{"gene":"CNOT6L","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2010,"finding":"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.","method":"X-ray crystallography (SAD), in vitro deadenylase assay, active-site mutagenesis, co-crystal structures with substrates","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation by in vitro assay and active-site mutagenesis, multiple orthogonal methods in a single rigorous study","pmids":["20628353"],"is_preprint":false},{"year":2007,"finding":"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.","method":"In vitro and in vivo deadenylase assay, RNAi knockdown, rescue with wild-type vs. catalytic mutant, poly(A) tail length assay, subcellular fractionation/localization","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro and in vivo enzymatic assays combined with mutagenesis rescue experiment and poly(A) tail analysis, multiple orthogonal methods in a single study","pmids":["17452450"],"is_preprint":false},{"year":2011,"finding":"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.","method":"RNAi knockdown, domain-deletion overexpression, cell viability/senescence assays, subcellular localization analysis, P-body microscopy, gene expression profiling","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple orthogonal cellular assays in a single study; localization tied to LRR domain deletion but no structural confirmation","pmids":["21233283"],"is_preprint":false},{"year":2018,"finding":"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.","method":"Conditional knockout mouse, poly(A) tail assay, transcriptome analysis, spindle/chromosome imaging, epistasis with ZFP36L2 and BTG4 adaptor proteins","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic knockout with defined molecular phenotype, epistasis with specific adaptors, poly(A) tail assays, replicated by independent lab (PMID:30456367)","pmids":["30478191"],"is_preprint":false},{"year":2018,"finding":"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.","method":"Cnot6l knockout mouse, RNA-seq transcriptome analysis, comparison with decapping inhibition, cross-species protein analysis","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo knockout with transcriptome analysis and cross-species validation; mechanism of selectivity inferred from transcriptome overlap, single lab","pmids":["30456367"],"is_preprint":false},{"year":2013,"finding":"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.","method":"Loss-of-function (Git2 knockout), miRNA target validation, poly(A) tail/mRNA stability assays, biochemical deadenylase assessment","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — miR-146a/CNOT6L/Zeb1 pathway established by loss-of-function and mRNA stability assays in a single study; CNOT6L's enzymatic role inferred from deadenylase function","pmids":["23591815"],"is_preprint":false},{"year":2015,"finding":"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.","method":"CNOT6L knockdown (siRNA), poly(A) tail length assay, gene expression profiling, gain- and loss-of-function assays for IL-8","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct poly(A) tail assay demonstrating CNOT6L targets IL-8 mRNA, supported by gain/loss-of-function; single lab, single study","pmids":["26608607"],"is_preprint":false},{"year":2021,"finding":"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.","method":"Cnot6l knockout mouse, computational inosine identification from RNA-seq, polysomal RNA-seq fractionation","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3–4 / Weak — primarily computational inosine identification from sequencing data in knockout oocytes; mechanistic link between CNOT6L and inosine modifications is correlative","pmids":["33530472"],"is_preprint":false},{"year":2024,"finding":"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.","method":"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","journal":"The Journal of pharmacology and experimental therapeutics","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — poly(A) tail assay plus 3'-UTR reporter assay identifying cis-element, confirmed by double-knockout genetic epistasis; multiple orthogonal methods in single study","pmids":["40023604"],"is_preprint":false},{"year":2025,"finding":"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.","method":"Mass spectrometry of CCR4-NOT immunoprecipitates, pull-down assay, in vitro ubiquitination assay, RNF219 knockdown with CNOT6L expression quantification, proteasome inhibitor treatment","journal":"FEBS open bio","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro ubiquitination assay plus pull-down identifying CNOT1-RNF219 interaction and functional knockdown linking RNF219 to CNOT6L protein levels; single lab","pmids":["40598799"],"is_preprint":false}],"current_model":"CNOT6L is a cytoplasmic EEP-family deadenylase subunit of the CCR4-NOT complex whose crystal structure reveals strict poly(A) specificity and Mg2+-dependent catalysis; it is recruited to target mRNAs via adaptor proteins (e.g., ZFP36L2 in oocytes) to deadenylate and degrade specific transcripts—including p27Kip1, IL-8, Zeb1, and tenascin-C mRNAs—thereby controlling cell cycle progression, meiotic maturation, myogenesis, and cardiac remodeling, and its own abundance is post-translationally regulated by RNF219-mediated ubiquitination and proteasomal degradation."