{"gene":"RNASE2","run_date":"2026-06-10T06:43:37","timeline":{"discoveries":[{"year":1998,"finding":"EDN/RNase2 mediates ribonucleolytic destruction of extracellular RSV virions; RNase activity is necessary but not sufficient for antiviral activity. A ribonucleolytically inactivated point mutant (rhEDNdK38) competitively inhibited wild-type EDN antiviral activity in a saturable, specific manner, while the related RNase k6 had no effect, demonstrating unique structural features beyond catalysis are required. An N-terminal segment of EDN was identified as containing sequence elements mediating specific interaction with RSV.","method":"In vitro antiviral assay with recombinant wild-type and point-mutant EDN (K38 active-site mutant), competitive inhibition assay, chimeric human/New World monkey EDN constructs","journal":"Nucleic Acids Research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with active-site mutagenesis and chimeric protein analysis in a single focused study with multiple orthogonal approaches","pmids":["9826755"],"is_preprint":false},{"year":2003,"finding":"EDN/RNase2 is selectively chemotactic for dendritic cells (DCs) but not other leukocytes. DC chemotactic activity was blocked by pertussis toxin pretreatment of DCs or by placental RNase inhibitor, implicating a Gi-protein-coupled receptor and requiring RNase activity. EDN induced p42/44 MAPK activation in DCs. Mouse ortholog mEAR2 was also chemotactic for DCs, and mutational analysis of mEAR2 identified the N-terminal region as important for chemotactic activity.","method":"Chemotaxis assay in vitro, pertussis toxin inhibition, RNase inhibitor inhibition, p42/44 MAPK activation assay, in vivo air pouch model, mutational analysis of mEAR2 N-terminus","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (inhibitor assays, signaling readout, in vivo model, mutagenesis) across two orthologs in a single focused study","pmids":["12855582"],"is_preprint":false},{"year":2002,"finding":"Crystal structure of a post-translationally modified form of EDN with four extra N-terminal residues ((-4)EDN) was solved at 1 Å resolution. The modified N-terminus influences the position of catalytically important His129, explaining diminished catalytic activity of this variant. Cytotoxicity toward Kaposi's sarcoma and endothelial cell lines was attributed to cellular recognition by the N-terminal extension rather than intrinsic RNase activity.","method":"X-ray crystallography at atomic resolution (1 Å), functional cytotoxicity assays","journal":"Journal of Molecular Biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — atomic-resolution crystal structure combined with functional cytotoxicity assays in a single study with multiple methods","pmids":["11916383"],"is_preprint":false},{"year":1996,"finding":"Optimal expression of the EDN/RNS2 gene requires interaction between the promoter region and an intronic enhancer element located in the single intron. The intron alone conferred 28-fold (uninduced) to 80-fold (butyric acid-induced) increase in reporter gene activity in eosinophilic HL-60 cells. The enhancer activity was tissue-specific (present in hematopoietic but not kidney cells). Consensus binding sites for AP-1 and NF-ATp were identified in the first 60 bp of the intron.","method":"Reporter gene (CAT) transfection assay in HL-60 clone 15 and other hematopoietic cell lines with promoter/exon1/intron constructs","journal":"Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean reporter assay with multiple constructs and cell lines, single lab","pmids":["8647842"],"is_preprint":false},{"year":1997,"finding":"The intronic enhancer activity of the EDN/RNS2 gene is mediated by an NFAT-1 consensus binding sequence (5'-GGAGAA-3'). Nuclear proteins from HL-60 cells bind specifically to this site, and disruption of the sequence reduced the 20-30-fold reporter activity to background levels. No supershift was observed with anti-NFAT-1 antiserum, suggesting a nuclear protein other than NFAT-1 may act at this consensus site.","method":"Reporter gene assay, gel shift assay, site-directed mutagenesis of NFAT-1 binding site, supershift assay","journal":"Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gel shift and mutagenesis in combination, single lab; binding factor identity remains uncertain (negative supershift)","pmids":["8999843"],"is_preprint":false},{"year":1998,"finding":"The transcription factor PU.1 binds to a tandem PU.1 binding site within the intronic enhancer of the EDN/RNS2 gene and is important for its expression in eosinophilic lineage cells. Point mutations in the PU.1 site drastically reduced intronic enhancer activity; multiple forms of PU.1 were shown to bind this region.","method":"Transfection reporter assay in differentiated eosinophilic HL-60 cells, gel-shift analysis, DNA affinity precipitation, site-directed mutagenesis","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gel shift, affinity precipitation, and mutagenesis in combination, single lab","pmids":["9490699"],"is_preprint":false},{"year":1999,"finding":"The C/EBP family of transcription factors regulates EDN promoter activity. A C/EBP binding site was identified at position -124 in the proximal promoter; mutation of this site decreased promoter activity in HL-60-eos cells and in eosinophils differentiated from CD34+ cells. Multiple C/EBP proteins were shown to bind this site.","method":"Reporter gene assay, gel shift assay, DNA affinity precipitation, site-directed mutagenesis, transfection in differentiated eosinophils","journal":"Journal of Leukocyte Biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis combined with binding assays and primary cell transfection, single lab","pmids":["10534126"],"is_preprint":false},{"year":2009,"finding":"The transcription factor HNF4 binds the -350/-329 region (ednR2) of the EDN/RNase2 promoter and acts as an activator when Sp1 is absent, but represses EDN promoter activity in the presence of Sp1 through direct HNF4-Sp1 interaction that abolishes Sp1 binding to a 34-nt segment. This explains differential transcriptional regulation between EDN (RNase2) and ECP (RNase3).","method":"Supershift