{"gene":"GLE1","run_date":"2026-04-28T18:06:53","timeline":{"discoveries":[{"year":2006,"finding":"Gle1 and inositol hexakisphosphate (InsP6/IP6) together stimulate the RNA-dependent ATPase activity of the DEAD-box protein Dbp5 at the nuclear pore complex cytoplasmic face to mediate directional mRNA export; Gle1 alone has minimal effect, InsP6 requires Gle1, and maximal InsP6 binding requires both Dbp5 and Gle1.","method":"In vitro ATPase kinetic assays, genetic epistasis (DBP5 overexpression suppresses ipk1 nup42 mutant defects), in vitro binding assays","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with kinetic analysis, replicated simultaneously by two independent labs (PMID:16783363 and PMID:16783364)","pmids":["16783363","16783364"],"is_preprint":false},{"year":2006,"finding":"Gle1 binds directly to InsP6, and this interaction potentiates Gle1-mediated stimulation of Dbp5 ATPase and RNA binding; a gle1 point mutant deficient for Dbp5 stimulation in vitro displays mRNA export defects in vivo.","method":"In vitro ATPase assays, direct binding assays, in vivo mRNA export analysis of gle1 point mutants","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution plus in vivo validation, replicated across two independent labs","pmids":["16783364","16783363"],"is_preprint":false},{"year":2008,"finding":"Gle1 and IP6 are required for efficient translation termination in S. cerevisiae and Gle1 physically interacts with translation termination factors; additionally, Gle1 has a conserved physical association with initiation factor eIF3 and gle1 mutants display genetic interactions with the eIF3 mutant nip1-1, indicating a role in translation initiation independent of IP6.","method":"Co-immunoprecipitation, genetic interaction analysis, in vivo translation assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP and genetic epistasis with multiple orthogonal methods in a high-impact study","pmids":["18724935"],"is_preprint":false},{"year":2010,"finding":"Specific Gle1 residues constituting the IP6 binding pocket are required for IP6-dependent Dbp5 stimulation in vitro; IP6-binding Gle1 mutants recapitulate all mRNA export and translation termination defects of IP6-depleted cells, establishing Gle1 as the primary target of IP6 for both processes.","method":"In vitro ATPase stimulation assays with Gle1 point mutants, in vivo mRNA export and translation termination assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — structure-guided mutagenesis combined with in vitro biochemistry and in vivo functional validation","pmids":["20371601"],"is_preprint":false},{"year":2011,"finding":"Nup159 is required for ADP release from Dbp5, while Gle1-IP6 stimulates ATP binding and primes Dbp5 for RNA loading, allowing multiple cycles of mRNP remodeling by a single Dbp5 at the NPC; Nup159 and Gle1-IP6 together regulate the nucleotide-bound state of Dbp5.","method":"In vitro reconstitution ATPase assays, dbp5 mutant analysis, in vivo mRNA export assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mutant bypass analysis and corresponding in vivo phenotypes, replicated in companion paper","pmids":["21576266","21576265"],"is_preprint":false},{"year":2011,"finding":"The Dbp5-Gle1 interaction is limiting for mRNA export and can be independent of Nup159; RNA-binding-deficient Dbp5 mutants act as dominant negatives by competing with wild-type Dbp5 for Gle1 at NPCs; ATP binding and hydrolysis are required for efficient Dbp5 association with NPCs; Dbp5 association with NPCs is very dynamic (<1 sec by FRAP).","method":"Dominant-negative mutant analysis, in vivo mRNA export assays, FRAP in yeast and human cells","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal approaches (genetics, FRAP, dominant-negative competition) in yeast and human cells","pmids":["21576265"],"is_preprint":false},{"year":2011,"finding":"Gle1 physically and genetically interacts with the DEAD-box protein Ded1 and inhibits Ded1 ATPase activity in vitro in an IP6-independent manner, thereby negatively regulating translation initiation; a gle1-4 mutant suppresses initiation defects in ded1-120.","method":"Co-immunoprecipitation, in vitro ATPase inhibition assays, genetic suppressor analysis, in vitro translation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro ATPase assay plus genetic epistasis plus in vitro translation, multiple orthogonal methods","pmids":["21949122"],"is_preprint":false},{"year":2009,"finding":"Crystal structure of the C-terminal domain of Dbp5 (1.8 Å) revealed the Gle1 interaction surface; structure-guided mutagenesis identified charged surface residues whose mutation weakens Gle1 binding and inhibits Gle1-stimulated ATPase activity, with the weakest mutants unable to support yeast growth.","method":"X-ray crystallography, structure-guided mutagenesis, in vitro ATPase assays, in vivo complementation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus mutagenesis plus in vitro and in vivo functional validation","pmids":["19805289"],"is_preprint":false},{"year":2013,"finding":"Gle1 self-associates via its coiled-coil domain to form ~26 nm diameter disk-shaped oligomeric particles visible by electron microscopy; the LCCS1 disease mutation FinMajor (PFQ insertion in the coiled-coil domain) causes malformed oligomeric particles and specifically impairs mRNA export but not translation functions of Gle1.","method":"Electron microscopy, in vitro self-association assays, in vivo functional assays for mRNA export and translation, FinMajor mutant analysis","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1–2 — EM structural analysis plus in vitro