wago-4 encodes a germline-restricted, worm-specific Argonaute (WAGO clade) of Caenorhabditis elegans. It is a secondary Argonaute that binds RNA-dependent RNA polymerase-derived 22G-RNAs (22-nucleotide small interfering RNAs bearing a 5' guanosine) together with their complementary target mRNAs, acting as an effector of small-RNA-directed post-transcriptional gene silencing rather than as a catalytic slicer; like other WAGO-subfamily proteins it lacks the conserved catalytic metal-binding residues of cleavage-competent Argonautes. WAGO-4 is essential for the germline RNA interference response and, in particular, for the transgenerational inheritance of RNAi-triggered silencing, operating in the cytoplasmic branch of the pathway downstream of primary Argonautes. It concentrates in perinuclear germ-granule compartments, transiently associating with P granules and, together with the helicase ZNFX-1, defining the Z granule, a liquid-like condensate positioned between P granules and Mutator foci, and it is required for ZNFX-1 to engage silenced mRNAs. Expression is confined to the hermaphrodite (and at low level the male) germline and oocytes, and the protein segregates asymmetrically with the germline lineage during early embryogenesis. Its endogenous 22G-RNA repertoire overlaps that of the Argonaute CSR-1.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: WAGO-4 is a cytoplasmic Argonaute; cytoplasmic activity is well supported. Reason: WAGO-4 functions in the cytoplasmic branch of the RNAi pathway and is experimentally shown to be cytoplasmic/perinuclear. The IBA is consistent with the experimental localization, though a more specific germ-granule location is captured in core_functions. Supporting Evidence: PMID:29791857 we identified a cytoplasmic Argonaute protein, WAGO-4, necessary for the inheritance of RNAi. |
| GO:0035194 regulatory ncRNA-mediated post-transcriptional gene silencing | IBA GO_REF:0000033 | ACCEPT | Summary: Core biological process. WAGO-4 uses 22G-RNAs to silence target mRNAs post-transcriptionally. Reason: WAGO-4 binds 22G-RNAs and their mRNA targets and is required for germline RNAi, placing it squarely in small-RNA-directed post-transcriptional gene silencing. Supporting Evidence: PMID:29791857 WAGO-4 binds to 22G-RNAs and their mRNA targets. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | REMOVE | Summary: WAGO-4 is a cytoplasmic Argonaute; there is no evidence it acts in the nucleus. This IBA is over-propagated from nuclear members of the Argonaute family. Reason: WAGO-4 is explicitly the cytoplasmic-branch Argonaute and is experimentally cytoplasmic/perinuclear/germ-granule; nuclear RNAi in C. elegans is executed by the distinct nuclear WAGOs (HRDE-1, NRDE-3). The nucleus term is inherited from nuclear Argonautes across the phylogenetic tree and is not warranted for this protein (argued against on biological grounds, not paralog confusion of an experimental annotation). Propagation Review Root cause: PROPAGATION BAD Failure modes: COMPARTMENT OR COMPLEX MISMATCH Supporting Evidence: PMID:29791857 we identified a cytoplasmic Argonaute protein, WAGO-4, necessary for the inheritance of RNAi. |
| GO:0004521 RNA endonuclease activity | IBA GO_REF:0000033 | REMOVE | Summary: WAGO-subfamily Argonautes lack the conserved catalytic/metal-binding residues needed for target cleavage; endonuclease (slicer) activity is not warranted for WAGO-4. Reason: This catalytic MF is propagated by IBA from cleavage-competent Argonautes, but WAGO-clade proteins including WAGO-4 lack the residues required for mRNA cleavage and probably do not slice. OpenScientist independently confirmed that WAGO-4 lacks three of four PIWI-domain catalytic tetrad positions (D1, D2, and H equivalents) relative to active slicer Argonautes, while the fetched GOA line shows the live IBA source as PANTHER:PTN008584027 within a broad Argonaute source set. Removing this