RDE-2 (also known as MUT-8) is an intrinsically disordered bridging adaptor protein of the C. elegans Mutator complex, the germline machinery that amplifies secondary small interfering RNAs (22G-RNAs) for RNA-mediated silencing. RDE-2 has no catalytic activity and no canonical folded domain; its structured C-terminal domain binds the C-terminal domain of the 3'-5' exoribonuclease MUT-7, while its partially structured N-terminal region contacts the intrinsically disordered scaffold MUT-16. By physically linking MUT-7 to MUT-16, RDE-2 recruits the MUT-7 exoribonuclease into perinuclear Mutator foci, the phase-separated condensates (adjacent to but distinct from P granules) in which the RNA-dependent RNA polymerase RRF-1 synthesizes secondary siRNAs. This function is required for exogenous and endogenous RNAi, transposon silencing, and germline genome surveillance; loss of rde-2 abolishes secondary siRNA accumulation and causes RNAi resistance, transposon mobilization and an X-chromosome non-disjunction (high-incidence-of-males) phenotype. RDE-2 and its MUT-7 interaction are restricted to the Caenorhabditis lineage.
Definition: The RNA-dependent RNA polymerase (RdRP)-mediated de novo synthesis of secondary small interfering RNAs (e.g. C. elegans 22G-RNAs) templated on target mRNAs recognized by primary small RNAs, occurring within a specialized RNA-processing compartment (Mutator focus). Distinguished from siRNA processing/Dicer-dependent primary siRNA generation.
Justification: RDE-2 and the Mutator complex act specifically in RdRP-dependent secondary siRNA amplification, a step for which no dedicated GO term exists; siRNA processing (GO:0030422) conflates Dicer-dependent primary siRNA generation with amplification.
Definition: A molecular function in which a protein provides a structural/organizational role as a subunit of the C. elegans Mutator complex, contributing to assembly of the siRNA-amplification compartment rather than catalysis.
Justification: RDE-2 is an obligate structural adaptor subunit with no catalytic activity; molecular adaptor activity (GO:0060090) captures the bridging aspect, but a complex-specific structural-constituent term would more precisely express its 'be part of the machine' role.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:15653635 RDE-2 interacts with MUT-7 to mediate RNA interference in Ca... | MODIFY | Summary: The IPI interaction with MUT-7 (P34607) is real and central, but 'protein binding' is uninformative. RDE-2's molecular function is to bridge the MUT-7 exoribonuclease to the MUT-16 scaffold, i.e. a molecular adaptor activity; this is the core molecular function of the protein. Reason: Replace the uninformative 'protein binding' with the more specific molecular adaptor activity. RDE-2 binds MUT-7 via its CTD and MUT-16 via its NTD, physically linking the two so that MUT-7 is recruited into Mutator foci (PMID:15653635, PMID:39188014). Proposed replacements: molecular adaptor activity Supporting Evidence: PMID:39188014 Caenorhabditis elegans MUT-7 contains a specific insertion within MUT7-C, which allows binding to MUT-8 and, consequently, MUT-7 recruitment to germ granules |
| GO:0005515 protein binding | IPI PMID:19123269 Empirically controlled mapping of the Caenorhabditis elegans... | MARK AS OVER ANNOTATED | Summary: A second IPI 'protein binding' annotation from a high-throughput interactome dataset recording the RDE-2/MUT-7 interaction. The interaction is genuine and corroborates the adaptor role, but the term itself is uninformative. Reason: 'protein binding' conveys no functional specificity; the informative molecular function (molecular adaptor activity) is captured from the dedicated interaction studies. Retained as supporting evidence for the MUT-7 interaction but not as a core function. Supporting Evidence: PMID:30036386 two proteins of unknown function, RDE-2 and MUT-15 |
| GO:0030422 siRNA processing | IDA PMID:15653635 RDE-2 interacts with MUT-7 to mediate RNA interference in Ca... | ACCEPT | Summary: RDE-2 is required for accumulation of siRNAs in vivo; the MUT-7/RDE-2 complex acts in the RdRP-dependent siRNA amplification step. GO:0030422 explicitly includes amplification of siRNA by RNA-directed RNA polymerase, so this is an appropriate core biological-process term. Reason: rde-2 mutants do not produce detectable siRNAs in vivo, and the complex functions downstream of primary siRNA production in amplification, consistent with the siRNA-processing term definition. Supporting Evidence: PMID:15653635 Together these data hint at a role for the MUT-7/RDE-2 complex in the amplification step of the RNAi pathway in C.elegans. |
