{"gene":"EIF4A2","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2013,"finding":"eIF4A2 is the key RNA helicase of the eIF4F initiation complex through which miRNAs exert translational repression; miRNA-mediated translational inhibition is the primary event required for subsequent mRNA degradation, and mRNAs with unstructured 5'UTRs are refractory to this repression.","method":"Knockdown/overexpression experiments, ribosome profiling, reporter assays, secondary structure analysis of 5'UTRs in miRNA target mRNAs","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (KD, reporters, structural analysis), replicated across mRNA targets, published in high-tier journal with broad mechanistic follow-up in subsequent studies","pmids":["23559250"],"is_preprint":false},{"year":2019,"finding":"eIF4A2 functions as a major effector of the repressive miRNA pathway by interacting with the Ccr4-Not complex, repressing mRNAs at translation initiation by binding purine-rich motifs enriched in the 5'UTR directly upstream of the AUG start codon; eIF4A2 has similar RNA selectivity to chemically inhibited eIF4A1.","method":"Co-immunoprecipitation, ribosome profiling, RNA-seq, motif enrichment analysis, comparison with hippuristanol-treated eIF4A1","journal":"Genome biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP with Ccr4-Not, ribosome profiling, and motif analysis, building on and confirming prior work with multiple orthogonal methods","pmids":["31791371"],"is_preprint":false},{"year":2016,"finding":"eIF4A2 is sumoylated on a single residue (K226); sumoylation is increased in response to arsenite and ionising radiation but decreased by heat shock or hippuristanol; sumoylation of eIF4A2 correlates with its recruitment to stress granules in arsenite-treated cells, and mutation of K226 (preventing sumoylation) impairs stress granule formation.","method":"Mass spectrometry identification of sumoylation site, site-directed mutagenesis (K226R), immunofluorescence microscopy of stress granule localization, arsenite/heat-shock/hippuristanol treatment","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — mutagenesis of specific sumoylation site combined with direct localization experiments and stress granule functional readout, single lab but multiple orthogonal methods","pmids":["27160682"],"is_preprint":false},{"year":2022,"finding":"eIF4A2 mediates translation initiation of mRNAs encoding pluripotency factors and histone variant H3.3 through ribosomal protein S26-independent and -dependent ribosomes; eIF4A2 also activates translation of Ddx6, which cooperates with eIF4A2 to restrict the totipotent two-cell transcription program in ESCs by repressing Zscan4 mRNA.","method":"RNAi screen in ESCs, ribosome fractionation, polysome profiling, knockdown with defined phenotypic readouts (loss of pluripotency markers, Zscan4 derepression)","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with defined cellular phenotype and ribosome fractionation, single lab","pmids":["35353581"],"is_preprint":false},{"year":2018,"finding":"eIF4A2 is required for efficient HIV-1 replication in human T cells; depletion of eIF4A2 reduces viral cDNA synthesis without affecting virion entry into target cells.","method":"Stable shRNA knockdown in MT4C5 cells, replication-competent reporter HIV-1 infection assay, viral cDNA quantification, entry assay","journal":"Microbes and infection","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KD with defined step-specific phenotype (cDNA synthesis, not entry), single lab, single study","pmids":["29842983"],"is_preprint":false},{"year":2018,"finding":"eIF4A2 directly interacts with the membrane (M) protein of transmissible gastroenteritis coronavirus (TGEV) in porcine intestinal cells, co-localizes with M protein in the cytoplasm, and siRNA-mediated knockdown of eIF4A2 markedly decreases M protein proliferation and TGEV replication.","method":"Yeast two-hybrid screening, GST pull-down, co-immunoprecipitation, confocal microscopy colocalization, siRNA knockdown with viral replication readout","journal":"Research in veterinary science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — GST pull-down and Co-IP confirm interaction, colocalization by confocal, KD phenotype; single lab but multiple orthogonal methods","pmids":["30583231"],"is_preprint":false},{"year":2024,"finding":"eIF4A1 controls global protein synthesis whereas eIF4A2 regulates the biogenesis of 18S ribosomal RNA and the 40S ribosome subunit during B-cell development; the two isoforms thus have distinct molecular functions despite exchanging freely within eIF4F complexes.","method":"Mouse conditional knockout genetic analysis, ribosome profiling, rRNA biogenesis assays, B-cell development flow cytometry","journal":"Cellular & molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO