id: O95628 gene_symbol: CNOT4 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:9606 label: Homo sapiens description: >- CNOT4 (CCR4-NOT transcription complex subunit 4, the human ortholog of yeast Not4) is a RING-type E3 ubiquitin-protein ligase. Its N-terminal atypical C4C4 RING domain pairs selectively with E2 ubiquitin-conjugating enzymes of the UBE2D/UbcH5 (UBC4/5) subfamily, most prominently UBE2D2 (UbcH5B), and catalyzes the transfer of ubiquitin from the E2 onto substrate lysines, building polyubiquitin chains; the enzyme is also autoubiquitinated. Beyond the RING, CNOT4 contains an RRM and a C3H1-type zinc finger and a large disordered C-terminus. CNOT4 is a peripheral, substoichiometric subunit of the CCR4-NOT complex, the major eukaryotic cytoplasmic mRNA deadenylase and a global regulator of mRNA turnover and translation; in human and Drosophila cells CNOT4 is only loosely/transiently associated with the holocomplex (it binds the CNOT1 scaffold and the CAF40/CNOT9 subunit via a CAF40-binding motif) rather than being a stable core subunit. Its ubiquitin-ligase activity is linked to co-translational and RNA quality control - it ubiquitinates ABCE1 (K48-linked) upon mitochondrial damage to trigger PINK1-directed mitophagy, and ubiquitinates methylated RBM15 to control RNA splicing and megakaryocyte differentiation. CNOT4 localizes to the cytoplasm (including P-bodies and stress granules) and nucleus. alternative_products: - name: '1' id: O95628-1 - name: '2' id: O95628-2 sequence_note: VSP_009924 - name: '3' id: O95628-3 sequence_note: VSP_009923 - name: '4' id: O95628-4 sequence_note: VSP_009928 - name: '5' id: O95628-5 sequence_note: VSP_009925, VSP_009926 - name: '6' id: O95628-6 sequence_note: VSP_009927 - name: '7' id: O95628-7 sequence_note: VSP_009929 - name: '8' id: O95628-8 sequence_note: VSP_009924, VSP_009928 - name: '9' id: O95628-9 sequence_note: VSP_009924, VSP_045469 - name: '10' id: O95628-10 sequence_note: VSP_045469 existing_annotations: - term: id: GO:0016567 label: protein ubiquitination evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in review: summary: Phylogenetic (PAN-GO) transfer asserting CNOT4 is involved in protein ubiquitination. This is well supported by direct experimental evidence in human CNOT4 (autoubiquitination and substrate ubiquitination of ABCE1 and RBM15) and is a core biological process for this RING E3 ligase. action: ACCEPT reason: Protein ubiquitination is a core, experimentally validated function of CNOT4 (see PMID:11823428, PMID:26575292, PMID:29861391). The IBA transfer is consistent with the directly supported annotations. supported_by: - reference_id: PMID:11823428 supporting_text: We demonstrate that CNOT4 functions as a ubiquitin-protein ligase (E3). - term: id: GO:0004842 label: ubiquitin-protein transferase activity evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables review: summary: Phylogenetic (PAN-GO) transfer of ubiquitin-protein transferase activity, the general parent of RING-type ubiquitin ligase activity. Well supported by direct biochemical assays of human CNOT4 E3 activity. action: ACCEPT reason: CNOT4 is an experimentally confirmed E2-dependent RING E3 ligase that catalyzes ubiquitin transfer; the more specific term GO:0061630 (ubiquitin protein ligase activity) is also annotated. supported_by: - reference_id: PMID:11823428 supporting_text: CNOT4 acts as a potent E3 ligase in vitro. - term: id: GO:0003676 label: nucleic acid binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: InterPro-based electronic annotation of generic nucleic acid binding, derived from the RRM domain. This is overly broad and uninformative; CNOT4 has a more specific RNA-binding annotation (GO:0003723) and there is no evidence it binds DNA. action: MARK_AS_OVER_ANNOTATED reason: Generic nucleic acid binding is the uninformative parent of RNA binding; CNOT4 has an RRM and is captured