},"narrative":{"mechanistic_narrative":"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].","teleology":[{"year":2007,"claim":"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","pmids":["17452450"],"confidence":"High","gaps":["Structural basis of catalysis and substrate specificity unresolved","Mechanism of recruitment to p27Kip1 mRNA unknown"]},{"year":2010,"claim":"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","pmids":["20628353"],"confidence":"High","gaps":["Structure of full-length protein with LRR not determined","No structure within the assembled CCR4-NOT complex"]},{"year":2011,"claim":"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","pmids":["21233283"],"confidence":"Medium","gaps":["No structural confirmation of LRR localization role","Molecular basis of differential P-body effects unresolved"]},{"year":2013,"claim":"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","pmids":["23591815"],"confidence":"Medium","gaps":["Direct biochemical demonstration of CNOT6L acting on Zeb1 limited","Single study, single system"]},{"year":2015,"claim":"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","pmids":["26608607"],"confidence":"Medium","gaps":["Adaptor mediating IL-8 mRNA recruitment unknown","Single lab, single study"]},{"year":2018,"claim":"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","pmids":["30478191","30456367"],"confidence":"High","gaps":["Structural basis of ZFP36L2-CNOT6L recruitment unresolved","Rules defining which transcripts each adaptor selects incomplete"]},{"year":2021,"claim":"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","pmids":["33530472"],"confidence":"Low","gaps":["Correlative computational inference, no direct mechanistic link","Cannot distinguish direct from indirect effects on inosine RNAs"]},{"year":2024,"claim":"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","pmids":["40023604"],"confidence":"High","gaps":["Adaptor/RBP recognizing the tenascin-C cis-element not identified","Signaling that modulates CNOT6L activity during pressure overload unknown"]},{"year":2025,"claim":"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","pmids":["40598799"],"confidence":"Medium","gaps":["Ubiquitination site(s) on CNOT6L not mapped","Physiological contexts where RNF219 regulates CNOT6L unknown","Single lab"]},{"year":null,"claim":"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.","evidence":"","pmids":[],"confidence":"Medium","gaps":["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":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,1,3,6,8]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,2]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[1,3,8]}],"complexes":["CCR4-NOT"],"partners":["CNOT1","RNF219","ZFP36L2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96LI5","full_name":"CCR4-NOT transcription complex subunit 6-like","aliases":["Carbon catabolite repressor protein 4 homolog B"],"length_aa":555,"mass_kda":63.0,"function":"Has 3'-5' poly(A) exoribonuclease activity for synthetic poly(A) RNA substrate. Catalytic 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. May be involved in the deadenylation-dependent degradation of mRNAs through the 3'-UTR AU-rich element-mediated mechanism. Involved in deadenylation-dependent degradation of CDKN1B mRNA. Its mRNA deadenylase activity can be inhibited by TOB1. Mediates cell proliferation and cell survival and prevents cellular senescence","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q96LI5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CNOT6L","classification":"Not Classified","n_dependent_lines":28,"n_total_lines":1208,"dependency_fraction":0.023178807947019868},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CAPZB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CNOT6L","total_profiled":1310},"omim":[{"mim_id":"618069","title":"CCR4-NOT TRANSCRIPTION COMPLEX, SUBUNIT 6-LIKE; CNOT6L","url":"https://www.omim.org/entry/618069"},{"mim_id":"146930","title":"CHEMOKINE, CXC MOTIF, LIGAND 8; CXCL8","url":"https://www.omim.org/entry/146930"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CNOT6L"},"hgnc":{"alias_symbol":["DKFZp434K098","Ccr4b"],"prev_symbol":[]},"alphafold":{"accession":"Q96LI5","domains":[{"cath_id":"3.80.10.10","chopping":"37-160","consensus_level":"high","plddt":96.6236,"start":37,"end":160},{"cath_id":"3.60.10.10","chopping":"173-537","consensus_level":"medium","plddt":92.1,"start":173,"end":537}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96LI5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96LI5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96LI5-F1-predicted_aligned_error_v6.png","plddt_mean":90.