assay, chromatin immunoprecipitation (ChIP), DNA affinity precipitation, HNF4 overexpression reporter assay, Sp1 depletion","journal":"Journal of Cellular Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP combined with overexpression and depletion experiments in a single lab study","pmids":["19115260"],"is_preprint":false},{"year":2003,"finding":"EDN/RNase2 is present at the surface of human granulocytes via a glycosylphosphatidylinositol (GPI) anchor, in addition to its storage in specific granules. Phosphatidylinositol-specific phospholipase C (PI-PLC) treatment reduced membrane EDN expression; lower expression was confirmed on granulocytes from paroxysmal nocturnal hemoglobinuria (PNH) patients lacking GPI-anchor proteins; metabolic labeling with GPI anchor components confirmed GPI anchoring.","method":"PI-PLC treatment, flow cytometry, PNH patient granulocyte comparison, metabolic labeling with GPI anchor components","journal":"FEBS Letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal biochemical approaches including PI-PLC, disease model (PNH), and metabolic labeling in a single study","pmids":["12606041"],"is_preprint":false},{"year":2013,"finding":"EPX/EDN (RNase2) localizes not only in specific granules but also in an extragranular low-equilibrium-density compartment of human eosinophils that partially overlaps with vesicular structures, cytosolic proteins, and plasma membranes, as demonstrated by sucrose density gradient fractionation and immuno-gold labeling electron microscopy.","method":"Sucrose density gradient fractionation, immuno-gold labeling electron microscopy","journal":"Inflammation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal localization methods (fractionation and EM immunolabeling), single lab","pmids":["23053729"],"is_preprint":false},{"year":2022,"finding":"RNase2 selectively cleaves tRNA-derived fragments (tRFs) and specific miRNAs in macrophages in response to RSV infection. Using RNase2 knockout THP-1 cells and cP-RNAseq, cleavage was found predominantly at anticodon and D-loops at U/C (B1) and A (B2) sites of tRNAs. In vitro cleavage selectivity was confirmed with recombinant protein and RNase2 KO significantly reduced cell survival after RSV infection.","method":"CRISPR/Cas9 knockout in THP-1 macrophages, cP-RNAseq (cyclic phosphate RNA sequencing), in vitro recombinant protein cleavage assay, tRF & tiRNA array screening","journal":"Cellular and Molecular Life Sciences","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — genetic knockout combined with in vitro biochemical assay and two sequencing approaches in a single focused study","pmids":["35347428"],"is_preprint":false},{"year":2024,"finding":"RNase2 selectively cleaves tRNA anticodon loops with base specificity at B1 (U/C) and B2 (A) positions. Structural determinants identified by MD simulations and in vitro cleavage of synthetic tRNA variants and anticodon-loop hairpins include residues Arg36/Asn39/Gln40/Asn65/Arg68/Arg132 in the α1, loop 3, loop 4, and β6 regions, spanning P-1 to P2 substrate-binding sites.","method":"In vitro cleavage assay with recombinant RNase2 and synthetic tRNA substrates/mutants/hairpins, tRF product sequencing, molecular dynamics (MD) simulations of protein-hairpin complexes","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis, substrate screening, product sequencing, and structural modeling in a single focused study","pmids":["38228145"],"is_preprint":false},{"year":2022,"finding":"RNASE2 silencing in monocytes down-regulated IL-10 production and consequently reduced age-associated B cell (ABC/CD11c+T-bet+ B cell) numbers in monocyte-B cell co-cultures; restoration with recombinant IL-10 rescued ABC numbers, placing RNASE2 upstream of monocyte-derived IL-10 in a pathway promoting autoreactive B cell expansion.","method":"siRNA knockdown in THP-1/primary monocytes, monocyte-B cell co-culture, cytokine measurement, recombinant IL-10 rescue experiment","journal":"Frontiers in Immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockdown with rescue experiment establishing pathway position, single lab","pmids":["35265065"],"is_preprint":false}],"current_model":"RNASE2/EDN is an eosinophil granule-derived secretory ribonuclease (RNase A superfamily) that exerts antiviral activity against single-stranded RNA viruses (notably RSV) through a combination of ribonucleolytic activity and unique structural features in its N-terminal region that mediate specific virion binding; it selectively cleaves tRNAs at anticodon loops (U/C at B1, A at B2 positions) via residues including Arg36/Asn39/Gln40/Asn65/Arg68/Arg132 to generate immunoregulatory tRFs; it acts as a chemoattractant for dendritic cells via a pertussis-toxin-sensitive (Gi-coupled) receptor and activates p42/44 MAPK signaling; it is surface-expressed on granulocytes via a GPI anchor in addition to granule storage; and its expression is transcriptionally controlled by a promoter-intron enhancer mechanism involving PU.1, C/EBP, NFAT-1 consensus sites, and HNF4/Sp1 interplay."},"narrative":{"mechanistic_narrative":"RNASE2 (EDN) is an eosinophil-derived secretory ribonuclease of the RNase A superfamily that functions at the interface of antiviral defense and immune signaling, combining catalytic RNA cleavage with non-catalytic recognition activities [PMID:9826755, PMID:35347428]. Its antiviral activity against single-stranded RNA virions, notably RSV, requires ribonucleolytic activity but is not explained by catalysis alone: an active-site mutant competitively inhibits wild-type EDN and an N-terminal segment confers specific virion interaction, establishing that unique structural features beyond the catalytic center mediate target recognition [PMID:9826755]. The contribution of the N-terminal region to non-catalytic recognition is reinforced by an atomic-resolution structure of an N-terminally extended variant, in which the modified terminus repositions the catalytic His129 and the extension itself drives cellular recognition independent of RNase activity [PMID:11916383]. As an enzyme, RNASE2 selectively cleaves tRNAs at anticodon- and D-loop positions with base specificity (U/C