over-propagated electronic inference on biological grounds; the supported molecular function is siRNA binding, captured separately. Propagation Review Root cause: PROPAGATION BAD Failure modes: PSEUDO OR SUBACTIVITY LOSS Sources checked: PANTHER:PTN008584027 · PAINT Argonaute/Piwi source node SUPPORTS SOURCE BUT NOT TARGET The fetched GOA line propagates RNA endonuclease activity through this broad Argonaute node, but WAGO-4 lacks the PIWI-domain catalytic tetrad required for slicer activity. Supporting Evidence: file:worm/wago-4/wago-4-goa.tsv UniProtKB O62275 wago-4 enables GO:0004521 RNA endonuclease activity molecular_function ECO:0000318 IBA GO_REF:0000033 AGI_LocusCode:AT1G48410|AGI_LocusCode:AT2G27040|FB:FBgn0000146|FB:FBgn0004872|FB:FBgn0087035|FB:FBgn0250816|FB:FBgn0262739|MGI:MGI:1928897|MGI:MGI:1930036|MGI:MGI:2446632|PANTHER:PTN008584027|PomBase:SPCC736.11|UniProtKB:O67434|UniProtKB:Q9H9G7|UniProtKB:Q9UKV8|WB:WBGene00000105|WB:WBGene00000106|WB:WBGene00004323|WB:WBGene00017641 6239 Caenorhabditis elegans GO_Central Argonaute protein wago-4 20241120 PMID:17110334 Interestingly, these AGO proteins lack key residues required for mRNA cleavage. file:worm/wago-4/wago-4-hypotheses/function-hypothesis-go-0004521/openscientist.md Two independent computational analyses confirm that WAGO-4 lacks the conserved DEDH catalytic tetrad at three of four critical positions in its PIWI domain file:worm/wago-4/wago-4-hypotheses/function-hypothesis-go-0004521/openscientist.md D1=G676 (score 31), D2=T756 (score 41), H=N913 (score 31) — non-conservative substitutions at three critical positions |
| GO:0016442 RISC complex | IBA GO_REF:0000033 | ACCEPT | Summary: As a small-RNA-loaded Argonaute that engages target mRNAs, WAGO-4 is the core of an RNA-induced silencing (effector) complex. Reason: WAGO-4 binds 22G-RNA guides and their target mRNAs, the defining composition of a RISC/effector complex. Supporting Evidence: PMID:29791857 WAGO-4 binds to 22G-RNAs and their mRNA targets. |
| GO:0036464 cytoplasmic ribonucleoprotein granule | IBA GO_REF:0000033 | ACCEPT | Summary: WAGO-4 localizes to cytoplasmic RNP germ granules (P granules and the Z granule); the IBA is correct and is refined to P granule in core_functions. Reason: Experimentally, WAGO-4 is a transient component of P granules and defines the Z granule, both cytoplasmic ribonucleoprotein granules. Supporting Evidence: PMID:29769721 ZNFX-1 and WAGO-4, that localize to Caenorhabditis elegans germ granules (P granules) in early germline blastomeres. |
| GO:0043186 P granule | IDA PMID:29769721 Spatiotemporal regulation of liquid-like condensates in epig... | NEW | Summary: WAGO-4 is experimentally shown to localize to P granules in early germline blastomeres; a more specific term than the IBA cytoplasmic ribonucleoprotein granule annotation. Reason: Direct imaging shows WAGO-4 (with ZNFX-1) at C. elegans germ granules (P granules) in early germline blastomeres, and WAGO-4 is a transient P-granule component in adult germ cells. GO:0043186 captures this experimentally supported localization, which is only implicit in the existing IBA GO:0036464 annotation. Supporting Evidence: PMID:29769721 ZNFX-1 and WAGO-4, that localize to Caenorhabditis elegans germ granules (P granules) in early germline blastomeres. |
| GO:0035198 miRNA binding | IBA GO_REF:0000033 | MODIFY | Summary: WAGO-4 binds 22G-RNAs (RdRP-derived endo-siRNAs), not miRNAs. The correct molecular function is siRNA binding. Reason: The miRNA-binding term is inherited by IBA from miRNA-class Argonautes, but the experimentally defined WAGO-4 guides are 22-nucleotide secondary siRNAs, not miRNAs. Replace with the class-appropriate siRNA binding term. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: FUNCTIONAL DIVERGENCE Proposed replacements: siRNA binding Supporting Evidence: PMID:29791857 WAGO-4 binds to 22G-RNAs and their mRNA targets. |
