RNF166 (RING finger protein 166) is a small (237 aa) RING-type E3 ubiquitin-protein ligase (EC 2.3.2.27) of the C3HC4 RING subfamily that also includes RNF114, RNF125 and RNF138. It is built from an N-terminal RING-type zinc finger (residues ~33-73; catalytic Cys33/Cys36) that recruits a ubiquitin-charged E2 conjugating enzyme (UBE2D2/UBE2D family), a central C2HC RNF-type zinc finger (~98-117) that mediates substrate/target binding, and a C-terminal ubiquitin-interacting motif (UIM). RNF166 catalyzes ubiquitin transfer to several substrates using different, largely non-degradative chain linkages. In antibacterial selective autophagy (xenophagy) it directly catalyzes Lys29- and Lys33-linked polyubiquitination of the autophagy adaptor SQSTM1/p62 (at K91 and K189), and is required for the early recruitment of ubiquitin and the autophagy adaptors p62 and NDP52 (and downstream LC3) to cytosol-invading bacteria such as Listeria monocytogenes and Shigella flexneri, thereby restricting their intracellular replication. In innate antiviral immunity RNF166 enhances RNA virus-induced Lys63-linked ubiquitination of the signaling adaptors TRAF3 and TRAF6, acting downstream of the mitochondrial adaptor MAVS/VISA and upstream of TBK1 to potentiate type I interferon (IFN-beta) production. RNF166 is a cytoplasmic protein that forms cytosolic puncta and relocalizes to intracellular bacteria. Its catalytic RING activity (lost in the C33A/C36A ligase-dead mutant) is required for both the xenophagy and antiviral functions.
Definition: The selective degradation of intracellular pathogen or some part of an intracellular pathogen by macroautophagy.
Justification: RNF166 is directly required for xenophagy of cytosol-adapted bacteria (Listeria, Shigella) - it ubiquitinates p62, recruits autophagy adaptors to bacteria, and restricts their replication - yet only generic catabolic/ubiquitination terms are present in the current GOA. The verified term GO:0098792 (xenophagy) should be annotated.
Parent term: xenophagy
Supporting Evidence:
Definition: Reactions triggered in response to the presence of a bacterium that act to protect the cell or organism.
Justification: RNF166 restricts intracellular replication of Listeria and Shigella through ligase-dependent xenophagy, a cell-autonomous antibacterial defense not captured by current GOA terms. GO:0042742 should be annotated.
Parent term: defense response to bacterium
Supporting Evidence:
Definition: Any process that activates or increases the frequency, rate, or extent of type I interferon production.
Justification: RNF166 potentiates RNA virus-induced IFN-beta production by enhancing K63-linked ubiquitination of TRAF3/TRAF6 downstream of MAVS; this innate antiviral signaling role is supported experimentally but absent from the curated GOA. GO:0032481 should be annotated.
Parent term: positive regulation of type I interferon production
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000209 protein polyubiquitination | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of protein polyubiquitination, the core catalytic process of RNF166, which assembles polyubiquitin chains on its substrates. Reason: Core biological process directly demonstrated experimentally - RNF166 catalyzes K29/K33-linked polyubiquitination of p62 and enhances K63-linked polyubiquitination of TRAF3/TRAF6. Supporting Evidence: PMID:27880896 RNF166 catalyzes K29- and K33-linked polyubiquitination of p62 at residues K91 and K189 PMID:26456228 K63-linked rather than K48-linked ubiquitination of TRAF3 and TRAF6 was decreased upon SeV infection when RNF166 was knocked down |
| GO:0006511 ubiquitin-dependent protein catabolic process | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Phylogenetic (family-level) inference of a degradative ubiquitin-dependent catabolic role. The experimentally established RNF166 ubiquitination events are non-degradative (K29/K33 on p62 promoting xenophagy; K63 on TRAF3/TRAF6 promoting signaling), so a proteasomal/catabolic framing is a family-default that does not match the gene-specific biology. Reason: RNF166's verified substrates are modified with non-degradative chain types serving autophagy and innate-immune signaling, not proteasomal degradation; the catabolic-process term is an over-propagated IBA default. No experimental evidence shows RNF166 targets a substrate for ubiquitin-dependent degradation. Supporting Evidence: PMID:27880896 these data suggest that RNF166 drives K29- and K33-linked ubiquitination of p62 |
| GO:0061630 ubiquitin protein ligase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of ubiquitin protein ligase activity, the core molecular function of RNF166 as a genuine RING-type E3 ligase. Reason: Core molecular function corroborated by direct in vitro and cell-based evidence; the ligase-dead C33A/C36A RING mutant abolishes activity. Supporting Evidence: PMID:27880896 a ligase-dead RNF166 mutant (RNF166 C33A, C36A), was unable to drive p62 ubiquitination under the same conditions |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic transfer of cytoplasmic localization from the UniProt subcellular location; the active compartment of RNF166, corroborated by experimental imaging. Reason: Correct core localization; RNF166 is cytoplasmic, forming cytosolic puncta and relocalizing to intracellular bacteria. Redundant with the EXP cytoplasm annotation. Supporting Evidence: PMID:26456228 Overexpressed RNF166 localized predominantly in the cytosol as dots which were probably aggregates of RNF166 protein |
