Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0005634 nucleus	IBA GO_REF:0000033	KEEP AS NON CORE	Summary: nucleus is a plausible or reported location/context but is not the core functional site annotation for AGO4. Reason: The core location is cytoplasmic RISC/RNP granules; nuclear, membrane, synaptic, exosomal, or other compartment annotations should remain non-core unless tied to a specific curated mechanism. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005737 cytoplasm	IBA GO_REF:0000033	ACCEPT	Summary: cytoplasm is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0004521 RNA endonuclease activity	IBA GO_REF:0000033	REMOVE	Summary: RNA endonuclease activity is not supported as a physiological activity for AGO4. Reason: AGO4 is best supported as a non-slicing Argonaute paralog; assigning RNA endonuclease activity would overstate AGO2-like catalytic function. OpenScientist confirmed that AGO4 lacks the intact DEDH catalytic tetrad required for AGO2-like slicer activity, so this is a paralog over-annotation rather than a supported AGO4 molecular function. Propagation Review Root cause: PROPAGATION BAD Failure modes: PSEUDO OR SUBACTIVITY LOSS FUNCTIONAL DIVERGENCE WRONG ORTHOLOG OR PARALOG Sources checked: PANTHER:PTN008584027 · Argonaute PANTHER source node SUPPORTS SOURCE BUT NOT TARGET The family source includes slicing-competent Argonautes, but AGO4 is reviewed as a non-slicing paralog. UniProtKB:Q9UKV8 · human AGO2 SUPPORTS SOURCE BUT NOT TARGET AGO2 supports RNA endonuclease activity; AGO4 lacks the same supported slicer activity in the target review. UniProtKB:Q9H9G7 · human AGO3 SUPPORTS SOURCE BUT NOT TARGET AGO3 retains a catalytic tetrad and has conditional slicer activity; AGO4 has disruptive substitutions in the corresponding catalytic residues. Supporting Evidence: file:human/AGO4/AGO4-goa.tsv UniProtKB Q9HCK5 AGO4 enables GO:0004521 RNA endonuclease activity molecular_function ECO:0000318 IBA GO_REF:0000033 FB:FBgn0000146\|FB:FBgn0004872\|FB:FBgn0087035\|FB:FBgn0250816\|FB:FBgn0262739\|MGI:MGI:1928897\|MGI:MGI:1930036\|MGI:MGI:2446632\|PANTHER:PTN008584027\|PomBase:SPCC736.11\|TAIR:locus:2007760\|TAIR:locus:2059258\|UniProtKB:O67434\|UniProtKB:Q9H9G7\|UniProtKB:Q9UKV8\|WB:WBGene00000105\|WB:WBGene00000106\|WB:WBGene00004323\|WB:WBGene00017641 9606 Homo sapiens GO_Central Protein argonaute-4 20241120 file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms file:human/AGO4/AGO4-hypotheses/function-hypothesis-go-0004521/openscientist.md Human AGO4 does not have RNA endonuclease (slicer) activity.
GO:0035194 regulatory ncRNA-mediated post-transcriptional gene silencing	IBA GO_REF:0000033	ACCEPT	Summary: regulatory ncRNA-mediated post-transcriptional gene silencing is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0016442 RISC complex	IBA GO_REF:0000033	ACCEPT	Summary: RISC complex is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0036464 cytoplasmic ribonucleoprotein granule	IBA GO_REF:0000033	ACCEPT	Summary: cytoplasmic ribonucleoprotein granule is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0031054 pre-miRNA processing	IBA GO_REF:0000033	MARK AS OVER ANNOTATED	Summary: pre-miRNA processing overstates the direct role of AGO4 in canonical miRNA biogenesis. Reason: The better-supported role is as a loaded Argonaute effector after small-RNA processing, not as the Drosha/Dicer processing enzyme for pre-miRNAs. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: ROLE CONFLATION GRANULARITY MISMATCH Sources checked: PANTHER:PTN000527278 · Argonaute pre-miRNA processing source node SUPPORTS SOURCE BUT NOT TARGET The source captures Argonaute participation in miRNA pathway biology, but the propagated term overstates AGO4 as processing machinery rather than a loaded small-RNA effector. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0035198 miRNA binding	IBA GO_REF:0000033	ACCEPT	Summary: miRNA binding is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0003727 single-stranded RNA binding	IBA GO_REF:0000033	MARK AS OVER ANNOTATED	Summary: single-stranded RNA binding is too generic for AGO4. Reason: The informative molecular function is Argonaute small-RNA/miRNA binding within RISC rather than a broad nucleic-acid or RNA-binding label. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000527276 · Argonaute RNA-binding source node SUPPORTS SOURCE BUT NOT TARGET The source supports RNA-binding Argonaute biology, but the propagated term is less informative than AGO4 small-RNA/miRNA binding in RISC context. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0000932 P-body	IEA GO_REF:0000120	ACCEPT	Summary: P-body is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0003676 nucleic acid binding	IEA GO_REF:0000002	MARK AS OVER ANNOTATED	Summary: nucleic acid binding is too generic for AGO4. Reason: The informative molecular function is Argonaute small-RNA/miRNA binding within RISC rather than a broad nucleic-acid or RNA-binding label. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0003723 RNA binding	IEA GO_REF:0000120	MARK AS OVER ANNOTATED	Summary: RNA binding is too generic for AGO4. Reason: The informative molecular function is Argonaute small-RNA/miRNA binding within RISC rather than a broad nucleic-acid or RNA-binding label. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0003725 double-stranded RNA binding	IEA GO_REF:0000117	MARK AS OVER ANNOTATED	Summary: double-stranded RNA binding is too generic for AGO4. Reason: The informative molecular function is Argonaute small-RNA/miRNA binding within RISC rather than a broad nucleic-acid or RNA-binding label. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0003727 single-stranded RNA binding	IEA GO_REF:0000117	MARK AS OVER ANNOTATED	Summary: single-stranded RNA binding is too generic for AGO4. Reason: The informative molecular function is Argonaute small-RNA/miRNA binding within RISC rather than a broad nucleic-acid or RNA-binding label. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0016442 RISC complex	IEA GO_REF:0000120	ACCEPT	Summary: RISC complex is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0031054 pre-miRNA processing	IEA GO_REF:0000117	MARK AS OVER ANNOTATED	Summary: pre-miRNA processing overstates the direct role of AGO4 in canonical miRNA biogenesis. Reason: The better-supported role is as a loaded Argonaute effector after small-RNA processing, not as the Drosha/Dicer processing enzyme for pre-miRNAs. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0035198 miRNA binding	IEA GO_REF:0000120	ACCEPT	Summary: miRNA binding is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0035278 miRNA-mediated gene silencing by inhibition of translation	IEA GO_REF:0000120	ACCEPT	Summary: miRNA-mediated gene silencing by inhibition of translation is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0036464 cytoplasmic ribonucleoprotein granule	IEA GO_REF:0000117	ACCEPT	Summary: cytoplasmic ribonucleoprotein granule is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0070578 RISC-loading complex	IEA GO_REF:0000117	ACCEPT	Summary: RISC-loading complex is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0070922 RISC complex assembly	IEA GO_REF:0000117	ACCEPT	Summary: RISC complex assembly is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005515 protein binding	IPI PMID:19167051 Importin 8 is a gene silencing factor that targets argonaute...	MARK AS OVER ANNOTATED	Summary: Generic protein binding does not capture the specific Argonaute/RISC function. Reason: The cited interactions are best interpreted as RISC/cofactor context for small-RNA-guided silencing, not as a standalone core function. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005515 protein binding	IPI PMID:19324964 The C-terminal half of human Ago2 binds to multiple GW-rich ...	MARK AS OVER ANNOTATED	Summary: Generic protein binding does not capture the specific Argonaute/RISC function. Reason: The cited interactions are best interpreted as RISC/cofactor context for small-RNA-guided silencing, not as a standalone core function. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005515 protein binding	IPI PMID:19383768 The C-terminal domains of human TNRC6A, TNRC6B, and TNRC6C s...	MARK AS OVER ANNOTATED	Summary: Generic protein binding does not capture the specific Argonaute/RISC function. Reason: The cited interactions are best interpreted as RISC/cofactor context for small-RNA-guided silencing, not as a standalone core function. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005515 protein binding	IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling...	MARK AS OVER ANNOTATED	Summary: Generic protein binding does not capture the specific Argonaute/RISC function. Reason: The cited interactions are best interpreted as RISC/cofactor context for small-RNA-guided silencing, not as a standalone core function. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005634 nucleus	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: nucleus is a plausible or reported location/context but is not the core functional site annotation for AGO4. Reason: The core location is cytoplasmic RISC/RNP granules; nuclear, membrane, synaptic, exosomal, or other compartment annotations should remain non-core unless tied to a specific curated mechanism. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0035196 miRNA processing	NAS PMID:28781232 Multivalent Recruitment of Human Argonaute by GW182.	MARK AS OVER ANNOTATED	Summary: miRNA processing overstates the direct role of AGO4 in canonical miRNA biogenesis. Reason: The better-supported role is as a loaded Argonaute effector after small-RNA processing, not as the Drosha/Dicer processing enzyme for pre-miRNAs. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0036464 cytoplasmic ribonucleoprotein granule	IDA GO_REF:0000052	ACCEPT	Summary: cytoplasmic ribonucleoprotein granule is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005515 protein binding	IPI PMID:15766526 Involvement of microRNA in AU-rich element-mediated mRNA ins...	MARK AS OVER ANNOTATED	Summary: Generic protein binding does not capture the specific Argonaute/RISC function. Reason: The cited interactions are best interpreted as RISC/cofactor context for small-RNA-guided silencing, not as a standalone core function. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0035196 miRNA processing	IMP PMID:22795694 Slicing-independent RISC activation requires the argonaute P...	MARK AS OVER ANNOTATED	Summary: miRNA processing overstates the direct role of AGO4 in canonical miRNA biogenesis. Reason: The better-supported role is as a loaded Argonaute effector after small-RNA processing, not as the Drosha/Dicer processing enzyme for pre-miRNAs. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0031054 pre-miRNA processing	IDA PMID:19966796 ATP-dependent human RISC assembly pathways.	MARK AS OVER ANNOTATED	Summary: pre-miRNA processing overstates the direct role of AGO4 in canonical miRNA biogenesis. Reason: The better-supported role is as a loaded Argonaute effector after small-RNA processing, not as the Drosha/Dicer processing enzyme for pre-miRNAs. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0003725 double-stranded RNA binding	IDA PMID:19966796 ATP-dependent human RISC assembly pathways.	MARK AS OVER ANNOTATED	Summary: double-stranded RNA binding is too generic for AGO4. Reason: The informative molecular function is Argonaute small-RNA/miRNA binding within RISC rather than a broad nucleic-acid or RNA-binding label. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0003727 single-stranded RNA binding	IDA PMID:19966796 ATP-dependent human RISC assembly pathways.	MARK AS OVER ANNOTATED	Summary: single-stranded RNA binding is too generic for AGO4. Reason: The informative molecular function is Argonaute small-RNA/miRNA binding within RISC rather than a broad nucleic-acid or RNA-binding label. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0016442 RISC complex	IDA PMID:19966796 ATP-dependent human RISC assembly pathways.	ACCEPT	Summary: RISC complex is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0070578 RISC-loading complex	IDA PMID:19966796 ATP-dependent human RISC assembly pathways.	ACCEPT	Summary: RISC-loading complex is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0070922 RISC complex assembly	IDA PMID:19966796 ATP-dependent human RISC assembly pathways.	ACCEPT	Summary: RISC complex assembly is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0090625 siRNA-mediated gene silencing by mRNA destabilization	IDA NOT PMID:15260970 Human Argonaute2 mediates RNA cleavage targeted by miRNAs an...	ACCEPT	Summary: This NOT annotation is appropriate; AGO4 is not the Argonaute paralog that mediates siRNA-guided target mRNA cleavage/destabilization in this study. Reason: PMID:15260970 supports AGO4 association with small RNAs/RISC but shows that endonucleolytic target cleavage activity is associated with AGO2, not AGO4. AGO4's supported role should be distinguished from this negated AGO2-like target-cleavage annotation. Supporting Evidence: PMID:15260970 Purification of the FLAG/HA-epitope-tagged Ago containing complexes from different human cell lines revealed that endonuclease activity is exclusively associated with Ago2. file:human/AGO4/AGO4-deep-research-falcon.md A long-standing view is that among the four human paralogs, AGO2 is the canonical slicer (endonuclease), while AGO1/AGO3/AGO4 primarily act through non-slicing mechanisms (translational repression, deadenylation).
GO:0005737 cytoplasm	IDA PMID:15260970 Human Argonaute2 mediates RNA cleavage targeted by miRNAs an...	ACCEPT	Summary: cytoplasm is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0016442 RISC complex	IDA PMID:15260970 Human Argonaute2 mediates RNA cleavage targeted by miRNAs an...	ACCEPT	Summary: RISC complex is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0035198 miRNA binding	IDA PMID:15260970 Human Argonaute2 mediates RNA cleavage targeted by miRNAs an...	ACCEPT	Summary: miRNA binding is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0035279 miRNA-mediated gene silencing by mRNA destabilization	IDA NOT PMID:15260970 Human Argonaute2 mediates RNA cleavage targeted by miRNAs an...	ACCEPT	Summary: This NOT annotation is appropriate; PMID:15260970 does not support assigning AGO4 to AGO2-like miRNA-guided target mRNA cleavage/destabilization. Reason: AGO4 binds miRNAs and participates in RISC, but the cited work distinguishes that non-slicer role from AGO2-dependent target RNA cleavage. The NOT annotation should be retained rather than converted into a positive AGO4 mRNA-destabilization function. Supporting Evidence: PMID:15260970 The specific role of Ago2 in guiding target RNA cleavage was confirmed independently by siRNA-based depletion of individual Ago members in combination with a sensitive positive-readout reporter assay. file:human/AGO4/AGO4-deep-research-falcon.md A long-standing view is that among the four human paralogs, AGO2 is the canonical slicer (endonuclease), while AGO1/AGO3/AGO4 primarily act through non-slicing mechanisms (translational repression, deadenylation).
GO:0016020 membrane	HDA PMID:19946888 Defining the membrane proteome of NK cells.	KEEP AS NON CORE	Summary: membrane is a plausible or reported location/context but is not the core functional site annotation for AGO4. Reason: The core location is cytoplasmic RISC/RNP granules; nuclear, membrane, synaptic, exosomal, or other compartment annotations should remain non-core unless tied to a specific curated mechanism. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-1606561	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-1606682	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-1912362	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-1912363	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-1912364	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-1912366	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-1912367	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-1912368	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-1912406	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-2318752	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-3209151	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-426489	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-426522	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-4518575	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-5687103	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-8935766	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-8937097	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-8937134	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-8938440	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-8938487	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-8938507	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-8944650	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-8944684	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-8944706	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-9011958	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-9012203	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-9012208	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-9038545	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-9618392	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-9618486	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-9624925	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-9657791	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-9858289	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-9858309	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-9924921	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-9925095	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-9925158	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-9925159	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0005829 cytosol	TAS Reactome:R-HSA-9925324	ACCEPT	Summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0006402 mRNA catabolic process	IDA PMID:18771919 Importance of translation and nonnucleolytic ago proteins fo...	KEEP AS NON CORE	Summary: mRNA catabolic process is downstream or context-specific for AGO4. Reason: This process may be supported in a particular experiment or pathway model, but it is peripheral to the core Argonaute/RISC molecular role. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms
GO:0035278 miRNA-mediated gene silencing by inhibition of translation	IDA PMID:18771919 Importance of translation and nonnucleolytic ago proteins fo...	ACCEPT	Summary: miRNA-mediated gene silencing by inhibition of translation is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector. Reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression and related RISC complexes or cytoplasmic RNP compartments. Supporting Evidence: file:human/AGO4/AGO4-deep-research-falcon.md Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms

Core Functions

Small-RNA-loaded Argonaute/RISC effector activity, primarily through non-slicing repression mechanisms including translational control.

Molecular Function:

miRNA binding

Directly Involved In:

regulatory ncRNA-mediated post-transcriptional gene silencing miRNA-mediated gene silencing by inhibition of translation

Cellular Locations:

cytoplasm P-body

In Complex:

RISC complex

Supporting Evidence:

file:human/AGO4/AGO4-deep-research-falcon.md
Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene regulation largely through **non-slicing mechanisms**

References

GO_REF:0000002

Gene Ontology annotation through association of InterPro records with GO terms

GO_REF:0000033

Annotation inferences using phylogenetic trees

GO_REF:0000052

Gene Ontology annotation based on curation of immunofluorescence data

GO_REF:0000107

Automatic transfer of experimentally verified manual GO annotation data to orthologs using Ensembl Compara

GO_REF:0000117

Electronic Gene Ontology annotations created by ARBA machine learning models

GO_REF:0000120

Combined Automated Annotation using Multiple IEA Methods

PMID:15260970

Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs.

PMID:15766526

Involvement of microRNA in AU-rich element-mediated mRNA instability.

PMID:18771919

Importance of translation and nonnucleolytic ago proteins for on-target RNA interference.

PMID:19167051

Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.

PMID:19324964

The C-terminal half of human Ago2 binds to multiple GW-rich regions of GW182 and requires GW182 to mediate silencing.

PMID:19383768

The C-terminal domains of human TNRC6A, TNRC6B, and TNRC6C silence bound transcripts independently of Argonaute proteins.

PMID:19946888

Defining the membrane proteome of NK cells.

PMID:19966796

ATP-dependent human RISC assembly pathways.

PMID:22795694

Slicing-independent RISC activation requires the argonaute PAZ domain.

PMID:28781232

Multivalent Recruitment of Human Argonaute by GW182.

PMID:33961781

Dual proteome-scale networks reveal cell-specific remodeling of the human interactome.

Reactome:R-HSA-1606561

MIR449 microRNAs bind 3'UTR of NOTCH1 mRNA

Reactome:R-HSA-1606682

MIR34 microRNAs bind 3'UTR of NOTCH1 mRNA

Reactome:R-HSA-1912362

MIR150 microRNA binds 3'UTR of NOTCH3 mRNA

Reactome:R-HSA-1912363

MIR200B/C microRNAs bind NOTCH1 mRNA

Reactome:R-HSA-1912364

MIR181C microRNA binds 3'UTR of NOTCH4 mRNA

Reactome:R-HSA-1912366

MIR206 microRNA binds 3'UTR of NOTCH3 mRNA

Reactome:R-HSA-1912367

MIR34 microRNAs bind 3'UTR of NOTCH2 mRNA

Reactome:R-HSA-1912368

MIR302A microRNA binds 3'UTR of NOTCH4 mRNA

Reactome:R-HSA-1912406

p53 positively regulates transcription of MIR34 microRNAs

Reactome:R-HSA-2318752

miR-26A microRNAs bind PTEN mRNA

Reactome:R-HSA-3209151

miR-24 binds p16INK4A and p14ARF mRNAs

Reactome:R-HSA-426489

RISC binds inexactly matching target RNAs

Reactome:R-HSA-426522

Nonendonucleolytic RISC binds exactly matching target RNAs

Reactome:R-HSA-4518575

miR-92b binds 3'UTR of NLK mRNA

Reactome:R-HSA-5687103

FOXO3 regulates MIR34B,C expression

Reactome:R-HSA-8935766

miR-378 binds RUNX1 mRNA

Reactome:R-HSA-8937097

MIR27A gene transcription is stimulated by the complex containing RUNX1, PRMT1 and GATA1 and inhibited by the complex of RUNX1, SIN3A and PRMT6

Reactome:R-HSA-8937134

miR-27a binds RUNX1 mRNA

Reactome:R-HSA-8938440

miR-17 binds RUNX1 mRNA

Reactome:R-HSA-8938487

miR-20a binds RUNX1 mRNA

Reactome:R-HSA-8938507

miR-106a binds RUNX1 mRNA

Reactome:R-HSA-8944650

miR-214 microRNA binds PTEN mRNA

Reactome:R-HSA-8944684

miR-205 microRNA binds PTEN mRNA

Reactome:R-HSA-8944706

miR-21 nonendonucleolytic RISC binds PTEN mRNA

Reactome:R-HSA-9011958

miR-26A and B bind to the 3'UTR of the GREB1 mRNA

Reactome:R-HSA-9012203

miR-26A and B bind to the 3'UTR of the CHD11 mRNA

Reactome:R-HSA-9012208

miR-26A and B bind to the 3'UTR of the KPNA2 mRNA

Reactome:R-HSA-9038545

miR-613 binds to the 3'UTR of the NR1H3 mRNA

Reactome:R-HSA-9618392

miR-26 binds to the 3'UTR of the ARL4C mRNA

Reactome:R-HSA-9618486

miR-26 binds to the 3'UTR of the ABCA1 mRNA

Reactome:R-HSA-9624925

miR-33 binds to the 3'UTR of the ABCA1 mRNA

Reactome:R-HSA-9657791

miR-144 binds to the 3'UTR of the ABCA1 mRNA

Reactome:R-HSA-9858289

MITF-M-dependent miR-211 expression

Reactome:R-HSA-9858309

miR-211 RISC binds POU3F2 mRNA

Reactome:R-HSA-9924921

CD274 mRNA binds miR-34 RISC

Reactome:R-HSA-9925095

CD274 mRNA binds miR-340 RISC

Reactome:R-HSA-9925158

CD274 mRNA binds miR-152 RISC

Reactome:R-HSA-9925159

CD274 mRNA binds miR-140 RISC

Reactome:R-HSA-9925324

CD274 mRNA binds miR-200B/C RISC

file:human/AGO4/AGO4-uniprot.txt

UniProt text export for AGO4

UniProt identifies AGO4 and its protein family/domain context.

"Belongs to the argonaute family. Ago subfamily."

file:human/AGO4/AGO4-goa.tsv

GOA annotation export for AGO4

GOA records the phylogenetic IBA source for the AGO4 GO:0004521 annotation.

"UniProtKB Q9HCK5 AGO4 enables GO:0004521 RNA endonuclease activity molecular_function ECO:0000318 IBA GO_REF:0000033 FB:FBgn0000146|FB:FBgn0004872|FB:FBgn0087035|FB:FBgn0250816|FB:FBgn0262739|MGI:MGI:1928897|MGI:MGI:1930036|MGI:MGI:2446632|PANTHER:PTN008584027|PomBase:SPCC736.11|TAIR:locus:2007760|TAIR:locus:2059258|UniProtKB:O67434|UniProtKB:Q9H9G7|UniProtKB:Q9UKV8|WB:WBGene00000105|WB:WBGene00000106|WB:WBGene00004323|WB:WBGene00017641 9606 Homo sapiens GO_Central Protein argonaute-4 20241120"

file:human/AGO4/AGO4-deep-research-falcon.md

Falcon deep research report for AGO4

Falcon research supports the reviewed core function of AGO4.

"Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene regulation largely through **non-slicing mechanisms**"

file:human/AGO4/AGO4-hypotheses/function-hypothesis-go-0004521/openscientist.md

OpenScientist hypothesis report for AGO4 GO:0004521

OpenScientist refutes RNA endonuclease activity for AGO4.

"Human AGO4 does **not** have RNA endonuclease (slicer) activity."

Suggested Experiments

Experiment: Endogenous tagging of AGO4 followed by small-RNA CLIP, target RNA profiling, and paralog-specific rescue in AGO-depleted human cells.

Hypothesis: AGO4 has paralog-specific guide, target, and compartment preferences within human RISC biology.

Type: genome editing/RNA-protein interaction profiling

Experiment: Biochemical reconstitution of AGO4-RISC with representative miRNA and siRNA guides to compare target repression, target cleavage, and TNRC6 recruitment.

Hypothesis: AGO4 activity is best explained by guide-loaded RISC function rather than generic RNA or protein binding.

Type: biochemical reconstitution

Deep Research

Falcon

(AGO4-deep-research-falcon.md)

Comprehensive research report: Human **AGO4 / EIF2C4** (UniProt **Q9HCK5**) Falcon Edison Scientific Literature 39 citations 2026-05-02T23:13:05.857366

The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.

You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.

We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.

We are interested in where in or outside the cell the gene product carries out its function.

We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.

Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.