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CNOT6L","jax_strain_url":"https://www.jax.org/strain/search?query=CNOT6L"},"sequence":{"accession":"Q96LI5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96LI5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96LI5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96LI5"}},"corpus_meta":[{"pmid":"30478191","id":"PMC_30478191","title":"CNOT6L couples the selective degradation of maternal transcripts to meiotic cell cycle progression in mouse oocyte.","date":"2018","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/30478191","citation_count":115,"is_preprint":false},{"pmid":"21233283","id":"PMC_21233283","title":"The Ccr4a (CNOT6) and Ccr4b (CNOT6L) deadenylase subunits of the human Ccr4-Not complex contribute to the prevention of cell death and senescence.","date":"2011","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/21233283","citation_count":99,"is_preprint":false},{"pmid":"17452450","id":"PMC_17452450","title":"Depletion of mammalian CCR4b deadenylase triggers elevation of the p27Kip1 mRNA level and impairs cell growth.","date":"2007","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/17452450","citation_count":98,"is_preprint":false},{"pmid":"20628353","id":"PMC_20628353","title":"Crystal structure of the human CNOT6L nuclease domain reveals strict poly(A) substrate specificity.","date":"2010","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/20628353","citation_count":82,"is_preprint":false},{"pmid":"30456367","id":"PMC_30456367","title":"Role of Cnot6l in maternal mRNA turnover.","date":"2018","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/30456367","citation_count":31,"is_preprint":false},{"pmid":"23591815","id":"PMC_23591815","title":"Loss of Git2 induces epithelial-mesenchymal transition by miR146a-Cnot6L-controlled expression of Zeb1.","date":"2013","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/23591815","citation_count":15,"is_preprint":false},{"pmid":"33530472","id":"PMC_33530472","title":"Loss of Cnot6l Impairs Inosine RNA Modifications in Mouse Oocytes.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33530472","citation_count":13,"is_preprint":false},{"pmid":"26608607","id":"PMC_26608607","title":"Post-transcriptional modulation of interleukin 8 by CNOT6L regulates skeletal muscle differentiation.","date":"2015","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/26608607","citation_count":11,"is_preprint":false},{"pmid":"40023604","id":"PMC_40023604","title":"CNOT6L deadenylase suppresses cardiac remodeling in heart failure through downregulation of tenascin-C mRNA.","date":"2024","source":"The Journal of pharmacology and experimental therapeutics","url":"https://pubmed.ncbi.nlm.nih.gov/40023604","citation_count":3,"is_preprint":false},{"pmid":"40598799","id":"PMC_40598799","title":"The E3 ubiquitin ligase, RNF219, suppresses CNOT6L expression to exhibit antiproliferative activity.","date":"2025","source":"FEBS open bio","url":"https://pubmed.ncbi.nlm.nih.gov/40598799","citation_count":2,"is_preprint":false},{"pmid":"39433858","id":"PMC_39433858","title":"High expression of CNOT6L contributes to the negative development of type 2 diabetes.","date":"2024","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/39433858","citation_count":1,"is_preprint":false},{"pmid":"40458121","id":"PMC_40458121","title":"CNOT6L regulates energy metabolism in the ovarian granulosa cells associated with polycystic ovary syndrome.","date":"2025","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/40458121","citation_count":1,"is_preprint":false},{"pmid":"40412792","id":"PMC_40412792","title":"Machine learning Reveals ATM and CNOT6L as critical factors in Cataract pathogenesis.","date":"2025","source":"Experimental eye research","url":"https://pubmed.ncbi.nlm.nih.gov/40412792","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8527,"output_tokens":3171,"usd":0.036573,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10639,"output_tokens":3162,"usd":0.066123,"stage2_stop_reason":"end_turn"},"total_usd":0.102696,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"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.\",\n      \"method\": \"X-ray crystallography (SAD), in vitro deadenylase assay, active-site mutagenesis, co-crystal structures with substrates\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation by in vitro assay and active-site mutagenesis, multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"20628353\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"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.\",\n      \"method\": \"In vitro and in vivo deadenylase assay, RNAi knockdown, rescue with wild-type vs. catalytic mutant, poly(A) tail length assay, subcellular fractionation/localization\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro and in vivo enzymatic assays combined with mutagenesis rescue experiment and poly(A) tail analysis, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"17452450\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"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.