as an mRNA-bound protein, so RNA binding (GO:0003723) is the appropriate specific term. DNA binding is not supported. supported_by: - reference_id: file:human/CNOT4/CNOT4-uniprot.txt supporting_text: DOMAIN 109..189 /note="RRM" - term: id: GO:0003723 label: RNA binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: InterPro RRM-based electronic annotation of RNA binding. CNOT4 contains an RRM domain and was experimentally captured in mRNA interactome studies (HDA, PMID:22681889), so RNA binding is plausible, though it is not a deeply characterized core function and is likely ancillary to the RING E3 activity. action: KEEP_AS_NON_CORE reason: RNA binding is supported by an RRM domain and orthogonal HDA evidence (PMID:22681889), but is not the characterized core molecular function of CNOT4. supported_by: - reference_id: file:human/CNOT4/CNOT4-uniprot.txt supporting_text: DOMAIN 109..189 /note="RRM" - term: id: GO:0004842 label: ubiquitin-protein transferase activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: InterPro-based electronic annotation of ubiquitin-protein transferase activity, redundant with the IBA and IDA/EXP annotations of the same/more specific activity. Accepted as supported by experimental evidence. action: ACCEPT reason: CNOT4 is an experimentally confirmed E3 ubiquitin transferase; the InterPro transfer is concordant with direct evidence. supported_by: - reference_id: PMID:11823428 supporting_text: CNOT4 acts as a potent E3 ligase in vitro. - term: id: GO:0005634 label: nucleus evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in review: summary: UniProt subcellular-location keyword mapping placing CNOT4 in the nucleus. UniProt reports both cytoplasmic and nuclear localization (ECO:0000305). Nuclear localization is plausible given reported transcription-related and RBM15-splicing roles, but is not strongly experimentally documented for the human protein. action: KEEP_AS_NON_CORE reason: Nuclear localization is asserted by UniProt but with weak (ECO:0000305) support; the predominant characterized site of action (E3 ligase, CCR4-NOT, P-bodies/stress granules) is cytoplasmic. supported_by: - reference_id: file:human/CNOT4/CNOT4-uniprot.txt supporting_text: 'SUBCELLULAR LOCATION: Cytoplasm {ECO:0000305}. Nucleus {ECO:0000305}.' - term: id: GO:0005737 label: cytoplasm evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in review: summary: UniProt subcellular-location keyword mapping placing CNOT4 in the cytoplasm. Consistent with its role as a cytoplasmic CCR4-NOT-associated mRNA-turnover factor and its localization to P-bodies and stress granules. action: ACCEPT reason: Cytoplasmic localization is well supported and consistent with CNOT4's mRNA-turnover and co-translational quality-control functions. supported_by: - reference_id: file:human/CNOT4/CNOT4-uniprot.txt supporting_text: 'SUBCELLULAR LOCATION: Cytoplasm {ECO:0000305}. Nucleus {ECO:0000305}.' - term: id: GO:0005829 label: cytosol evidence_type: IEA original_reference_id: GO_REF:0000117 qualifier: located_in review: summary: ARBA machine-learning electronic annotation of cytosol, consistent with the cytoplasmic localization and with Reactome TAS cytosol annotations. action: ACCEPT reason: Cytosolic localization is consistent with the experimentally supported cytoplasmic localization and CCR4-NOT-associated function. supported_by: - reference_id: file:human/CNOT4/CNOT4-uniprot.txt supporting_text: 'SUBCELLULAR LOCATION: Cytoplasm {ECO:0000305}.' - term: id: GO:0030014 label: CCR4-NOT complex evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: part_of review: summary: InterPro-based electronic annotation of CCR4-NOT complex membership. CNOT4 is the human ortholog of yeast Not4, a CCR4-NOT subunit, and associates with the complex via CNOT1 and CAF40/CNOT9, though in human cells it is only loosely/substoichiometrically associated rather than a stable core subunit. action: ACCEPT reason: CCR4-NOT complex membership is real and corroborated by NAS and IDA evidence, though the association is peripheral/transient in human cells. supported_by: - reference_id: file:human/CNOT4/CNOT4-uniprot.txt supporting_text: The RING finger protein CNOT4 is a component of the CCR4-NOT complex. - term: id: GO:0046872 label: metal ion binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: InterPro-based electronic annotation of metal ion binding (parent of zinc ion binding), derived from the RING and C3H1 zinc-finger domains. CNOT4's C4C4 RING coordinates two zinc atoms (NMR structure). The more specific term is zinc ion binding (GO:0008270). action: MARK_AS_OVER_ANNOTATED reason: Metal ion binding is the uninformative parent of zinc ion binding; CNOT4 specifically coordinates zinc via its RING/zinc-finger domains, so GO:0008270 would be more appropriate. proposed_replacement_terms: - id: GO:0008270 label: zinc ion binding supported_by: - reference_id: file:human/CNOT4/CNOT4-uniprot.txt supporting_text: ZN_FING 14..57 /note="RING-type; degenerate" - term: id: GO:0061630 label: ubiquitin protein ligase activity evidence_type: IEA original_reference_id: GO_REF:0000003 qualifier: enables review: summary: EC-mapping (EC 2.3.2.27) electronic annotation of ubiquitin protein ligase activity. This is the core molecular function of CNOT4 and is independently supported by multiple EXP/IDA annotations. action: ACCEPT reason: Ubiquitin protein ligase activity is the experimentally confirmed core molecular function of CNOT4. supported_by: - reference_id: PMID:11823428 supporting_text: We demonstrate that CNOT4 functions as a ubiquitin-protein ligase (E3). - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:32296183 qualifier: enables isoform: O95628-2 review: summary: High-throughput binary yeast two-hybrid interaction (HuRI reference interactome) between CNOT4 isoform O95628-2 and IL36RN (Q9UBH0). This is a generic 'protein binding' annotation with no functional/biological context and no demonstrated biological relevance. action: MARK_AS_OVER_ANNOTATED reason: Bare 'protein binding' from a high-throughput binary screen is uninformative about CNOT4 molecular function; the IL36RN interaction has no supporting biological context. supported_by: - reference_id: PMID:32296183 supporting_text: A reference map of the human binary protein interactome. - term: id: GO:0016567 label: protein ubiquitination evidence_type: IEA original_reference_id: GO_REF:0000041 qualifier: involved_in review: summary: UniPathway-based electronic annotation of protein ubiquitination, redundant with the IBA annotation of the same term and supported by direct experimental evidence. action: ACCEPT reason: Protein ubiquitination is a core, experimentally validated biological process for CNOT4. supported_by: - reference_id: PMID:29861391 supporting_text: NOT4 was able to directly ubiquitinate ABCE1 in vitro - term: id: GO:0000289 label: nuclear-transcribed mRNA poly(A) tail shortening evidence_type: NAS original_reference_id: PMID:31320642 qualifier: involved_in review: summary: ComplexPortal NAS annotation derived from CCR4-NOT complex membership. Poly(A) tail shortening (deadenylation) is the catalytic activity of the CCR4-NOT deadenylase subunits (CNOT6/6L and CNOT7/8), not of CNOT4. The reconstituted recombinant human CCR4-NOT in PMID:31320642 did not even include CNOT4, and CNOT4's C-terminal CAF40-binding motif inhibits deadenylation in vitro. This is a complex-level annotation that does not reflect a CNOT4 molecular activity. action: KEEP_AS_NON_CORE reason: CNOT4 is a peripheral CCR4-NOT subunit and participates in mRNA turnover at the complex level, but deadenylation/poly(A) shortening is catalyzed by other subunits; this is a membership-derived process annotation, not a CNOT4 catalytic function. supported_by: - reference_id: PMID:31320642 supporting_text: the NOT4 