mouse genetics with defined cellular and molecular phenotype, single lab","pmids":["39516355"],"is_preprint":false},{"year":2023,"finding":"EIF4A2 haploinsufficiency (frameshift deletion causing ~50% protein reduction) results in dominant dystonia-tremor syndrome; reduced eIF4A2 is associated with increased levels of IMP1 (a target of Ccr4-Not) and diminished colocalization of eIF4A2 with Ccr4-Not in patient-derived fibroblasts, consistent with loss of miRNA-dependent translational repression.","method":"Western blotting and immunocytochemistry in patient-derived fibroblasts, quantification of IMP1 as pathway readout, colocalization analysis of eIF4A2 and Ccr4-Not","journal":"Movement disorders","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient fibroblast biochemistry with multiple readouts (protein level, pathway target, colocalization), single lab","pmids":["37485550"],"is_preprint":false},{"year":2025,"finding":"eIF4A1 has higher ATP-binding affinity than eIF4A2 (Km 6.55 μM vs 11.61 μM); leucine 98 (L98) and alanine 100 (A100) are important for ATPase activity differences between isoforms; BLF1 toxin treatment significantly enhances eIF4A2-mediated ATP hydrolysis whereas it inhibits eIF4A1, revealing isoform-specific sensitivity.","method":"In vitro ATPase assay, N-terminal domain swapping, site-directed mutagenesis (L98, A100), BLF1 treatment of purified proteins","journal":"Toxins","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic reconstitution with mutagenesis and domain swapping; single lab but multiple orthogonal biochemical methods directly testing mechanism","pmids":["40423315"],"is_preprint":false},{"year":2022,"finding":"Rare de novo mono-allelic and inherited bi-allelic variants in EIF4A2 cause a neurodevelopmental syndrome; in vivo rescue assays in Drosophila showed that human EIF4A2 wild-type cDNA fully rescues pupal lethality caused by loss of the fly ortholog eIF4A, whereas disease-associated variant cDNAs failed or incompletely rescued, demonstrating both loss-of-function and gain-of-function mechanisms for different variants.","method":"Drosophila loss-of-function rescue assay, molecular modeling of variant structural effects, clinical variant analysis","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo complementation assay in Drosophila with multiple variants tested, single study","pmids":["36528028"],"is_preprint":false},{"year":2025,"finding":"Deletion of eIF4A2 in early KRAS-driven lung adenocarcinoma leads to dysregulated protein synthesis (upregulated secretome, enlarged secretory compartments, increased oxidative metabolism) and acquisition of senescence-like characteristics (p21-positive non-proliferative cells); rapamycin-mediated reduction of mRNA translation suppresses senescence and restores tumorigenesis, placing eIF4A2 upstream of mTOR-regulated translation and senescence induction.","method":"Conditional eIF4A2 knockout in KRAS mouse lung cancer model, rapamycin treatment rescue experiment, proteomics/secretome analysis, p21 immunostaining, MEK inhibitor sensitivity assay","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse model with defined phenotype plus pharmacological rescue with rapamycin establishing pathway position; preprint, single lab","pmids":["bio_10.1101_2025.03.19.644135"],"is_preprint":true}],"current_model":"eIF4A2 is an ATP-dependent DEAD-box RNA helicase that functions as the key translational repressor within the eIF4F cap-binding complex, acting downstream of miRNA-loaded RISC by interacting with the Ccr4-Not deadenylase complex to repress translation initiation at purine-rich motifs in the 5'UTR of target mRNAs; it is sumoylated at K226 (promoting stress granule recruitment), regulates 40S ribosome biogenesis in B cells, and its haploinsufficiency or variant-specific dysfunction causes neurodevelopmental disorders and dystonia through deregulation of miRNA-dependent translational repression."},"narrative":{"mechanistic_narrative":"EIF4A2 is an ATP-dependent DEAD-box RNA helicase of the eIF4F cap-binding initiation complex that serves as a principal effector of miRNA-mediated translational repression [PMID:23559250, PMID:31791371]. It executes repression downstream of miRNA-loaded effectors by physically associating with the Ccr4-Not deadenylase complex and binding purine-rich motifs enriched in the 5'UTR immediately upstream of the start codon, blocking initiation as a primary event that precedes mRNA degradation; mRNAs with unstructured 5'UTRs are refractory to this control [PMID:23559250, PMID:31791371]. Although eIF4A1 and eIF4A2 exchange freely within eIF4F, they are functionally distinct: eIF4A2 specifically governs 18S rRNA processing and 40S ribosome subunit biogenesis during B-cell development [PMID:39516355], and biochemically