at B1, A at B2), generating tRNA-derived fragments during RSV infection, and its loss reduces macrophage survival after infection [PMID:35347428]; the substrate selectivity is governed by a defined set of binding-site residues including Arg36, Asn39, Gln40, Asn65, Arg68, and Arg132 [PMID:38228145]. Beyond direct antiviral action, RNASE2 acts in immune signaling: it is a selective chemoattractant for dendritic cells acting through a pertussis-toxin-sensitive (Gi-coupled) receptor and p42/44 MAPK activation [PMID:12855582], and it operates upstream of monocyte-derived IL-10 to promote age-associated B cell expansion [PMID:35265065]. The protein is both stored in eosinophil granules and displayed at the granulocyte surface via a GPI anchor, distributing across granular and extragranular compartments [PMID:12606041, PMID:23053729]. Its lineage-restricted expression is controlled by a promoter–intronic enhancer architecture engaging PU.1, C/EBP, and an NFAT-1 consensus site, with HNF4–Sp1 interplay providing differential regulation [PMID:8647842, PMID:9490699, PMID:10534126, PMID:19115260].","teleology":[{"year":1996,"claim":"Established how the EDN gene achieves high, lineage-restricted expression, identifying an intronic enhancer cooperating with the promoter rather than promoter elements alone.","evidence":"CAT reporter transfection of promoter/exon1/intron constructs in eosinophilic HL-60 and other hematopoietic cell lines","pmids":["8647842"],"confidence":"Medium","gaps":["Specific factors binding the AP-1 and NF-ATp consensus sites not yet identified","Single-lab reporter system"]},{"year":1997,"claim":"Mapped the intronic enhancer activity to an NFAT-1 consensus sequence, but showed the responsible binding factor is not NFAT-1 itself, leaving the trans-acting protein unresolved.","evidence":"Reporter assay, gel shift, site-directed mutagenesis, and negative supershift with anti-NFAT-1 in HL-60 cells","pmids":["8999843"],"confidence":"Medium","gaps":["Identity of the nuclear protein binding the GGAGAA site unknown","Negative supershift leaves the factor uncharacterized"]},{"year":1998,"claim":"Identified PU.1 as a direct activator binding a tandem site in the intronic enhancer, linking EDN expression to myeloid/eosinophilic transcription factor programs.","evidence":"Reporter assay, gel shift, DNA affinity precipitation, and mutagenesis in differentiated eosinophilic HL-60 cells","pmids":["9490699"],"confidence":"Medium","gaps":["Combinatorial logic with other intronic factors not resolved","Single-lab study"]},{"year":1998,"claim":"Resolved that EDN's antiviral activity against RSV requires but is not sufficiently explained by ribonucleolysis, pointing to N-terminal structural elements that mediate specific virion recognition.","evidence":"In vitro antiviral assay with active-site mutant (K38), competitive inhibition, and chimeric human/monkey constructs","pmids":["9826755"],"confidence":"High","gaps":["Virion target/receptor of the N-terminal segment not identified","Mechanism coupling binding to virion destruction not detailed"]},{"year":1999,"claim":"Extended transcriptional control of EDN to the proximal promoter, defining a functional C/EBP site, confirmed in primary eosinophils.","evidence":"Reporter assay, gel shift, DNA affinity precipitation, mutagenesis, and transfection in eosinophils differentiated from CD34+ cells","pmids":["10534126"],"confidence":"Medium","gaps":["Which C/EBP family member dominates in vivo unclear","Interplay with intronic enhancer not dissected"]},{"year":2002,"claim":"Provided atomic-resolution structural insight showing how an N-terminal extension repositions the catalytic His129 and drives cellular recognition independent of catalysis, mechanistically separating EDN's enzymatic and recognition functions.","evidence":"1 Å X-ray crystallography of (-4)EDN plus cytotoxicity assays on Kaposi's sarcoma and endothelial cell lines","pmids":["11916383"],"confidence":"High","gaps":["Physiological prevalence of the N-terminally extended form unclear","Cellular receptor recognizing the extension not identified"]},{"year":2003,"claim":"Defined a non-antiviral immune role for EDN as a selective dendritic cell chemoattractant signaling through a Gi-coupled receptor and MAPK, requiring RNase activity.","evidence":"In vitro chemotaxis with pertussis toxin and RNase inhibitor blockade, p42/44 MAPK assay, in vivo air pouch, and mEAR2 N-terminal mutagenesis","pmids":["12855582"],"confidence":"High","gaps":["The Gi-coupled receptor on dendritic cells not molecularly identified","How RNase activity is mechanistically required for chemotaxis unresolved"]},{"year":2003,"claim":"Showed EDN is not solely a granule protein but is displayed at the granulocyte surface via a GPI anchor, expanding its potential sites of action.","evidence":"PI-PLC treatment, flow cytometry, PNH patient granulocyte comparison, and metabolic labeling with GPI anchor components","pmids":["12606041"],"confidence":"Medium","gaps":["Functional consequence of surface display not established","Mechanism of GPI attachment to a secretory ribonuclease unclear"]},{"year":2009,"claim":"Explained differential regulation between RNase2 (EDN) and RNase3 (ECP) through an HNF4–Sp1 switch acting on the promoter.","evidence":"Supershift, ChIP, DNA affinity precipitation, HNF4 overexpression reporter, and Sp1 depletion","pmids":["19115260"],"confidence":"Medium","gaps":["In vivo relevance of HNF4 in eosinophils not established","Single-lab study"]},{"year":2013,"claim":"Refined the subcellular distribution of EDN, demonstrating an extragranular vesicular/cytosolic/membrane-associated pool beyond specific granules.","evidence":"Sucrose density gradient fractionation and immuno-gold electron microscopy of human eosinophils","pmids":["23053729"],"confidence":"Medium","gaps":["Functional role of the extragranular pool not defined","Trafficking route to this compartment unknown"]},{"year":2022,"claim":"Established RNase2 as a generator of tRNA-derived fragments in macrophages during RSV infection and showed its loss compromises infected-cell survival, linking catalytic RNA cleavage to antiviral