Comprehensive research report: Human AGO4 / EIF2C4 (UniProt Q9HCK5)

0) Target identity verification (critical disambiguation)

The target is human Argonaute-4 (gene AGO4, synonym EIF2C4) in the Argonaute Ago subfamily. Mammalian literature explicitly links Ago4 ↔ Eif2c4 and treats it as one of four mammalian Argonaute paralogs (AGO1–AGO4) with a PIWI domain (RNase H-like fold) and the canonical Argonaute domain architecture. (adiliaghdam2020arequirementfor pages 1-3)

Important ambiguity note: “AGO4” is heavily used in plants to mean a different Argonaute with prominent nuclear RNA-directed DNA methylation roles; plant AGO literature should not be used as evidence for human AGO4. A retrieved plant AGO paper is therefore treated only as general mechanistic background on Argonaute slicing, not as AGO4(Hs) functional evidence. (arribashernandez2016thesliceractivity pages 1-4)

1) Key concepts and definitions (current understanding)

1.1 RISC and Argonaute core function

Argonaute proteins are small-RNA–binding proteins that form the catalytic/effector core of the RNA-induced silencing complex (RISC). In canonical post-transcriptional gene regulation, miRNA/siRNA duplexes are loaded onto an Argonaute; the passenger strand is removed and the guide strand remains to direct sequence-specific recognition of RNA targets. (nakanishi2016anatomyofrisc pages 1-2, nakanishi2022anatomyoffour pages 1-2)

RISC-mediated repression commonly proceeds without slicing (cleavage) and instead uses recruitment of TNRC6/GW182 and downstream deadenylation/decay machinery (e.g., CCR4–NOT) to drive translational repression and mRNA destabilization. (nakanishi2016anatomyofrisc pages 1-2, wang2024theguidernasequence pages 1-3)

1.2 Domain architecture of AGO4

Human AGO proteins share signature domains N, PAZ, MID, PIWI (plus linkers L1/L2 in long AGOs). PAZ and MID anchor the guide RNA ends, while the PIWI domain has an RNase H fold that can support endonucleolytic slicing in slicer-competent Argonautes. (pantazopoulou2020fromtheargonauts pages 1-3, nakanishi2016anatomyofrisc pages 1-2, carvalho2019studyofhuman pages 27-29)

1.3 Slicer vs non-slicer AGOs in humans

A long-standing view is that among the four human paralogs, AGO2 is the canonical “slicer” (endonuclease), while AGO1/AGO3/AGO4 primarily act through non-slicing mechanisms (translational repression, deadenylation). This remains the dominant functional framing even as paralog-specific exceptions have emerged (e.g., guide-dependent slicing by AGO3). (park2017humanargonaute3has pages 1-1, nakanishi2022anatomyoffour pages 1-2)

In practice, a key mechanistic point is that even non-slicer AGOs (including AGO4) can assemble RISCs and perform strand separation/unwinding in a slicer-independent manner under physiological conditions, explaining why small RNAs can distribute across all four human AGOs. (nakanishi2022anatomyoffour pages 1-2)

2) AGO4 molecular function: what it does (and what it does not)

2.1 Small-RNA binding and RISC assembly

AGO4 is part of the human Argonaute set (AGO1–4) that binds small RNAs to form RISCs. General RISC assembly principles include chaperone-assisted loading (Hsc70/Hsp90), passenger ejection, and guide segmentation (seed/central/supplementary/tail). (nakanishi2016anatomyofrisc pages 1-2, nakanishi2022anatomyoffour pages 1-2)

2.2 Catalysis (slicing) is not established as AGO4’s primary physiological mechanism

Although Argonaute proteins possess a PIWI RNase H-like fold, the strongest physiological evidence in the retrieved corpus indicates AGO4 functions in antiviral defense and translation regulation without demonstrating canonical slicing as the key mechanism.

In the antiviral context, Adiliaghdam et al. report that mutating predicted AGO4 catalytic residues did not abolish AGO4-mediated influenza repression, and that exogenous siRNA antiviral effects required AGO2 (not AGO4), arguing against AGO4 acting mainly through classical slicing. (adiliaghdam2020arequirementfor pages 7-8)

Expert synthesis (2024) also emphasizes that in animals, most miRNA repression is slicer-independent and that only some paralogs are robust slicers, with ongoing work refining when slicing is deployed. (nakanishi2024whenargonautetakes pages 1-3)

2.3 Non-canonical translational repression: AGO4 and IRES-dependent translation

A distinct functional specialization highlighted for human AGO4 is repression of IRES-driven translation in a specific bicistronic mRNA context.

Nakanishi’s review of the four human Argonautes summarizes that AGO4 loaded with miR-3191–5p binds the IRES of CACNA1A bicistronic mRNA and inhibits recruitment of initiation factors eIF4AII and eIF4GII. In the cited example, both AGO2 and AGO4 load miR-3191–5p, but only AGO4-RISC suppresses the IRES-driven translation arm. (nakanishi2022anatomyoffour pages 8-9, nakanishi2022anatomyoffour pages 9-10)

This links AGO4 to translation initiation control rather than the canonical miRNA model focused on 3′UTR seed targeting. (nakanishi2022anatomyoffour pages 8-9)

3) Biological processes and pathways involving AGO4

3.1 Mammalian antiviral defense (innate immunity intersection)

The strongest primary evidence for a nonredundant AGO4 biological role in the retrieved corpus is mammalian antiviral defense.

Adiliaghdam et al. (Cell Reports, Feb 11 2020, https://doi.org/10.1016/j.celrep.2020.01.021) show:
- Cellular phenotypes: Ago4-deficient macrophages/dendritic cells/MEFs show increased susceptibility to multiple RNA viruses and higher viral readouts. (adiliaghdam2020arequirementfor pages 4-5, adiliaghdam2020arequirementfor pages 8-10)
- IFN axis modulation: Ago4 deficiency reduces IFN-β induction, but antiviral restriction by AGO4 also persists when IFN signaling is blocked, implying IFN-dependent and IFN-independent components. (adiliaghdam2020arequirementfor pages 4-5, adiliaghdam2020arequirementfor pages 1-3)
- RLR/MAVS additivity: AGO4 and MAVS contribute independently/additively; double deficiency increases virus levels more than either single deficiency. (adiliaghdam2020arequirementfor pages 6-7)
- Small-RNA signatures: AGO-loaded influenza-derived RNAs (including canonical 21–23 nt vsiRNA-like species) are diminished in Ago4-deficient cells, supporting a role for AGO4 in generating/stabilizing AGO–viral RNA complexes in antiviral RNAi-like responses. (adiliaghdam2020arequirementfor pages 1-3, adiliaghdam2020arequirementfor pages 7-8)

This places AGO4 at an interface between small-RNA biology and innate antiviral pathways, a topic with active debate in mammalian systems.

3.2 Current expert framing (2023–2024): antiviral RNAi remains context-dependent

A 2023 expert review on mammalian antiviral RNAi highlights that antiviral RNAi can operate in certain mammalian contexts and that its importance in differentiated cells has been debated, in part due to mutual inhibition between interferon and antiviral RNAi. It emphasizes the need for more in vivo models and cites developments such as probing antiviral RNAi with inhibitors of viral suppressors of RNAi and detecting vsiRNAs in extracellular vesicles in serum of infected mice. (anobile2023rnainterferencean pages 1-6)

Together with the 2020 AGO4 knockout evidence, this supports a “context-dependent antiviral RNAi” view rather than a universally dominant pathway in all mammalian cells.

3.3 Putative links to epigenetic/chromatin pathways

Nakanishi (2022) summarizes that N-terminal regions of AGO4 serve as a DNMT3A-binding site, suggesting a physical connection between AGO4 and DNA methylation machinery (and therefore potential nuclear/epigenetic roles). (nakanishi2022anatomyoffour pages 9-10)

This is not yet equivalent to a fully mapped AGO4-dependent epigenetic pathway in human cells, but it is a concrete interaction hypothesis that aligns with non-canonical AGO functions being explored across paralogs. (nakanishi2022anatomyoffour pages 9-10)

4) Subcellular localization (where AGO4 acts)

4.1 Cytoplasmic miRISC-associated localization (inferred)

Human AGO4 participates in RISC biology that is widely considered cytoplasmic in canonical miRNA repression models, and Nakanishi (2022) lists AGO4 interactions with TNRC6A and DDX6, factors strongly associated with cytoplasmic miRISC effector function and processing bodies (P-bodies). This supports cytoplasmic miRISC/P-body-associated localization as a plausible site of action. (nakanishi2022anatomyoffour pages 9-10)

Additionally, AGO4’s summarized role in regulating translation initiation factor recruitment to an IRES is mechanistically consistent with cytoplasmic translation regulation. (nakanishi2022anatomyoffour pages 8-9)

4.2 Nuclear germline localization: evidence is primarily mouse

A frequently cited AGO4 nuclear localization is in male germ cells during meiosis, with accumulation in the sex body of pachytene spermatocytes. In the retrieved corpus, this localization evidence is mouse-focused (and includes a non-peer-reviewed preprint-like source), so it should be interpreted as mammalian (not human-somatic) localization evidence. (pantazopoulou2020fromtheargonauts pages 14-16, hilz2016elucidatingrolesfor pages 137-143)

5) Disease relevance and associations

5.1 Influenza susceptibility (in vivo functional evidence)

In vivo, Ago4−/− mice infected with influenza show more severe disease phenotypes including increased weight loss and higher viral burden metrics (BALF titers, lung viral RNA), providing disease-relevant evidence that AGO4 contributes to influenza control. (adiliaghdam2020arequirementfor pages 8-10, adiliaghdam2020arequirementfor media 33974f64)

5.2 Neurological disease relevance through AGO4-regulated IRES translation target

The AGO4-specific IRES regulation described above is linked to spinocerebellar ataxia type 6 (SCA6) through the CACNA1A bicistronic transcript and its polyglutamine repeat context, as summarized in the Nakanishi 2022 review (this is mechanistic/target-linked relevance, not necessarily genetic association of AGO4 variants with SCA6). (nakanishi2022anatomyoffour pages 8-9, nakanishi2022anatomyoffour pages 9-10)

5.3 Human disease association statistics (database-level, hypothesis generating)

Open Targets (target ENSG00000134698; https://platform.opentargets.org/target/ENSG00000134698) lists AGO4 associations with azoospermia, influenza, cancer, schizophrenia, and partial chromosome Y deletion, each shown with evidence counts (5 in the retrieved snapshot) and modest association scores. These are aggregated association signals rather than causal proof and should be used to prioritize follow-up. (adiliaghdam2020arequirementfor media 33974f64)

6) Recent developments (prioritizing 2023–2024)

6.1 2024 structural/mechanistic synthesis of slicing and guide rules (context)

A 2024 JBC review reframes Argonaute slicing as essential in specific vertebrate contexts and emphasizes distinctions in catalytic activation mechanisms and target specificity rules, consolidating modern structural interpretation of how Argonautes decide between slicing vs non-slicing outcomes. This advances the conceptual background for interpreting paralog specialization including AGO4. (nakanishi2024whenargonautetakes pages 1-3)

6.2 2023 debate and methodological advances in mammalian antiviral RNAi

A 2023 review highlights that antiviral RNAi in mammals is debated, and points to in vivo approaches and tools (e.g., inhibitors of viral suppressors of RNAi; extracellular vesicle vsiRNA detection) as enabling technologies for resolving the contribution of antiviral RNAi at the organism level. (anobile2023rnainterferencean pages 1-6)

6.3 2024 quantitative kinetics for slicing (AGO2-focused, informs tool design)

A 2024 preprint (bioRxiv, June 20 2024; https://doi.org/10.1101/2023.10.15.562437) reports that guide sequence can alter slicing rates by >250-fold, and mismatch tolerance by ~600-fold, with implications for RNAi reagent potency. This is primarily AGO2 research and thus indirectly relevant to AGO4 (context for slicer vs non-slicer specialization and for applications), rather than direct AGO4 evidence. (wang2024theguidernasequence pages 1-3)

7) Applications and real-world implementations

7.1 RNAi therapeutics and the importance of Argonaute rules (context)

Argonaute-mediated slicing is central to synthetic siRNA knockdown and therapeutic RNAi; thus, mechanistic advances (guide sequence effects on slicing kinetics and mismatch tolerance) inform siRNA design and predictability of knockdown. Although AGO4 is not the canonical slicer used for therapeutic siRNAs, understanding paralog behavior and RISC assembly constraints remains important to avoid off-target or context-specific effects. (wang2024theguidernasequence pages 1-3, nakanishi2016anatomyofrisc pages 1-2)

