{"gene":"SNF8","run_date":"2026-06-10T07:46:37","timeline":{"discoveries":[{"year":1999,"finding":"EAP30 (SNF8) was identified as a subunit of the ELL complex; it interacts directly with ELL and derepresses ELL's inhibitory activity on RNA polymerase II transcription in vitro. EAP30 has sequence homology to S. cerevisiae SNF8, whose genetic analysis links it to derepression of gene expression.","method":"In vitro transcription assay, protein interaction studies, sequence homology analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1–2 / Weak — in vitro functional assay demonstrating derepression, but single lab, limited mechanistic detail in abstract","pmids":["10419521"],"is_preprint":false},{"year":1995,"finding":"In S. cerevisiae, SNF8 is required for derepression of the SUC2 gene in response to glucose limitation; genetic epistasis showed SNF8 and SNF7 are functionally related (double mutant showed no additive impairment), and snf8 is genetically distinct from SNF1, SNF4, SNF2, SNF5, SNF6 groups.","method":"Gene disruption, genetic epistasis, complementation cloning, invertase derepression assay","journal":"Yeast (Chichester, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with multiple allele combinations, clean loss-of-function phenotype, but in yeast and single lab","pmids":["7785322"],"is_preprint":false},{"year":2007,"finding":"Vps22/EAP30 (SNF8), as an ESCRT-II subunit, localizes to endosomes containing EGFR, Hrs, ESCRT-I and ESCRT-III; siRNA depletion of Vps22 greatly reduced degradation of EGFR and CXCR4, caused EGFR accumulation on limiting membranes of early endosomes and formation of aberrantly small multivesicular bodies. Notably, EGF-activated ERK1/2 signaling was not sustained upon Vps22 depletion (unlike Hrs or Tsg101 depletion), placing ESCRT-II engagement after termination of EGF signaling.","method":"siRNA knockdown, fluorescence microscopy, receptor degradation assays, ERK phosphorylation/nuclear translocation assays","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean siRNA knockdown with defined cellular phenotypes, multiple orthogonal readouts (receptor degradation, endosome morphology, signaling), pathway placement via epistasis","pmids":["17714434"],"is_preprint":false},{"year":2006,"finding":"RILP interacts with VPS22 (SNF8) of ESCRT-II; the N-terminal half of RILP mediates interaction with VPS22. RILP regulates membrane recruitment of ESCRT-II subunits VPS22 and VPS36, integrating the late endocytic (Rab7/RILP) machinery with the early sorting (ESCRT) machinery.","method":"Co-immunoprecipitation, domain mapping, overexpression, endosomal trafficking assays (EGF sorting, LBPA/EEA1 markers)","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP with domain mapping confirmed in two independent papers (PMID 17010938 and 16857164), single method type","pmids":["17010938","16857164"],"is_preprint":false},{"year":2006,"finding":"Using yeast two-hybrid and co-immunoprecipitation, VPS22 (EAP30/SNF8) was confirmed as a binding partner of RILP; the interaction is with the N-terminal half of RILP. Confocal immunofluorescence showed colocalization of GFP-RILP and HA-VPS22.","method":"Yeast two-hybrid, co-immunoprecipitation, confocal immunofluorescence","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid plus co-IP plus colocalization, replicated in companion paper, but single interaction type","pmids":["16857164"],"is_preprint":false},{"year":2005,"finding":"The fission yeast homolog of EAP30/SNF8 (Dot2) negatively regulates meiotic spindle pole body (SPB) maturation; dot2 mutants accumulate excess electron-dense material near SPBs (AMtOCs), assemble multipolar spindles, and missegregate chromosomes. Elevated Pcp1 (pericentrin ortholog) levels were associated with SPB aberrations, and reducing pcp1 expression suppressed AMtOCs, placing Dot2 upstream of Pcp1 in SPB maturation control.","method":"Genetic mutation analysis, electron microscopy, epistasis (pcp1 suppression of dot2 mutant), chromosome segregation assays","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with suppressor analysis, electron microscopy, clean loss-of-function phenotype in fission yeast ortholog","pmids":["15992541"],"is_preprint":false},{"year":2012,"finding":"SNF8 interacts with the amino-terminal cytoplasmic domain (first 107 amino acids) of TRPC6; overexpression of SNF8 enhances TRPC6-mediated whole-cell currents and NFAT-mediated transcription activation by gain-of-function TRPC6 mutants, while RNAi knockdown partially inhibits NFAT activation. SNF8 overexpression does not alter cell-surface levels of TRPC6, suggesting the effect is on channel activity rather than trafficking.","method":"Yeast two-hybrid, co-immunoprecipitation, whole-cell patch-clamp electrophysiology, NFAT reporter assay, RNAi knockdown, cell-surface