self-association plus orthogonal in vivo functional dissection","pmids":["24243016"],"is_preprint":false},{"year":2005,"finding":"The hGle1B isoform-specific C-terminal 43 amino acid region mediates binding to the nucleoporin hCG1/NPL1; hNup155, hGle1B, and hCG1 form a heterotrimeric complex in vitro; siRNA-mediated depletion of hCG1 results in hGle1 accumulation in cytoplasmic foci and inhibition of Hsp70 mRNA export.","method":"In vitro binding assays, heterotrimeric complex reconstitution, siRNA knockdown, mRNA export assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — in vitro complex reconstitution plus siRNA functional validation with specific mRNA export readout","pmids":["16000379"],"is_preprint":false},{"year":2003,"finding":"hGle1 (hGle1B isoform) localizes to the nuclear envelope/NPC; hGle1 shuttles between nucleus and cytoplasm via an internal 39-amino acid shuttling domain; disruption of shuttling by a cell-permeable peptide impairs bulk poly(A)+ mRNA export and specific mRNA export, reducing hGle1 localization at the NE.","method":"Live cell FRAP, microinjection of recombinant protein, cell-permeable shuttling domain peptide, mRNA export assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — FRAP localization tied to functional consequence, peptide competition with specific export readout","pmids":["12668658"],"is_preprint":false},{"year":2003,"finding":"hGle1 physically interacts with the nucleoporin hNup155; the N-terminal 29 residues of hGle1 constitute the hNup155-binding domain and are required for nuclear rim/NPC localization of hGle1B.","method":"Yeast two-hybrid screen, in vitro binding assays, deletion analysis, localization studies in HeLa cells","journal":"Molecular & cellular proteomics","confidence":"Medium","confidence_rationale":"Tier 3 — yeast two-hybrid confirmed by in vitro pulldown plus localization; single lab","pmids":["14645504"],"is_preprint":false},{"year":2004,"finding":"Cytoplasmic IP6 production is sufficient to mediate the Gle1-mRNA export pathway; genetic epistasis placed IP6 action at a cytoplasmic step involving Nup42, Nup159, Dbp5, and Gle2 but not FG-repeat domains of nucleoporins.","method":"Genetic epistasis with ipk1 null double mutants, spatial restriction of Ipk1 to cytoplasm, complementation assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with spatial restriction experiments, multiple mutant combinations","pmids":["15459192"],"is_preprint":false},{"year":2004,"finding":"Nab2, Gfd1, and Gle1 associate in a complex; Gfd1 directly binds Nab2 at its N-terminal domain (distinct from RNA-binding domains), and Gfd1 bridges Gle1 and Nab2-bound mRNA during export; Nab2 export is blocked in gle1 mutants.","method":"Pulldown with recombinant proteins, co-immunoprecipitation from yeast lysates, two-hybrid assays, in vivo export analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal pulldown plus co-IP plus genetic in vivo analysis","pmids":["15208322"],"is_preprint":false},{"year":2017,"finding":"Nup42's C-terminal domain binds Gle1/hGle1B at an interface distinct from the Gle1-Dbp5 interaction site; a trimeric Nup42-CTD/Gle1-CTD/Dbp5 complex forms in the presence of IP6; Nup42-CTD and IP6 stimulate Gle1/hGle1B activation of Dbp5/DDX19B in vitro in a non-additive manner; deletion of NUP42 abrogates Gle1-Dbp5 interaction and disruption of Nup42 or IP6 binding sites causes mRNA export defects in yeast and human cells.","method":"In vitro ATPase stimulation assays, trimeric complex formation, structure-function mutagenesis in yeast and human cells, mRNA export assays","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro reconstitution of trimeric complex plus ATPase assays plus in vivo validation in both yeast and human cells","pmids":["28869701"],"is_preprint":false},{"year":2018,"finding":"Human Gle1 is phosphorylated during heat shock stress by MAPKs ERK and JNK, which phosphorylate the Gle1A N-terminal domain and prime it for further phosphorylation by GSK3; phosphomimetic gle1A6D perturbs self-association and inhibits DDX3 ATPase activity; phosphodeficient GFP-gle1A6A promotes stress granule (SG) assembly whereas phosphomimetic GFP-gle1A6D enhances SG disassembly.","method":"MS identification of phosphorylation sites, in vitro kinase assays, ATPase inhibition assays, GFP-tagged phosphomimetic/phosphodeficient mutant expression with SG phenotype readout","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — MS-identified sites validated by in vitro kinase assays plus ATPase assays plus in vivo SG functional readout, multiple orthogonal methods","pmids":["30429220"],"is_preprint":false},{"year":2022,"finding":"Gle1 activates Dbp5 ATPase by two mechanisms: (1) thermodynamically coupling Gle1 binding to ATP binding on Dbp5 (Gle1 binds Dbp5-ATP >100-fold more tightly than other nucleotide states; Gle1 increases ATP equilibrium binding >150-fold by slowing ATP dissociation), and (2) accelerating the rate-limiting Pi release step ~20-fold.","method":"Kinetic and equilibrium ATPase analyses (stopped-flow and steady-state), fluorescence-based nucleotide binding assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — rigorous in vitro kinetic mechanistic dissection with multiple equilibrium and transient-state measurements","pmids":["35286399"],"is_preprint":false},{"year":2018,"finding":"Nup159 does not accelerate ADP release from Dbp5; instead, Gle1 slows ADP release from Dbp5 independent of Mg2+; in the presence of Nup159, Gle1 interaction with ADP-bound Dbp5 is reduced ~18-fold, suggesting Nup159 modulates the Dbp5-Gle1 interaction to aid Gle1 release rather