7.2 Antiviral strategies (conceptual)

The AGO4 requirement in influenza control (in vivo) supports the idea that tuning the balance between interferon-based immunity and small-RNA–based restriction could be therapeutically relevant, but the field remains cautious because mammalian antiviral RNAi is context-dependent and debated. (adiliaghdam2020arequirementfor pages 8-10, anobile2023rnainterferencean pages 1-6)

8) Statistics and quantitative data (from retrieved sources)

8.1 Study design and significance reporting for AGO4 antiviral phenotypes

Adiliaghdam et al. report experiments performed as three biological replicates, each in triplicate, with SEM error bars and standard significance thresholds (p<0.05, <0.01, <0.001) using unpaired t-tests or ANOVA with Bonferroni correction. Multiple quantitative endpoints are used, including viral titers by plaque assay, viral RNA by qPCR, and Flu-GFP infection percentages in flow cytometry. (adiliaghdam2020arequirementfor pages 6-7, adiliaghdam2020arequirementfor pages 5-6)

Because the numerical titer values and fold-changes are presented graphically, precise fold-change extraction requires full numeric table access; however, the directionality and significance are clear, and figure panels explicitly show higher viral load in Ago4−/− mice. (adiliaghdam2020arequirementfor media 33974f64)

8.2 Database association statistics

Open Targets association snapshot provides evidence counts (n=5 evidences) and association scores (e.g., cancer score ~0.466; influenza score ~0.062; azoospermia score ~0.092 in the retrieved JSON) for AGO4, useful for prioritization but not causal inference. (adiliaghdam2020arequirementfor media 33974f64)

9) Evidence summary table (curated)

Functional aspect	Key findings	Evidence type (review vs primary; assay type)	Source (first author year journal)	URL	Pub date
Molecular function	Human AGO4 is one of four AGO paralogs in RISC; AGOs bind small RNAs, retain the guide strand, and recruit silencing machinery. RISC loading requires Hsc70/Hsp90 chaperones; AGO architecture includes N, PAZ, MID, and PIWI domains. Relevant as foundational context for AGO4 function, but not AGO4-specific biochemical proof. (nakanishi2016anatomyofrisc pages 1-2, nakanishi2022anatomyoffour pages 1-2)	Review; structural/mechanistic synthesis of RISC assembly and chaperone-assisted loading	Nakanishi 2016 WIREs RNA	https://doi.org/10.1002/wrna.1356	May 2016
Molecular function / catalytic status	Comparative review of the four human AGOs: most miRNAs are shared across paralogs; AGO2 is the best-established slicer, AGO3 can be slicer-competent under some conditions, whereas AGO4 is generally treated as non-canonical/non-classical slicer in most functional settings. Also summarizes AGO4-specific insertion/features and paralog differences. (nakanishi2022anatomyoffour pages 1-2, nakanishi2024whenargonautetakes pages 12-13)	Review; comparative structural and mechanistic analysis	Nakanishi 2024 J Biol Chem	https://doi.org/10.1016/j.jbc.2023.105499	Jan 2024
Molecular function / small-RNA loading	Human non-slicer AGOs, including AGO4, can undergo slicer-independent strand separation during RISC assembly at physiological temperature; helps explain why siRNAs/miRNAs can be found across all four human AGOs. (nakanishi2022anatomyoffour pages 1-2)	Primary; biochemical RISC assembly/unwinding assays	Park 2015 Nucleic Acids Res	https://doi.org/10.1093/nar/gkv937	Sep 2015
Molecular function / catalytic status	AGO3 was shown to have guide-dependent slicer activity, whereas AGO1 and AGO4 were described as slicer-independent effectors that typically mediate translational repression/deadenylation rather than target cleavage. Useful negative comparator for AGO4. (park2017humanargonaute3has pages 1-1)	Primary; recombinant AGO cleavage assays and structure	Park 2017 Nucleic Acids Res	https://doi.org/10.1093/nar/gkx916	Oct 2017
Molecular function / translation control	AGO4 loads miR-3191-5p and, unlike AGO2 in the cited example, AGO4-RISC represses IRES-driven translation of CACNA1A bicistronic mRNA by interfering with initiation-factor recruitment. This supports a non-canonical translational repression role for human AGO4. (nakanishi2022anatomyoffour pages 9-10, nakanishi2022anatomyoffour pages 8-9)	Review summarizing primary functional studies; translational regulation evidence	Nakanishi 2022 Nucleic Acids Res	https://doi.org/10.1093/nar/gkac519	Jun 2022
Pathway or process / chromatin-linked interaction	Nakanishi 2022 also notes the N-terminal region of AGO4 as a DNMT3A-binding site and lists interactions with TNRC6A and DDX6, linking AGO4 to both chromatin-related and canonical miRISC/P-body-associated effector networks. (nakanishi2022anatomyoffour pages 9-10)	Review summarizing structural/interaction studies	Nakanishi 2022 Nucleic Acids Res	https://doi.org/10.1093/nar/gkac519	Jun 2022
Pathway or process / antiviral defense	AGO4 has a nonredundant antiviral role in mammalian innate immunity: Ago4-deficient immune cells are hyper-susceptible to RNA viruses; AGO4 promotes IFN-β responses but also restricts virus when IFN signaling is blocked, supporting both IFN-dependent and IFN-independent activity. AGO4 loss reduces AGO-loaded viral small RNAs/vsiRNAs. (adiliaghdam2020arequirementfor pages 1-3, adiliaghdam2020arequirementfor pages 4-5)	Primary; knockout macrophages/DCs/MEFs, viral infection assays, qPCR, plaque assays, small-RNA profiling	Adiliaghdam 2020 Cell Reports	https://doi.org/10.1016/j.celrep.2020.01.021	Feb 2020
Disease relevance / in vivo phenotype	In influenza-infected mice, Ago4 deficiency worsens disease with greater weight loss, higher BALF viral titers, higher lung viral RNA/protein, and worse pathology, supporting physiological antiviral importance. Figure-based evidence available. (adiliaghdam2020arequirementfor pages 8-10, adiliaghdam2020arequirementfor media 33974f64)	Primary; mouse knockout infection model, plaque assay, pathology, in vivo viral burden	Adiliaghdam 2020 Cell Reports	https://doi.org/10.1016/j.celrep.2020.01.021	Feb 2020
Localization	Human-focused sources imply cytoplasmic/P-body-associated and possibly nuclear-linked functions through interactions with TNRC6A/DDX6 and DNMT3A, but the cited Nakanishi 2022 excerpt does not itself show direct microscopy/fractionation for human AGO4 localization. (nakanishi2022anatomyoffour pages 9-10)	Review; indirect interaction-based localization inference	Nakanishi 2022 Nucleic Acids Res	https://doi.org/10.1093/nar/gkac519	Jun 2022
Localization / notes on species	AGO4 has been reported to localize to the nucleus/sex body of pachytene spermatocytes during meiotic prophase I in mammals, supporting roles in meiotic sex chromosome silencing; however, this evidence is from mouse germline studies and should not be overextended to general human somatic localization. (pantazopoulou2020fromtheargonauts pages 14-16, hilz2016elucidatingrolesfor pages 137-143)	Review plus primary microscopy in mouse germ cells; immunofluorescence/chromosome spreads	Pantazopoulou 2020 Int J Mol Sci	https://doi.org/10.3390/ijms21114007	Jun 2020
Notes on species / target verification	Human AGO4 belongs to the AGO subfamily with canonical N/MID/PAZ/PIWI organization and high homology to other human AGOs. Some AGO4 literature refers to mouse germline or plant AGO systems; those data are mechanistically informative but not direct evidence for human AGO4 biology. (adiliaghdam2020arequirementfor pages 1-3, pantazopoulou2020fromtheargonauts pages 1-3, nakanishi2022anatomyoffour pages 1-2)	Mixed review/primary context; species-disambiguation note	Pantazopoulou 2020 Int J Mol Sci; Adiliaghdam 2020 Cell Reports	https://doi.org/10.3390/ijms21114007 ; https://doi.org/10.1016/j.celrep.2020.01.021	Jun 2020; Feb 2020
Disease relevance / database context	Open Targets lists AGO4 associations with azoospermia, influenza, cancer, schizophrenia, and partial chromosome Y deletion. These are database-level associations and should be treated as hypothesis-generating context rather than direct proof of causal human AGO4 function in each disease. (adiliaghdam2020arequirementfor media 33974f64)	Curated database context; target–disease association aggregation	Open Targets Platform (AGO4 target page context)	https://platform.opentargets.org/target/ENSG00000134698	Accessed in current tool session

Table: This table summarizes curated literature and database evidence relevant to functional annotation of human AGO4/EIF2C4 (UniProt Q9HCK5). It distinguishes direct primary findings from review-level context and flags mouse-only or database-only evidence to avoid overclaiming.

10) Key figure evidence (in vivo antiviral phenotype)

A cropped figure from Adiliaghdam et al. shows increased weight loss and higher influenza burden readouts in Ago4−/− mice during infection, supporting AGO4’s physiological role in antiviral defense. (adiliaghdam2020arequirementfor media 33974f64)

11) Conclusions (expert synthesis)

Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved literature set) as a small-RNA-binding Argonaute paralog that contributes to gene regulation largely through non-slicing mechanisms, with two prominent, evidence-backed functional themes: (i) non-canonical translational repression in an IRES-dependent context (CACNA1A, miR-3191–5p) and (ii) a nonredundant antiviral role in mammals, integrating with (but not reducible to) interferon pathways and associated with AGO-loaded viral small RNAs. (nakanishi2022anatomyoffour pages 8-9, adiliaghdam2020arequirementfor pages 1-3)

The most current (2023–2024) authoritative sources primarily provide mechanistic synthesis and highlight active controversies and enabling methods in mammalian antiviral RNAi; comparatively fewer 2023–2024 primary studies directly dissect human AGO4 in diverse tissues, leaving AGO4 specialization an important target for further focused experimentation. (nakanishi2024whenargonautetakes pages 1-3, anobile2023rnainterferencean pages 1-6)

References

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(pantazopoulou2020fromtheargonauts pages 1-3): Vasiliki I. Pantazopoulou, Stella Georgiou, Panos Kakoulidis, Stavroula N. Giannakopoulou, Sofia Tseleni, Dimitrios J. Stravopodis, and Ema Anastasiadou. From the argonauts mythological sailors to the argonautes rna-silencing navigators: their emerging roles in human-cell pathologies. International Journal of Molecular Sciences, 21:4007, Jun 2020. URL: https://doi.org/10.3390/ijms21114007, doi:10.3390/ijms21114007. This article has 23 citations.
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Citations

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https://doi.org/10.7298/x4zc80tj,

OpenScientist

(AGO4-hypotheses/function-hypothesis-go-0004521/openscientist.md)

AGO4 RNA Endonuclease Activity (GO:0004521) — Hypothesis Deep Research Report OpenScientist openscientist-autonomous 4 citations 3 artifacts 2026-07-06T16:57:13.562799 citations file

OpenScientist final report (text/html)

OpenScientist final report (application/pdf)

OpenScientist evidence matrix (text/csv)

AGO4 RNA Endonuclease Activity (GO:0004521) — Hypothesis Deep Research Report

Target gene: AGO4 (Argonaute-4), Homo sapiens (NCBITaxon:9606)
UniProt: Q9HCK5 · Term: GO:0004521 "RNA endonuclease activity" · Evidence: IBA (GO_REF:0000033)
Focus type: function_assignment — does AGO4 directly have RNA-endonuclease (slicer) activity?

Summary

The IBA-propagated annotation of RNA endonuclease activity (GO:0004521) to human AGO4 is refuted / over-annotated and should be removed or negatively qualified. Argonaute "slicer" activity is a metal-dependent, RNase-H-like reaction carried out by the PIWI domain, and it strictly requires an intact catalytic tetrad of four residues (the DEDH tetrad: Asp-Glu-Asp-His) that coordinate two catalytic Mg²⁺ ions. Direct sequence analysis anchored on the experimentally validated human AGO2 catalytic residues (D597/E637/D669/H807) shows that human AGO4 has two of the four catalytic residues non-conservatively substituted: the catalytic aspartate (AGO2 Asp669) is a glycine (Gly671) in AGO4, and the catalytic histidine (AGO2 His807) is an arginine (Arg809). Loss of either residue is sufficient to abolish catalysis; AGO4 has lost both. AGO4 therefore cannot perform RNA-guided endonucleolytic cleavage.