the isoforms differ in ATP affinity and ATPase behavior, with residues L98 and A100 underlying these differences and opposite sensitivity to the BLF1 toxin [PMID:40423315]. Beyond its repressive role, eIF4A2 promotes translation of specific transcripts—pluripotency factors, histone variant H3.3, and Ddx6—and cooperates with Ddx6 to restrain the two-cell totipotency program in embryonic stem cells [PMID:35353581]. The protein is sumoylated at K226, a modification induced by arsenite and ionizing radiation that drives its recruitment to stress granules [PMID:27160682]. EIF4A2 is required for efficient HIV-1 cDNA synthesis and for transmissible gastroenteritis coronavirus replication via direct binding of the viral M protein [PMID:29842983, PMID:30583231]. Human genetics establishes disease relevance: EIF4A2 haploinsufficiency causes a dominant dystonia-tremor syndrome associated with elevated IMP1 and diminished eIF4A2–Ccr4-Not colocalization [PMID:37485550], and rare de novo and bi-allelic variants cause a neurodevelopmental syndrome through both loss- and gain-of-function mechanisms [PMID:36528028].","teleology":[{"year":2013,"claim":"Established that eIF4A2, rather than acting only in general initiation, is the specific eIF4F helicase through which miRNAs repress translation, and that repression—not degradation—is the primary event.","evidence":"Knockdown/overexpression, ribosome profiling, reporter assays, and 5'UTR structure analysis of miRNA targets","pmids":["23559250"],"confidence":"High","gaps":["Did not define the RNA sequence determinants of eIF4A2 selectivity","Mechanistic link to deadenylation machinery not yet established"]},{"year":2016,"claim":"Identified a single SUMO acceptor site (K226) and connected its modification to stress-induced relocalization, linking eIF4A2 to stress granule dynamics.","evidence":"Mass spectrometry, K226R mutagenesis, and stress granule immunofluorescence under arsenite/heat-shock/hippuristanol","pmids":["27160682"],"confidence":"High","gaps":["SUMO ligase responsible for K226 modification not identified","Functional consequence of stress granule recruitment for translation not defined"]},{"year":2018,"claim":"Showed eIF4A2 is a host factor co-opted by viruses, with step-specific requirements distinguishing entry from downstream replication.","evidence":"shRNA knockdown in T cells with viral cDNA/entry assays (HIV-1); yeast two-hybrid, GST pull-down, Co-IP, and siRNA knockdown (TGEV M protein)","pmids":["29842983","30583231"],"confidence":"Medium","gaps":["Molecular role of eIF4A2 in viral cDNA synthesis unresolved","Whether helicase activity is required for viral co-option untested"]},{"year":2019,"claim":"Defined the molecular basis of eIF4A2-mediated repression by demonstrating direct Ccr4-Not association and binding to purine-rich 5'UTR motifs upstream of the AUG.","evidence":"Reciprocal Co-IP, ribosome profiling, RNA-seq, motif enrichment, and comparison with hippuristanol-inhibited eIF4A1","pmids":["31791371"],"confidence":"High","gaps":["Stoichiometry and architecture of the eIF4A2–Ccr4-Not interaction not resolved","How RISC hands off to eIF4A2 not defined"]},{"year":2022,"claim":"Revealed an activating, transcript-selective role for eIF4A2 in stem cell translation, beyond its repressive function, including control of the totipotency program.","evidence":"RNAi screen, ribosome fractionation, and polysome profiling in ESCs with pluripotency/Zscan4 readouts","pmids":["35353581"],"confidence":"Medium","gaps":["Determinants directing eIF4A2 to activation versus repression unknown","RPS26-dependent versus -independent ribosome usage mechanism unresolved"]},{"year":2022,"claim":"Demonstrated that EIF4A2 variants cause a neurodevelopmental syndrome and that disease arises by both loss- and gain-of-function, using cross-species complementation.","evidence":"Drosophila loss-of-function rescue with human wild-type and variant cDNAs, plus molecular modeling","pmids":["36528028"],"confidence":"Medium","gaps":["Molecular nature of the gain-of-function variants not biochemically defined","Affected neuronal mRNA targets not identified"]},{"year":2023,"claim":"Linked EIF4A2 haploinsufficiency to a dystonia-tremor syndrome and connected the disease to the Ccr4-Not repression pathway through a patient-cell target readout.","evidence":"Western blot, immunocytochemistry, IMP1 quantification, and eIF4A2–Ccr4-Not colocalization in patient fibroblasts","pmids":["37485550"],"confidence":"Medium","gaps":["Causality between IMP1 derepression and disease phenotype not established","Neuronal cell-type-specific consequences untested"]},{"year":2024,"claim":"Distinguished eIF4A2 from eIF4A1 functionally, assigning eIF4A2 a specific role in 18S rRNA and 40S subunit biogenesis despite free exchange within eIF4F.","evidence":"Conditional