biotinylation","journal":"BMC cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by Co-IP, functional electrophysiology assay, RNAi, and surface expression measurement; multiple orthogonal methods in single lab","pmids":["23171048"],"is_preprint":false},{"year":2009,"finding":"MCM2 binds to EAP30 (SNF8) and competes with ELL for binding to EAP30, thereby potentially modulating the stability and activity of the Holo-ELL complex.","method":"Co-immunoprecipitation, competitive binding assay","journal":"FEBS letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP/competition assay, single lab, limited mechanistic follow-up","pmids":["19819239"],"is_preprint":false},{"year":2017,"finding":"EAP30 (SNF8/VPS22) is essential for IRF3-dependent induction of type I and III interferons and antiviral defense. A fraction of EAP30 localizes to the nucleus where, in a virus-inducible manner, it forms a complex with IRF3 and its co-activator CBP, promoting IRF3 binding to target gene promoters (IFN-β, IFN-λ1, ISG56). EAP30 acts downstream of IRF3 phosphorylation/activation, not upstream. This function is specific to EAP30 among ESCRT-II subunits.","method":"siRNA knockdown, co-immunoprecipitation (EAP30-IRF3-CBP complex), chromatin immunoprecipitation (IRF3 promoter binding), nuclear fractionation, antiviral assays (VSV, HCV)","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ChIP, nuclear fractionation, functional antiviral assays) in single rigorous study establishing pathway position and nuclear mechanism","pmids":["29084253"],"is_preprint":false},{"year":2020,"finding":"In C. elegans, VPS-22/SNF8 acts in the same pathway as DAF-16 (FOXO transcription factor) to regulate longevity; epistasis analysis showed that knockdown of vps-22/snf8 in daf-16 null worms did not further shorten lifespan, and overexpression of daf-16 rescued the short-lived phenotype of vps-22/snf8 knockdown. Mechanistically, downregulation of vps-22/snf8 decreased nuclear localization of DAF-16 and altered expression of DAF-16 target longevity genes.","method":"RNAi knockdown, genetic epistasis (double mutant/knockdown), nuclear localization assay (DAF-16::GFP), lifespan assay, gene expression analysis","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis plus nuclear localization assay plus target gene expression, multiple methods, single lab, C. elegans ortholog","pmids":["32829877"],"is_preprint":false}],"current_model":"SNF8/EAP30/VPS22 is a multifunctional ESCRT-II subunit that: (1) acts on endosomes to sort ubiquitinated receptors (EGFR, CXCR4) into multivesicular bodies for lysosomal degradation, where its membrane recruitment is regulated by the Rab7 effector RILP; (2) functions in the nucleus as part of the ELL transcription elongation complex (where it derepresses ELL-mediated inhibition of RNA Pol II and its interaction with ELL is modulated by MCM2) and, independently, forms a virus-inducible complex with IRF3 and CBP to drive antiviral interferon gene transcription; (3) interacts with and enhances TRPC6 channel activity independently of receptor trafficking; and in model organisms, its ortholog regulates meiotic spindle pole body maturation (fission yeast) and modulates longevity via the DAF-16/FOXO pathway (C. elegans)."},"narrative":{"mechanistic_narrative":"SNF8 (EAP30/VPS22) is a subunit of the ESCRT-II complex that drives sorting of ubiquitinated cell-surface receptors into multivesicular bodies for lysosomal degradation, and which has been repeatedly captured acting in the nucleus to control gene transcription [PMID:17714434, PMID:10419521]. On endosomes, SNF8 localizes to compartments containing EGFR, Hrs and other ESCRT components, and its depletion blocks degradation of EGFR and CXCR4, causes receptor accumulation on the limiting membrane of early endosomes, and yields aberrantly small multivesicular bodies; this engagement occurs after termination of EGF-stimulated ERK signaling [PMID:17714434]. Membrane recruitment of the ESCRT-II subunits SNF8 (VPS22) and VPS36 is directed by the Rab7 effector RILP, whose N-terminal half binds SNF8, linking the late endocytic machinery to early ESCRT sorting [PMID:17010938, PMID:16857164]. Independently of trafficking, SNF8 is a subunit of the ELL transcription elongation complex, where it binds ELL directly and derepresses ELL-mediated inhibition of RNA polymerase II [PMID:10419521], and a nuclear pool of SNF8 forms a virus-inducible complex with IRF3 and the co-activator CBP, acting downstream of IRF3 activation to promote IRF3 binding at interferon and ISG promoters and drive antiviral type I and III interferon responses [PMID:29084253]. SNF8 also binds the N-terminal cytoplasmic domain of TRPC6 and enhances TRPC6 channel current and downstream NFAT activation without changing channel surface levels [PMID:23171048]. Work in model organisms shows the ortholog negatively regulates meiotic spindle pole body maturation upstream of the pericentrin ortholog Pcp1 in fission yeast [PMID:15992541] and acts in the DAF-16/FOXO longevity pathway in C. elegans by supporting DAF-16 nuclear localization [PMID:32829877].","teleology":[{"year":1995,"claim":"Established the founding loss-of-function phenotype: SNF8 was unknown in function until yeast genetics showed it is required for glucose-derepression of SUC2 and is functionally related to SNF7, defining a discrete gene group distinct from the SNF1/SNF4 cluster.","evidence":"Gene disruption, genetic epistasis, and invertase derepression assay in S. cerevisiae","pmids":["7785322"],"confidence":"Medium","gaps":["No molecular mechanism for how SNF8 derepresses transcription","Relationship to membrane trafficking not yet appreciated","Mammalian ortholog function untested"]},{"year":1999,"claim":"Connected the yeast gene to a mammalian transcription mechanism: EAP30/SNF8 was found to be an ELL-complex subunit that binds ELL and relieves ELL's inhibition of RNA Pol II, giving a biochemical activity to the derepression phenotype.","evidence":"In vitro transcription assay, protein interaction studies, and sequence homology analysis","pmids":["10419521"],"confidence":"Medium","gaps":["In vitro only; nuclear role not shown in cells","Stoichiometry/structure of EAP30 within Holo-ELL unresolved","Does not address any membrane function"]},{"year":2005,"claim":"Revealed an organelle-biogenesis role: the fission yeast ortholog Dot2 negatively regulates meiotic spindle pole body maturation, placing it upstream of the pericentrin ortholog Pcp1.","evidence":"Genetic mutation, electron microscopy, and pcp1-suppression epistasis in S. pombe meiosis","pmids":["15992541"],"confidence":"Medium","gaps":["Mechanistic link between ESCRT-II function and SPB control unclear","Conservation to mammalian centrosomes untested","Whether this depends on ESCRT membrane activity unknown"]},{"year":2006,"claim":"Identified the recruitment cue for ESCRT-II to endosomes: RILP, the Rab7 effector, binds SNF8/VPS22 via its N-terminal half and controls membrane recruitment of ESCRT-II, bridging late-endosomal and early-sorting machinery.","evidence":"Yeast two-hybrid, co-immunoprecipitation, domain mapping, and confocal colocalization (two companion papers)","pmids":["17010938","16857164"],"confidence":"Medium","gaps":["Single interaction-method class (no structure of the complex)","Functional consequence of disrupting RILP-VPS22 binding not quantified","How Rab7 activity gates the interaction unresolved"]},{"year":2007,"claim":"Defined SNF8's core cell-biological role and pathway position: as an ESCRT-II subunit it is required for MVB sorting and lysosomal degradation of EGFR and CXCR4, acting after EGF signaling has terminated.","evidence":"siRNA knockdown with receptor degradation, endosome morphology, and ERK signaling readouts","pmids":["17714434"],"confidence":"High","gaps":["Does not resolve how ESCRT-II selects ubiquitinated cargo at this step","Structural role within the ESCRT cascade not addressed","Specificity for EGFR/CXCR4 vs other receptors untested"]},{"year":2009,"claim":"Probed regulation of the nuclear ELL complex: MCM2 binds EAP30 and competes with ELL, indicating a route to modulate Holo-ELL stability/activity.","evidence":"Co-immunoprecipitation and competitive binding assay","pmids":["19819239"],"confidence":"Low","gaps":["Single Co-IP/competition assay without functional or transcriptional readout","Cellular relevance of competition not demonstrated","Not independently confirmed"]},{"year":2012,"claim":"Uncovered a trafficking-independent ion-channel function: SNF8 binds the TRPC6 N-terminal cytoplasmic domain and enhances channel current and NFAT signaling without altering surface channel levels.","evidence":"Yeast two-hybrid, co-IP, whole-cell patch-clamp, NFAT reporter, RNAi, and surface biotinylation","pmids":["23171048"],"confidence":"Medium","gaps":["Mechanism by which SNF8 potentiates channel gating unknown","Physiological context of TRPC6 regulation untested","Single lab"]},{"year":2017,"claim":"Established a distinct nuclear antiviral role: a fraction of EAP30 forms a virus-inducible IRF3-CBP complex acting downstream of IRF3 activation to promote promoter binding and induction of type I/III interferons, a function specific to EAP30 among ESCRT-II subunits.","evidence":"siRNA, co-IP, ChIP, nuclear fractionation, and VSV/HCV antiviral assays","pmids":["29084253"],"confidence":"High","gaps":["How EAP30 partitions between endosomal ESCRT-II and nuclear pools is unresolved","Whether this role requires ESCRT-II assembly unknown","Signal that triggers nuclear localization not defined"]},{"year":2020,"claim":"Linked