| GO:1990633 mutator focus | IDA PMID:22713602 MUT-16 promotes formation of perinuclear mutator foci requir... | ACCEPT | Summary: RDE-2 is one of the six mutator proteins that localize to perinuclear Mutator foci in the germline, the siRNA-amplification compartment. This is the informative, species-appropriate cellular-component annotation and represents where RDE-2 carries out its function. Reason: Direct localization of the mutator proteins (including RDE-2) to perinuclear Mutator foci; RDE-2 localization to these foci depends on MUT-16 (PMID:30036386). Supporting Evidence: PMID:22713602 each of the six mutator proteins localizes to punctate foci at the periphery of germline nuclei. The Mutator foci are adjacent to P granules |
| GO:0016441 post-transcriptional gene silencing | IMP PMID:10535731 The rde-1 gene, RNA interference, and transposon silencing i... | KEEP AS NON CORE | Summary: Loss of rde-2 causes RNAi resistance and transposon de-silencing, i.e. defective post-transcriptional gene silencing. This is a defining phenotype of the gene, though it is a parent of the more specific regulatory-ncRNA-mediated PTGS term. Reason: Correct but general; the more specific GO:0035194 (regulatory ncRNA-mediated post-transcriptional gene silencing) better captures the mechanism and is retained as core. Kept as valid non-core context. Supporting Evidence: PMID:15653635 This allele, removing the last three-quarters of the gene, is viable and is RNAi resistant |
| GO:0045132 meiotic chromosome segregation | IMP PMID:10535731 The rde-1 gene, RNA interference, and transposon silencing i... | KEEP AS NON CORE | Summary: rde-2 mutants show a high-incidence-of-males (Him) phenotype caused by X-chromosome non-disjunction. This is a downstream, pleiotropic consequence of losing germline small-RNA silencing rather than evidence that RDE-2 acts directly in the meiotic chromosome-segregation machinery. Reason: The segregation defect is an indirect consequence of the RNAi/silencing deficiency (shared with mut-7), not a direct molecular role of RDE-2 in meiosis. Retained as a non-core phenotype-based annotation. Supporting Evidence: PMID:15653635 both mut-7 and rde-2 mutants show a high incidence of males (him) phenotype |
| GO:0035194 regulatory ncRNA-mediated post-transcriptional gene silencing | IMP PMID:10535731 The rde-1 gene, RNA interference, and transposon silencing i... | ACCEPT | Summary: RDE-2 is required for small-RNA (siRNA/22G-RNA)-guided silencing of endogenous and exogenous targets and of transposons; this term captures the specific mechanism of RDE-2's biological role and is a core process annotation. Reason: Loss of rde-2 abolishes RdRP-dependent secondary siRNA accumulation and silencing, the defining regulatory-ncRNA-mediated PTGS function. Supporting Evidence: PMID:30036386 mut-15, rde-2, rde-8, or rrf-1) result in a substantial loss of the RdRP-dependent secondary siRNAs |
| GO:0005829 cytosol | IDA PMID:15653635 RDE-2 interacts with MUT-7 to mediate RNA interference in Ca... | KEEP AS NON CORE | Summary: Biochemical fractionation placed the MUT-7/RDE-2 complex in the cytosolic (S100) fraction. This is correct but generic; the informative, function-relevant localization is the perinuclear Mutator focus (GO:1990633), which is a specialized cytoplasmic compartment. Reason: Valid subcellular fraction data, but subsumed functionally by the Mutator-focus annotation. Kept as non-core supporting localization. Supporting Evidence: PMID:15653635 the MUT-7 and RDE-2 proteins are associated with each other in the cytosol, but not in the nucleus |
Loading supporting content…
Download this section (compressed HTML)Q: Does RDE-2/MUT-8 possess any activity beyond bridging MUT-7 and MUT-16 - for example intrinsic RNA binding or a role in nucleating Mutator-focus condensation?
Suggested experts: René F. Ketting, Sebastian Falk, Carolyn M. Phillips
Experiment: Generate separation-of-function rde-2 alleles that retain MUT-16 binding but disrupt the MUT-7 CTD interface (guided by the PDB 8Q66 interface), assay MUT-7 focus localization, secondary 22G-RNA levels, and RNAi/transposon-silencing competence.
