 Genetic deletion of Cnot6l in mouse oocytes impaired deadenylation and degradation of a specific subset of maternal mRNAs during meiotic maturation, causing microtubule-chromosome organization defects, spindle assembly checkpoint activation, and arrest at prometaphase. CNOT6L function in oocytes is mediated through the RNA-binding protein ZFP36L2 (not BTG4, which recruits CNOT7/CNOT8), establishing that different adaptor proteins recruit different CCR4-NOT catalytic subunits for stage-specific maternal mRNA decay. Conditional knockout mouse, poly(A) tail assay, transcriptome analysis, spindle/chromosome imaging, epistasis with ZFP36L2 and BTG4 adaptor proteins The EMBO journal High 30478191
2018 CNOT6L supplies the majority of CCR4 deadenylase activity in the maternal CCR4-NOT complex in mouse, hamster, and bovine oocytes. Loss of Cnot6l caused major transcriptome changes in ovulated eggs and one-cell zygotes but minimal changes in preovulatory oocytes, consistent with dormancy of Cnot6l mRNA before oocyte activation. Transcripts sensitive to decapping inhibition and those sensitive to Cnot6l loss showed minimal overlap, indicating that decapping and CNOT6L-mediated deadenylation target distinct mRNA subsets during oocyte-to-embryo transition. Cnot6l knockout mouse, RNA-seq transcriptome analysis, comparison with decapping inhibition, cross-species protein analysis Life science alliance Medium 30456367
2013 CNOT6L deadenylase activity regulates the stability of Zeb1 mRNA downstream of a miR-146a/CNOT6L axis in epithelial-mesenchymal transition (EMT). miR-146a targets CNOT6L as a validated target, and CNOT6L in turn controls Zeb1 mRNA stability through its deadenylase activity, linking Git2 loss to EMT induction. Loss-of-function (Git2 knockout), miRNA target validation, poly(A) tail/mRNA stability assays, biochemical deadenylase assessment Journal of cell science Medium 23591815
2015 CNOT6L directly targets IL-8 mRNA for deadenylation in human skeletal muscle myoblasts, as shown by poly(A) tail length assays and biochemical approaches. CNOT6L knockdown elevated IL-8 mRNA levels, and gain- and loss-of-function experiments established IL-8 as a functional effector of CNOT6L-regulated myogenesis. CNOT6L knockdown (siRNA), poly(A) tail length assay, gene expression profiling, gain- and loss-of-function assays for IL-8 Biochimica et biophysica acta Medium 26608607
2021 Loss of Cnot6l in mouse oocytes caused pronounced depletion of inosine RNA modifications in total and polysomal RNA compared to controls, whereas Btg4 knockout did not. Ribosome-associated RNA analysis revealed clearance of inosine-modified mRNAs, suggesting that inosine-containing transcripts are degraded in a parallel but independent mechanism to CCR4-NOT deadenylation during oocyte maturation. Cnot6l knockout mouse, computational inosine identification from RNA-seq, polysomal RNA-seq fractionation International journal of molecular sciences Low 33530472
2024 CNOT6L deadenylase directly targets tenascin-C mRNA via a cis-element in its 3'-UTR in cardiac fibroblasts, as shown by poly(A) tail length assays and luciferase reporter assays. Genetic deletion of Cnot6l in mice subjected to transverse aortic constriction led to marked cardiac fibrosis and dysfunction, and double knockout of tenascin-C and Cnot6l ameliorated these phenotypes, establishing a CNOT6L–tenascin-C axis in cardiac remodeling. Cnot6l knockout mouse, poly(A) tail length assay, luciferase reporter assay (3'-UTR cis-element), double-knockout epistasis (Cnot6l/tenascin-C), transverse aortic constriction model The Journal of pharmacology and experimental therapeutics High 40023604
2025 The E3 ubiquitin ligase RNF219 directly ubiquitinates CNOT6L in vitro and suppresses CNOT6L protein levels through proteasome-mediated degradation. RNF219 binds to the CNOT1 DUF3819 domain (direct pull-down), and RNF219 knockdown in HEK293T cells elevated CNOT6L expression accompanied by increased cell proliferation, indicating that RNF219 controls CNOT6L abundance post-translationally. Mass spectrometry of CCR4-NOT immunoprecipitates, pull-down assay, in vitro ubiquitination assay, RNF219 knockdown with CNOT6L expression quantification, proteasome inhibitor treatment FEBS open bio Medium 40598799

Source papers

Stage 0 corpus · 13 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2018 CNOT6L couples the selective degradation of maternal transcripts to meiotic cell cycle progression in mouse oocyte. The EMBO journal 115 30478191
2011 The Ccr4a (CNOT6) and Ccr4b (CNOT6L) deadenylase subunits of the human Ccr4-Not complex contribute to the prevention of cell death and senescence. Molecular biology of the cell 99 21233283
2007 Depletion of mammalian CCR4b deadenylase triggers elevation of the p27Kip1 mRNA level and impairs cell growth. Molecular and cellular biology 98 17452450
2010 Crystal structure of the human CNOT6L nuclease domain reveals strict poly(A) substrate specificity. The EMBO journal 82 20628353
2018 Role of Cnot6l in maternal mRNA turnover. Life science alliance 31 30456367
2013 Loss of Git2 induces epithelial-mesenchymal transition by miR146a-Cnot6L-controlled expression of Zeb1. Journal of cell science 15 23591815
2021 Loss of Cnot6l Impairs Inosine RNA Modifications in Mouse Oocytes. International journal of molecular sciences 13 33530472
2015 Post-transcriptional modulation of interleukin 8 by CNOT6L regulates skeletal muscle differentiation. Biochimica et biophysica acta 11 26608607
2024 CNOT6L deadenylase suppresses cardiac remodeling in heart failure through downregulation of tenascin-C mRNA. The Journal of pharmacology and experimental therapeutics 3 40023604
2025 The E3 ubiquitin ligase, RNF219, suppresses CNOT6L expression to exhibit antiproliferative activity. FEBS open bio 2 40598799
2025 Machine learning Reveals ATM and CNOT6L as critical factors in Cataract pathogenesis. Experimental eye research 1 40412792
2025 CNOT6L regulates energy metabolism in the ovarian granulosa cells associated with polycystic ovary syndrome. Frontiers in cell and developmental biology 1 40458121
2024 High expression of CNOT6L contributes to the negative development of type 2 diabetes. Scientific reports 1 39433858

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