subunit, which functions as an E2-dependent RING E3 ligase. It is stably incorporated within the yeast Ccr4-Not but not in Drosophila S2 and human cells - term: id: GO:0030014 label: CCR4-NOT complex evidence_type: NAS original_reference_id: PMID:19558367 qualifier: part_of review: summary: ComplexPortal NAS annotation of CCR4-NOT complex membership. PMID:19558367 shows human CNOT4 is in a separate ~200 kDa complex distinct from the stable CCR4-NOT core, indicating peripheral/substoichiometric association rather than stable core membership. action: ACCEPT reason: CCR4-NOT membership is supported, although in human cells CNOT4 is only loosely associated; part_of membership is appropriate to retain. supported_by: - reference_id: PMID:19558367 supporting_text: human CNOT4 is in a separate approximately 200 kDa complex - term: id: GO:0061630 label: ubiquitin protein ligase activity evidence_type: EXP original_reference_id: PMID:11823428 qualifier: enables review: summary: Direct biochemical demonstration that CNOT4 is an E2-dependent RING E3 ubiquitin-protein ligase whose C4C4 RING binds UbcH5-family E2s and catalyzes polyubiquitin chain assembly in vitro. This is the core molecular function of CNOT4. action: ACCEPT reason: Strong direct experimental evidence for the defining molecular function of CNOT4 as a RING E3 ubiquitin ligase. supported_by: - reference_id: PMID:11823428 supporting_text: CNOT4 acts as a potent E3 ligase in vitro. Mutations that destabilize the E2-E3 interface abolish this activity. - term: id: GO:0061630 label: ubiquitin protein ligase activity evidence_type: EXP original_reference_id: PMID:26575292 qualifier: enables review: summary: Experimental demonstration that CNOT4 acts as the E3 ligase ubiquitinating methylated RBM15, confirming its ubiquitin protein ligase activity on a physiological substrate. action: ACCEPT reason: Direct experimental support for CNOT4 ubiquitin ligase activity acting on RBM15. supported_by: - reference_id: PMID:26575292 supporting_text: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing. - term: id: GO:0061630 label: ubiquitin protein ligase activity evidence_type: EXP original_reference_id: PMID:29861391 qualifier: enables review: summary: Experimental demonstration that NOT4 (CNOT4) directly ubiquitinates ABCE1 in vitro (primarily K48-linked), confirming its E3 ubiquitin ligase activity in a co-translational quality-control context. action: ACCEPT reason: Direct experimental support for CNOT4 ubiquitin ligase activity on ABCE1. supported_by: - reference_id: PMID:29861391 supporting_text: NOT4 was able to directly ubiquitinate ABCE1 in vitro - term: id: GO:0004842 label: ubiquitin-protein transferase activity evidence_type: IDA original_reference_id: PMID:26575292 qualifier: enables review: summary: Direct assay of CNOT4 ubiquitin-protein transferase activity in the context of RBM15 ubiquitination. Redundant with the more specific GO:0061630 annotation from the same study; supports the core E3 activity. action: ACCEPT reason: Direct experimental support for CNOT4 ubiquitin transferase activity. supported_by: - reference_id: PMID:26575292 supporting_text: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing. - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:26575292 qualifier: enables review: summary: IPI 'protein binding' annotation recording the interaction of CNOT4 with its substrate RBM15 (Q96T37). While the interaction is biologically meaningful (RBM15 is a CNOT4 ubiquitination substrate), bare 'protein binding' is uninformative; the substrate relationship is better captured by the RBM15 ubiquitination annotations. action: MARK_AS_OVER_ANNOTATED reason: Bare 'protein binding' is uninformative; the underlying CNOT4-RBM15 substrate relationship is already captured by the ubiquitination/ubiquitin-dependent catabolism annotations. supported_by: - reference_id: PMID:26575292 