\",\n      \"method\": \"RNAi knockdown, domain-deletion overexpression, cell viability/senescence assays, subcellular localization analysis, P-body microscopy, gene expression profiling\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple orthogonal cellular assays in a single study; localization tied to LRR domain deletion but no structural confirmation\",\n      \"pmids\": [\"21233283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"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.\",\n      \"method\": \"Conditional knockout mouse, poly(A) tail assay, transcriptome analysis, spindle/chromosome imaging, epistasis with ZFP36L2 and BTG4 adaptor proteins\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic knockout with defined molecular phenotype, epistasis with specific adaptors, poly(A) tail assays, replicated by independent lab (PMID:30456367)\",\n      \"pmids\": [\"30478191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"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.\",\n      \"method\": \"Cnot6l knockout mouse, RNA-seq transcriptome analysis, comparison with decapping inhibition, cross-species protein analysis\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo knockout with transcriptome analysis and cross-species validation; mechanism of selectivity inferred from transcriptome overlap, single lab\",\n      \"pmids\": [\"30456367\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"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.\",\n      \"method\": \"Loss-of-function (Git2 knockout), miRNA target validation, poly(A) tail/mRNA stability assays, biochemical deadenylase assessment\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — miR-146a/CNOT6L/Zeb1 pathway established by loss-of-function and mRNA stability assays in a single study; CNOT6L's enzymatic role inferred from deadenylase function\",\n      \"pmids\": [\"23591815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"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.\",\n      \"method\": \"CNOT6L knockdown (siRNA), poly(A) tail length assay, gene expression profiling, gain- and loss-of-function assays for IL-8\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct poly(A) tail assay demonstrating CNOT6L targets IL-8 mRNA, supported by gain/loss-of-function; single lab, single study\",\n      \"pmids\": [\"26608607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"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.\",\n      \"method\": \"Cnot6l knockout mouse, computational inosine identification from RNA-seq, polysomal RNA-seq fractionation\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3–4 / Weak — primarily computational inosine identification from sequencing data in knockout oocytes; mechanistic link between CNOT6L and inosine modifications is correlative\",\n      \"pmids\": [\"33530472\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"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.\",\n      \"method\": \"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\",\n      \"journal\": \"The Journal of pharmacology and experimental therapeutics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — poly(A) tail assay plus 3'-UTR reporter assay identifying cis-element, confirmed by double-knockout genetic epistasis; multiple orthogonal methods in single study\",\n      \"pmids\": [\"40023604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"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.\",\n      \"method\": \"Mass spectrometry of CCR4-NOT immunoprecipitates, pull-down assay, in vitro ubiquitination assay, RNF219 knockdown with CNOT6L expression quantification, proteasome inhibitor treatment\",\n      \"journal\": \"FEBS open bio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro ubiquitination assay plus pull-down identifying CNOT1-RNF219 interaction and functional knockdown linking RNF219 to CNOT6L protein levels; single lab\",\n      \"pmids\": [\"40598799\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CNOT6L is a cytoplasmic EEP-family deadenylase subunit of the CCR4-NOT complex whose crystal structure reveals strict poly(A) specificity and Mg2+-dependent catalysis; it is recruited to target mRNAs via adaptor proteins (e.g., ZFP36L2 in oocytes) to deadenylate and degrade specific transcripts—including p27Kip1, IL-8, Zeb1, and tenascin-C mRNAs—thereby controlling cell cycle progression, meiotic maturation, myogenesis, and cardiac remodeling, and its own abundance is post-translationally regulated by RNF219-mediated ubiquitination and proteasomal degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"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 [#1, #0]. 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 [#0, #2]. 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 [#1, #6, #5, #8]. 