SNF8 to longevity signaling: the C. elegans ortholog acts in the DAF-16/FOXO pathway, with knockdown reducing DAF-16 nuclear localization and shortening lifespan in a daf-16-dependent manner.","evidence":"RNAi, genetic epistasis, DAF-16::GFP nuclear localization, lifespan and target-gene expression assays","pmids":["32829877"],"confidence":"Medium","gaps":["Molecular link between ESCRT-II and DAF-16 trafficking unknown","Whether endosomal or nuclear SNF8 function mediates the effect unclear","Conservation to mammalian FOXO untested"]},{"year":null,"claim":"How a single ESCRT-II subunit is partitioned and regulated across its membrane-sorting, transcription-elongation, antiviral, channel-modulatory, and spindle/longevity roles remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model integrating SNF8's endosomal vs nuclear functions","Signals governing nuclear translocation undefined","Whether non-ESCRT roles require the intact ESCRT-II complex unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,8]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,3]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[2,3]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,8]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,3]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,8]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[8]}],"complexes":["ESCRT-II","ELL transcription elongation complex"],"partners":["ELL","RILP","VPS36","TRPC6","IRF3","CBP","MCM2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96H20","full_name":"Vacuolar-sorting protein SNF8","aliases":["ELL-associated protein of 30 kDa","ESCRT-II complex subunit VPS22","hVps22"],"length_aa":258,"mass_kda":28.9,"function":"Component of the endosomal sorting complex required for transport II (ESCRT-II), which is required for multivesicular body (MVB) formation and sorting of endosomal cargo proteins into MVBs, and plays a role in autophagy (PubMed:38423010). The MVB pathway mediates delivery of transmembrane proteins into the lumen of the lysosome for degradation. The ESCRT-II complex is probably involved in the recruitment of the ESCRT-III complex. The ESCRT-II complex may also play a role in transcription regulation by participating in derepression of transcription by RNA polymerase II, possibly via its interaction with ELL. Required for degradation of both endocytosed EGF and EGFR, but not for the EGFR ligand-mediated internalization. It is also required for the degradation of CXCR4. Required for the exosomal release of SDCBP, CD63 and syndecan (PubMed:22660413)","subcellular_location":"Cytoplasm; Endosome membrane; Nucleus; Late endosome membrane","url":"https://www.uniprot.org/uniprotkb/Q96H20/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/SNF8","classification":"Common Essential","n_dependent_lines":1157,"n_total_lines":1208,"dependency_fraction":0.9577814569536424},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000159210","cell_line_id":"CID000783","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"VPS36","stoichiometry":10.0},{"gene":"VPS25","stoichiometry":10.0},{"gene":"UBE3B","stoichiometry":0.2},{"gene":"TNPO2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000783","total_profiled":1310},"omim":[{"mim_id":"620784","title":"NEURODEVELOPMENTAL DISORDER PLUS OPTIC ATROPHY; NEDOA","url":"https://www.omim.org/entry/620784"},{"mim_id":"620783","title":"DEVELOPMENTAL AND EPILEPTIC ENCEPHALOPATHY 115; DEE115","url":"https://www.omim.org/entry/620783"},{"mim_id":"610907","title":"VACUOLAR PROTEIN SORTING 25 HOMOLOG; VPS25","url":"https://www.omim.org/entry/610907"},{"mim_id":"610904","title":"SNF8 SUBUNIT OF ESCRIT-II; SNF8","url":"https://www.omim.org/entry/610904"},{"mim_id":"610903","title":"VACUOLAR PROTEIN SORTING 36 HOMOLOG; VPS36","url":"https://www.omim.org/entry/610903"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SNF8"},"hgnc":{"alias_symbol":["EAP30","VPS22","Dot3"],"prev_symbol":[]},"alphafold":{"accession":"Q96H20","domains":[{"cath_id":"1.10.10.10","chopping":"44-155","consensus_level":"medium","plddt":85.2311,"start":44,"end":155},{"cath_id":"1.10.10.10","chopping":"174-246","consensus_level":"medium","plddt":89.8823,"start":174,"end":246}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96H20","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96H20-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96H20-F1-predicted_aligned_error_v6.png","plddt_mean":84.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SNF8","jax_strain_url":"https://www.jax.org/strain/search?query=SNF8"},"sequence":{"accession":"Q96H20","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96H20.