Hypothesis: RDE-2 acts purely as a structural adaptor whose sole essential role is to recruit and position MUT-7 within Mutator foci.
Type: structure-guided mutagenesis with small-RNA sequencing and localization
Experiment: Reconstitute recombinant RDE-2 with each mutator component and RRF-1 and test for direct binary interactions by pulldown/SEC and crosslinking mass spectrometry, and attempt cryo-EM of the assembled complex.
Hypothesis: RDE-2 directly contacts additional mutator components beyond MUT-7 and MUT-16.
Type: in vitro interaction mapping / structural biology
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: Beyond acting as a passive structural bridge between MUT-7 and MUT-16, it is unknown whether the intrinsically disordered regions of RDE-2/MUT-8 have any additional molecular activity (e.g. RNA binding, condensate nucleation, or allosteric regulation of MUT-7 nuclease activity). RDE-2 has no catalytic activity and no canonical folded domain, so its full biochemical contribution to the complex is undefined.
OPEN BIOLOGY MF_DARK
What is known: It is firmly established that RDE-2's structured C-terminal domain binds the MUT-7 CTD (crystal structure PDB 8Q66) and that its partially structured N-terminal region contacts the MUT-16 scaffold, recruiting MUT-7 to Mutator foci. What is unknown is whether RDE-2 does anything mechanistically beyond this bridging.
Significance: Because RDE-2 is essential for secondary siRNA amplification but is not itself an enzyme, distinguishing 'pure scaffold/recruiter' from 'active participant' would determine whether the amplification defect in rde-2 mutants is purely a mislocalization/assembly failure or also a loss of a direct biochemical step.
What would resolve it: In vitro reconstitution of the MUT-16/RDE-2/MUT-7 module with siRNA-amplification assays, RNA-binding assays on isolated RDE-2 domains, and separation-of-function RDE-2 alleles that retain MUT-7/MUT-16 binding but disrupt any additional activity would resolve this.
Provenance (the field's own admissions):
Gap: The complete set of RDE-2 direct binding partners within the Mutator complex is unresolved. Direct interactions are established only with MUT-7 (CTD-CTD) and MUT-16 (via the RDE-2 N-terminal region); whether RDE-2 directly contacts other mutator components (MUT-2/RDE-3, MUT-14, MUT-15, NYN-1/2, RDE-8) or the RdRP RRF-1, or merely co-resides with them in the focus, has not been tested biochemically.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: RDE-2 localization to Mutator foci depends on MUT-16, and RDE-2 is in turn required for MUT-7 localization (a MUT-16 -> RDE-2 -> MUT-7 recruitment axis). Other mutator components localize via separate MUT-16 branches, but their direct/indirect relationship to RDE-2 is inferred from localization dependencies, not from binary interaction data.
Significance: Knowing which contacts are direct defines the true architecture of the amplification compartment and whether RDE-2 is a dedicated MUT-7 adaptor or a more central hub.
What would resolve it: Systematic binary interaction mapping (e.g. reconstituted pulldowns, crosslinking mass spectrometry, or cryo-EM of the assembled complex) of RDE-2 against each mutator component.
Provenance (the field's own admissions):
Gap: How RDE-2 mechanistically contributes to the secondary siRNA (22G-RNA) amplification reaction itself is unknown. It is established that rde-2 loss abolishes RdRP-dependent secondary siRNA accumulation, but whether RDE-2's only role is to recruit and position MUT-7, or whether it also participates in target-mRNA capture or templating for RRF-1, is undetermined.
OPEN BIOLOGYONTOLOGY MF_DARK
What is known: The genetic requirement is clear (no detectable secondary siRNAs without rde-2) and the focus-assembly role is structurally defined, but the biochemical step at which RDE-2 acts within amplification is not.
Significance: Secondary siRNA amplification is the amplifying core of C. elegans RNA silencing and heritable epigenetic inheritance; resolving RDE-2's step would clarify a rate-limiting node. There is also no GO term for 'secondary siRNA amplification' as a distinct process or for a 'structural constituent of the Mutator complex', so RDE-2's specific contribution cannot currently be expressed precisely.
What would resolve it: Mechanistic in vitro amplification assays with defined mutator subcomplexes, plus ontology development for a secondary-siRNA-amplification process term and/or a Mutator-complex structural-constituent term.
Provenance (the field's own admissions):
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)