knockout mouse genetics, ribosome profiling, rRNA biogenesis assays, and B-cell flow cytometry","pmids":["39516355"],"confidence":"Medium","gaps":["Mechanism by which eIF4A2 acts in rRNA processing unknown","Whether this role extends beyond B cells untested"]},{"year":2025,"claim":"Resolved isoform-specific biochemistry by quantifying differential ATP affinity/ATPase activity and mapping the responsible residues, plus opposite BLF1 toxin sensitivity.","evidence":"In vitro ATPase assays, N-terminal domain swapping, L98/A100 mutagenesis, and BLF1 treatment of purified proteins","pmids":["40423315"],"confidence":"High","gaps":["How ATPase differences translate to distinct cellular functions not shown","Structural basis for BLF1 isoform selectivity not determined"]},{"year":2025,"claim":"Placed eIF4A2 upstream of mTOR-regulated translation in tumor biology, where its loss triggers senescence that pharmacological translation reduction reverses.","evidence":"Conditional eIF4A2 knockout in KRAS lung cancer mouse model, rapamycin rescue, secretome proteomics, and p21 staining (preprint)","pmids":["bio_10.1101_2025.03.19.644135"],"confidence":"Medium","gaps":["Direct mRNA targets driving senescence not identified","Generalizability beyond KRAS-driven lung adenocarcinoma untested"]},{"year":null,"claim":"How a single helicase reconciles its opposing roles—miRNA/Ccr4-Not-dependent repression versus transcript-selective translational activation and 40S biogenesis—remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of eIF4A2 engaged with Ccr4-Not","No unified rule predicting repression versus activation of a given mRNA","Connection between SUMO/stress regulation and target selection undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,8]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,1]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,8]},{"term_id":"GO:0045182","term_label":"translation regulator activity","supporting_discovery_ids":[0,1,3]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[8]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,5]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,1,6]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,6]}],"complexes":["eIF4F","Ccr4-Not"],"partners":["CNOT1","DDX6","IGF2BP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q14240","full_name":"Eukaryotic initiation factor 4A-II","aliases":["ATP-dependent RNA helicase eIF4A-2"],"length_aa":407,"mass_kda":46.4,"function":"ATP-dependent RNA helicase which is a subunit of the eIF4F complex involved in cap recognition and is required for mRNA binding to ribosome. In the current model of translation initiation, eIF4A unwinds RNA secondary structures in the 5'-UTR of mRNAs which is necessary to allow efficient binding of the small ribosomal subunit, and subsequent scanning for the initiator codon","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q14240/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EIF4A2","classification":"Not Classified","n_dependent_lines":19,"n_total_lines":1208,"dependency_fraction":0.015728476821192054},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CAPZB","stoichiometry":0.2},{"gene":"CYP51A1","stoichiometry":0.2},{"gene":"DDX6","stoichiometry":0.2},{"gene":"DRG1","stoichiometry":0.2},{"gene":"EIF3G","stoichiometry":0.2},{"gene":"NPM1","stoichiometry":0.2},{"gene":"PSPC1","stoichiometry":0.2},{"gene":"RBM42","stoichiometry":0.2},{"gene":"RBM8A","stoichiometry":0.2},{"gene":"RPS16","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/EIF4A2","total_profiled":1310},"omim":[{"mim_id":"620455","title":"NEURODEVELOPMENTAL DISORDER WITH HYPOTONIA AND SPEECH DELAY, WITH OR WITHOUT SEIZURES; NEDHSS","url":"https://www.omim.org/entry/620455"},{"mim_id":"611269","title":"NUCLEOLAR PROTEIN WITH MIF4G DOMAIN 1; NOM1","url":"https://www.omim.org/entry/611269"},{"mim_id":"608546","title":"EUKARYOTIC TRANSLATION INITIATION FACTOR 4A, ISOFORM 3; EIF4A3","url":"https://www.omim.org/entry/608546"},{"mim_id":"602641","title":"EUKARYOTIC TRANSLATION INITIATION FACTOR 4A, ISOFORM 1; EIF4A1","url":"https://www.omim.org/entry/602641"},{"mim_id":"601102","title":"EUKARYOTIC TRANSLATION INITIATION FACTOR 4A, ISOFORM 2; EIF4A2","url":"https://www.omim.org/entry/601102"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/EIF4A2"},"hgnc":{"alias_symbol":["DDX2B","EIF4A","BM-010"],"prev_symbol":["EIF4F"]},"alphafold":{"accession":"Q14240","domains":[{"cath_id":"3.40.50.300","chopping":"23-240","consensus_level":"high","plddt":91.8683,"start":23,"end":240},{"cath_id":"3.40.50.300","chopping":"246-404","consensus_level":"high","plddt":87.2733,"start":246,"end":404}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14240","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q14240-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q14240-F1-predicted_aligned_error_v6.png","plddt_mean":86.