than acting as a nucleotide exchange factor.","method":"In vitro solution-based kinetic and thermodynamic ATPase/nucleotide binding assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinetic reconstitution with quantitative binding measurements","pmids":["29782832"],"is_preprint":false},{"year":2017,"finding":"Gle1 inhibits Ded1 by reducing Ded1's affinity for RNA; this inhibition counteracts excess Ded1-mediated translation repression, positioning Gle1 as a gatekeeper to optimize Ded1 activity; both Ded1 and Gle1 affect preinitiation complex assembly.","method":"RNA binding assays (mutation analysis), in vitro translation assays, in vivo genetic suppressor analysis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — RNA binding assays plus in vitro and in vivo translation data, single lab","pmids":["28784717"],"is_preprint":false},{"year":2016,"finding":"Gle1 is enriched at the centrosome and basal body, assembling into the toroid-shaped pericentriolar material around the mother centriole; reduced Gle1 levels correlate with decreased pericentrin at the centrosome and microtubule organization defects not caused by loss of mRNA export; Gle1 depletion in zebrafish compromises ciliary beating in Kupffer's vesicle.","method":"Immunofluorescence localization, siRNA knockdown with microtubule phenotype readout, zebrafish morpholino depletion","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2–3 — direct localization experiments with functional consequence (microtubule defects, cilia), but mechanism at centrosome not fully defined","pmids":["28035044"],"is_preprint":false},{"year":2020,"finding":"Gle1 self-association is driven by two distinct regions: a coiled-coil domain and a novel 10-amino acid aggregation-prone region; both are required for proper mRNA export and stress granule formation; Gle1 oligomerization is dispensable for non-stressed translation initiation but both self-association domains are independently required for translation regulation under cellular stress.","method":"Mutagenesis of oligomerization domains, functional assays in HeLa cells for mRNA export, stress granule formation, and translation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — structure-function mutagenesis with multiple functional readouts, single lab","pmids":["32981894"],"is_preprint":false},{"year":2020,"finding":"Disruption of Gle1 nucleocytoplasmic shuttling causes nuclear accumulation of specific mRNAs with elongated 3'-UTRs due to changes in transcription state (not altered nuclear export); shuttling inhibition results in increased DDX1 nucleoplasmic localization, decreased DDX1 interaction with Gle1 and the pre-mRNA cleavage factor CstF-64, and increased nuclear R-loop signal; Gle1 colocalizes with R-loops, revealing a nuclear role in coordinating DDX1 function in transcription termination.","method":"Peptide-mediated shuttling disruption, RNA sequencing, immunofluorescence, co-immunoprecipitation, R-loop detection","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2–3 — multiple orthogonal approaches but mechanistic link to DDX1/transcription termination based partly on correlative evidence","pmids":["32755435"],"is_preprint":false},{"year":2020,"finding":"The RNA-binding E3 ubiquitin ligase MKRN2 physically interacts with GLE1; MKRN2 knockdown or knockout in zebrafish partially rescues retinal developmental defects caused by GLE1 depletion, indicating MKRN2 is epistatic to GLE1; MKRN2 knockdown enhances nuclear export of MKRN2-associated mRNAs.","method":"Affinity-purification mass spectrometry, zebrafish morpholino knockdown and CRISPR knockout with epistasis analysis, ribonomic (RIP) assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 — AP-MS interaction confirmed by zebrafish genetic epistasis with specific developmental phenotype readout","pmids":["32460013"],"is_preprint":false},{"year":2024,"finding":"Gle1 binding to Dbp5 is required for Dbp5 to direct tRNA export in vivo; tRNA (or dsRNA) does not itself activate Dbp5 ATPase activity but acts synergistically with Gle1 to fully activate Dbp5; Dbp5 is recruited to tRNA independently of canonical mRNA export factors Los1, Msn5, or Mex67.","method":"Co-immunoprecipitation (in vivo), Dbp5 dominant-negative overexpression epistasis, in vitro ATPase assays with tRNA","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro biochemistry plus in vivo co-IP and genetic epistasis, single lab","pmids":["38189406"],"is_preprint":false},{"year":2023,"finding":"Phosphorylation of Gle1 at threonine 102 (T102) serves as a priming event for further phosphorylation in Gle1's N-terminal low complexity cluster; basally phosphorylated (pT102) Gle1 is predominantly nuclear with punctate distribution; under sodium arsenite stress, pT102-Gle1 shifts to cytoplasm; pT102-Gle1 co-immunoprecipitates with DDX1 in a phosphatase-sensitive manner, linking phosphorylation to Gle1's role in regulating DDX1 during transcription termination.","method":"In vitro kinase assays, phospho-specific antibody immunofluorescence microscopy, co-immunoprecipitation","journal":"Advances in biological regulation","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro kinase assays plus phospho-specific antibody immunofluorescence and co-IP, single lab","pmids":["37801910"],"is_preprint":false},{"year":2025,"finding":"Crystal structure of N-terminally truncated Gle1 from Debaryomyces hansenii (DhGle1ΔN) was determined at 1.5 Å resolution, revealing 13 α-helices and a RecA-like fold; comparison with homolog structures in complexes identifies distinct secondary structural elements in α1, α3, α4, and α8 that