RDE-8 is a germline-enriched NYN-domain (Zc3h12a/Regnase-like) endoribonuclease required for RNA interference in C. elegans. RDE-8 is recruited to RNAi target mRNAs downstream of the primary Argonaute RDE-1, which itself does not need slicer activity for silencing. With its interactors MUT-16, MUT-15, the NYN-domain proteins NYN-1/NYN-2 and the nucleotidyltransferase RDE-3/MUT-2, RDE-8 works in perinuclear germline Mutator foci. There it cleaves target mRNA, which promotes silencing, and its 5' cleavage fragments are 3'-uridylated by RDE-3 to act as templates for RRF-1 RNA-dependent RNA polymerase. RDE-8 catalytic activity (D76) is needed for WAGO 22G-RNA amplification and RNAi. RDE-8 is also required for RRF-3-dependent 26G-RNA accumulation in the ERI pathway, but this does not need its nuclease activity.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003729 mRNA binding | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: NYN/Zc3h12 family IBA for mRNA binding. RDE-8 associates with RNAi target mRNA, but the binding is transient or indirect. Reason: RDE-8 has no recognizable RNA-binding domain. Its association with target mRNA is detected only with the catalytic-dead D76N mutant and depends on RDE-1 and RDE-8 interactors. RDE-8 does engage its mRNA substrate, but direct sequence binding is not established. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN001767779 · PTN001767779 SUPPORTS TRANSFER Zc3h12/Regnase-type NYN node. RDE-8 engages target mRNA, though perhaps indirectly. Supporting Evidence: PMID:25635455 RDE-8 contains no recognizable RNA-binding domain, and we only detected target binding when the conserved catalytic residues of RDE-8 were mutated, suggesting a transient or indirect interaction between RDE-8 and the target mRNA. |
| GO:0003729 mRNA binding | IDA PMID:25635455 A ribonuclease coordinates siRNA amplification and mRNA clea... | KEEP AS NON CORE | Summary: RIP of catalytic-dead GFP::RDE-8(D76N) specifically captures the sel-1 target mRNA after sel-1 RNAi. Reason: The RIP shows that RDE-8 associates with its substrate mRNA, but the authors note the interaction may be transient or indirect. Kept, but not core. Supporting Evidence: PMID:25635455 Strikingly, we found that RDE-8(D76N) specifically captured the sel-1 transcript when animals were exposed to sel-1 dsRNA (Figure 6A). |
| GO:0004521 RNA endonuclease activity | IBA GO_REF:0000033 | ACCEPT | Summary: NYN/Zc3h12 family IBA for RNA endonuclease activity. Confirmed experimentally for RDE-8. Reason: Core MF. RDE-8 keeps the catalytic NYN residues and cleaves RNA in vitro, in a D76-dependent way. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN001767779 · PTN001767779 SUPPORTS TRANSFER NYN-domain endoribonuclease node. RDE-8 itself is a confirmed catalytic member. Supporting Evidence: PMID:25635455 These data indicate that RDE-8 encodes an endoribonuclease required for RNAi. |
| GO:0004521 RNA endonuclease activity | IDA PMID:25635455 A ribonuclease coordinates siRNA amplification and mRNA clea... | ACCEPT | Summary: Recombinant RDE-8 cleaves single-stranded RNA in vitro; the D76N mutant does not. Reason: Direct biochemical evidence for the core activity. Supporting Evidence: PMID:25635455 RDE-8 can cleave RNA in vitro and is needed for the production of 3' uridylated fragments of target mRNA in vivo. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: NYN/Zc3h12 family IBA for nucleus, from Regnase/ZC3H12-family members with nuclear roles. Reason: GFP::RDE-8 is mainly in the germline cytoplasm and in perinuclear Mutator foci next to nuclear pores. No nuclear pool has been reported. The nuclear assertion reflects other family members. Propagation Review Root cause: PROPAGATION BAD Failure modes: COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN001767779 · PTN001767779 SUPPORTS SOURCE BUT NOT TARGET Nuclear localization comes from ZC3H12-family members. RDE-8 is seen only in the cytoplasm and perinuclear foci. Supporting Evidence: PMID:25635455 GFP::RDE-8 was primarily observed in the germline cytoplasm and in prominent perinuclear foci associated with nuclear pores in the germline (Figure 4B). |