supporting_text: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing. - term: id: GO:0006511 label: ubiquitin-dependent protein catabolic process evidence_type: IDA original_reference_id: PMID:26575292 qualifier: involved_in review: summary: CNOT4-mediated ubiquitination of methylated RBM15 controls RBM15 turnover, i.e. targets it for ubiquitin-dependent degradation. This is a supported biological process downstream of CNOT4's E3 activity, though a substrate-specific rather than core annotation. action: KEEP_AS_NON_CORE reason: CNOT4 mediates ubiquitin-dependent turnover of substrates such as RBM15, a real but substrate-specific biological process rather than the single core function. supported_by: - reference_id: PMID:26575292 supporting_text: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing. - term: id: GO:0045652 label: regulation of megakaryocyte differentiation evidence_type: IDA original_reference_id: PMID:26575292 qualifier: involved_in review: summary: CNOT4-mediated ubiquitination of RBM15 regulates RNA splicing and megakaryocyte differentiation. This is a downstream, cell-type-specific physiological consequence of CNOT4's E3 activity rather than a core molecular function. action: KEEP_AS_NON_CORE reason: Regulation of megakaryocyte differentiation is a specific downstream developmental role mediated via RBM15 ubiquitination, not a core/general function of CNOT4. supported_by: - reference_id: PMID:26575292 supporting_text: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing. - term: id: GO:0003723 label: RNA binding evidence_type: HDA original_reference_id: PMID:22681889 qualifier: enables review: summary: High-throughput mRNA interactome capture identified CNOT4 as an mRNA-bound protein in HeLa cells. Consistent with its RRM domain, supporting plausible direct RNA binding, though not a deeply characterized core function. action: KEEP_AS_NON_CORE reason: RNA binding is supported by orthogonal HDA evidence and an RRM domain but is ancillary to CNOT4's characterized E3 ligase function. supported_by: - reference_id: PMID:22681889 supporting_text: The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. - term: id: GO:0005829 label: cytosol evidence_type: TAS original_reference_id: Reactome:R-HSA-429955 qualifier: located_in review: summary: Reactome TAS cytosol annotation associated with the CCR4-NOT deadenylation reaction. Consistent with the cytoplasmic localization of CNOT4 and CCR4-NOT. action: ACCEPT reason: Cytosolic localization is consistent with CNOT4's cytoplasmic CCR4-NOT-associated function. supported_by: - reference_id: file:human/CNOT4/CNOT4-uniprot.txt supporting_text: 'SUBCELLULAR LOCATION: Cytoplasm {ECO:0000305}.' - term: id: GO:0005829 label: cytosol evidence_type: TAS original_reference_id: Reactome:R-HSA-6798044 qualifier: located_in review: summary: Reactome TAS cytosol annotation associated with BTG2 binding to the CCR4-NOT complex. Redundant with the other cytosol annotations and consistent with CNOT4's cytoplasmic localization. action: ACCEPT reason: Cytosolic localization is consistent with CNOT4's cytoplasmic CCR4-NOT-associated function. supported_by: - reference_id: file:human/CNOT4/CNOT4-uniprot.txt supporting_text: 'SUBCELLULAR LOCATION: Cytoplasm {ECO:0000305}.' - term: id: GO:0030014 label: CCR4-NOT complex evidence_type: IDA original_reference_id: PMID:19558367 qualifier: colocalizes_with review: summary: IDA colocalizes_with annotation reflecting that human CNOT4 co-purifies/colocalizes with CCR4-NOT components but is found in a separate ~200 kDa complex. The colocalizes_with qualifier appropriately reflects the loose/peripheral association rather than stable core membership. action: ACCEPT reason: The colocalizes_with qualifier accurately captures CNOT4's peripheral association with the CCR4-NOT complex in human cells. supported_by: - reference_id: PMID:19558367 supporting_text: human CNOT4 is in a separate