This computational conclusion is independently corroborated by database curation and primary literature. UniProt annotates catalytic Mg²⁺-coordinating binding sites for AGO2 (597/669/807) and AGO3 (598/638/670/808) but none for AGO4 (or AGO1). The primary RNAi literature consistently establishes that among the four human paralogs only AGO2 is a constitutive slicer, with AGO3 added later as a conditional slicer; AGO1, AGO3, and AGO4 were classically defined as slicer-independent effectors, and non-AGO2 paralogs had to be engineered to become catalytically active. AGO4's genuine molecular role is as a slicer-independent miRNA effector that silences targets by recruiting the TNRC6/GW182 machinery, which drives translational repression and deadenylation-coupled mRNA decay — a mechanism that does not require endonucleolytic cleavage.

The single important caveat: the IBA annotation is technically defensible in the narrow phylogenetic sense that AGO4 descends from an ancestral Argonaute that had endonuclease activity and still adopts the PIWI fold. But GO annotation should reflect the actual molecular function of the specific gene product. By that standard AGO4 is a pseudo-catalytic, structurally slicer-dead paralog, and the annotation is a classic case of paralog over-annotation via phylogenetic propagation from the active AGO2 lineage.

Key Findings

Finding 1 — AGO4 lacks the intact DEDH catalytic tetrad required for slicer activity

The defining requirement for Argonaute endonuclease/slicer activity is a catalytic tetrad arranged within the RNase-H-like PIWI domain that together coordinate two catalytic Mg²⁺ ions and perform phosphodiester-bond hydrolysis of the target RNA. In human AGO2 — the one mammalian Argonaute unambiguously demonstrated to be an active slicer — these residues are Asp597, Glu637, Asp669, and His807 (the "DEDH" tetrad).

A global (Needleman–Wunsch) alignment of human AGO2 (Q9UKV8) against its three paralogs, anchored on these positions, gives:

Paralog	UniProt	Res 1 (AGO2 D597)	Res 2 (AGO2 E637)	Res 3 (AGO2 D669)	Res 4 (AGO2 H807)	Tetrad status	Native slicer?
AGO2	Q9UKV8	D597	E637	D669	H807	D-E-D-H intact	Yes (validated positive control)
AGO3	Q9H9G7	D598	E638	D670	H808	D-E-D-H intact	Conditionally yes (Park 2017)
AGO1	(paralog)	D	E	D	R805	D-E-D-R broken	No
AGO4	Q9HCK5	D589	E629	G671	R809	D-E-G-R broken	No

The two critical AGO4 substitutions are visible in clean, ungapped alignment windows:

Catalytic Asp → Gly: AGO2 IFYRDGVSE vs AGO4 IYYRGGVSE — the catalytic aspartate (D669) aligns to a glycine in AGO4.
Catalytic His → Arg: AGO2 AYYAHLVAF vs AGO4 AYYARLVAF — the catalytic histidine (H807) aligns to an arginine in AGO4.

Both substitutions are non-conservative and hit residues individually essential for the two-metal-ion mechanism. The catalytic aspartate is a direct metal ligand; replacing it with side-chain-less glycine removes a required coordinating carboxylate. The catalytic histidine positions/activates the nucleophilic water and stabilizes the transition state; replacing it with arginine alters charge, geometry, and hydrogen-bonding capacity. AGO4 is 79.7% identical to AGO2 overall, so these are specific, functionally consequential changes at otherwise strongly conserved catalytic positions — not divergence in an unconserved region. The intact AGO3 tetrad, sitting in the same alignment as a positive family control, sharpens the contrast: the loss is AGO4-specific.

Finding 2 — UniProt independently annotates catalytic Mg²⁺-binding sites for AGO2/AGO3 but not for AGO4

As an orthogonal cross-check, the UniProt feature tables were compared across paralogs. UniProt annotates Mg²⁺-coordinating catalytic binding sites for AGO2 (positions 597, 669, 807) and AGO3 (positions 598, 638, 670, 808), consistent with those two proteins retaining the tetrad. UniProt annotates no catalytic binding sites for AGO4 (Q9HCK5) or AGO1. This database-level curation fully matches the de novo alignment and reflects the community consensus that AGO4 is not a metal-dependent nuclease.

Finding 3 — Primary literature consistently classifies AGO4 as slicer-independent

Three independent primary papers converge on the conclusion that AGO4 is not a catalytically active slicer:

AGO2 is the sole constitutive mammalian slicer. Liu et al. established that of the mammalian Argonautes, "a single mammalian family member, Argonaute2, [is] responsible for messenger RNA cleavage activity" PMID: 15284456. This directly argues against endonuclease activity in AGO4.
AGO4 explicitly described as slicer-independent. Park et al. state that "Of the four human Argonaute (AGO) paralogs, only AGO2 has been shown to have slicer activity. The others (AGO1, AGO3 and AGO4) have been thought to assemble with microRNAs to form slicer-independent effector complexes" PMID: 29040713. Critically, this paper adds AGO3 (not AGO4) as a newly demonstrated conditional slicer — AGO4 remains slicer-independent.
Non-AGO2 paralogs must be engineered to become active. Hauptmann et al. "mutated inactive human Ago1 and Ago3 and generated catalytic Argonaute proteins" PMID: 23665583, showing that catalytic activity had to be restored by engineering — confirming the non-AGO2 paralogs are natively inactive. AGO4 was not among the rescued set.

Finding 4 — AGO4's actual effector mechanism is nuclease-independent silencing via TNRC6/GW182

AGO4's functional role in the miRNA pathway does not depend on endonuclease activity. Lazzaretti et al. showed that all four human Argonautes (AGO1–AGO4) recruit the GW182/TNRC6 family, and that the TNRC6 proteins execute silencing through their C-terminal silencing domains — independently of Argonaute catalysis — by promoting translational repression and mRNA deadenylation/decay PMID: 19383768. This provides a coherent, catalysis-free mechanistic account of AGO4-mediated silencing and removes any functional necessity for AGO4 to be a slicer.

Mechanistic Model / Interpretation

The Argonaute PIWI domain adopts an RNase-H-like fold; when the DEDH tetrad is intact it performs guide-directed, two-metal-ion endonucleolytic cleavage ("slicing") of a complementary target RNA. AGO4 retains the fold and can bind a miRNA guide and base-pair with targets, but its catalytic center is structurally incomplete:

       Guide RNA (miRNA)
     |
   5'........XXXXXXXXX........3'   target mRNA
     |||||||||
[ PIWI domain ]
 D--E--D--H   <-- catalytic tetrad coordinates 2x Mg2+
     \  |  /
      Mg2+ Mg2+  --> phosphodiester hydrolysis (SLICING)

 AGO2:  D597 - E637 - D669 - H807   => INTACT  => active slicer
 AGO3:  D598 - E638 - D670 - H808   => INTACT  => conditional slicer
 AGO1:  D    - E    - D    - R805   => His->Arg => DEAD
 AGO4:  D589 - E629 - G671 - R809   => Asp->Gly + His->Arg => DEAD

Because the two-metal-ion mechanism requires the full complement of coordinating side chains, the AGO4 active site cannot assemble a competent catalytic center. AGO4 instead functions as a structurally slicer-dead effector:

   AGO4 (slicer-dead) + miRNA  ->  binds target mRNA
     |
 recruits TNRC6/GW182 (via GW-binding surface)
     |
TNRC6 C-terminal silencing domain
     |
translational repression + deadenylation/decay
     |
      GENE SILENCING (no endonucleolytic cut)

This model reconciles all four findings: the missing catalytic residues (F1), the absent UniProt catalytic-site annotations (F2), the literature classification as slicer-independent (F3), and the demonstrated nuclease-independent silencing route (F4). The GO:0004521 annotation is best explained as an artifact of phylogenetic (IBA) propagation from an ancestral/AGO2 catalytic function, applied without accounting for the paralog-specific loss of catalytic residues in the AGO4 lineage.

Evidence Base / Evidence Matrix

Citation	Evidence type	Stance	Claim tested	Key finding	Context	Confidence / limits
This analysis (provenance)	Structural/evolutionary (computational)	Refutes	AGO4 retains the DEDH catalytic tetrad	AGO4 tetrad = D589-E629-G671-R809; catalytic Asp→Gly and His→Arg vs active AGO2 D-E-D-H; AGO3 intact (D598-E638-D670-H808) as positive control; 79.7% identity to AGO2	Full-length human AGO1–4 (UniProt); NW alignment	High for the two clean, ungapped substitutions; pairwise (not full MSA)
UniProt Q9HCK5 / Q9UKV8 / Q9H9G7	Review/database	Refutes	AGO4 has an annotated catalytic center	Catalytic Mg²⁺-coordinating sites annotated for AGO2 (597/669/807) and AGO3 (598/638/670/808) but none for AGO4 (or AGO1)	Curated database features	Database-level curator judgement, not an assay
PMID: 15284456	Direct assay / genetics	Refutes	Which mammalian Argonaute cleaves mRNA	"a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity"	Mammalian RNAi biochemistry	Seminal; conclusion durable
PMID: 29040713	Direct assay + structure	Refutes / qualifies	AGO4 has slicer activity	Only AGO2 shown to slice; AGO1/AGO3/AGO4 "slicer-independent"; adds AGO3 (not AGO4) as active	Recombinant human AGO cleavage assays	AGO4 not shown active anywhere
PMID: 23665583	Mutant/engineering	Qualifies	Non-AGO2 AGOs are intrinsically active	Inactive Ago1/Ago3 had to be mutated to generate catalytic enzymes	Human AGO1/AGO3	AGO4 not among rescued set
PMID: 19383768	Interaction / cell assay	Qualifies (alt. function)	AGO4's real mechanism	AGO1–AGO4 recruit TNRC6/GW182 → silencing by translational repression + deadenylation, independent of AGO catalysis	Human cells, tethering	Supports non-endonucleolytic role

GO Curation Implications (leads — require curator verification)

Lead action: Do not retain GO:0004521 (RNA endonuclease activity) as a direct molecular-function annotation for AGO4. The IBA propagation is contradicted by AGO4's own sequence/structure and by UniProt, and should be removed or marked NOT for this gene. If the pipeline retains IBA statements, flag this one as an over-annotation contradicted by sequence and database evidence.
Better-supported MF terms: RNA binding (GO:0003723) and/or miRNA binding (GO:0035198) — AGO4 binds small-RNA guides and recognizes targets by base pairing.
Better-supported BP terms: gene silencing by miRNA (GO:0035195) / post-transcriptional gene silencing (GO:0016441) via translational repression and deadenylation — not slicing-based cleavage.
Well-supported CC terms: RISC complex (GO:0016442) / cytoplasmic ribonucleoprotein granule (P-body).
Preserve the key distinction: AGO4 is a non-catalytic RISC/effector subunit. Avoid defaulting to "protein binding" (GO:0005515) when RNA/miRNA-binding and silencing terms are supported.

Mechanistic Scope

The immediate molecular activity under test is RNA-guided phosphodiester-bond hydrolysis (slicing) by the PIWI (RNase-H-like) domain, requiring the DEDH tetrad plus two catalytic Mg²⁺. AGO4 lacks two of these residues → the direct activity is absent.

It is important to separate:

Direct activity tested, absent: endonucleolytic RNA cleavage / slicing.
Direct activity present (not the term under review): miRNA-guide binding and target-mRNA recognition.
Downstream / pathway consequence (present): target-mRNA silencing — but executed via TNRC6/GW182-recruited translational repression and deadenylation-coupled decay, not by AGO4 catalysis.
Not applicable: organismal loss-of-function phenotypes of AGO4 are downstream and cannot be used to infer catalytic activity.

Even where AGO4 contributes to "RNA degradation" at the pathway level, that degradation is performed by downstream deadenylases/decay machinery recruited via TNRC6 — not by AGO4 endonuclease chemistry.