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96H20/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96H20"}},"corpus_meta":[{"pmid":"17714434","id":"PMC_17714434","title":"Vps22/EAP30 in ESCRT-II mediates endosomal sorting of growth factor and chemokine receptors destined for lysosomal degradation.","date":"2007","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/17714434","citation_count":97,"is_preprint":false},{"pmid":"17010938","id":"PMC_17010938","title":"RILP interacts with VPS22 and VPS36 of ESCRT-II and regulates their membrane recruitment.","date":"2006","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/17010938","citation_count":49,"is_preprint":false},{"pmid":"16857164","id":"PMC_16857164","title":"RILP interacts with the VPS22 component of the ESCRT-II complex.","date":"2006","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/16857164","citation_count":43,"is_preprint":false},{"pmid":"10419521","id":"PMC_10419521","title":"Cloning and characterization of the EAP30 subunit of the ELL complex that confers derepression of transcription by RNA polymerase II.","date":"1999","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10419521","citation_count":35,"is_preprint":false},{"pmid":"15992541","id":"PMC_15992541","title":"The fission yeast homolog of the human transcription factor EAP30 blocks meiotic spindle pole body amplification.","date":"2005","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/15992541","citation_count":22,"is_preprint":false},{"pmid":"29084253","id":"PMC_29084253","title":"Pivotal role for the ESCRT-II complex subunit EAP30/SNF8 in IRF3-dependent innate antiviral defense.","date":"2017","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/29084253","citation_count":20,"is_preprint":false},{"pmid":"7785322","id":"PMC_7785322","title":"Molecular analysis of the SNF8 gene of Saccharomyces cerevisiae.","date":"1995","source":"Yeast (Chichester, England)","url":"https://pubmed.ncbi.nlm.nih.gov/7785322","citation_count":20,"is_preprint":false},{"pmid":"23171048","id":"PMC_23171048","title":"SNF8, a member of the ESCRT-II complex, interacts with TRPC6 and enhances its channel activity.","date":"2012","source":"BMC cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/23171048","citation_count":11,"is_preprint":false},{"pmid":"19819239","id":"PMC_19819239","title":"The interaction between EAP30 and ELL is modulated by MCM2.","date":"2009","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/19819239","citation_count":4,"is_preprint":false},{"pmid":"32829877","id":"PMC_32829877","title":"VPS-22/SNF8 regulates longevity via modulating the activity of DAF-16 in C. elegans.","date":"2020","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/32829877","citation_count":3,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6250,"output_tokens":2876,"usd":0.030945,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10134,"output_tokens":3565,"usd":0.069898,"stage2_stop_reason":"end_turn"},"total_usd":0.100843,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"EAP30 (SNF8) was identified as a subunit of the ELL complex; it interacts directly with ELL and derepresses ELL's inhibitory activity on RNA polymerase II transcription in vitro. EAP30 has sequence homology to S. cerevisiae SNF8, whose genetic analysis links it to derepression of gene expression.\",\n      \"method\": \"In vitro transcription assay, protein interaction studies, sequence homology analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Weak — in vitro functional assay demonstrating derepression, but single lab, limited mechanistic detail in abstract\",\n      \"pmids\": [\"10419521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"In S. cerevisiae, SNF8 is required for derepression of the SUC2 gene in response to glucose limitation; genetic epistasis showed SNF8 and SNF7 are functionally related (double mutant showed no additive impairment), and snf8 is genetically distinct from SNF1, SNF4, SNF2, SNF5, SNF6 groups.\",\n      \"method\": \"Gene disruption, genetic epistasis, complementation cloning, invertase derepression assay\",\n      \"journal\": \"Yeast (Chichester, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with multiple allele combinations, clean loss-of-function phenotype, but in yeast and single lab\",\n      \"pmids\": [\"7785322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Vps22/EAP30 (SNF8), as an ESCRT-II subunit, localizes to endosomes containing EGFR, Hrs, ESCRT-I and ESCRT-III; siRNA depletion of Vps22 greatly reduced degradation of EGFR and CXCR4, caused EGFR accumulation on limiting membranes of early endosomes and formation of aberrantly small multivesicular bodies. Notably, EGF-activated ERK1/2 signaling was not sustained upon Vps22 depletion (unlike Hrs or Tsg101 depletion), placing ESCRT-II engagement after termination of EGF signaling.