outcome.","evidence":"CRISPR knockout in THP-1 macrophages, cP-RNAseq, recombinant protein cleavage, and tRF/tiRNA array screening","pmids":["35347428"],"confidence":"High","gaps":["Downstream immunoregulatory function of the tRFs not defined","Mechanism linking tRF generation to survival unresolved"]},{"year":2022,"claim":"Placed RNASE2 upstream of monocyte-derived IL-10 in a pathway driving age-associated B cell expansion, adding an autoimmunity-relevant function.","evidence":"siRNA knockdown in THP-1/primary monocytes, monocyte-B cell co-culture, cytokine measurement, and recombinant IL-10 rescue","pmids":["35265065"],"confidence":"Medium","gaps":["Mechanism by which RNASE2 controls IL-10 production unknown","Whether catalytic activity is required not tested"]},{"year":2024,"claim":"Defined the structural basis of RNase2 tRNA anticodon-loop cleavage selectivity, identifying the substrate-binding residues that confer B1/B2 base specificity.","evidence":"In vitro cleavage of synthetic tRNA/hairpin substrates and mutants, tRF product sequencing, and MD simulations of protein-hairpin complexes","pmids":["38228145"],"confidence":"High","gaps":["No experimental high-resolution co-structure of protein bound to tRNA substrate","Physiological tRF targets in cells not mapped to these residues"]},{"year":null,"claim":"The molecular identity of the Gi-coupled dendritic cell receptor and of the RSV virion target recognized by the N-terminal segment, and the mechanism linking RNase2-generated tRFs to antiviral and immunoregulatory outcomes, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No receptor identified for chemotactic or virion-binding activity","Functional pathway from tRFs to phenotype not established","Catalytic-vs-recognition contributions to each phenotype not separately quantified in vivo"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,10,11]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[10,11]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,2,11]}],"localization":[{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[9]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[8,9]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,12]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[0,10]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[10,11]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[3,5,6,7]}],"complexes":[],"partners":["SPI1","HNF4A","SP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P10153","full_name":"Non-secretory ribonuclease","aliases":["Eosinophil-derived neurotoxin","RNase UpI-2","Ribonuclease 2","RNase 2","Ribonuclease US"],"length_aa":161,"mass_kda":18.4,"function":"This is a non-secretory ribonuclease. It is a pyrimidine specific nuclease with a slight preference for U. Cytotoxin and helminthotoxin. Selectively chemotactic for dendritic cells. Possesses a wide variety of biological activities","subcellular_location":"Lysosome; Cytoplasmic granule","url":"https://www.uniprot.org/uniprotkb/P10153/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RNASE2","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RNASE2","total_profiled":1310},"omim":[{"mim_id":"615985","title":"BARDET-BIEDL SYNDROME 8; BBS8","url":"https://www.omim.org/entry/615985"},{"mim_id":"613464","title":"RETINITIS PIGMENTOSA 51; RP51","url":"https://www.omim.org/entry/613464"},{"mim_id":"606790","title":"CD300A ANTIGEN; CD300A","url":"https://www.omim.org/entry/606790"},{"mim_id":"601981","title":"RIBONUCLEASE A FAMILY, MEMBER 6; RNASE6","url":"https://www.omim.org/entry/601981"},{"mim_id":"131410","title":"RIBONUCLEASE A FAMILY, MEMBER 2; RNASE2","url":"https://www.omim.org/entry/131410"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"bone marrow","ntpm":1614.8}],"url":"https://www.proteinatlas.org/search/RNASE2"},"hgnc":{"alias_symbol":["EDN","RAF3"],"prev_symbol":["RNS2"]},"alphafold":{"accession":"P10153","domains":[{"cath_id":"3.10.130.10","chopping":"29-159","consensus_level":"high","plddt":97.9878,"start":29,"end":159}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P10153","model_url":"https://alphafold.ebi.ac.uk/files/AF-P10153-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P10153-F1-predicted_aligned_error_v6.png","plddt_mean":91.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RNASE2","jax_strain_url":"https://www.jax.org/strain/search?query=RNASE2"},"sequence":{"accession":"P10153","fasta_url":"https://rest.uniprot.org/uniprotkb/P10153.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P10153/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P10153"}},"corpus_meta":[{"pmid":"12855582","id":"PMC_12855582","title":"Eosinophil-derived neurotoxin (EDN), an antimicrobial protein with chemotactic activities for dendritic cells.","date":"2003","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/12855582","citation_count":129,"is_preprint":false},{"pmid":"21199950","id":"PMC_21199950","title":"RNS2, a conserved member of the RNase T2 family, is necessary for ribosomal RNA decay in plants.","date":"2011","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/21199950","citation_count":118,"is_preprint":false},{"pmid":"9826755","id":"PMC_9826755","title":"Evolution of antiviral activity in the ribonuclease A gene superfamily: evidence for a specific interaction between eosinophil-derived neurotoxin (EDN/RNase 2) and respiratory syncytial virus.","date":"1998","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/9826755","citation_count":67,"is_preprint":false},{"pmid":"26184157","id":"PMC_26184157","title":"Eosinophil-Derived Neurotoxin (EDN/RNase 2) and the Mouse Eosinophil-Associated RNases (mEars): Expanding Roles in Promoting Host Defense.","date":"2015","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/26184157","citation_count":65,"is_preprint":false},{"pmid":"8999843","id":"PMC_8999843","title":"Intronic enhancer activity of the eosinophil-derived neurotoxin (RNS2) and eosinophil cationic protein (RNS3) genes is mediated by an NFAT-1 consensus binding sequence.","date":"1997","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8999843","citation_count":58,"is_preprint":false},{"pmid":"8288872","id":"PMC_8288872","title":"Measurement of eosinophil cationic protein (ECP) and eosinophil protein X/eosinophil derived neurotoxin (EPX/EDN). 