CNOT4

Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0016567 protein ubiquitination	IBA GO_REF:0000033	ACCEPT	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. 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. Supporting Evidence: PMID:11823428 We demonstrate that CNOT4 functions as a ubiquitin-protein ligase (E3).
GO:0004842 ubiquitin-protein transferase activity	IBA GO_REF:0000033	ACCEPT	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. 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. Supporting Evidence: PMID:11823428 CNOT4 acts as a potent E3 ligase in vitro.
GO:0003676 nucleic acid binding	IEA GO_REF:0000002	MARK AS OVER ANNOTATED	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. 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. Supporting Evidence: file:human/CNOT4/CNOT4-uniprot.txt DOMAIN 109..189 /note="RRM"
GO:0003723 RNA binding	IEA GO_REF:0000002	KEEP AS NON CORE	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. 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. Supporting Evidence: file:human/CNOT4/CNOT4-uniprot.txt DOMAIN 109..189 /note="RRM"
GO:0004842 ubiquitin-protein transferase activity	IEA GO_REF:0000002	ACCEPT	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. Reason: CNOT4 is an experimentally confirmed E3 ubiquitin transferase; the InterPro transfer is concordant with direct evidence. Supporting Evidence: PMID:11823428 CNOT4 acts as a potent E3 ligase in vitro.
GO:0005634 nucleus	IEA GO_REF:0000044	KEEP AS NON CORE	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. 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. Supporting Evidence: file:human/CNOT4/CNOT4-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000305}. Nucleus {ECO:0000305}.
GO:0005737 cytoplasm	IEA GO_REF:0000044	ACCEPT	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. Reason: Cytoplasmic localization is well supported and consistent with CNOT4's mRNA-turnover and co-translational quality-control functions. Supporting Evidence: file:human/CNOT4/CNOT4-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000305}. Nucleus {ECO:0000305}.
GO:0005829 cytosol	IEA GO_REF:0000117	ACCEPT	Summary: ARBA machine-learning electronic annotation of cytosol, consistent with the cytoplasmic localization and with Reactome TAS cytosol annotations. Reason: Cytosolic localization is consistent with the experimentally supported cytoplasmic localization and CCR4-NOT-associated function. Supporting Evidence: file:human/CNOT4/CNOT4-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000305}.
GO:0030014 CCR4-NOT complex	IEA GO_REF:0000002	ACCEPT	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. Reason: CCR4-NOT complex membership is real and corroborated by NAS and IDA evidence, though the association is peripheral/transient in human cells. Supporting Evidence: file:human/CNOT4/CNOT4-uniprot.txt The RING finger protein CNOT4 is a component of the CCR4-NOT complex.
GO:0046872 metal ion binding	IEA GO_REF:0000002	MARK AS OVER ANNOTATED	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). 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 replacements: zinc ion binding Supporting Evidence: file:human/CNOT4/CNOT4-uniprot.txt ZN_FING 14..57 /note="RING-type; degenerate"
GO:0061630 ubiquitin protein ligase activity	IEA GO_REF:0000003	ACCEPT	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. Reason: Ubiquitin protein ligase activity is the experimentally confirmed core molecular function of CNOT4. Supporting Evidence: PMID:11823428 We demonstrate that CNOT4 functions as a ubiquitin-protein ligase (E3).
GO:0005515 protein binding	IPI O95628-2 PMID:32296183 A reference map of the human binary protein interactome.	MARK AS OVER ANNOTATED	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. Reason: Bare 'protein binding' from a high-throughput binary screen is uninformative about CNOT4 molecular function; the IL36RN interaction has no supporting biological context. Supporting Evidence: PMID:32296183 A reference map of the human binary protein interactome.
GO:0016567 protein ubiquitination	IEA GO_REF:0000041	ACCEPT	Summary: UniPathway-based electronic annotation of protein ubiquitination, redundant with the IBA annotation of the same term and supported by direct experimental evidence. Reason: Protein ubiquitination is a core, experimentally validated biological process for CNOT4. Supporting Evidence: PMID:29861391 NOT4 was able to directly ubiquitinate ABCE1 in vitro
GO:0000289 nuclear-transcribed mRNA poly(A) tail shortening	NAS PMID:31320642 Reconstitution of recombinant human CCR4-NOT reveals molecul...	KEEP AS NON CORE	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. 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. Supporting Evidence: PMID:31320642 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
GO:0030014 CCR4-NOT complex	NAS PMID:19558367 Human Ccr4-Not complexes contain variable deadenylase subuni...	ACCEPT	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. Reason: CCR4-NOT membership is supported, although in human cells CNOT4 is only loosely associated; part_of membership is appropriate to retain. Supporting Evidence: PMID:19558367 human CNOT4 is in a separate approximately 200 kDa complex
GO:0061630 ubiquitin protein ligase activity	EXP PMID:11823428 Identification of a ubiquitin-protein ligase subunit within ...	ACCEPT	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. Reason: Strong direct experimental evidence for the defining molecular function of CNOT4 as a RING E3 ubiquitin ligase. Supporting Evidence: PMID:11823428 CNOT4 acts as a potent E3 ligase in vitro. Mutations that destabilize the E2-E3 interface abolish this activity.