may relate to Dbp5 interaction.","method":"X-ray crystallography at 1.5 Å resolution","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 1 for structure — high-resolution crystal structure, but limited functional validation beyond structural comparison","pmids":["40004126"],"is_preprint":false}],"current_model":"GLE1 is a conserved, multifunctional regulator of DEAD-box RNA helicases that localizes to the cytoplasmic face of the nuclear pore complex (NPC) via interactions with Nup155, hCG1, and Nup42; at the NPC, IP6-bound Gle1 activates the DEAD-box ATPase Dbp5/DDX19B by thermodynamically coupling ATP binding to Gle1 binding and accelerating rate-limiting phosphate release, thereby driving mRNP remodeling and directional mRNA (and tRNA) export; Gle1 also regulates translation initiation (by inhibiting Ded1/DDX3 ATPase and interacting with eIF3), translation termination (in complex with termination factors), stress granule dynamics (controlled by MAPK/GSK3-mediated phosphorylation), and transcription termination (by coordinating nuclear DDX1 function at 3'-end processing complexes), with Gle1 oligomerization via its coiled-coil domain required specifically for mRNA export but not all translation functions, and human disease mutations (LCCS1, LAAHD, ALS) disrupting NPC localization, oligomerization, or IP6-dependent Dbp5 activation."},"narrative":{"teleology":[{"year":2003,"claim":"Establishing that hGle1 localizes to the NPC via Nup155 binding and shuttles between nucleus and cytoplasm answered how Gle1 is positioned to function in mRNA export.","evidence":"FRAP, microinjection, cell-permeable peptide competition in HeLa cells; yeast two-hybrid and in vitro pulldowns for Nup155 interaction","pmids":["12668658","14645504"],"confidence":"High","gaps":["Structural basis of Gle1-Nup155 interface not resolved","Whether shuttling is cargo-associated or autonomous was unclear"]},{"year":2005,"claim":"Identification of the hGle1B–hCG1–Nup155 heterotrimeric complex at the NPC cytoplasmic face defined the molecular platform through which Gle1 is tethered for mRNA export.","evidence":"In vitro reconstitution of trimeric complex, siRNA depletion of hCG1 with mRNA export readout","pmids":["16000379"],"confidence":"High","gaps":["Stoichiometry of the complex at the intact NPC not determined","Contribution of Nup42 to human NPC tethering not yet tested"]},{"year":2006,"claim":"Demonstrating that Gle1 and IP6 together stimulate Dbp5 ATPase activity revealed the enzymatic mechanism by which Gle1 drives directional mRNA export at the NPC.","evidence":"In vitro ATPase kinetic assays and genetic epistasis, independently replicated by two labs","pmids":["16783363","16783364"],"confidence":"High","gaps":["Precise kinetic mechanism of stimulation (binding vs. catalysis) not yet dissected","Whether Gle1-IP6 activates other DEAD-box proteins was unknown"]},{"year":2008,"claim":"Discovery that Gle1 functions in both translation termination (IP6-dependent, with termination factors) and translation initiation (via eIF3, IP6-independent) expanded Gle1's role beyond mRNA export to cytoplasmic gene expression control.","evidence":"Co-immunoprecipitation with termination factors and eIF3, genetic interactions with nip1-1, in vivo translation assays in S. cerevisiae","pmids":["18724935"],"confidence":"High","gaps":["Direct target helicase for translation termination role not identified","Mechanism of eIF3 interaction not structurally resolved"]},{"year":2009,"claim":"Crystal structure of Dbp5 C-terminal domain mapped the Gle1-interaction surface and showed that charge-complementary residues are essential for Gle1-stimulated ATPase activity and viability.","evidence":"X-ray crystallography at 1.8 Å, structure-guided mutagenesis with in vitro and in vivo validation","pmids":["19805289"],"confidence":"High","gaps":["Full Gle1-Dbp5 co-crystal structure not obtained","Conformational changes upon Gle1 binding not visualized"]},{"year":2010,"claim":"Mapping the IP6 binding pocket on Gle1 and showing that IP6-binding mutants phenocopy IP6-depleted cells established Gle1 as the primary effector of IP6 signaling in both mRNA export and translation termination.","evidence":"Structure-guided Gle1 point mutants tested in vitro (ATPase stimulation) and in vivo (export and translation assays)","pmids":["20371601"],"confidence":"High","gaps":["Whether IP6 binding induces a conformational change in Gle1 was not determined"]},{"year":2011,"claim":"Reconstitution of the Dbp5 catalytic cycle showed that Gle1-IP6 stimulates ATP binding and primes RNA loading while Nup159 modulates nucleotide state, explaining how a single Dbp5 molecule performs multiple remodeling cycles at the NPC.","evidence":"In vitro ATPase reconstitution, Dbp5 mutant bypass analysis, FRAP dynamics, dominant-negative competition","pmids":["21576266","21576265"],"confidence":"High","gaps":["Precise role of Nup159 in ADP release versus Gle1 dissociation debated","Real-time single-molecule visualization of the cycle not achieved"]},{"year":2011,"claim":"Finding that Gle1 inhibits Ded1 ATPase activity (IP6-independently) revealed that Gle1 can both activate and inhibit distinct DEAD-box helicases, with opposing effects tuning translation initiation.","evidence":"In vitro ATPase inhibition assays, co-IP, genetic suppression of ded1-120 by gle1-4, in vitro translation","pmids":["21949122"],"confidence":"High","gaps":["Structural basis for differential activation (Dbp5) versus inhibition (Ded1) unknown","Whether inhibition occurs on polysomes or free mRNPs not resolved"]},{"year":2013,"claim":"Demonstration that