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1993)","url":"https://pubmed.ncbi.nlm.nih.gov/38228145","citation_count":4,"is_preprint":false},{"pmid":"23053729","id":"PMC_23053729","title":"An extragranular compartment of blood eosinophils contains eosinophil protein X/eosinophil-derived neurotoxin (EPX/EDN).","date":"2013","source":"Inflammation","url":"https://pubmed.ncbi.nlm.nih.gov/23053729","citation_count":4,"is_preprint":false},{"pmid":"12606041","id":"PMC_12606041","title":"Evidence for glycosylphosphatidylinositol (GPI)-anchored eosinophil-derived neurotoxin (EDN) on human granulocytes.","date":"2003","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/12606041","citation_count":4,"is_preprint":false},{"pmid":"19115260","id":"PMC_19115260","title":"Transcriptional regulation of human eosinophil RNase2 by the liver-enriched hepatocyte nuclear factor 4.","date":"2009","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19115260","citation_count":3,"is_preprint":false},{"pmid":"34762913","id":"PMC_34762913","title":"The commutability of enzyme linked immunosorbent assays for the quantification of serum eosinophil-derived neurotoxin (EDN).","date":"2021","source":"Journal of immunological methods","url":"https://pubmed.ncbi.nlm.nih.gov/34762913","citation_count":3,"is_preprint":false},{"pmid":"36829141","id":"PMC_36829141","title":"Potential role of eNOS and EDN-1 gene polymorphisms in the development and progression of retinopathy of prematurity.","date":"2023","source":"BMC ophthalmology","url":"https://pubmed.ncbi.nlm.nih.gov/36829141","citation_count":2,"is_preprint":false},{"pmid":"24738159","id":"PMC_24738159","title":"The production of the eosinophil proteins ECP and EPX/EDN are regulated in a reciprocal manner.","date":"2014","source":"APMIS : acta pathologica, microbiologica, et immunologica Scandinavica","url":"https://pubmed.ncbi.nlm.nih.gov/24738159","citation_count":2,"is_preprint":false},{"pmid":"8492433","id":"PMC_8492433","title":"[Eosinophil granule proteins (MBP, ECP, EPX/EDN, EPO)--a possible process of eosinophil activation and degranulation].","date":"1993","source":"Nihon rinsho. Japanese journal of clinical medicine","url":"https://pubmed.ncbi.nlm.nih.gov/8492433","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14544,"output_tokens":3721,"usd":0.049723,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11328,"output_tokens":4293,"usd":0.081982,"stage2_stop_reason":"end_turn"},"total_usd":0.131705,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"EDN/RNase2 mediates ribonucleolytic destruction of extracellular RSV virions; RNase activity is necessary but not sufficient for antiviral activity. A ribonucleolytically inactivated point mutant (rhEDNdK38) competitively inhibited wild-type EDN antiviral activity in a saturable, specific manner, while the related RNase k6 had no effect, demonstrating unique structural features beyond catalysis are required. An N-terminal segment of EDN was identified as containing sequence elements mediating specific interaction with RSV.\",\n      \"method\": \"In vitro antiviral assay with recombinant wild-type and point-mutant EDN (K38 active-site mutant), competitive inhibition assay, chimeric human/New World monkey EDN constructs\",\n      \"journal\": \"Nucleic Acids Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with active-site mutagenesis and chimeric protein analysis in a single focused study with multiple orthogonal approaches\",\n      \"pmids\": [\"9826755\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"EDN/RNase2 is selectively chemotactic for dendritic cells (DCs) but not other leukocytes. DC chemotactic activity was blocked by pertussis toxin pretreatment of DCs or by placental RNase inhibitor, implicating a Gi-protein-coupled receptor and requiring RNase activity. EDN induced p42/44 MAPK activation in DCs. Mouse ortholog mEAR2 was also chemotactic for DCs, and mutational analysis of mEAR2 identified the N-terminal region as important for chemotactic activity.\",\n      \"method\": \"Chemotaxis assay in vitro, pertussis toxin inhibition, RNase inhibitor inhibition, p42/44 MAPK activation assay, in vivo air pouch model, mutational analysis of mEAR2 N-terminus\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (inhibitor assays, signaling readout, in vivo model, mutagenesis) across two orthologs in a single focused study\",\n      \"pmids\": [\"12855582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Crystal structure of a post-translationally modified form of EDN with four extra N-terminal residues ((-4)EDN) was solved at 1 Å resolution. The modified N-terminus influences the position of catalytically important His129, explaining diminished catalytic activity of this variant. Cytotoxicity toward Kaposi's sarcoma and endothelial cell lines was attributed to cellular recognition by the N-terminal extension rather than intrinsic RNase activity.\",\n      \"method\": \"X-ray crystallography at atomic resolution (1 Å), functional cytotoxicity assays\",\n      \"journal\": \"Journal of Molecular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — atomic-resolution crystal structure combined with functional cytotoxicity assays in a single study with multiple methods\",\n      \"pmids\": [\"11916383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Optimal expression of the EDN/RNS2 gene requires interaction between the promoter region and an intronic enhancer element located in the single intron. The intron alone conferred 28-fold (uninduced) to 80-fold (butyric acid-induced) increase in reporter gene activity in eosinophilic HL-60 cells. The enhancer activity was tissue-specific (present in hematopoietic but not kidney cells). Consensus binding sites for AP-1 and NF-ATp were identified in the first 60 bp of the intron.