\",\n      \"method\": \"siRNA knockdown, fluorescence microscopy, receptor degradation assays, ERK phosphorylation/nuclear translocation assays\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean siRNA knockdown with defined cellular phenotypes, multiple orthogonal readouts (receptor degradation, endosome morphology, signaling), pathway placement via epistasis\",\n      \"pmids\": [\"17714434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RILP interacts with VPS22 (SNF8) of ESCRT-II; the N-terminal half of RILP mediates interaction with VPS22. RILP regulates membrane recruitment of ESCRT-II subunits VPS22 and VPS36, integrating the late endocytic (Rab7/RILP) machinery with the early sorting (ESCRT) machinery.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, overexpression, endosomal trafficking assays (EGF sorting, LBPA/EEA1 markers)\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP with domain mapping confirmed in two independent papers (PMID 17010938 and 16857164), single method type\",\n      \"pmids\": [\"17010938\", \"16857164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Using yeast two-hybrid and co-immunoprecipitation, VPS22 (EAP30/SNF8) was confirmed as a binding partner of RILP; the interaction is with the N-terminal half of RILP. Confocal immunofluorescence showed colocalization of GFP-RILP and HA-VPS22.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, confocal immunofluorescence\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid plus co-IP plus colocalization, replicated in companion paper, but single interaction type\",\n      \"pmids\": [\"16857164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The fission yeast homolog of EAP30/SNF8 (Dot2) negatively regulates meiotic spindle pole body (SPB) maturation; dot2 mutants accumulate excess electron-dense material near SPBs (AMtOCs), assemble multipolar spindles, and missegregate chromosomes. Elevated Pcp1 (pericentrin ortholog) levels were associated with SPB aberrations, and reducing pcp1 expression suppressed AMtOCs, placing Dot2 upstream of Pcp1 in SPB maturation control.\",\n      \"method\": \"Genetic mutation analysis, electron microscopy, epistasis (pcp1 suppression of dot2 mutant), chromosome segregation assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with suppressor analysis, electron microscopy, clean loss-of-function phenotype in fission yeast ortholog\",\n      \"pmids\": [\"15992541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SNF8 interacts with the amino-terminal cytoplasmic domain (first 107 amino acids) of TRPC6; overexpression of SNF8 enhances TRPC6-mediated whole-cell currents and NFAT-mediated transcription activation by gain-of-function TRPC6 mutants, while RNAi knockdown partially inhibits NFAT activation. SNF8 overexpression does not alter cell-surface levels of TRPC6, suggesting the effect is on channel activity rather than trafficking.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, whole-cell patch-clamp electrophysiology, NFAT reporter assay, RNAi knockdown, cell-surface biotinylation\",\n      \"journal\": \"BMC cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by Co-IP, functional electrophysiology assay, RNAi, and surface expression measurement; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"23171048\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"MCM2 binds to EAP30 (SNF8) and competes with ELL for binding to EAP30, thereby potentially modulating the stability and activity of the Holo-ELL complex.\",\n      \"method\": \"Co-immunoprecipitation, competitive binding assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP/competition assay, single lab, limited mechanistic follow-up\",\n      \"pmids\": [\"19819239\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"EAP30 (SNF8/VPS22) is essential for IRF3-dependent induction of type I and III interferons and antiviral defense. A fraction of EAP30 localizes to the nucleus where, in a virus-inducible manner, it forms a complex with IRF3 and its co-activator CBP, promoting IRF3 binding to target gene promoters (IFN-β, IFN-λ1, ISG56). EAP30 acts downstream of IRF3 phosphorylation/activation, not upstream. This function is specific to EAP30 among ESCRT-II subunits.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation (EAP30-IRF3-CBP complex), chromatin immunoprecipitation (IRF3 promoter binding), nuclear fractionation, antiviral assays (VSV, HCV)\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ChIP, nuclear fractionation, functional antiviral assays) in single rigorous study establishing pathway position and nuclear mechanism\",\n      \"pmids\": [\"29084253\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In C. elegans, VPS-22/SNF8 acts in the same pathway as DAF-16 (FOXO transcription factor) to regulate longevity; epistasis analysis showed that knockdown of vps-22/snf8 in daf-16 null worms did not further shorten lifespan, and overexpression of daf-16 rescued the short-lived phenotype of vps-22/snf8 knockdown. Mechanistically, downregulation of vps-22/snf8 decreased nuclear localization of DAF-16 and altered expression of DAF-16 target longevity genes.