approximately 200 kDa complex - term: id: GO:0004842 label: ubiquitin-protein transferase activity evidence_type: IDA original_reference_id: PMID:15001359 qualifier: enables review: summary: Direct assay of CNOT4 ubiquitin-protein transferase activity using the CNOT4 RING-UbcH5B (UBE2D2) E2-E3 pair. Supports the core E3 ligase function; redundant with GO:0061630. action: ACCEPT reason: Direct experimental support for CNOT4 ubiquitin transferase activity with its cognate E2. supported_by: - reference_id: PMID:15001359 supporting_text: binding of the CNOT4 RING finger to the ubiquitin-conjugating enzyme (E2) UbcH5B is highly selective - term: id: GO:0051865 label: protein autoubiquitination evidence_type: IDA original_reference_id: PMID:15001359 qualifier: involved_in review: summary: CNOT4 is autoubiquitinated in vitro in a UbcH5B-dependent manner, a hallmark assay of active RING E3 ligases. Supported and consistent with the UniProt PTM annotation. action: ACCEPT reason: Autoubiquitination is directly demonstrated and is a characteristic readout of CNOT4's intrinsic E3 activity. supported_by: - reference_id: file:human/CNOT4/CNOT4-uniprot.txt supporting_text: 'PTM: Autoubiquitinated. {ECO:0000269|PubMed:11823428, ECO:0000269|PubMed:15001359}.' - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:15001359 qualifier: enables review: summary: IPI 'protein binding' recording the highly selective interaction of the CNOT4 RING finger with the E2 ubiquitin-conjugating enzyme UbcH5B/UBE2D2 (P62837). This is a specific, functionally informative interaction (E2 binding by an E3 RING) and should be captured by the specific term ubiquitin conjugating enzyme binding rather than bare 'protein binding'. action: MODIFY reason: The interaction partner (P62837/UBE2D2) is an E2 ubiquitin-conjugating enzyme bound via the CNOT4 RING domain; the specific term GO:0031624 (ubiquitin conjugating enzyme binding) is far more informative than generic protein binding. proposed_replacement_terms: - id: GO:0031624 label: ubiquitin conjugating enzyme binding supported_by: - reference_id: PMID:15001359 supporting_text: binding of the CNOT4 RING finger to the ubiquitin-conjugating enzyme (E2) UbcH5B is highly selective core_functions: - description: RING-type (C4C4) E3 ubiquitin-protein ligase that selectively binds UBE2D/UbcH5-family E2 ubiquitin-conjugating enzymes via its RING domain and catalyzes transfer of ubiquitin onto substrate lysines to assemble polyubiquitin chains. supported_by: - reference_id: PMID:11823428 supporting_text: CNOT4 acts as a potent E3 ligase in vitro. Mutations that destabilize the E2-E3 interface abolish this activity. molecular_function: id: GO:0061630 label: ubiquitin protein ligase activity directly_involved_in: - id: GO:0016567 label: protein ubiquitination in_complex: id: GO:0030014 label: CCR4-NOT complex - description: Selective binding of UBE2D2 (UbcH5B) and related UBC4/5-subfamily E2 ubiquitin-conjugating enzymes through the CNOT4 C4C4 RING domain, the molecular recognition step that enables CNOT4 E3 ligase activity. supported_by: - reference_id: PMID:15001359 supporting_text: binding of the CNOT4 RING finger to the ubiquitin-conjugating enzyme (E2) UbcH5B is highly selective molecular_function: id: GO:0031624 label: ubiquitin conjugating enzyme binding - description: Ubiquitination of substrates in co-translational and RNA quality-control pathways, including K48-linked ubiquitination of ABCE1 upon mitochondrial damage to trigger PINK1-directed mitophagy, and ubiquitination of methylated RBM15 to control RNA splicing and megakaryocyte differentiation. supported_by: - reference_id: PMID:29861391 supporting_text: NOT4 was able to directly ubiquitinate ABCE1 in vitro - reference_id: PMID:26575292 supporting_text: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing. molecular_function: id: GO:0061630 label: ubiquitin protein ligase activity