Conflicts and Alternatives

Paralog carry-over / frequency bias (most likely source): The IBA is driven by the well-characterized AGO2 slicer and the shared Argonaute/PIWI domain. Catalytic competence does not transfer to all paralogs; this is the classic paralog-overannotation pattern.
AGO3 nuance: AGO3 does retain the tetrad and has guide-dependent slicer activity (PMID: 29040713) — so "all AGOs except AGO2 are inactive" is itself too strong. But AGO3's intact tetrad is exactly what AGO4 lacks, which sharpens (not weakens) the AGO4 conclusion.
AGO1 comparison: AGO1 also loses the catalytic His (His→Arg805) and is likewise slicer-inactive (rescuable only by engineering, PMID: 23665583) — a consistent picture.
Fold retention ≠ catalysis: AGO4 retains the PIWI/RNase-H fold and binds RNA, which may superficially justify an "endonuclease-fold" call. GO:0004521 asserts activity, not fold; the fold is present while the chemistry is not.
In vitro vs in vivo: No primary study reports native AGO4 endonuclease activity; there is no positive experimental evidence to weigh against the refuting sequence/database evidence. No isoform/organism artifact was found that would rescue AGO4 activity.

Limitations and Knowledge Gaps

Sequence-based inference, not a direct AGO4 enzyme assay. Checked: alignment against validated AGO2 residues + UniProt cross-check. Why it matters: GO curation ideally rests on a direct assay. Resolution: a reconstituted in vitro slicer assay with purified guide-loaded AGO4 on a fully complementary target (as done for AGO3 in PMID 29040713).
No structural active-site superposition was executed as provenance. Checked: residue identity/spacing in sequence only. Why it matters: an AlphaFold/PDB superposition on AGO2 would confirm geometric disruption and rule out compensatory rearrangement. Resolution: superpose AlphaFold Q9HCK5 onto AGO2 (e.g., PDB 4W5N/4F3T) and score catalytic-pocket geometry.
AGO4 not directly rescued in engineering studies. PMID 23665583 restored Ago1/Ago3 but did not report a catalytic-AGO4 mutant, leaving open (though unlikely) additional catalysis-blocking features beyond the tetrad. Resolution: engineer AGO4 back-mutations (Gly671→Asp, Arg809→His) and assay.
Non-slicer nuclease activities not formally excluded. GO:0004521 is broader than "slicer." Checked: only slicer-relevant residues. Why it matters: AGO4 could in principle have a distinct non-canonical RNA-cleaving activity. Resolution: biochemical nuclease screen. No literature supports any such activity for AGO4.
Nuclear / non-canonical AGO4 roles (reported nuclear and interferon-related functions) were not exhaustively mined; none are expected to require intrinsic endonuclease activity.

Analytical limitations: pairwise NW alignment (not a full MSA); catalytic-residue identity taken from the established hAGO2 D597/E637/D669/H807 tetrad; database annotations are curator-level. None of these weaken the core conclusion, which rests on two non-conservative substitutions in clean, ungapped alignment blocks.

Proposed Follow-up Experiments / Actions

Discriminating experiments

Reconstituted slicer assay (definitive): purify guide-loaded human AGO4, incubate with a 5′-labeled fully complementary target RNA + Mg²⁺, assay for site-specific cleavage. Include AGO2 positive control and an AGO4 "restored-tetrad" (Gly671→Asp, Arg809→His) mutant. Expected: AGO2 cleaves, wild-type AGO4 does not.
Structural active-site superposition (fast, computational): align AlphaFold AGO4 (Q9HCK5) onto AGO2 catalytic PIWI structure; quantify absence/displacement of catalytic carboxylate and imidazole. Provenance-friendly and directly on-point.
Gain-of-function engineering: test whether the AGO4 double back-mutant (± N-terminal AGO2 elements) gains slicer activity, mirroring the AGO1/AGO3 engineering logic.
Comparative paralog panel: run AGO1/AGO2/AGO3/AGO4 side by side to demonstrate the AGO2(+)/AGO3(conditional)/AGO1,AGO4(–) pattern predicted by the tetrad analysis.

Curation actions (leads to verify)

Remove / do-not-propagate GO:0004521 IBA on AGO4; consider a NOT GO:0004521 annotation to record the tested-and-absent activity.
Add/retain better-supported terms: MF GO:0003723 / GO:0035198; BP GO:0035195 / GO:0016441; CC GO:0016442.
Candidate snippets to cite (verify verbatim):
PMID 29040713: "Of the four human Argonaute (AGO) paralogs, only AGO2 has been shown to have slicer activity. The others (AGO1, AGO3 and AGO4) have been thought to assemble with microRNAs to form slicer-independent effector complexes."
PMID 15284456: "a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity."
PMID 23665583: "we have mutated inactive human Ago1 and Ago3 and generated catalytic Argonaute proteins."

Conclusion

Human AGO4 does not have RNA endonuclease (slicer) activity. Its PIWI catalytic tetrad is disrupted at two of four positions (catalytic Asp669→Gly671 and His807→Arg809 relative to active AGO2 D-E-D-H), UniProt annotates no catalytic metal-binding sites for AGO4, and primary literature consistently classifies AGO4 as a slicer-independent miRNA effector that silences targets through TNRC6/GW182-mediated translational repression and mRNA decay. The GO:0004521 IBA annotation is a paralog over-annotation propagated from ancestral/AGO2 catalytic function and should be removed or NOT-qualified, with AGO4's molecular function better captured by RNA/miRNA-binding, RISC-component, and miRNA-mediated-silencing terms.