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 [#3, #8]. CNOT6L abundance is itself regulated post-translationally by the E3 ubiquitin ligase RNF219, which ubiquitinates CNOT6L and promotes its proteasomal degradation [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"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.\",\n      \"evidence\": \"in vitro/in vivo deadenylase assays, RNAi with catalytic-mutant rescue, and poly(A) tail analysis of p27Kip1 in NIH 3T3 cells\",\n      \"pmids\": [\"17452450\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of catalysis and substrate specificity unresolved\", \"Mechanism of recruitment to p27Kip1 mRNA unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined the structural and chemical basis of CNOT6L activity, explaining how it achieves Mg2+-dependent catalysis and strict poly(A) specificity.\",\n      \"evidence\": \"X-ray crystallography of the nuclease domain, co-crystals with AMP and poly(A), in vitro deadenylase assays with active-site mutagenesis\",\n      \"pmids\": [\"20628353\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of full-length protein with LRR not determined\", \"No structure within the assembled CCR4-NOT complex\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"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.\",\n      \"evidence\": \"RNAi knockdown, domain-deletion overexpression, senescence/viability assays, and P-body microscopy\",\n      \"pmids\": [\"21233283\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural confirmation of LRR localization role\", \"Molecular basis of differential P-body effects unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed CNOT6L within a regulatory circuit by showing it is itself a miRNA target whose deadenylase output controls an EMT effector mRNA.\",\n      \"evidence\": \"Git2 loss-of-function, miR-146a target validation, and Zeb1 mRNA stability/poly(A) assays\",\n      \"pmids\": [\"23591815\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical demonstration of CNOT6L acting on Zeb1 limited\", \"Single study, single system\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extended CNOT6L target repertoire to inflammatory signaling in differentiation by identifying IL-8 mRNA as a direct substrate controlling myogenesis.\",\n      \"evidence\": \"siRNA knockdown, poly(A) tail length assays, and IL-8 gain/loss-of-function in human myoblasts\",\n      \"pmids\": [\"26608607\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Adaptor mediating IL-8 mRNA recruitment unknown\", \"Single lab, single study\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"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.\",\n      \"evidence\": \"Cnot6l conditional/constitutive knockout mice, poly(A) and transcriptome analysis, spindle imaging, and ZFP36L2 vs BTG4 epistasis; cross-species oocyte analysis\",\n      \"pmids\": [\"30478191\", \"30456367\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of ZFP36L2-CNOT6L recruitment unresolved\", \"Rules defining which transcripts each adaptor selects incomplete\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linked CNOT6L loss to depletion of inosine-modified transcripts, raising the question of how deadenylation relates to clearance of modified mRNAs.\",\n      \"evidence\": \"computational inosine identification from RNA-seq and polysomal fractionation in Cnot6l-knockout oocytes\",\n      \"pmids\": [\"33530472\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Correlative computational inference, no direct mechanistic link\", \"Cannot distinguish direct from indirect effects on inosine RNAs\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"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.\",\n      \"evidence\": \"Cnot6l knockout under transverse aortic constriction, poly(A) and 3'-UTR luciferase reporter assays, and Cnot6l/tenascin-C double-knockout epistasis\",\n      \"pmids\": [\"40023604\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Adaptor/RBP recognizing the tenascin-C cis-element not identified\", \"Signaling that modulates CNOT6L activity during pressure overload unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified post-translational control of CNOT6L abundance, answering how its protein levels are regulated independently of transcription.\",\n      \"evidence\": \"MS of CCR4-NOT immunoprecipitates, in vitro ubiquitination, RNF219-CNOT1 pull-down, and RNF219 knockdown with proteasome inhibition in HEK293T\",\n      \"pmids\": [\"40598799\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination site(s) on CNOT6L not mapped\", \"Physiological contexts where RNF219 regulates CNOT6L unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"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.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"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\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 1, 3, 6, 8]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [1, 3, 8]}\n    ],\n    \"complexes\": [\"CCR4-NOT\"],\n    \"partners\": [\"CNOT1\", \"RNF219\", \"ZFP36L2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}