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EIF4A2","jax_strain_url":"https://www.jax.org/strain/search?query=EIF4A2"},"sequence":{"accession":"Q14240","fasta_url":"https://rest.uniprot.org/uniprotkb/Q14240.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q14240/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14240"}},"corpus_meta":[{"pmid":"23559250","id":"PMC_23559250","title":"Translational repression and eIF4A2 activity are critical for microRNA-mediated gene regulation.","date":"2013","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/23559250","citation_count":262,"is_preprint":false},{"pmid":"31308851","id":"PMC_31308851","title":"MicroRNA-5195-3p enhances the chemosensitivity of triple-negative breast cancer to paclitaxel by downregulating EIF4A2.","date":"2019","source":"Cellular & molecular biology letters","url":"https://pubmed.ncbi.nlm.nih.gov/31308851","citation_count":48,"is_preprint":false},{"pmid":"31791371","id":"PMC_31791371","title":"eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5'UTR.","date":"2019","source":"Genome biology","url":"https://pubmed.ncbi.nlm.nih.gov/31791371","citation_count":47,"is_preprint":false},{"pmid":"27160682","id":"PMC_27160682","title":"Sumoylation of eIF4A2 affects stress granule formation.","date":"2016","source":"Journal of cell 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Framingham Heart Study (FHS) Offspring Cohort (OS)","date":"2024-06-19","source":"bioRxiv","url":"https://doi.org/10.1101/2024.06.18.24309120","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11778,"output_tokens":2881,"usd":0.039274,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10234,"output_tokens":3637,"usd":0.071047,"stage2_stop_reason":"end_turn"},"total_usd":0.110321,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"eIF4A2 is the key RNA helicase of the eIF4F initiation complex through which miRNAs exert translational repression; miRNA-mediated translational inhibition is the primary event required for subsequent mRNA degradation, and mRNAs with unstructured 5'UTRs are refractory to this repression.\",\n      \"method\": \"Knockdown/overexpression experiments, ribosome profiling, reporter assays, secondary structure analysis of 5'UTRs in miRNA target mRNAs\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (KD, reporters, structural analysis), replicated across mRNA targets, published in high-tier journal with broad mechanistic follow-up in subsequent studies\",\n      \"pmids\": [\"23559250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"eIF4A2 functions as a major effector of the repressive miRNA pathway by interacting with the Ccr4-Not complex, repressing mRNAs at translation initiation by binding purine-rich motifs enriched in the 5'UTR directly upstream of the AUG start codon; eIF4A2 has similar RNA selectivity to chemically inhibited eIF4A1.\",\n      \"method\": \"Co-immunoprecipitation, ribosome profiling, RNA-seq, motif enrichment analysis, comparison with hippuristanol-treated eIF4A1\",\n      \"journal\": \"Genome biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP with Ccr4-Not, ribosome profiling, and motif analysis, building on and confirming prior work with multiple orthogonal methods\",\n      \"pmids\": [\"31791371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"eIF4A2 is sumoylated on a single residue (K226); sumoylation is increased in response to arsenite and ionising radiation but decreased by heat shock or hippuristanol; sumoylation of eIF4A2 correlates with its recruitment to stress granules in arsenite-treated cells, and mutation of K226 (preventing sumoylation) impairs stress granule formation.\",\n      \"method\": \"Mass spectrometry identification of sumoylation site, site-directed mutagenesis (K226R), immunofluorescence microscopy of stress granule localization, arsenite/heat-shock/hippuristanol treatment\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis of specific sumoylation site combined with direct localization experiments and stress granule functional readout, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"27160682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"eIF4A2 mediates translation initiation of mRNAs encoding pluripotency factors and histone variant H3.3 through ribosomal protein S26-independent and -dependent ribosomes; eIF4A2 also activates translation of Ddx6, which cooperates with eIF4A2 to restrict the totipotent two-cell transcription program in ESCs by repressing Zscan4 mRNA.