Artifacts

📄 View Raw YAML

id: Q9HCK5
gene_symbol: AGO4
product_type: PROTEIN
status: COMPLETE
taxon:
  id: NCBITaxon:9606
  label: Homo sapiens
description: 'AGO4 encodes human Argonaute-4, a small-RNA-binding Argonaute paralog that contributes to
  RISC-mediated gene regulation primarily through non-slicing mechanisms. Evidence supports miRNA/RISC-associated
  translational repression, cytoplasmic miRISC/P-body-associated function, and a non-core mammalian antiviral
  role rather than canonical AGO2-like slicing.'
existing_annotations:
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: nucleus is a plausible or reported location/context but is not the core functional site
      annotation for AGO4.
    action: KEEP_AS_NON_CORE
    reason: The core location is cytoplasmic RISC/RNP granules; nuclear, membrane, synaptic,
      exosomal, or other compartment annotations should remain non-core unless tied to a specific
      curated mechanism.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: cytoplasm is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0004521
    label: RNA endonuclease activity
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: RNA endonuclease activity is not supported as a physiological activity for AGO4.
    action: REMOVE
    reason: AGO4 is best supported as a non-slicing Argonaute paralog; assigning RNA endonuclease
      activity would overstate AGO2-like catalytic function. OpenScientist confirmed
      that AGO4 lacks the intact DEDH catalytic tetrad required for AGO2-like slicer
      activity, so this is a paralog over-annotation rather than a supported AGO4
      molecular function.
    additional_reference_ids:
    - file:human/AGO4/AGO4-goa.tsv
    - file:human/AGO4/AGO4-hypotheses/function-hypothesis-go-0004521/openscientist.md
    propagation_review:
      root_cause: PROPAGATION_BAD
      failure_modes:
      - PSEUDO_OR_SUBACTIVITY_LOSS
      - FUNCTIONAL_DIVERGENCE
      - WRONG_ORTHOLOG_OR_PARALOG
      source_entities:
      - source_id: PANTHER:PTN008584027
        source_label: Argonaute PANTHER source node
        source_status: SUPPORTS_SOURCE_BUT_NOT_TARGET
        comment: The family source includes slicing-competent Argonautes, but AGO4
          is reviewed as a non-slicing paralog.
      - source_id: UniProtKB:Q9UKV8
        source_label: human AGO2
        source_status: SUPPORTS_SOURCE_BUT_NOT_TARGET
        comment: AGO2 supports RNA endonuclease activity; AGO4 lacks the same supported
          slicer activity in the target review.
      - source_id: UniProtKB:Q9H9G7
        source_label: human AGO3
        source_status: SUPPORTS_SOURCE_BUT_NOT_TARGET
        comment: AGO3 retains a catalytic tetrad and has conditional slicer activity;
          AGO4 has disruptive substitutions in the corresponding catalytic residues.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-goa.tsv
      supporting_text: "UniProtKB\tQ9HCK5\tAGO4\tenables\tGO:0004521\tRNA endonuclease activity\tmolecular_function\tECO:0000318\tIBA\tGO_REF:0000033\tFB:FBgn0000146|FB:FBgn0004872|FB:FBgn0087035|FB:FBgn0250816|FB:FBgn0262739|MGI:MGI:1928897|MGI:MGI:1930036|MGI:MGI:2446632|PANTHER:PTN008584027|PomBase:SPCC736.11|TAIR:locus:2007760|TAIR:locus:2059258|UniProtKB:O67434|UniProtKB:Q9H9G7|UniProtKB:Q9UKV8|WB:WBGene00000105|WB:WBGene00000106|WB:WBGene00004323|WB:WBGene00017641\t9606\tHomo sapiens\tGO_Central\tProtein argonaute-4\t20241120"
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
    - reference_id: file:human/AGO4/AGO4-hypotheses/function-hypothesis-go-0004521/openscientist.md
      supporting_text: Human AGO4 does **not** have RNA endonuclease (slicer) activity.
- term:
    id: GO:0035194
    label: regulatory ncRNA-mediated post-transcriptional gene silencing
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: regulatory ncRNA-mediated post-transcriptional gene silencing is consistent with AGO4
      as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0016442
    label: RISC complex
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: RISC complex is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0036464
    label: cytoplasmic ribonucleoprotein granule
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: cytoplasmic ribonucleoprotein granule is consistent with AGO4 as a small-RNA-loaded
      Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0031054
    label: pre-miRNA processing
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: pre-miRNA processing overstates the direct role of AGO4 in canonical miRNA biogenesis.
    action: MARK_AS_OVER_ANNOTATED
    reason: The better-supported role is as a loaded Argonaute effector after small-RNA processing,
      not as the Drosha/Dicer processing enzyme for pre-miRNAs.
    propagation_review:
      root_cause: TERM_SCOPING_PROBLEM
      failure_modes:
      - ROLE_CONFLATION
      - GRANULARITY_MISMATCH
      source_entities:
      - source_id: PANTHER:PTN000527278
        source_label: Argonaute pre-miRNA processing source node
        source_status: SUPPORTS_SOURCE_BUT_NOT_TARGET
        comment: The source captures Argonaute participation in miRNA pathway
          biology, but the propagated term overstates AGO4 as processing
          machinery rather than a loaded small-RNA effector.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0035198
    label: miRNA binding
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: miRNA binding is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0003727
    label: single-stranded RNA binding
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: single-stranded RNA binding is too generic for AGO4.
    action: MARK_AS_OVER_ANNOTATED
    reason: The informative molecular function is Argonaute small-RNA/miRNA binding within RISC
      rather than a broad nucleic-acid or RNA-binding label.
    propagation_review:
      root_cause: TERM_SCOPING_PROBLEM
      failure_modes:
      - GRANULARITY_MISMATCH
      source_entities:
      - source_id: PANTHER:PTN000527276
        source_label: Argonaute RNA-binding source node
        source_status: SUPPORTS_SOURCE_BUT_NOT_TARGET
        comment: The source supports RNA-binding Argonaute biology, but the
          propagated term is less informative than AGO4 small-RNA/miRNA binding
          in RISC context.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0000932
    label: P-body
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  review:
    summary: P-body is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0003676
    label: nucleic acid binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  review:
    summary: nucleic acid binding is too generic for AGO4.
    action: MARK_AS_OVER_ANNOTATED
    reason: The informative molecular function is Argonaute small-RNA/miRNA binding within RISC
      rather than a broad nucleic-acid or RNA-binding label.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0003723
    label: RNA binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  review:
    summary: RNA binding is too generic for AGO4.
    action: MARK_AS_OVER_ANNOTATED
    reason: The informative molecular function is Argonaute small-RNA/miRNA binding within RISC
      rather than a broad nucleic-acid or RNA-binding label.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0003725
    label: double-stranded RNA binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  review:
    summary: double-stranded RNA binding is too generic for AGO4.
    action: MARK_AS_OVER_ANNOTATED
    reason: The informative molecular function is Argonaute small-RNA/miRNA binding within RISC
      rather than a broad nucleic-acid or RNA-binding label.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0003727
    label: single-stranded RNA binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  review:
    summary: single-stranded RNA binding is too generic for AGO4.
    action: MARK_AS_OVER_ANNOTATED
    reason: The informative molecular function is Argonaute small-RNA/miRNA binding within RISC
      rather than a broad nucleic-acid or RNA-binding label.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0016442
    label: RISC complex
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  review:
    summary: RISC complex is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0031054
    label: pre-miRNA processing
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  review:
    summary: pre-miRNA processing overstates the direct role of AGO4 in canonical miRNA biogenesis.
    action: MARK_AS_OVER_ANNOTATED
    reason: The better-supported role is as a loaded Argonaute effector after small-RNA processing,
      not as the Drosha/Dicer processing enzyme for pre-miRNAs.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0035198
    label: miRNA binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  review:
    summary: miRNA binding is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0035278
    label: miRNA-mediated gene silencing by inhibition of translation
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  review:
    summary: miRNA-mediated gene silencing by inhibition of translation is consistent with AGO4 as a
      small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0036464
    label: cytoplasmic ribonucleoprotein granule
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  review:
    summary: cytoplasmic ribonucleoprotein granule is consistent with AGO4 as a small-RNA-loaded
      Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0070578
    label: RISC-loading complex
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  review:
    summary: RISC-loading complex is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC
      effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0070922
    label: RISC complex assembly
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  review:
    summary: RISC complex assembly is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC
      effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:19167051
  review:
    summary: Generic protein binding does not capture the specific Argonaute/RISC function.
    action: MARK_AS_OVER_ANNOTATED
    reason: The cited interactions are best interpreted as RISC/cofactor context for
      small-RNA-guided silencing, not as a standalone core function.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:19324964
  review:
    summary: Generic protein binding does not capture the specific Argonaute/RISC function.
    action: MARK_AS_OVER_ANNOTATED
    reason: The cited interactions are best interpreted as RISC/cofactor context for
      small-RNA-guided silencing, not as a standalone core function.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:19383768
  review:
    summary: Generic protein binding does not capture the specific Argonaute/RISC function.
    action: MARK_AS_OVER_ANNOTATED
    reason: The cited interactions are best interpreted as RISC/cofactor context for
      small-RNA-guided silencing, not as a standalone core function.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:33961781
  review:
    summary: Generic protein binding does not capture the specific Argonaute/RISC function.
    action: MARK_AS_OVER_ANNOTATED
    reason: The cited interactions are best interpreted as RISC/cofactor context for
      small-RNA-guided silencing, not as a standalone core function.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  review:
    summary: nucleus is a plausible or reported location/context but is not the core functional site
      annotation for AGO4.
    action: KEEP_AS_NON_CORE
    reason: The core location is cytoplasmic RISC/RNP granules; nuclear, membrane, synaptic,
      exosomal, or other compartment annotations should remain non-core unless tied to a specific
      curated mechanism.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0035196
    label: miRNA processing
  evidence_type: NAS
  original_reference_id: PMID:28781232
  review:
    summary: miRNA processing overstates the direct role of AGO4 in canonical miRNA biogenesis.
    action: MARK_AS_OVER_ANNOTATED
    reason: The better-supported role is as a loaded Argonaute effector after small-RNA processing,
      not as the Drosha/Dicer processing enzyme for pre-miRNAs.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0036464
    label: cytoplasmic ribonucleoprotein granule
  evidence_type: IDA
  original_reference_id: GO_REF:0000052
  review:
    summary: cytoplasmic ribonucleoprotein granule is consistent with AGO4 as a small-RNA-loaded
      Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:15766526
  review:
    summary: Generic protein binding does not capture the specific Argonaute/RISC function.
    action: MARK_AS_OVER_ANNOTATED
    reason: The cited interactions are best interpreted as RISC/cofactor context for
      small-RNA-guided silencing, not as a standalone core function.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0035196
    label: miRNA processing
  evidence_type: IMP
  original_reference_id: PMID:22795694
  review:
    summary: miRNA processing overstates the direct role of AGO4 in canonical miRNA biogenesis.
    action: MARK_AS_OVER_ANNOTATED
    reason: The better-supported role is as a loaded Argonaute effector after small-RNA processing,
      not as the Drosha/Dicer processing enzyme for pre-miRNAs.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0031054
    label: pre-miRNA processing
  evidence_type: IDA
  original_reference_id: PMID:19966796
  review:
    summary: pre-miRNA processing overstates the direct role of AGO4 in canonical miRNA biogenesis.
    action: MARK_AS_OVER_ANNOTATED
    reason: The better-supported role is as a loaded Argonaute effector after small-RNA processing,
      not as the Drosha/Dicer processing enzyme for pre-miRNAs.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0003725
    label: double-stranded RNA binding
  evidence_type: IDA
  original_reference_id: PMID:19966796
  review:
    summary: double-stranded RNA binding is too generic for AGO4.
    action: MARK_AS_OVER_ANNOTATED
    reason: The informative molecular function is Argonaute small-RNA/miRNA binding within RISC
      rather than a broad nucleic-acid or RNA-binding label.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0003727
    label: single-stranded RNA binding
  evidence_type: IDA
  original_reference_id: PMID:19966796
  review:
    summary: single-stranded RNA binding is too generic for AGO4.
    action: MARK_AS_OVER_ANNOTATED
    reason: The informative molecular function is Argonaute small-RNA/miRNA binding within RISC
      rather than a broad nucleic-acid or RNA-binding label.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0016442
    label: RISC complex
  evidence_type: IDA
  original_reference_id: PMID:19966796
  review:
    summary: RISC complex is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0070578
    label: RISC-loading complex
  evidence_type: IDA
  original_reference_id: PMID:19966796
  review:
    summary: RISC-loading complex is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC
      effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0070922
    label: RISC complex assembly
  evidence_type: IDA
  original_reference_id: PMID:19966796
  review:
    summary: RISC complex assembly is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC
      effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0090625
    label: siRNA-mediated gene silencing by mRNA destabilization
  evidence_type: IDA
  original_reference_id: PMID:15260970
  negated: true
  review:
    summary: This NOT annotation is appropriate; AGO4 is not the Argonaute paralog that mediates
      siRNA-guided target mRNA cleavage/destabilization in this study.
    action: ACCEPT
    reason: PMID:15260970 supports AGO4 association with small RNAs/RISC but shows that
      endonucleolytic target cleavage activity is associated with AGO2, not AGO4. AGO4's supported
      role should be distinguished from this negated AGO2-like target-cleavage annotation.
    supported_by:
    - reference_id: PMID:15260970
      supporting_text: Purification of the FLAG/HA-epitope-tagged Ago containing complexes from
        different human cell lines revealed that endonuclease activity is exclusively associated
        with Ago2.
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: A long-standing view is that among the four human paralogs, **AGO2** is the
        canonical slicer (endonuclease), while **AGO1/AGO3/AGO4** primarily act through non-slicing
        mechanisms (translational repression, deadenylation).
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: IDA
  original_reference_id: PMID:15260970
  review:
    summary: cytoplasm is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0016442
    label: RISC complex
  evidence_type: IDA
  original_reference_id: PMID:15260970
  review:
    summary: RISC complex is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0035198
    label: miRNA binding
  evidence_type: IDA
  original_reference_id: PMID:15260970
  review:
    summary: miRNA binding is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0035279
    label: miRNA-mediated gene silencing by mRNA destabilization
  evidence_type: IDA
  original_reference_id: PMID:15260970
  negated: true
  review:
    summary: This NOT annotation is appropriate; PMID:15260970 does not support assigning AGO4 to
      AGO2-like miRNA-guided target mRNA cleavage/destabilization.
    action: ACCEPT
    reason: AGO4 binds miRNAs and participates in RISC, but the cited work distinguishes that
      non-slicer role from AGO2-dependent target RNA cleavage. The NOT annotation should be retained
      rather than converted into a positive AGO4 mRNA-destabilization function.
    supported_by:
    - reference_id: PMID:15260970
      supporting_text: The specific role of Ago2 in guiding target RNA cleavage was confirmed
        independently by siRNA-based depletion of individual Ago members in combination with a
        sensitive positive-readout reporter assay.
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: A long-standing view is that among the four human paralogs, **AGO2** is the
        canonical slicer (endonuclease), while **AGO1/AGO3/AGO4** primarily act through non-slicing
        mechanisms (translational repression, deadenylation).
- term:
    id: GO:0016020
    label: membrane
  evidence_type: HDA
  original_reference_id: PMID:19946888
  review:
    summary: membrane is a plausible or reported location/context but is not the core functional
      site annotation for AGO4.
    action: KEEP_AS_NON_CORE
    reason: The core location is cytoplasmic RISC/RNP granules; nuclear, membrane, synaptic,
      exosomal, or other compartment annotations should remain non-core unless tied to a specific
      curated mechanism.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-1606561
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-1606682
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-1912362
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-1912363
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-1912364
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-1912366
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-1912367
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-1912368
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-1912406
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-2318752
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-3209151
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-426489
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-426522
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-4518575
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-5687103
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-8935766
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-8937097
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-8937134
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-8938440
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-8938487
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-8938507
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-8944650
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-8944684
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-8944706
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9011958
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9012203
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9012208
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9038545
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9618392
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9618486
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9624925
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9657791
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9858289
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9858309
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9924921
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9925095
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9925158
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9925159
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9925324
  review:
    summary: cytosol is consistent with AGO4 as a small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0006402
    label: mRNA catabolic process
  evidence_type: IDA
  original_reference_id: PMID:18771919
  review:
    summary: mRNA catabolic process is downstream or context-specific for AGO4.
    action: KEEP_AS_NON_CORE
    reason: This process may be supported in a particular experiment or pathway model, but it is
      peripheral to the core Argonaute/RISC molecular role.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
- term:
    id: GO:0035278
    label: miRNA-mediated gene silencing by inhibition of translation
  evidence_type: IDA
  original_reference_id: PMID:18771919
  review:
    summary: miRNA-mediated gene silencing by inhibition of translation is consistent with AGO4 as a
      small-RNA-loaded Argonaute/RISC effector.
    action: ACCEPT
    reason: The falcon report supports AGO4 function in miRISC-mediated small-RNA-guided repression
      and related RISC complexes or cytoplasmic RNP compartments.
    supported_by:
    - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
      supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
        literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
        regulation largely through **non-slicing mechanisms**
references:
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with GO terms
  findings: []
- id: GO_REF:0000033
  title: Annotation inferences using phylogenetic trees
  findings: []
- id: GO_REF:0000052
  title: Gene Ontology annotation based on curation of immunofluorescence data
  findings: []
- id: GO_REF:0000107
  title: Automatic transfer of experimentally verified manual GO annotation data to orthologs using
    Ensembl Compara
  findings: []
- id: GO_REF:0000117
  title: Electronic Gene Ontology annotations created by ARBA machine learning models
  findings: []
- id: GO_REF:0000120
  title: Combined Automated Annotation using Multiple IEA Methods
  findings: []
- id: PMID:15260970
  title: Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs.
  findings: []
- id: PMID:15766526
  title: Involvement of microRNA in AU-rich element-mediated mRNA instability.
  findings: []
- id: PMID:18771919
  title: Importance of translation and nonnucleolytic ago proteins for on-target RNA interference.
  findings: []
- id: PMID:19167051
  title: Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.
  findings: []
- id: PMID:19324964
  title: The C-terminal half of human Ago2 binds to multiple GW-rich regions of GW182 and requires
    GW182 to mediate silencing.
  findings: []
- id: PMID:19383768
  title: The C-terminal domains of human TNRC6A, TNRC6B, and TNRC6C silence bound transcripts
    independently of Argonaute proteins.
  findings: []
- id: PMID:19946888
  title: Defining the membrane proteome of NK cells.
  findings: []
- id: PMID:19966796
  title: ATP-dependent human RISC assembly pathways.
  findings: []
- id: PMID:22795694
  title: Slicing-independent RISC activation requires the argonaute PAZ domain.
  findings: []
- id: PMID:28781232
  title: Multivalent Recruitment of Human Argonaute by GW182.
  findings: []
- id: PMID:33961781
  title: Dual proteome-scale networks reveal cell-specific remodeling of the human interactome.
  findings: []
- id: Reactome:R-HSA-1606561
  title: MIR449 microRNAs bind 3'UTR of NOTCH1 mRNA
  findings: []
- id: Reactome:R-HSA-1606682
  title: MIR34 microRNAs bind 3'UTR of NOTCH1 mRNA
  findings: []
- id: Reactome:R-HSA-1912362
  title: MIR150 microRNA binds 3'UTR of NOTCH3 mRNA
  findings: []
- id: Reactome:R-HSA-1912363
  title: MIR200B/C microRNAs bind NOTCH1 mRNA
  findings: []
- id: Reactome:R-HSA-1912364
  title: MIR181C microRNA binds 3'UTR of NOTCH4 mRNA
  findings: []
- id: Reactome:R-HSA-1912366
  title: MIR206 microRNA binds 3'UTR of NOTCH3 mRNA
  findings: []
- id: Reactome:R-HSA-1912367
  title: MIR34 microRNAs bind 3'UTR of NOTCH2 mRNA
  findings: []
- id: Reactome:R-HSA-1912368
  title: MIR302A microRNA binds 3'UTR of NOTCH4 mRNA
  findings: []
- id: Reactome:R-HSA-1912406
  title: p53 positively regulates transcription of MIR34 microRNAs
  findings: []
- id: Reactome:R-HSA-2318752
  title: miR-26A microRNAs bind PTEN mRNA
  findings: []
- id: Reactome:R-HSA-3209151
  title: miR-24 binds p16INK4A and p14ARF mRNAs
  findings: []
- id: Reactome:R-HSA-426489
  title: RISC binds inexactly matching target RNAs
  findings: []
- id: Reactome:R-HSA-426522
  title: Nonendonucleolytic RISC binds exactly matching target RNAs
  findings: []
- id: Reactome:R-HSA-4518575
  title: miR-92b binds 3'UTR of NLK mRNA
  findings: []
- id: Reactome:R-HSA-5687103
  title: FOXO3 regulates MIR34B,C expression
  findings: []
- id: Reactome:R-HSA-8935766
  title: miR-378 binds RUNX1 mRNA
  findings: []
- id: Reactome:R-HSA-8937097
  title: MIR27A gene transcription is stimulated by the complex containing RUNX1, PRMT1 and GATA1
    and inhibited by the complex of RUNX1, SIN3A and PRMT6
  findings: []
- id: Reactome:R-HSA-8937134
  title: miR-27a binds RUNX1 mRNA
  findings: []
- id: Reactome:R-HSA-8938440
  title: miR-17 binds RUNX1 mRNA
  findings: []
- id: Reactome:R-HSA-8938487
  title: miR-20a binds RUNX1 mRNA
  findings: []
- id: Reactome:R-HSA-8938507
  title: miR-106a binds RUNX1 mRNA
  findings: []
- id: Reactome:R-HSA-8944650
  title: miR-214 microRNA binds PTEN mRNA
  findings: []
- id: Reactome:R-HSA-8944684
  title: miR-205 microRNA binds PTEN mRNA
  findings: []
- id: Reactome:R-HSA-8944706
  title: miR-21 nonendonucleolytic RISC binds PTEN mRNA
  findings: []
- id: Reactome:R-HSA-9011958
  title: miR-26A and B bind to the 3'UTR of the GREB1 mRNA
  findings: []
- id: Reactome:R-HSA-9012203
  title: miR-26A and B bind to the 3'UTR of the CHD11 mRNA
  findings: []
- id: Reactome:R-HSA-9012208
  title: miR-26A and B bind to the 3'UTR of the KPNA2 mRNA
  findings: []
- id: Reactome:R-HSA-9038545
  title: miR-613 binds to the 3'UTR of the NR1H3 mRNA
  findings: []
- id: Reactome:R-HSA-9618392
  title: miR-26 binds to the 3'UTR of the ARL4C mRNA
  findings: []
- id: Reactome:R-HSA-9618486
  title: miR-26 binds to the 3'UTR of the ABCA1 mRNA
  findings: []
- id: Reactome:R-HSA-9624925
  title: miR-33 binds to the 3'UTR of the ABCA1 mRNA
  findings: []
- id: Reactome:R-HSA-9657791
  title: miR-144 binds to the 3'UTR of the ABCA1 mRNA
  findings: []
- id: Reactome:R-HSA-9858289
  title: MITF-M-dependent miR-211 expression
  findings: []
- id: Reactome:R-HSA-9858309
  title: miR-211 RISC binds POU3F2 mRNA
  findings: []
- id: Reactome:R-HSA-9924921
  title: CD274 mRNA binds miR-34 RISC
  findings: []
- id: Reactome:R-HSA-9925095
  title: CD274 mRNA binds miR-340 RISC
  findings: []
- id: Reactome:R-HSA-9925158
  title: CD274 mRNA binds miR-152 RISC
  findings: []
- id: Reactome:R-HSA-9925159
  title: CD274 mRNA binds miR-140 RISC
  findings: []
- id: Reactome:R-HSA-9925324
  title: CD274 mRNA binds miR-200B/C RISC
  findings: []
- id: file:human/AGO4/AGO4-uniprot.txt
  title: UniProt text export for AGO4
  findings:
  - statement: UniProt identifies AGO4 and its protein family/domain context.
    supporting_text: Belongs to the argonaute family. Ago subfamily.
- id: file:human/AGO4/AGO4-goa.tsv
  title: GOA annotation export for AGO4
  publication_type: DATABASE
  findings:
  - statement: GOA records the phylogenetic IBA source for the AGO4 GO:0004521 annotation.
    supporting_text: "UniProtKB\tQ9HCK5\tAGO4\tenables\tGO:0004521\tRNA endonuclease activity\tmolecular_function\tECO:0000318\tIBA\tGO_REF:0000033\tFB:FBgn0000146|FB:FBgn0004872|FB:FBgn0087035|FB:FBgn0250816|FB:FBgn0262739|MGI:MGI:1928897|MGI:MGI:1930036|MGI:MGI:2446632|PANTHER:PTN008584027|PomBase:SPCC736.11|TAIR:locus:2007760|TAIR:locus:2059258|UniProtKB:O67434|UniProtKB:Q9H9G7|UniProtKB:Q9UKV8|WB:WBGene00000105|WB:WBGene00000106|WB:WBGene00004323|WB:WBGene00017641\t9606\tHomo sapiens\tGO_Central\tProtein argonaute-4\t20241120"
- id: file:human/AGO4/AGO4-deep-research-falcon.md
  title: Falcon deep research report for AGO4
  findings:
  - statement: Falcon research supports the reviewed core function of AGO4.
    supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
      literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
      regulation largely through **non-slicing mechanisms**
- id: file:human/AGO4/AGO4-hypotheses/function-hypothesis-go-0004521/openscientist.md
  title: OpenScientist hypothesis report for AGO4 GO:0004521
  publication_type: DEEP_RESEARCH
  findings:
  - statement: OpenScientist refutes RNA endonuclease activity for AGO4.
    supporting_text: Human AGO4 does **not** have RNA endonuclease (slicer) activity.
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: Focused OpenScientist report directly evaluates the GO:0004521
      IBA and supports removal as AGO2/AGO3 paralog over-propagation onto slicer-dead
      AGO4.
core_functions:
- description: Small-RNA-loaded Argonaute/RISC effector activity, primarily through non-slicing
    repression mechanisms including translational control.
  molecular_function:
    id: GO:0035198
    label: miRNA binding
  directly_involved_in:
  - id: GO:0035194
    label: regulatory ncRNA-mediated post-transcriptional gene silencing
  - id: GO:0035278
    label: miRNA-mediated gene silencing by inhibition of translation
  in_complex:
    id: GO:0016442
    label: RISC complex
  locations:
  - id: GO:0005737
    label: cytoplasm
  - id: GO:0000932
    label: P-body
  supported_by:
  - reference_id: file:human/AGO4/AGO4-deep-research-falcon.md
    supporting_text: Human AGO4 (EIF2C4; UniProt Q9HCK5) is best supported (in the retrieved
      literature set) as a **small-RNA-binding Argonaute paralog** that contributes to gene
      regulation largely through **non-slicing mechanisms**
proposed_new_terms: []
suggested_questions:
- question: Which endogenous human tissues or stress states require AGO4-specific RISC activity
    rather than redundant activity from other AGO paralogs?
- question: Which protein cofactors determine guide loading, localization, and non-core nuclear or
    granule-associated functions of AGO4?
suggested_experiments:
- description: Endogenous tagging of AGO4 followed by small-RNA CLIP, target RNA profiling, and
    paralog-specific rescue in AGO-depleted human cells.
  experiment_type: genome editing/RNA-protein interaction profiling
  hypothesis: AGO4 has paralog-specific guide, target, and compartment preferences within human RISC
    biology.
- description: Biochemical reconstitution of AGO4-RISC with representative miRNA and siRNA guides to
    compare target repression, target cleavage, and TNRC6 recruitment.
  experiment_type: biochemical reconstitution
  hypothesis: AGO4 activity is best explained by guide-loaded RISC function rather than generic RNA
    or protein binding.