\",\n      \"method\": \"RNAi knockdown, genetic epistasis (double mutant/knockdown), nuclear localization assay (DAF-16::GFP), lifespan assay, gene expression analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis plus nuclear localization assay plus target gene expression, multiple methods, single lab, C. elegans ortholog\",\n      \"pmids\": [\"32829877\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SNF8/EAP30/VPS22 is a multifunctional ESCRT-II subunit that: (1) acts on endosomes to sort ubiquitinated receptors (EGFR, CXCR4) into multivesicular bodies for lysosomal degradation, where its membrane recruitment is regulated by the Rab7 effector RILP; (2) functions in the nucleus as part of the ELL transcription elongation complex (where it derepresses ELL-mediated inhibition of RNA Pol II and its interaction with ELL is modulated by MCM2) and, independently, forms a virus-inducible complex with IRF3 and CBP to drive antiviral interferon gene transcription; (3) interacts with and enhances TRPC6 channel activity independently of receptor trafficking; and in model organisms, its ortholog regulates meiotic spindle pole body maturation (fission yeast) and modulates longevity via the DAF-16/FOXO pathway (C. elegans).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SNF8 (EAP30/VPS22) is a subunit of the ESCRT-II complex that drives sorting of ubiquitinated cell-surface receptors into multivesicular bodies for lysosomal degradation, and which has been repeatedly captured acting in the nucleus to control gene transcription [#2, #0]. On endosomes, SNF8 localizes to compartments containing EGFR, Hrs and other ESCRT components, and its depletion blocks degradation of EGFR and CXCR4, causes receptor accumulation on the limiting membrane of early endosomes, and yields aberrantly small multivesicular bodies; this engagement occurs after termination of EGF-stimulated ERK signaling [#2]. Membrane recruitment of the ESCRT-II subunits SNF8 (VPS22) and VPS36 is directed by the Rab7 effector RILP, whose N-terminal half binds SNF8, linking the late endocytic machinery to early ESCRT sorting [#3, #4]. Independently of trafficking, SNF8 is a subunit of the ELL transcription elongation complex, where it binds ELL directly and derepresses ELL-mediated inhibition of RNA polymerase II [#0], and a nuclear pool of SNF8 forms a virus-inducible complex with IRF3 and the co-activator CBP, acting downstream of IRF3 activation to promote IRF3 binding at interferon and ISG promoters and drive antiviral type I and III interferon responses [#8]. SNF8 also binds the N-terminal cytoplasmic domain of TRPC6 and enhances TRPC6 channel current and downstream NFAT activation without changing channel surface levels [#6]. Work in model organisms shows the ortholog negatively regulates meiotic spindle pole body maturation upstream of the pericentrin ortholog Pcp1 in fission yeast [#5] and acts in the DAF-16/FOXO longevity pathway in C. elegans by supporting DAF-16 nuclear localization [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Established the founding loss-of-function phenotype: SNF8 was unknown in function until yeast genetics showed it is required for glucose-derepression of SUC2 and is functionally related to SNF7, defining a discrete gene group distinct from the SNF1/SNF4 cluster.\",\n      \"evidence\": \"Gene disruption, genetic epistasis, and invertase derepression assay in S. cerevisiae\",\n      \"pmids\": [\"7785322\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No molecular mechanism for how SNF8 derepresses transcription\", \"Relationship to membrane trafficking not yet appreciated\", \"Mammalian ortholog function untested\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Connected the yeast gene to a mammalian transcription mechanism: EAP30/SNF8 was found to be an ELL-complex subunit that binds ELL and relieves ELL's inhibition of RNA Pol II, giving a biochemical activity to the derepression phenotype.\",\n      \"evidence\": \"In vitro transcription assay, protein interaction studies, and sequence homology analysis\",\n      \"pmids\": [\"10419521\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro only; nuclear role not shown in cells\", \"Stoichiometry/structure of EAP30 within Holo-ELL unresolved\", \"Does not address any membrane function\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Revealed an organelle-biogenesis role: the fission yeast ortholog Dot2 negatively regulates meiotic spindle pole body maturation, placing it upstream of the pericentrin ortholog Pcp1.