directly_involved_in: - id: GO:0006511 label: ubiquitin-dependent protein catabolic process - description: Peripheral/substoichiometric subunit of the CCR4-NOT complex, the major eukaryotic cytoplasmic mRNA deadenylase and a global regulator of mRNA turnover and translation; CNOT4 associates loosely with the holocomplex via CNOT1 and the CAF40/CNOT9 subunit. supported_by: - reference_id: PMID:19558367 supporting_text: human CNOT4 is in a separate approximately 200 kDa complex in_complex: id: GO:0030014 label: CCR4-NOT complex proposed_new_terms: [] suggested_questions: - question: In human cells, what fraction of cellular CNOT4 is associated with the CCR4-NOT holocomplex versus free or in distinct assemblies, and is its E3 ligase activity regulated by this association? - question: Does human CNOT4 act as a ribosome-associated E3 ligase that ubiquitinates nascent chains or ribosomal proteins during co-translational quality control, as shown for yeast/Drosophila Not4, and what are the direct substrates? - question: What is the physiological substrate repertoire and ubiquitin chain-type specificity (K48 vs K63 etc.) of human CNOT4, and how is substrate selection achieved given the absence of a dedicated substrate-recognition module beyond the RRM/zinc fingers? suggested_experiments: - description: Define the in vivo CNOT4 substrate landscape using proximity-labeling (BioID/TurboID) combined with ubiquitin-remnant (diGly) proteomics in cells expressing WT versus RING-dead (e.g. E49 mutant) CNOT4. - description: Reconstitute CNOT4 E3 activity in vitro with UBE2D-family E2s and candidate substrates (ABCE1, RBM15, ribosomal proteins) to determine ubiquitin linkage specificity and the contribution of the RRM and C3H1 zinc finger to substrate selection. - description: Use selective ribosome profiling and ubiquitination assays on stalled ribosomes to test whether human CNOT4 ubiquitinates nascent chains/ribosomal proteins during ribosome-associated quality control. - description: Quantify CNOT4 association with the CCR4-NOT holocomplex by quantitative mass spectrometry and size-exclusion/IP across conditions to determine stoichiometry and whether stress or mitochondrial damage modulates its recruitment. references: - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: [] - id: GO_REF:0000003 title: Gene Ontology annotation based on Enzyme Commission mapping findings: [] - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees findings: [] - id: GO_REF:0000041 title: Gene Ontology annotation based on UniPathway vocabulary mapping findings: [] - id: GO_REF:0000044 title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt findings: [] - id: GO_REF:0000117 title: Electronic Gene Ontology annotations created by ARBA machine learning models findings: [] - id: PMID:11823428 title: Identification of a ubiquitin-protein ligase subunit within the CCR4-NOT transcription repressor complex. findings: [] - id: PMID:15001359 title: An altered-specificity ubiquitin-conjugating enzyme/ubiquitin-protein ligase pair. findings: [] - id: PMID:19558367 title: Human Ccr4-Not complexes contain variable deadenylase subunits. findings: [] - id: PMID:22681889 title: The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. findings: [] - id: PMID:26575292 title: Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing. findings: [] - id: PMID:29861391 title: Ubiquitination of ABCE1 by NOT4 in Response to Mitochondrial Damage Links Co-translational Quality Control to PINK1-Directed Mitophagy. findings: [] - id: PMID:31320642 title: Reconstitution of recombinant human CCR4-NOT reveals molecular insights into regulated deadenylation. findings: [] - id: PMID:32296183 title: A reference map of the human binary protein interactome. findings: [] - id: Reactome:R-HSA-429955 title: CCR4-NOT complex deadenylates mRNA findings: [] - id: Reactome:R-HSA-6798044 title: BTG2 binds CCR4-NOT complex findings: []