Gle1 forms disk-shaped oligomers via its coiled-coil domain and that the LCCS1 FinMajor mutation disrupts oligomerization and specifically impairs mRNA export (but not translation) linked a human disease mechanism to a discrete structural feature of Gle1.","evidence":"Electron microscopy of oligomeric particles, in vitro self-association assays, in vivo dissection of export versus translation in FinMajor mutants","pmids":["24243016"],"confidence":"High","gaps":["Oligomer stoichiometry and arrangement within the NPC not resolved","Whether other LAAHD/ALS mutations also disrupt oligomerization not systematically tested"]},{"year":2017,"claim":"Reconstitution of the Nup42-Gle1-Dbp5 trimeric complex and demonstration that Nup42 and IP6 non-additively enhance Gle1-mediated Dbp5 activation clarified how NPC-tethered Gle1 achieves maximal helicase stimulation in situ.","evidence":"In vitro trimeric complex reconstitution, ATPase stimulation, structure-function mutagenesis in yeast and human cells","pmids":["28869701"],"confidence":"High","gaps":["Cryo-EM or crystal structure of the intact trimeric complex not obtained"]},{"year":2017,"claim":"Showing that Gle1 inhibits Ded1 by reducing its RNA affinity defined the molecular mechanism through which Gle1 gates Ded1-mediated translation repression.","evidence":"RNA binding assays with Ded1 mutants, in vitro translation, genetic suppressor analysis","pmids":["28784717"],"confidence":"Medium","gaps":["Binding interface between Gle1 and Ded1 not mapped","In vivo transcriptome-wide impact on Ded1 targets not profiled"]},{"year":2018,"claim":"Quantitative kinetic analysis revised the Dbp5 catalytic cycle: Gle1 slows ADP release rather than Nup159 accelerating it, and Nup159 instead weakens Gle1-Dbp5(ADP) interaction to promote Gle1 release, redefining the roles of both cofactors.","evidence":"Solution-based kinetic and thermodynamic nucleotide binding assays","pmids":["29782832"],"confidence":"High","gaps":["Whether this revised model holds under NPC-proximal crowding conditions not tested"]},{"year":2018,"claim":"Discovery that MAPK (ERK/JNK) and GSK3 phosphorylate Gle1A's N-terminal domain during stress, modulating self-association, DDX3 inhibition, and stress granule dynamics, revealed a signaling input that toggles Gle1's cytoplasmic functions.","evidence":"Mass spectrometry of phosphorylation sites, in vitro kinase assays, phosphomimetic/phosphodeficient mutant SG phenotypes","pmids":["30429220"],"confidence":"High","gaps":["Phosphatase(s) responsible for dephosphorylation not identified","Whether phosphorylation also affects mRNA export function not tested"]},{"year":2020,"claim":"Identification of two independent self-association regions (coiled-coil and a 10-aa aggregation-prone motif), both required for mRNA export and stress granule formation but dispensable for basal translation, dissected which Gle1 functions require oligomerization.","evidence":"Mutagenesis of oligomerization domains with functional assays in HeLa cells for export, SG formation, and translation","pmids":["32981894"],"confidence":"Medium","gaps":["Relative contribution of each domain to oligomer architecture not structurally resolved"]},{"year":2020,"claim":"Revealing that Gle1 shuttling disruption causes nuclear mRNA accumulation through altered transcription state (not export block), via reduced DDX1 interaction with CstF-64 and increased R-loops, established a nuclear role for Gle1 in transcription termination and 3′-end processing.","evidence":"Peptide-mediated shuttling inhibition, RNA-seq, co-IP of DDX1/CstF-64, R-loop immunofluorescence","pmids":["32755435"],"confidence":"Medium","gaps":["Direct enzymatic activity of Gle1 on DDX1 not biochemically demonstrated","Genome-wide sites of Gle1-dependent transcription termination not mapped"]},{"year":2022,"claim":"Definitive kinetic dissection showed Gle1 activates Dbp5 by two synergistic mechanisms—coupling Gle1 binding to ATP binding (>100-fold selectivity) and accelerating Pi release ~20-fold—providing the most complete mechanistic picture of Gle1-dependent helicase activation.","evidence":"Stopped-flow and steady-state fluorescence-based nucleotide binding and ATPase assays","pmids":["35286399"],"confidence":"High","gaps":["Whether these kinetic parameters change when Dbp5 is RNA-loaded at the NPC not tested","Structural basis for preferential ATP-state binding not visualized"]},{"year":2024,"claim":"Demonstrating that Gle1-Dbp5 interaction is required for tRNA export and that tRNA synergizes with Gle1 to activate Dbp5 expanded Gle1's role beyond mRNA to a broader RNA export regulator.","evidence":"In vivo co-IP, dominant-negative Dbp5 overexpression epistasis, in vitro ATPase assays with tRNA substrates","pmids":["38189406"],"confidence":"Medium","gaps":["Whether Gle1-Dbp5 acts on tRNA at the NPC or in the nucleoplasm not resolved","Specific tRNA species affected not profiled"]},{"year":null,"claim":"A high-resolution structure of the full Gle1-Dbp5 complex (ideally with IP6, Nup42, and RNA) is needed to explain how Gle1 differentially activates Dbp5 while inhibiting Ded1/DDX3, and how disease mutations structurally disrupt these interactions.","evidence":"","pmids":[],"confidence":"High","gaps":["No co-crystal or cryo-EM structure of the full Gle1-Dbp5-Nup42-IP6 complex","Structural basis for Gle1 activation versus inhibition of different DEAD-box helicases unknown","How Gle1 oligomeric architecture integrates into the intact NPC scaffold not