| GO:0003727 single-stranded RNA binding | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Correct but general. WAGO-4 binds single-stranded RNA (its 22G-RNA guide and target mRNA); the more informative term is siRNA binding. Reason: Accurate at the ssRNA level but subsumed by the specific siRNA-binding activity recorded as the core molecular function; retained as a true, non-core general term. Propagation Review Root cause: NO FAILURE NON CORE Failure modes: GRANULARITY MISMATCH Supporting Evidence: PMID:29791857 WAGO-4 binds to 22G-RNAs and their mRNA targets. |
| GO:0003676 nucleic acid binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Generic InterPro-derived parent term; true but uninformative. Reason: High-level ancestor of the specific RNA/siRNA-binding activity of WAGO-4; correct but not informative of the actual function. |
| GO:0003723 RNA binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Generic InterPro-derived term; accurate but subsumed by siRNA binding. Reason: WAGO-4 is an RNA-binding protein, but the informative molecular function is siRNA (22G-RNA) binding; retained as a correct general parent. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Cytoplasmic localization, consistent with experimental data. Reason: UniProt SubCell mapping agrees with the experimental cytoplasmic/perinuclear localization of WAGO-4. Supporting Evidence: PMID:29791857 accumulates at the perinuclear foci in the germline |
| GO:0048471 perinuclear region of cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Well-supported specific localization; WAGO-4 accumulates at germline perinuclear foci. Reason: Directly corroborated by experimental imaging of WAGO-4 at perinuclear foci in the germline. Supporting Evidence: PMID:29791857 accumulates at the perinuclear foci in the germline |
| GO:0005737 cytoplasm | EXP PMID:29791857 A Cytoplasmic Argonaute Protein Promotes the Inheritance of ... | ACCEPT | Summary: Experimental cytoplasmic localization of WAGO-4. Reason: Direct experimental support; WAGO-4 is the cytoplasmic Argonaute of the RNAi pathway. Supporting Evidence: PMID:29791857 we identified a cytoplasmic Argonaute protein, WAGO-4, necessary for the inheritance of RNAi. |
| GO:0060966 regulation of gene silencing by regulatory ncRNA | IMP PMID:30728462 MINA-1 and WAGO-4 are part of regulatory network coordinatin... | ACCEPT | Summary: WAGO-4 level positively regulates RNAi efficacy; its overexpression causes RNAi hypersensitivity, supporting a positive-regulator role in ncRNA-mediated silencing. Reason: Experimental (IMP) evidence that WAGO-4 dosage governs silencing efficiency; consistent with secondary Argonautes being limiting for RNAi. Supporting Evidence: PMID:30728462 upregulation of WAGO-4 in mina-1 mutant animals causes hypersensitivity to exogenous RNAi. |
| GO:0031048 regulatory ncRNA-mediated heterochromatin formation | IGI PMID:22231482 Amplification of siRNA in Caenorhabditis elegans generates a... | KEEP AS NON CORE | Summary: wago-4 was assayed only within a six-gene MAGO secondary-Argonaute group whose collective loss impairs RNAi-triggered H3K9me3; a redundant, indirect contribution for this cytoplasmic Argonaute. Reason: Experimental (IGI) and therefore retained, but the phenotype reflects the combined loss of six secondary Argonautes, not a WAGO-4-specific nuclear function. WAGO-4's core role is cytoplasmic 22G-RNA-directed post-transcriptional silencing; its input to heterochromatin formation is as part of the redundant secondary-siRNA machinery that feeds the nuclear pathway. Supporting Evidence: PMID:22231482 MAGO (ppw-1(tm914), sago-1(tm1195), sago-2(tm894), F58G1.1(tm1019), C06A1.4(tm887), and M03D4.6(tm1144)] |
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Download this section (compressed HTML)Q: Is WAGO-4 catalytically inactive in vivo, or does it retain a cryptic slicer or other nuclease-recruiting activity on its target mRNAs?