GO:0061630 ubiquitin protein ligase activity	EXP PMID:26575292 Cross-talk between PRMT1-mediated methylation and ubiquityla...	ACCEPT	Summary: Experimental demonstration that CNOT4 acts as the E3 ligase ubiquitinating methylated RBM15, confirming its ubiquitin protein ligase activity on a physiological substrate. Reason: Direct experimental support for CNOT4 ubiquitin ligase activity acting on RBM15. Supporting Evidence: PMID:26575292 Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.
GO:0061630 ubiquitin protein ligase activity	EXP PMID:29861391 Ubiquitination of ABCE1 by NOT4 in Response to Mitochondrial...	ACCEPT	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. Reason: Direct experimental support for CNOT4 ubiquitin ligase activity on ABCE1. Supporting Evidence: PMID:29861391 NOT4 was able to directly ubiquitinate ABCE1 in vitro
GO:0004842 ubiquitin-protein transferase activity	IDA PMID:26575292 Cross-talk between PRMT1-mediated methylation and ubiquityla...	ACCEPT	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. Reason: Direct experimental support for CNOT4 ubiquitin transferase activity. Supporting Evidence: PMID:26575292 Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.
GO:0005515 protein binding	IPI PMID:26575292 Cross-talk between PRMT1-mediated methylation and ubiquityla...	MARK AS OVER ANNOTATED	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. Reason: Bare 'protein binding' is uninformative; the underlying CNOT4-RBM15 substrate relationship is already captured by the ubiquitination/ubiquitin-dependent catabolism annotations. Supporting Evidence: PMID:26575292 Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.
GO:0006511 ubiquitin-dependent protein catabolic process	IDA PMID:26575292 Cross-talk between PRMT1-mediated methylation and ubiquityla...	KEEP AS NON CORE	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. Reason: CNOT4 mediates ubiquitin-dependent turnover of substrates such as RBM15, a real but substrate-specific biological process rather than the single core function. Supporting Evidence: PMID:26575292 Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.
GO:0045652 regulation of megakaryocyte differentiation	IDA PMID:26575292 Cross-talk between PRMT1-mediated methylation and ubiquityla...	KEEP AS NON CORE	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. Reason: Regulation of megakaryocyte differentiation is a specific downstream developmental role mediated via RBM15 ubiquitination, not a core/general function of CNOT4. Supporting Evidence: PMID:26575292 Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.
GO:0003723 RNA binding	HDA PMID:22681889 The mRNA-bound proteome and its global occupancy profile on ...	KEEP AS NON CORE	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. Reason: RNA binding is supported by orthogonal HDA evidence and an RRM domain but is ancillary to CNOT4's characterized E3 ligase function. Supporting Evidence: PMID:22681889 The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.
GO:0005829 cytosol	TAS Reactome:R-HSA-429955	ACCEPT	Summary: Reactome TAS cytosol annotation associated with the CCR4-NOT deadenylation reaction. Consistent with the cytoplasmic localization of CNOT4 and CCR4-NOT. Reason: Cytosolic localization is consistent with CNOT4's cytoplasmic CCR4-NOT-associated function. Supporting Evidence: file:human/CNOT4/CNOT4-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000305}.
GO:0005829 cytosol	TAS Reactome:R-HSA-6798044	ACCEPT	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. Reason: Cytosolic localization is consistent with CNOT4's cytoplasmic CCR4-NOT-associated function. Supporting Evidence: file:human/CNOT4/CNOT4-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000305}.
GO:0030014 CCR4-NOT complex	IDA PMID:19558367 Human Ccr4-Not complexes contain variable deadenylase subuni...	ACCEPT	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. Reason: The colocalizes_with qualifier accurately captures CNOT4's peripheral association with the CCR4-NOT complex in human cells. Supporting Evidence: PMID:19558367 human CNOT4 is in a separate approximately 200 kDa complex
GO:0004842 ubiquitin-protein transferase activity	IDA PMID:15001359 An altered-specificity ubiquitin-conjugating enzyme/ubiquiti...	ACCEPT	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. Reason: Direct experimental support for CNOT4 ubiquitin transferase activity with its cognate E2. Supporting Evidence: PMID:15001359 binding of the CNOT4 RING finger to the ubiquitin-conjugating enzyme (E2) UbcH5B is highly selective
GO:0051865 protein autoubiquitination	IDA PMID:15001359 An altered-specificity ubiquitin-conjugating enzyme/ubiquiti...	ACCEPT	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. Reason: Autoubiquitination is directly demonstrated and is a characteristic readout of CNOT4's intrinsic E3 activity. Supporting Evidence: file:human/CNOT4/CNOT4-uniprot.txt PTM: Autoubiquitinated. {ECO:0000269\|PubMed:11823428, ECO:0000269\|PubMed:15001359}.
GO:0005515 protein binding	IPI PMID:15001359 An altered-specificity ubiquitin-conjugating enzyme/ubiquiti...	MODIFY	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'. 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 replacements: ubiquitin conjugating enzyme binding Supporting Evidence: PMID:15001359 binding of the CNOT4 RING finger to the ubiquitin-conjugating enzyme (E2) UbcH5B is highly selective

Gene Description

Existing Annotations Review

Core Functions

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.

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.

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.

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.

References

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Notes

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