visualized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,6,16,17,18]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,9,13]}],"localization":[{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[10,11]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[15,20]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[21,24]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[19]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,1,4,14,23]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2,6,18]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,10,14]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[21]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[15,20]}],"complexes":["Nup155–hCG1–Gle1B NPC subcomplex","Nup42–Gle1–Dbp5 trimeric complex","Gle1–Gfd1–Nab2 export complex"],"partners":["DBP5","DDX19B","DDX3","DDX1","NUP155","NUP42","NUPL2","MKRN2"],"other_free_text":[]},"mechanistic_narrative":"GLE1 is a conserved regulator of DEAD-box RNA helicases that couples nuclear pore complex (NPC) function to mRNA export, translation, and transcription termination. At the cytoplasmic face of the NPC, where it is anchored via interactions with Nup155, hCG1, and Nup42, IP6-bound Gle1 activates Dbp5/DDX19B by thermodynamically coupling its own binding to ATP loading on Dbp5 (>150-fold increase in ATP affinity) and accelerating rate-limiting phosphate release ~20-fold, driving iterative mRNP remodeling for directional mRNA and tRNA export [PMID:16783363, PMID:35286399, PMID:38189406]. Gle1 also inhibits the DEAD-box helicase Ded1/DDX3 in an IP6-independent manner to modulate translation initiation, interacts with eIF3 and translation termination factors for broader translational control, and coordinates DDX1 function in nuclear transcription termination and 3′-end processing [PMID:21949122, PMID:18724935, PMID:32755435]. Gle1 oligomerization via its coiled-coil domain is specifically required for mRNA export and stress granule dynamics—the latter regulated by MAPK/GSK3-mediated phosphorylation—and disease-causing mutations in LCCS1/LAAHD disrupt oligomer architecture and NPC localization [PMID:24243016, PMID:30429220]."},"prefetch_data":{"uniprot":{"accession":"Q53GS7","full_name":"mRNA export factor GLE1","aliases":["GLE1 RNA export mediator","GLE1-like protein","Nucleoporin GLE1"],"length_aa":698,"mass_kda":79.8,"function":"Required for the export of mRNAs containing poly(A) tails from the nucleus into the cytoplasm. May be involved in the terminal step of the mRNA transport through the nuclear pore complex (NPC)","subcellular_location":"Cytoplasm; Nucleus, nuclear pore complex","url":"https://www.uniprot.org/uniprotkb/Q53GS7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/GLE1","classification":"Common Essential","n_dependent_lines":973,"n_total_lines":1208,"dependency_fraction":0.8054635761589404},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"RAN","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/GLE1","total_profiled":1310},"omim":[{"mim_id":"621395","title":"DEAD-BOX HELICASE 19A; DDX19A","url":"https://www.omim.org/entry/621395"},{"mim_id":"616886","title":"GSE1 COILED-COIL PROTEIN; GSE1","url":"https://www.omim.org/entry/616886"},{"mim_id":"611890","title":"CONGENITAL ARTHROGRYPOSIS WITH ANTERIOR HORN CELL DISEASE; 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Dbp5 at the nuclear pore complex cytoplasmic face to mediate directional mRNA export; Gle1 alone has minimal effect, InsP6 requires Gle1, and maximal InsP6 binding requires both Dbp5 and Gle1.\",\n      \"method\": \"In vitro ATPase kinetic assays, genetic epistasis (DBP5 overexpression suppresses ipk1 nup42 mutant defects), in vitro binding assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with kinetic analysis, replicated simultaneously by two independent labs (PMID:16783363 and PMID:16783364)\",\n      \"pmids\": [\"16783363\", \"16783364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Gle1 binds directly to InsP6, and this interaction potentiates Gle1-mediated stimulation of Dbp5 ATPase and RNA binding; a gle1 point mutant deficient for Dbp5 stimulation in vitro displays mRNA export defects in vivo.\",\n      \"method\": \"In vitro ATPase assays, direct binding assays, in vivo mRNA export analysis of gle1 point mutants\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution plus in vivo validation, replicated across two independent labs\",\n      \"pmids\": [\"16783364\", \"16783363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Gle1 and IP6 are required for efficient translation termination in S. cerevisiae and Gle1 physically interacts with translation termination factors; additionally, Gle1 has a conserved physical association with initiation factor eIF3 and gle1 mutants display genetic interactions with the eIF3 mutant nip1-1, indicating a role in translation initiation independent of IP6.\",\n      \"method\": \"Co-immunoprecipitation, genetic interaction analysis, in vivo translation assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP and genetic epistasis with multiple orthogonal methods in a high-impact study\",\n      \"pmids\": [\"18724935\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Specific Gle1 residues constituting the IP6 binding pocket are required for IP6-dependent Dbp5 stimulation in vitro; IP6-binding Gle1 mutants recapitulate all mRNA export and translation termination defects of IP6-depleted cells, establishing Gle1 as the primary target of IP6 for both processes.