| GO:0005737 cytoplasm | IDA PMID:25635455 A ribonuclease coordinates siRNA amplification and mRNA clea... | ACCEPT | Summary: GFP::RDE-8 is mainly in the germline cytoplasm. Reason: Directly observed. The more specific location is Mutator foci. Supporting Evidence: PMID:25635455 GFP::RDE-8 was primarily observed in the germline cytoplasm and in prominent perinuclear foci associated with nuclear pores in the germline (Figure 4B). |
| GO:0030422 siRNA processing | IMP PMID:25635455 A ribonuclease coordinates siRNA amplification and mRNA clea... | ACCEPT | Summary: rde-8 mutants fail to accumulate RdRP-dependent 22G-RNAs and 26G-RNAs. RDE-8 is needed for efficient RRF-1 RdRP activity in vitro. Reason: Core process. RDE-8 cleavage produces the target-mRNA fragments that, after RDE-3 uridylation, act as RdRP templates for secondary siRNA amplification. Supporting Evidence: PMID:25635455 We further show that RDE-8 is required for efficient RRF-1 RdRP activity in vitro. PMID:25635455 Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3' uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal. |
| GO:0036464 cytoplasmic ribonucleoprotein granule | IBA GO_REF:0000033 | ACCEPT | Summary: NYN/Zc3h12 family IBA for cytoplasmic RNP granule. RDE-8 is in Mutator foci, which are a kind of cytoplasmic RNP granule. Reason: Correct. Mutator focus (GO:1990633, IDA below) is a child of this term and is the more precise location. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN001767779 · PTN001767779 SUPPORTS TRANSFER RNP-granule localization holds for RDE-8 (Mutator foci). Supporting Evidence: PMID:25635455 We identify RDE-8 homologs and RNAi and transposon-silencing factors as RDE-8–interacting proteins, and show that RDE-8 localizes to germline Mutator foci. |
| GO:0061158 3'-UTR-mediated mRNA destabilization | IBA GO_REF:0000033 | MODIFY | Summary: NYN/Zc3h12 family IBA for 3'-UTR-mediated mRNA destabilization, from Regnase-type ribonucleases that recognize 3'-UTR stem-loops. Reason: RDE-8 does destabilize mRNA, but it is directed to targets by RDE-1/siRNA complexes binding the dsRNA-homologous region, not by 3'-UTR elements. siRNA-mediated gene silencing by mRNA destabilization describes this better. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN001767779 · PTN001767779 SUPPORTS SOURCE BUT NOT TARGET 3'-UTR-directed decay fits Regnase/ZC3H12A. RDE-8 targets are chosen by siRNA-loaded RDE-1. Proposed replacements: siRNA-mediated gene silencing by mRNA destabilization Supporting Evidence: PMID:25635455 We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. PMID:25635455 Consistent with the idea that RDE-8 activity is important for target cleavage, we found that target-mRNA fragments tended to be higher in gfp::rde-8(+) worms than in gfp::rde-8(D76N) worms |
| GO:1990633 mutator focus | IDA PMID:25635455 A ribonuclease coordinates siRNA amplification and mRNA clea... | ACCEPT | Summary: GFP::RDE-8 forms perinuclear germline foci, adjacent to P granules, that are Mutator foci. Reason: Core location, where RDE-8 works with MUT-16, MUT-15 and RDE-3. Supporting Evidence: PMID:25635455 We identify RDE-8 homologs and RNAi and transposon-silencing factors as RDE-8–interacting proteins, and show that RDE-8 localizes to germline Mutator foci. |
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Download this section (compressed HTML)Q: What decides where along a target mRNA RDE-8 cleaves, and is its in vivo activity specific to Argonaute-paired RNA (the activity described by endoribonuclease activity, cleaving siRNA-paired mRNA)?
Q: How does RDE-8 support ERI-pathway 26G-RNA accumulation without its nuclease activity? Is this a structural role in the RRF-3 complex?
Experiment: Map RDE-8 cleavage sites on RNAi target mRNAs in vivo by degradome sequencing in wild-type and D76N animals. Reconstitute cleavage with purified RDE-1/siRNA complexes to test whether pairing to a guide stimulates or directs RDE-8 activity.
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