| GO:0061630 ubiquitin protein ligase activity | IEA GO_REF:0000003 | ACCEPT | Summary: Enzyme Commission-based electronic assignment of ubiquitin protein ligase activity (EC 2.3.2.27), the core catalytic molecular function. Reason: Core molecular function; EC 2.3.2.27 was experimentally established for RNF166. Redundant with the IBA/EXP ligase activity annotations. Supporting Evidence: file:human/RNF166/RNF166-uniprot.txt EC=2.3.2.27 {ECO:0000269|PubMed:27880896} |
| GO:0005515 protein binding | IPI PMID:19549727 Analysis of the human E2 ubiquitin conjugating enzyme protei... | KEEP AS NON CORE | Summary: Interaction from the human E2 ubiquitin-conjugating enzyme interaction network (E2 binding). Bare protein binding is uninformative. Reason: Records a real interaction (consistent with RNF166 binding E2 enzymes such as UBE2D4/UBE2K, central to its RING mechanism) but bare protein binding is uninformative per curation guidelines. Supporting Evidence: file:human/RNF166/RNF166-uniprot.txt Q96A37; Q9Y2X8: UBE2D4; NbExp=4; IntAct=EBI-2130320, EBI-745527 |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | KEEP AS NON CORE | Summary: High-throughput binary interactome interaction. Bare protein binding is uninformative. Reason: High-throughput interactome; bare protein binding is uninformative per curation guidelines. Supporting Evidence: file:human/RNF166/RNF166-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:26456228} |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | KEEP AS NON CORE | Summary: Neurodegeneration interactome interaction (TARDBP). Bare protein binding is uninformative. Reason: Records a real interaction (TARDBP) but bare protein binding is uninformative per curation guidelines. Supporting Evidence: file:human/RNF166/RNF166-uniprot.txt Q96A37; Q13148: TARDBP; NbExp=3; IntAct=EBI-2130320, EBI-372899 |
| GO:0005515 protein binding | IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling... | KEEP AS NON CORE | Summary: Cell-specific interactome interaction. Bare protein binding is uninformative. Reason: High-throughput interactome; bare protein binding is uninformative per curation guidelines. Supporting Evidence: file:human/RNF166/RNF166-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:26456228} |
| GO:0016567 protein ubiquitination | IEA GO_REF:0000041 | KEEP AS NON CORE | Summary: UniPathway-derived general protein ubiquitination process, a parent of the specific polyubiquitination RNF166 catalyzes. Reason: Correct but generic; the specific protein polyubiquitination annotation (GO:0000209) better captures the role. Supporting Evidence: file:human/RNF166/RNF166-uniprot.txt PATHWAY: Protein modification; protein ubiquitination. |
| GO:0005737 cytoplasm | EXP PMID:26456228 Ring finger protein 166 potentiates RNA virus-induced interf... | ACCEPT | Summary: Experimental evidence (immunofluorescence) that RNF166 localizes to the cytoplasm, forming cytosolic dots. The active compartment for its xenophagy and antiviral functions. Reason: Core localization with direct experimental support; RNF166 acts in the cytoplasm where it ubiquitinates p62 on bacteria and TRAF3/TRAF6 in antiviral signaling. Supporting Evidence: PMID:26456228 Overexpressed RNF166 localized predominantly in the cytosol as dots which were probably aggregates of RNF166 protein |
| GO:0061630 ubiquitin protein ligase activity | EXP PMID:27880896 RNF166 Determines Recruitment of Adaptor Proteins during Ant... | ACCEPT | Summary: Experimental evidence that RNF166 is a ubiquitin protein ligase - reconstituted in vitro with E1, UBE2D2 (E2) and ubiquitin to ubiquitinate p62, with activity abolished in the RING ligase-dead mutant. Core molecular function. Reason: Core molecular function directly demonstrated; RNF166 is a genuine catalytic RING-type E3 ligase whose activity requires an intact RING (C33/C36). Supporting Evidence: PMID:27880896 we used an in vitro ubiquitination assay with recombinant UBA1 (E1), E2 enzymes, HA-ubiquitin, GST-RNF166, and SUMO-p62 |
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Download this section (compressed HTML)Q: Does RNF166 act as a stand-alone monomeric RING E3 or does it require partners/cofactors for substrate selection between p62 (xenophagy) and TRAF3/TRAF6 (antiviral signaling)?
Q: What determines the atypical chain-linkage specificity of RNF166 (K29/K33 on p62 versus K63 on TRAF3/TRAF6), and is this dictated by the E2 used or by substrate context?
Experiment: Reconstitute RNF166-mediated ubiquitination in vitro with a panel of E2 enzymes and chain-linkage-specific ubiquitin mutants to define the intrinsic chain specificity for p62 versus TRAF3/TRAF6, comparing wild-type and C33A/C36A RING-dead RNF166.
Experiment: Use RNF166-knockout cells reconstituted with wild-type or ligase-dead RNF166 to test antibacterial (Listeria/Shigella) and antiviral (SeV/EMCV type I IFN) phenotypes in parallel, distinguishing catalytic from scaffolding contributions.
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