Q: What distinguishes a WAGO-4-silenced target from a CSR-1-protected target when both share the same 22G-RNA cohort?
Experiment: Reconstitute recombinant WAGO-4 loaded with a defined synthetic 22G-RNA and assay cleavage of a perfectly complementary target RNA in vitro, alongside a cleavage-competent control Argonaute; combine with structure-guided mutagenesis of the putative catalytic tetrad.
Hypothesis: WAGO-4 silences targets without slicing them, acting through target sequestration and recruitment of the RdRP amplification/inheritance machinery.
Type: in vitro biochemistry / slicer assay
Experiment: Perform WAGO-4 small-RNA and mRNA CLIP/IP-seq in parallel with CSR-1 in wild-type, wago-4(-), and WAGO-4-overexpressing germlines, integrating transcript stability and ribosome-profiling readouts to define target-specific outcomes.
Hypothesis: WAGO-4 and CSR-1 act on overlapping 22G-RNA targets but produce opposite transcript-level outcomes.
Type: comparative CLIP-seq / functional genomics
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: Whether WAGO-4 has any catalytic (slicer/endonuclease) activity is undetermined. It is inferred to be a non-catalytic, siRNA-guided mRNA-binding effector, but no biochemical assay has directly tested WAGO-4 for target cleavage.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: It is firmly established that WAGO-4 binds 22G-RNA guides and their target mRNAs and is required for germline RNAi and its inheritance, and that WAGO-subfamily Argonautes lack the conserved catalytic metal-binding residues of cleavage-competent Argonautes.
Significance: Whether silencing is achieved by slicing, by recruiting downstream nucleases or the RdRP amplification loop, or purely by target sequestration/marking determines the molecular mechanism of WAGO-4 and how it differs from cleavage-competent Argonautes.
What would resolve it: In vitro slicer assays with recombinant WAGO-4 loaded with a defined 22G-RNA on a complementary target, and structural confirmation of the (in)complete catalytic tetrad, would settle the activity.
Provenance (the field's own admissions):
Gap: The direct molecular consequence of WAGO-4 engaging a target mRNA, and how the shared 22G-RNA target space with CSR-1 yields opposite (silencing versus licensing) outcomes, is unresolved. The full endogenous target-mRNA repertoire that WAGO-4 functionally silences is not defined.
OPEN BIOLOGY BP_DARK
What is known: It is established that WAGO-4-associated 22G-RNAs target the same cohort of germline genes as CSR-1 and carry 3' untemplated uridylation, and that WAGO-4 is a dosage-sensitive positive regulator of silencing.
Significance: Distinguishing which targets WAGO-4 silences (versus those CSR-1 protects), and the readout (transcript destabilization, translational repression, or a heritable mark), is required to place WAGO-4 precisely within the germline 22G-RNA network.
What would resolve it: Target-resolved WAGO-4 IP-seq/CLIP paired with transcript-level and translational profiling in wago-4 loss- and gain-of-function backgrounds, contrasted with CSR-1, would define the functional target set and outcome.
Provenance (the field's own admissions):
Gap: The mechanism by which WAGO-4 (with ZNFX-1 in the Z granule) transports and transmits 22G-RNA/mRNA silencing information across generations is unknown, including how material is handed between P granules, Z granules, and Mutator foci.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: It is established that WAGO-4 is required for RNAi inheritance, that it physically associates with ZNFX-1, that WAGO-4 is needed for ZNFX-1 to engage silenced mRNA, and that WAGO-4/ZNFX-1 define the Z granule between P granules and Mutator foci.
Significance: How a cytoplasmic small-RNA/mRNA complex is physically routed through germ-granule sub-compartments and loaded into progeny germ cells is the central mechanistic question of transgenerational epigenetic inheritance in this pathway.
What would resolve it: Live imaging of tagged WAGO-4/ZNFX-1 with sub-granule resolution during the germline-to-embryo transition, plus separation-of-function alleles that uncouple condensate residence from RNA binding, would test the transport model.
Provenance (the field's own admissions):
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