\",\n      \"method\": \"Reporter gene (CAT) transfection assay in HL-60 clone 15 and other hematopoietic cell lines with promoter/exon1/intron constructs\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean reporter assay with multiple constructs and cell lines, single lab\",\n      \"pmids\": [\"8647842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"The intronic enhancer activity of the EDN/RNS2 gene is mediated by an NFAT-1 consensus binding sequence (5'-GGAGAA-3'). Nuclear proteins from HL-60 cells bind specifically to this site, and disruption of the sequence reduced the 20-30-fold reporter activity to background levels. No supershift was observed with anti-NFAT-1 antiserum, suggesting a nuclear protein other than NFAT-1 may act at this consensus site.\",\n      \"method\": \"Reporter gene assay, gel shift assay, site-directed mutagenesis of NFAT-1 binding site, supershift assay\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gel shift and mutagenesis in combination, single lab; binding factor identity remains uncertain (negative supershift)\",\n      \"pmids\": [\"8999843\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The transcription factor PU.1 binds to a tandem PU.1 binding site within the intronic enhancer of the EDN/RNS2 gene and is important for its expression in eosinophilic lineage cells. Point mutations in the PU.1 site drastically reduced intronic enhancer activity; multiple forms of PU.1 were shown to bind this region.\",\n      \"method\": \"Transfection reporter assay in differentiated eosinophilic HL-60 cells, gel-shift analysis, DNA affinity precipitation, site-directed mutagenesis\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gel shift, affinity precipitation, and mutagenesis in combination, single lab\",\n      \"pmids\": [\"9490699\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The C/EBP family of transcription factors regulates EDN promoter activity. A C/EBP binding site was identified at position -124 in the proximal promoter; mutation of this site decreased promoter activity in HL-60-eos cells and in eosinophils differentiated from CD34+ cells. Multiple C/EBP proteins were shown to bind this site.\",\n      \"method\": \"Reporter gene assay, gel shift assay, DNA affinity precipitation, site-directed mutagenesis, transfection in differentiated eosinophils\",\n      \"journal\": \"Journal of Leukocyte Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis combined with binding assays and primary cell transfection, single lab\",\n      \"pmids\": [\"10534126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The transcription factor HNF4 binds the -350/-329 region (ednR2) of the EDN/RNase2 promoter and acts as an activator when Sp1 is absent, but represses EDN promoter activity in the presence of Sp1 through direct HNF4-Sp1 interaction that abolishes Sp1 binding to a 34-nt segment. This explains differential transcriptional regulation between EDN (RNase2) and ECP (RNase3).\",\n      \"method\": \"Supershift assay, chromatin immunoprecipitation (ChIP), DNA affinity precipitation, HNF4 overexpression reporter assay, Sp1 depletion\",\n      \"journal\": \"Journal of Cellular Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP combined with overexpression and depletion experiments in a single lab study\",\n      \"pmids\": [\"19115260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"EDN/RNase2 is present at the surface of human granulocytes via a glycosylphosphatidylinositol (GPI) anchor, in addition to its storage in specific granules. Phosphatidylinositol-specific phospholipase C (PI-PLC) treatment reduced membrane EDN expression; lower expression was confirmed on granulocytes from paroxysmal nocturnal hemoglobinuria (PNH) patients lacking GPI-anchor proteins; metabolic labeling with GPI anchor components confirmed GPI anchoring.\",\n      \"method\": \"PI-PLC treatment, flow cytometry, PNH patient granulocyte comparison, metabolic labeling with GPI anchor components\",\n      \"journal\": \"FEBS Letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal biochemical approaches including PI-PLC, disease model (PNH), and metabolic labeling in a single study\",\n      \"pmids\": [\"12606041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"EPX/EDN (RNase2) localizes not only in specific granules but also in an extragranular low-equilibrium-density compartment of human eosinophils that partially overlaps with vesicular structures, cytosolic proteins, and plasma membranes, as demonstrated by sucrose density gradient fractionation and immuno-gold labeling electron microscopy.\",\n      \"method\": \"Sucrose density gradient fractionation, immuno-gold labeling electron microscopy\",\n      \"journal\": \"Inflammation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal localization methods (fractionation and EM immunolabeling), single lab\",\n      \"pmids\": [\"23053729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNase2 selectively cleaves tRNA-derived fragments (tRFs) and specific miRNAs in macrophages in response to RSV infection. Using RNase2 knockout THP-1 cells and cP-RNAseq, cleavage was found predominantly at anticodon and D-loops at U/C (B1) and A (B2) sites of tRNAs. In vitro cleavage selectivity was confirmed with recombinant protein and RNase2 KO significantly reduced cell survival after RSV infection.\",\n      \"method\": \"CRISPR/Cas9 knockout in THP-1 macrophages, cP-RNAseq (cyclic phosphate RNA sequencing), in vitro recombinant protein cleavage assay, tRF & tiRNA array screening\",\n      \"journal\": \"Cellular and Molecular Life Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — genetic knockout combined with in vitro biochemical assay and two sequencing approaches in a single focused study\",\n      \"pmids\": [\"35347428\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNase2 selectively cleaves tRNA anticodon loops with base specificity at B1 (U/C) and B2 (A) positions. Structural determinants identified by MD simulations and in vitro cleavage of synthetic tRNA variants and anticodon-loop hairpins include residues Arg36/Asn39/Gln40/Asn65/Arg68/Arg132 in the α1, loop 3, loop 4, and β6 regions, spanning P-1 to P2 substrate-binding sites.