\",\n      \"method\": \"RNAi screen in ESCs, ribosome fractionation, polysome profiling, knockdown with defined phenotypic readouts (loss of pluripotency markers, Zscan4 derepression)\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with defined cellular phenotype and ribosome fractionation, single lab\",\n      \"pmids\": [\"35353581\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"eIF4A2 is required for efficient HIV-1 replication in human T cells; depletion of eIF4A2 reduces viral cDNA synthesis without affecting virion entry into target cells.\",\n      \"method\": \"Stable shRNA knockdown in MT4C5 cells, replication-competent reporter HIV-1 infection assay, viral cDNA quantification, entry assay\",\n      \"journal\": \"Microbes and infection\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KD with defined step-specific phenotype (cDNA synthesis, not entry), single lab, single study\",\n      \"pmids\": [\"29842983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"eIF4A2 directly interacts with the membrane (M) protein of transmissible gastroenteritis coronavirus (TGEV) in porcine intestinal cells, co-localizes with M protein in the cytoplasm, and siRNA-mediated knockdown of eIF4A2 markedly decreases M protein proliferation and TGEV replication.\",\n      \"method\": \"Yeast two-hybrid screening, GST pull-down, co-immunoprecipitation, confocal microscopy colocalization, siRNA knockdown with viral replication readout\",\n      \"journal\": \"Research in veterinary science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — GST pull-down and Co-IP confirm interaction, colocalization by confocal, KD phenotype; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"30583231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"eIF4A1 controls global protein synthesis whereas eIF4A2 regulates the biogenesis of 18S ribosomal RNA and the 40S ribosome subunit during B-cell development; the two isoforms thus have distinct molecular functions despite exchanging freely within eIF4F complexes.\",\n      \"method\": \"Mouse conditional knockout genetic analysis, ribosome profiling, rRNA biogenesis assays, B-cell development flow cytometry\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO mouse genetics with defined cellular and molecular phenotype, single lab\",\n      \"pmids\": [\"39516355\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EIF4A2 haploinsufficiency (frameshift deletion causing ~50% protein reduction) results in dominant dystonia-tremor syndrome; reduced eIF4A2 is associated with increased levels of IMP1 (a target of Ccr4-Not) and diminished colocalization of eIF4A2 with Ccr4-Not in patient-derived fibroblasts, consistent with loss of miRNA-dependent translational repression.\",\n      \"method\": \"Western blotting and immunocytochemistry in patient-derived fibroblasts, quantification of IMP1 as pathway readout, colocalization analysis of eIF4A2 and Ccr4-Not\",\n      \"journal\": \"Movement disorders\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient fibroblast biochemistry with multiple readouts (protein level, pathway target, colocalization), single lab\",\n      \"pmids\": [\"37485550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"eIF4A1 has higher ATP-binding affinity than eIF4A2 (Km 6.55 μM vs 11.61 μM); leucine 98 (L98) and alanine 100 (A100) are important for ATPase activity differences between isoforms; BLF1 toxin treatment significantly enhances eIF4A2-mediated ATP hydrolysis whereas it inhibits eIF4A1, revealing isoform-specific sensitivity.\",\n      \"method\": \"In vitro ATPase assay, N-terminal domain swapping, site-directed mutagenesis (L98, A100), BLF1 treatment of purified proteins\",\n      \"journal\": \"Toxins\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic reconstitution with mutagenesis and domain swapping; single lab but multiple orthogonal biochemical methods directly testing mechanism\",\n      \"pmids\": [\"40423315\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Rare de novo mono-allelic and inherited bi-allelic variants in EIF4A2 cause a neurodevelopmental syndrome; in vivo rescue assays in Drosophila showed that human EIF4A2 wild-type cDNA fully rescues pupal lethality caused by loss of the fly ortholog eIF4A, whereas disease-associated variant cDNAs failed or incompletely rescued, demonstrating both loss-of-function and gain-of-function mechanisms for different variants.\",\n      \"method\": \"Drosophila loss-of-function rescue assay, molecular modeling of variant structural effects, clinical variant analysis\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo complementation assay in Drosophila with multiple variants tested, single study\",\n      \"pmids\": [\"36528028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Deletion of eIF4A2 in early KRAS-driven lung adenocarcinoma leads to dysregulated protein synthesis (upregulated secretome, enlarged secretory compartments, increased oxidative metabolism) and acquisition of senescence-like characteristics (p21-positive non-proliferative cells); rapamycin-mediated reduction of mRNA translation suppresses senescence and restores tumorigenesis, placing eIF4A2 upstream of mTOR-regulated translation and senescence induction.