\",\n      \"evidence\": \"Genetic mutation, electron microscopy, and pcp1-suppression epistasis in S. pombe meiosis\",\n      \"pmids\": [\"15992541\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link between ESCRT-II function and SPB control unclear\", \"Conservation to mammalian centrosomes untested\", \"Whether this depends on ESCRT membrane activity unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identified the recruitment cue for ESCRT-II to endosomes: RILP, the Rab7 effector, binds SNF8/VPS22 via its N-terminal half and controls membrane recruitment of ESCRT-II, bridging late-endosomal and early-sorting machinery.\",\n      \"evidence\": \"Yeast two-hybrid, co-immunoprecipitation, domain mapping, and confocal colocalization (two companion papers)\",\n      \"pmids\": [\"17010938\", \"16857164\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single interaction-method class (no structure of the complex)\", \"Functional consequence of disrupting RILP-VPS22 binding not quantified\", \"How Rab7 activity gates the interaction unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined SNF8's core cell-biological role and pathway position: as an ESCRT-II subunit it is required for MVB sorting and lysosomal degradation of EGFR and CXCR4, acting after EGF signaling has terminated.\",\n      \"evidence\": \"siRNA knockdown with receptor degradation, endosome morphology, and ERK signaling readouts\",\n      \"pmids\": [\"17714434\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not resolve how ESCRT-II selects ubiquitinated cargo at this step\", \"Structural role within the ESCRT cascade not addressed\", \"Specificity for EGFR/CXCR4 vs other receptors untested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Probed regulation of the nuclear ELL complex: MCM2 binds EAP30 and competes with ELL, indicating a route to modulate Holo-ELL stability/activity.\",\n      \"evidence\": \"Co-immunoprecipitation and competitive binding assay\",\n      \"pmids\": [\"19819239\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP/competition assay without functional or transcriptional readout\", \"Cellular relevance of competition not demonstrated\", \"Not independently confirmed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Uncovered a trafficking-independent ion-channel function: SNF8 binds the TRPC6 N-terminal cytoplasmic domain and enhances channel current and NFAT signaling without altering surface channel levels.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, whole-cell patch-clamp, NFAT reporter, RNAi, and surface biotinylation\",\n      \"pmids\": [\"23171048\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which SNF8 potentiates channel gating unknown\", \"Physiological context of TRPC6 regulation untested\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established a distinct nuclear antiviral role: a fraction of EAP30 forms a virus-inducible IRF3-CBP complex acting downstream of IRF3 activation to promote promoter binding and induction of type I/III interferons, a function specific to EAP30 among ESCRT-II subunits.\",\n      \"evidence\": \"siRNA, co-IP, ChIP, nuclear fractionation, and VSV/HCV antiviral assays\",\n      \"pmids\": [\"29084253\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How EAP30 partitions between endosomal ESCRT-II and nuclear pools is unresolved\", \"Whether this role requires ESCRT-II assembly unknown\", \"Signal that triggers nuclear localization not defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked SNF8 to longevity signaling: the C. elegans ortholog acts in the DAF-16/FOXO pathway, with knockdown reducing DAF-16 nuclear localization and shortening lifespan in a daf-16-dependent manner.\",\n      \"evidence\": \"RNAi, genetic epistasis, DAF-16::GFP nuclear localization, lifespan and target-gene expression assays\",\n      \"pmids\": [\"32829877\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between ESCRT-II and DAF-16 trafficking unknown\", \"Whether endosomal or nuclear SNF8 function mediates the effect unclear\", \"Conservation to mammalian FOXO untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single ESCRT-II subunit is partitioned and regulated across its membrane-sorting, transcription-elongation, antiviral, channel-modulatory, and spindle/longevity roles remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model integrating SNF8's endosomal vs nuclear functions\", \"Signals governing nuclear translocation undefined\", \"Whether non-ESCRT roles require the intact ESCRT-II complex unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [\"ESCRT-II\", \"ELL transcription elongation complex\"],\n    \"partners\": [\"ELL\", \"RILP\", \"VPS36\", \"TRPC6\", \"IRF3\", \"CBP\", \"MCM2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}