\",\n      \"method\": \"In vitro cleavage assay with recombinant RNase2 and synthetic tRNA substrates/mutants/hairpins, tRF product sequencing, molecular dynamics (MD) simulations of protein-hairpin complexes\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis, substrate screening, product sequencing, and structural modeling in a single focused study\",\n      \"pmids\": [\"38228145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNASE2 silencing in monocytes down-regulated IL-10 production and consequently reduced age-associated B cell (ABC/CD11c+T-bet+ B cell) numbers in monocyte-B cell co-cultures; restoration with recombinant IL-10 rescued ABC numbers, placing RNASE2 upstream of monocyte-derived IL-10 in a pathway promoting autoreactive B cell expansion.\",\n      \"method\": \"siRNA knockdown in THP-1/primary monocytes, monocyte-B cell co-culture, cytokine measurement, recombinant IL-10 rescue experiment\",\n      \"journal\": \"Frontiers in Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockdown with rescue experiment establishing pathway position, single lab\",\n      \"pmids\": [\"35265065\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RNASE2/EDN is an eosinophil granule-derived secretory ribonuclease (RNase A superfamily) that exerts antiviral activity against single-stranded RNA viruses (notably RSV) through a combination of ribonucleolytic activity and unique structural features in its N-terminal region that mediate specific virion binding; it selectively cleaves tRNAs at anticodon loops (U/C at B1, A at B2 positions) via residues including Arg36/Asn39/Gln40/Asn65/Arg68/Arg132 to generate immunoregulatory tRFs; it acts as a chemoattractant for dendritic cells via a pertussis-toxin-sensitive (Gi-coupled) receptor and activates p42/44 MAPK signaling; it is surface-expressed on granulocytes via a GPI anchor in addition to granule storage; and its expression is transcriptionally controlled by a promoter-intron enhancer mechanism involving PU.1, C/EBP, NFAT-1 consensus sites, and HNF4/Sp1 interplay.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RNASE2 (EDN) is an eosinophil-derived secretory ribonuclease of the RNase A superfamily that functions at the interface of antiviral defense and immune signaling, combining catalytic RNA cleavage with non-catalytic recognition activities [#0, #10]. Its antiviral activity against single-stranded RNA virions, notably RSV, requires ribonucleolytic activity but is not explained by catalysis alone: an active-site mutant competitively inhibits wild-type EDN and an N-terminal segment confers specific virion interaction, establishing that unique structural features beyond the catalytic center mediate target recognition [#0]. The contribution of the N-terminal region to non-catalytic recognition is reinforced by an atomic-resolution structure of an N-terminally extended variant, in which the modified terminus repositions the catalytic His129 and the extension itself drives cellular recognition independent of RNase activity [#2]. As an enzyme, RNASE2 selectively cleaves tRNAs at anticodon- and D-loop positions with base specificity (U/C at B1, A at B2), generating tRNA-derived fragments during RSV infection, and its loss reduces macrophage survival after infection [#10]; the substrate selectivity is governed by a defined set of binding-site residues including Arg36, Asn39, Gln40, Asn65, Arg68, and Arg132 [#11]. Beyond direct antiviral action, RNASE2 acts in immune signaling: it is a selective chemoattractant for dendritic cells acting through a pertussis-toxin-sensitive (Gi-coupled) receptor and p42/44 MAPK activation [#1], and it operates upstream of monocyte-derived IL-10 to promote age-associated B cell expansion [#12]. The protein is both stored in eosinophil granules and displayed at the granulocyte surface via a GPI anchor, distributing across granular and extragranular compartments [#8, #9]. Its lineage-restricted expression is controlled by a promoter–intronic enhancer architecture engaging PU.1, C/EBP, and an NFAT-1 consensus site, with HNF4–Sp1 interplay providing differential regulation [#3, #5, #6, #7].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established how the EDN gene achieves high, lineage-restricted expression, identifying an intronic enhancer cooperating with the promoter rather than promoter elements alone.\",\n      \"evidence\": \"CAT reporter transfection of promoter/exon1/intron constructs in eosinophilic HL-60 and other hematopoietic cell lines\",\n      \"pmids\": [\"8647842\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific factors binding the AP-1 and NF-ATp consensus sites not yet identified\", \"Single-lab reporter system\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Mapped the intronic enhancer activity to an NFAT-1 consensus sequence, but showed the responsible binding factor is not NFAT-1 itself, leaving the trans-acting protein unresolved.\",\n      \"evidence\": \"Reporter assay, gel shift, site-directed mutagenesis, and negative supershift with anti-NFAT-1 in HL-60 cells\",\n      \"pmids\": [\"8999843\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the nuclear protein binding the GGAGAA site unknown\", \"Negative supershift leaves the factor uncharacterized\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Identified PU.1 as a direct activator binding a tandem site in the intronic enhancer, linking EDN expression to myeloid/eosinophilic transcription factor programs.\",\n      \"evidence\": \"Reporter assay, gel shift, DNA affinity precipitation, and mutagenesis in differentiated eosinophilic HL-60 cells\",\n      \"pmids\": [\"9490699\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Combinatorial logic with other intronic factors not resolved\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Resolved that EDN's antiviral activity against RSV requires but is not sufficiently explained by ribonucleolysis, pointing to N-terminal structural elements that mediate specific virion recognition.\",\n      \"evidence\": \"In vitro antiviral assay with active-site mutant (K38), competitive inhibition, and chimeric human/monkey constructs\",\n      \"pmids\": [\"9826755\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Virion target/receptor of the N-terminal segment not identified\", \"Mechanism coupling binding to virion destruction not detailed\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Extended transcriptional control of EDN to the proximal promoter, defining a functional C/EBP site, confirmed in primary eosinophils.