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: []