\",\n      \"method\": \"Conditional eIF4A2 knockout in KRAS mouse lung cancer model, rapamycin treatment rescue experiment, proteomics/secretome analysis, p21 immunostaining, MEK inhibitor sensitivity assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse model with defined phenotype plus pharmacological rescue with rapamycin establishing pathway position; preprint, single lab\",\n      \"pmids\": [\"bio_10.1101_2025.03.19.644135\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"eIF4A2 is an ATP-dependent DEAD-box RNA helicase that functions as the key translational repressor within the eIF4F cap-binding complex, acting downstream of miRNA-loaded RISC by interacting with the Ccr4-Not deadenylase complex to repress translation initiation at purine-rich motifs in the 5'UTR of target mRNAs; it is sumoylated at K226 (promoting stress granule recruitment), regulates 40S ribosome biogenesis in B cells, and its haploinsufficiency or variant-specific dysfunction causes neurodevelopmental disorders and dystonia through deregulation of miRNA-dependent translational repression.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EIF4A2 is an ATP-dependent DEAD-box RNA helicase of the eIF4F cap-binding initiation complex that serves as a principal effector of miRNA-mediated translational repression [#0, #1]. It executes repression downstream of miRNA-loaded effectors by physically associating with the Ccr4-Not deadenylase complex and binding purine-rich motifs enriched in the 5'UTR immediately upstream of the start codon, blocking initiation as a primary event that precedes mRNA degradation; mRNAs with unstructured 5'UTRs are refractory to this control [#0, #1]. Although eIF4A1 and eIF4A2 exchange freely within eIF4F, they are functionally distinct: eIF4A2 specifically governs 18S rRNA processing and 40S ribosome subunit biogenesis during B-cell development [#6], and biochemically the isoforms differ in ATP affinity and ATPase behavior, with residues L98 and A100 underlying these differences and opposite sensitivity to the BLF1 toxin [#8]. Beyond its repressive role, eIF4A2 promotes translation of specific transcripts—pluripotency factors, histone variant H3.3, and Ddx6—and cooperates with Ddx6 to restrain the two-cell totipotency program in embryonic stem cells [#3]. The protein is sumoylated at K226, a modification induced by arsenite and ionizing radiation that drives its recruitment to stress granules [#2]. EIF4A2 is required for efficient HIV-1 cDNA synthesis and for transmissible gastroenteritis coronavirus replication via direct binding of the viral M protein [#4, #5]. Human genetics establishes disease relevance: EIF4A2 haploinsufficiency causes a dominant dystonia-tremor syndrome associated with elevated IMP1 and diminished eIF4A2–Ccr4-Not colocalization [#7], and rare de novo and bi-allelic variants cause a neurodevelopmental syndrome through both loss- and gain-of-function mechanisms [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"Established that eIF4A2, rather than acting only in general initiation, is the specific eIF4F helicase through which miRNAs repress translation, and that repression—not degradation—is the primary event.\",\n      \"evidence\": \"Knockdown/overexpression, ribosome profiling, reporter assays, and 5'UTR structure analysis of miRNA targets\",\n      \"pmids\": [\"23559250\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the RNA sequence determinants of eIF4A2 selectivity\", \"Mechanistic link to deadenylation machinery not yet established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified a single SUMO acceptor site (K226) and connected its modification to stress-induced relocalization, linking eIF4A2 to stress granule dynamics.\",\n      \"evidence\": \"Mass spectrometry, K226R mutagenesis, and stress granule immunofluorescence under arsenite/heat-shock/hippuristanol\",\n      \"pmids\": [\"27160682\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SUMO ligase responsible for K226 modification not identified\", \"Functional consequence of stress granule recruitment for translation not defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed eIF4A2 is a host factor co-opted by viruses, with step-specific requirements distinguishing entry from downstream replication.\",\n      \"evidence\": \"shRNA knockdown in T cells with viral cDNA/entry assays (HIV-1); yeast two-hybrid, GST pull-down, Co-IP, and siRNA knockdown (TGEV M protein)\",\n      \"pmids\": [\"29842983\", \"30583231\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular role of eIF4A2 in viral cDNA synthesis unresolved\", \"Whether helicase activity is required for viral co-option untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined the molecular basis of eIF4A2-mediated repression by demonstrating direct Ccr4-Not association and binding to purine-rich 5'UTR motifs upstream of the AUG.