\",\n      \"evidence\": \"Reporter assay, gel shift, DNA affinity precipitation, mutagenesis, and transfection in eosinophils differentiated from CD34+ cells\",\n      \"pmids\": [\"10534126\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which C/EBP family member dominates in vivo unclear\", \"Interplay with intronic enhancer not dissected\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Provided atomic-resolution structural insight showing how an N-terminal extension repositions the catalytic His129 and drives cellular recognition independent of catalysis, mechanistically separating EDN's enzymatic and recognition functions.\",\n      \"evidence\": \"1 Å X-ray crystallography of (-4)EDN plus cytotoxicity assays on Kaposi's sarcoma and endothelial cell lines\",\n      \"pmids\": [\"11916383\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological prevalence of the N-terminally extended form unclear\", \"Cellular receptor recognizing the extension not identified\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined a non-antiviral immune role for EDN as a selective dendritic cell chemoattractant signaling through a Gi-coupled receptor and MAPK, requiring RNase activity.\",\n      \"evidence\": \"In vitro chemotaxis with pertussis toxin and RNase inhibitor blockade, p42/44 MAPK assay, in vivo air pouch, and mEAR2 N-terminal mutagenesis\",\n      \"pmids\": [\"12855582\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The Gi-coupled receptor on dendritic cells not molecularly identified\", \"How RNase activity is mechanistically required for chemotaxis unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showed EDN is not solely a granule protein but is displayed at the granulocyte surface via a GPI anchor, expanding its potential sites of action.\",\n      \"evidence\": \"PI-PLC treatment, flow cytometry, PNH patient granulocyte comparison, and metabolic labeling with GPI anchor components\",\n      \"pmids\": [\"12606041\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of surface display not established\", \"Mechanism of GPI attachment to a secretory ribonuclease unclear\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Explained differential regulation between RNase2 (EDN) and RNase3 (ECP) through an HNF4–Sp1 switch acting on the promoter.\",\n      \"evidence\": \"Supershift, ChIP, DNA affinity precipitation, HNF4 overexpression reporter, and Sp1 depletion\",\n      \"pmids\": [\"19115260\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance of HNF4 in eosinophils not established\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Refined the subcellular distribution of EDN, demonstrating an extragranular vesicular/cytosolic/membrane-associated pool beyond specific granules.\",\n      \"evidence\": \"Sucrose density gradient fractionation and immuno-gold electron microscopy of human eosinophils\",\n      \"pmids\": [\"23053729\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional role of the extragranular pool not defined\", \"Trafficking route to this compartment unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established RNase2 as a generator of tRNA-derived fragments in macrophages during RSV infection and showed its loss compromises infected-cell survival, linking catalytic RNA cleavage to antiviral outcome.\",\n      \"evidence\": \"CRISPR knockout in THP-1 macrophages, cP-RNAseq, recombinant protein cleavage, and tRF/tiRNA array screening\",\n      \"pmids\": [\"35347428\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream immunoregulatory function of the tRFs not defined\", \"Mechanism linking tRF generation to survival unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed RNASE2 upstream of monocyte-derived IL-10 in a pathway driving age-associated B cell expansion, adding an autoimmunity-relevant function.\",\n      \"evidence\": \"siRNA knockdown in THP-1/primary monocytes, monocyte-B cell co-culture, cytokine measurement, and recombinant IL-10 rescue\",\n      \"pmids\": [\"35265065\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which RNASE2 controls IL-10 production unknown\", \"Whether catalytic activity is required not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the structural basis of RNase2 tRNA anticodon-loop cleavage selectivity, identifying the substrate-binding residues that confer B1/B2 base specificity.\",\n      \"evidence\": \"In vitro cleavage of synthetic tRNA/hairpin substrates and mutants, tRF product sequencing, and MD simulations of protein-hairpin complexes\",\n      \"pmids\": [\"38228145\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No experimental high-resolution co-structure of protein bound to tRNA substrate\", \"Physiological tRF targets in cells not mapped to these residues\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular identity of the Gi-coupled dendritic cell receptor and of the RSV virion target recognized by the N-terminal segment, and the mechanism linking RNase2-generated tRFs to antiviral and immunoregulatory outcomes, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No receptor identified for chemotactic or virion-binding activity\", \"Functional pathway from tRFs to phenotype not established\", \"Catalytic-vs-recognition contributions to each phenotype not separately quantified in vivo\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 10, 11]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [10, 11]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 2, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [8, 9]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 12]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 10]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [10, 11]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [3, 5, 6, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SPI1\", \"HNF4A\", \"SP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}