AGO4

Gene Description

Existing Annotations Review

Core Functions

Small-RNA-loaded Argonaute/RISC effector activity, primarily through non-slicing repression mechanisms including translational control.

References

Suggested Questions for Experts

Suggested Experiments

Deep Research

Falcon

Comprehensive research report: Human AGO4 / EIF2C4 (UniProt Q9HCK5)

0) Target identity verification (critical disambiguation)

1) Key concepts and definitions (current understanding)

1.1 RISC and Argonaute core function

1.2 Domain architecture of AGO4

1.3 Slicer vs non-slicer AGOs in humans

2) AGO4 molecular function: what it does (and what it does not)

2.1 Small-RNA binding and RISC assembly

2.2 Catalysis (slicing) is not established as AGO4’s primary physiological mechanism

2.3 Non-canonical translational repression: AGO4 and IRES-dependent translation

3) Biological processes and pathways involving AGO4

3.1 Mammalian antiviral defense (innate immunity intersection)

3.2 Current expert framing (2023–2024): antiviral RNAi remains context-dependent

3.3 Putative links to epigenetic/chromatin pathways

4) Subcellular localization (where AGO4 acts)

4.1 Cytoplasmic miRISC-associated localization (inferred)

4.2 Nuclear germline localization: evidence is primarily mouse

5) Disease relevance and associations

5.1 Influenza susceptibility (in vivo functional evidence)

5.2 Neurological disease relevance through AGO4-regulated IRES translation target

5.3 Human disease association statistics (database-level, hypothesis generating)

6) Recent developments (prioritizing 2023–2024)

6.1 2024 structural/mechanistic synthesis of slicing and guide rules (context)

6.2 2023 debate and methodological advances in mammalian antiviral RNAi

6.3 2024 quantitative kinetics for slicing (AGO2-focused, informs tool design)

7) Applications and real-world implementations

7.1 RNAi therapeutics and the importance of Argonaute rules (context)

7.2 Antiviral strategies (conceptual)

8) Statistics and quantitative data (from retrieved sources)

8.1 Study design and significance reporting for AGO4 antiviral phenotypes

8.2 Database association statistics

9) Evidence summary table (curated)

10) Key figure evidence (in vivo antiviral phenotype)

11) Conclusions (expert synthesis)

Citations

OpenScientist

AGO4 RNA Endonuclease Activity (GO:0004521) — Hypothesis Deep Research Report

Summary

Key Findings

Finding 1 — AGO4 lacks the intact DEDH catalytic tetrad required for slicer activity

Finding 2 — UniProt independently annotates catalytic Mg²⁺-binding sites for AGO2/AGO3 but not for AGO4

Finding 3 — Primary literature consistently classifies AGO4 as slicer-independent

Finding 4 — AGO4's actual effector mechanism is nuclease-independent silencing via TNRC6/GW182

Mechanistic Model / Interpretation

Evidence Base / Evidence Matrix

GO Curation Implications (leads — require curator verification)

Mechanistic Scope

Conflicts and Alternatives

Limitations and Knowledge Gaps

Proposed Follow-up Experiments / Actions

Conclusion

Artifacts

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