\",\n      \"evidence\": \"Reciprocal Co-IP, ribosome profiling, RNA-seq, motif enrichment, and comparison with hippuristanol-inhibited eIF4A1\",\n      \"pmids\": [\"31791371\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and architecture of the eIF4A2–Ccr4-Not interaction not resolved\", \"How RISC hands off to eIF4A2 not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed an activating, transcript-selective role for eIF4A2 in stem cell translation, beyond its repressive function, including control of the totipotency program.\",\n      \"evidence\": \"RNAi screen, ribosome fractionation, and polysome profiling in ESCs with pluripotency/Zscan4 readouts\",\n      \"pmids\": [\"35353581\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Determinants directing eIF4A2 to activation versus repression unknown\", \"RPS26-dependent versus -independent ribosome usage mechanism unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated that EIF4A2 variants cause a neurodevelopmental syndrome and that disease arises by both loss- and gain-of-function, using cross-species complementation.\",\n      \"evidence\": \"Drosophila loss-of-function rescue with human wild-type and variant cDNAs, plus molecular modeling\",\n      \"pmids\": [\"36528028\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular nature of the gain-of-function variants not biochemically defined\", \"Affected neuronal mRNA targets not identified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Linked EIF4A2 haploinsufficiency to a dystonia-tremor syndrome and connected the disease to the Ccr4-Not repression pathway through a patient-cell target readout.\",\n      \"evidence\": \"Western blot, immunocytochemistry, IMP1 quantification, and eIF4A2–Ccr4-Not colocalization in patient fibroblasts\",\n      \"pmids\": [\"37485550\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causality between IMP1 derepression and disease phenotype not established\", \"Neuronal cell-type-specific consequences untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Distinguished eIF4A2 from eIF4A1 functionally, assigning eIF4A2 a specific role in 18S rRNA and 40S subunit biogenesis despite free exchange within eIF4F.\",\n      \"evidence\": \"Conditional knockout mouse genetics, ribosome profiling, rRNA biogenesis assays, and B-cell flow cytometry\",\n      \"pmids\": [\"39516355\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which eIF4A2 acts in rRNA processing unknown\", \"Whether this role extends beyond B cells untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved isoform-specific biochemistry by quantifying differential ATP affinity/ATPase activity and mapping the responsible residues, plus opposite BLF1 toxin sensitivity.\",\n      \"evidence\": \"In vitro ATPase assays, N-terminal domain swapping, L98/A100 mutagenesis, and BLF1 treatment of purified proteins\",\n      \"pmids\": [\"40423315\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ATPase differences translate to distinct cellular functions not shown\", \"Structural basis for BLF1 isoform selectivity not determined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Placed eIF4A2 upstream of mTOR-regulated translation in tumor biology, where its loss triggers senescence that pharmacological translation reduction reverses.\",\n      \"evidence\": \"Conditional eIF4A2 knockout in KRAS lung cancer mouse model, rapamycin rescue, secretome proteomics, and p21 staining (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.03.19.644135\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mRNA targets driving senescence not identified\", \"Generalizability beyond KRAS-driven lung adenocarcinoma untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single helicase reconciles its opposing roles—miRNA/Ccr4-Not-dependent repression versus transcript-selective translational activation and 40S biogenesis—remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of eIF4A2 engaged with Ccr4-Not\", \"No unified rule predicting repression versus activation of a given mRNA\", \"Connection between SUMO/stress regulation and target selection undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0045182\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-72613\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 1, 6]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"complexes\": [\"eIF4F\", \"Ccr4-Not\"],\n    \"partners\": [\"CNOT1\", \"DDX6\", \"IGF2BP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":7,"faith_total":7,"faith_pct":100.0}}