\",\n      \"method\": \"In vitro ATPase stimulation assays with Gle1 point mutants, in vivo mRNA export and translation termination assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structure-guided mutagenesis combined with in vitro biochemistry and in vivo functional validation\",\n      \"pmids\": [\"20371601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Nup159 is required for ADP release from Dbp5, while Gle1-IP6 stimulates ATP binding and primes Dbp5 for RNA loading, allowing multiple cycles of mRNP remodeling by a single Dbp5 at the NPC; Nup159 and Gle1-IP6 together regulate the nucleotide-bound state of Dbp5.\",\n      \"method\": \"In vitro reconstitution ATPase assays, dbp5 mutant analysis, in vivo mRNA export assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutant bypass analysis and corresponding in vivo phenotypes, replicated in companion paper\",\n      \"pmids\": [\"21576266\", \"21576265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The Dbp5-Gle1 interaction is limiting for mRNA export and can be independent of Nup159; RNA-binding-deficient Dbp5 mutants act as dominant negatives by competing with wild-type Dbp5 for Gle1 at NPCs; ATP binding and hydrolysis are required for efficient Dbp5 association with NPCs; Dbp5 association with NPCs is very dynamic (<1 sec by FRAP).\",\n      \"method\": \"Dominant-negative mutant analysis, in vivo mRNA export assays, FRAP in yeast and human cells\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches (genetics, FRAP, dominant-negative competition) in yeast and human cells\",\n      \"pmids\": [\"21576265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Gle1 physically and genetically interacts with the DEAD-box protein Ded1 and inhibits Ded1 ATPase activity in vitro in an IP6-independent manner, thereby negatively regulating translation initiation; a gle1-4 mutant suppresses initiation defects in ded1-120.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ATPase inhibition assays, genetic suppressor analysis, in vitro translation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro ATPase assay plus genetic epistasis plus in vitro translation, multiple orthogonal methods\",\n      \"pmids\": [\"21949122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Crystal structure of the C-terminal domain of Dbp5 (1.8 Å) revealed the Gle1 interaction surface; structure-guided mutagenesis identified charged surface residues whose mutation weakens Gle1 binding and inhibits Gle1-stimulated ATPase activity, with the weakest mutants unable to support yeast growth.\",\n      \"method\": \"X-ray crystallography, structure-guided mutagenesis, in vitro ATPase assays, in vivo complementation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus mutagenesis plus in vitro and in vivo functional validation\",\n      \"pmids\": [\"19805289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Gle1 self-associates via its coiled-coil domain to form ~26 nm diameter disk-shaped oligomeric particles visible by electron microscopy; the LCCS1 disease mutation FinMajor (PFQ insertion in the coiled-coil domain) causes malformed oligomeric particles and specifically impairs mRNA export but not translation functions of Gle1.\",\n      \"method\": \"Electron microscopy, in vitro self-association assays, in vivo functional assays for mRNA export and translation, FinMajor mutant analysis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — EM structural analysis plus in vitro self-association plus orthogonal in vivo functional dissection\",\n      \"pmids\": [\"24243016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The hGle1B isoform-specific C-terminal 43 amino acid region mediates binding to the nucleoporin hCG1/NPL1; hNup155, hGle1B, and hCG1 form a heterotrimeric complex in vitro; siRNA-mediated depletion of hCG1 results in hGle1 accumulation in cytoplasmic foci and inhibition of Hsp70 mRNA export.\",\n      \"method\": \"In vitro binding assays, heterotrimeric complex reconstitution, siRNA knockdown, mRNA export assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vitro complex reconstitution plus siRNA functional validation with specific mRNA export readout\",\n      \"pmids\": [\"16000379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"hGle1 (hGle1B isoform) localizes to the nuclear envelope/NPC; hGle1 shuttles between nucleus and cytoplasm via an internal 39-amino acid shuttling domain; disruption of shuttling by a cell-permeable peptide impairs bulk poly(A)+ mRNA export and specific mRNA export, reducing hGle1 localization at the NE.\",\n      \"method\": \"Live cell FRAP, microinjection of recombinant protein, cell-permeable shuttling domain peptide, mRNA export assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — FRAP localization tied to functional consequence, peptide competition with specific export readout\",\n      \"pmids\": [\"12668658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"hGle1 physically interacts with the nucleoporin hNup155; the N-terminal 29 residues of hGle1 constitute the hNup155-binding domain and are required for nuclear rim/NPC localization of hGle1B.\",\n      \"method\": \"Yeast two-hybrid screen, in vitro binding assays, deletion analysis, localization studies in HeLa cells\",\n      \"journal\": \"Molecular & cellular proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — yeast two-hybrid confirmed by in vitro pulldown plus localization; single lab\",\n      \"pmids\": [\"14645504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Cytoplasmic IP6 production is sufficient to mediate the Gle1-mRNA export pathway; genetic epistasis placed IP6 action at a cytoplasmic step involving Nup42, Nup159, Dbp5, and Gle2 but not FG-repeat domains of nucleoporins.\",\n      \"method\": \"Genetic epistasis with ipk1 null double mutants, spatial restriction of Ipk1 to cytoplasm, complementation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with spatial restriction experiments, multiple mutant combinations\",\n      \"pmids\": [\"15459192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Nab2, Gfd1, and Gle1 associate in a complex; Gfd1 directly binds Nab2 at its N-terminal domain (distinct from RNA-binding domains), and Gfd1 bridges Gle1 and Nab2-bound mRNA during export; Nab2 export is blocked in gle1 mutants.\",\n      \"method\": \"Pulldown with recombinant proteins, co-immunoprecipitation from yeast lysates, two-hybrid assays, in vivo export analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal pulldown plus co-IP plus genetic in vivo analysis\",\n      \"pmids\": [\"15208322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Nup42's C-terminal domain binds Gle1/hGle1B at an interface distinct from the Gle1-Dbp5 interaction site; a trimeric Nup42-CTD/Gle1-CTD/Dbp5 complex forms in the presence of IP6; Nup42-CTD and IP6 stimulate Gle1/hGle1B activation of Dbp5/DDX19B in vitro in a non-additive manner; deletion of NUP42 abrogates Gle1-Dbp5 interaction and disruption of Nup42 or IP6 binding sites causes mRNA export defects in yeast and human cells.\",\n      \"method\": \"In vitro ATPase stimulation assays, trimeric complex formation, structure-function mutagenesis in yeast and human cells, mRNA export assays\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro reconstitution of trimeric complex plus ATPase assays plus in vivo validation in both yeast and human cells\",\n      \"pmids\": [\"28869701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Human Gle1 is phosphorylated during heat shock stress by MAPKs ERK and JNK, which phosphorylate the Gle1A N-terminal domain and prime it for further phosphorylation by GSK3; phosphomimetic gle1A6D perturbs self-association and inhibits DDX3 ATPase activity; phosphodeficient GFP-gle1A6A promotes stress granule (SG) assembly whereas phosphomimetic GFP-gle1A6D enhances SG disassembly.\",\n      \"method\": \"MS identification of phosphorylation sites, in vitro kinase assays, ATPase inhibition assays, GFP-tagged phosphomimetic/phosphodeficient mutant expression with SG phenotype readout\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — MS-identified sites validated by in vitro kinase assays plus ATPase assays plus in vivo SG functional readout, multiple orthogonal methods\",\n      \"pmids\": [\"30429220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Gle1 activates Dbp5 ATPase by two mechanisms: (1) thermodynamically coupling Gle1 binding to ATP binding on Dbp5 (Gle1 binds Dbp5-ATP >100-fold more tightly than other nucleotide states; Gle1 increases ATP equilibrium binding >150-fold by slowing ATP dissociation), and (2) accelerating the rate-limiting Pi release step ~20-fold.\",\n      \"method\": \"Kinetic and equilibrium ATPase analyses (stopped-flow and steady-state), fluorescence-based nucleotide binding assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — rigorous in vitro kinetic mechanistic dissection with multiple equilibrium and transient-state measurements\",\n      \"pmids\": [\"35286399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Nup159 does not accelerate ADP release from Dbp5; instead, Gle1 slows ADP release from Dbp5 independent of Mg2+; in the presence of Nup159, Gle1 interaction with ADP-bound Dbp5 is reduced ~18-fold, suggesting Nup159 modulates the Dbp5-Gle1 interaction to aid Gle1 release rather than acting as a nucleotide exchange factor.\",\n      \"method\": \"In vitro solution-based kinetic and thermodynamic ATPase/nucleotide binding assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinetic reconstitution with quantitative binding measurements\",\n      \"pmids\": [\"29782832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Gle1 inhibits Ded1 by reducing Ded1's affinity for RNA; this inhibition counteracts excess Ded1-mediated translation repression, positioning Gle1 as a gatekeeper to optimize Ded1 activity; both Ded1 and Gle1 affect preinitiation complex assembly.\",\n      \"method\": \"RNA binding assays (mutation analysis), in vitro translation assays, in vivo genetic suppressor analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNA binding assays plus in vitro and in vivo translation data, single lab\",\n      \"pmids\": [\"28784717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Gle1 is enriched at the centrosome and basal body, assembling into the toroid-shaped pericentriolar material around the mother centriole; reduced Gle1 levels correlate with decreased pericentrin at the centrosome and microtubule organization defects not caused by loss of mRNA export; Gle1 depletion in zebrafish compromises ciliary beating in Kupffer's vesicle.\",\n      \"method\": \"Immunofluorescence localization, siRNA knockdown with microtubule phenotype readout, zebrafish morpholino depletion\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — direct localization experiments with functional consequence (microtubule defects, cilia), but mechanism at centrosome not fully defined\",\n      \"pmids\": [\"28035044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Gle1 self-association is driven by two distinct regions: a coiled-coil domain and a novel 10-amino acid aggregation-prone region; both are required for proper mRNA export and stress granule formation; Gle1 oligomerization is dispensable for non-stressed translation initiation but both self-association domains are independently required for translation regulation under cellular stress.\",\n      \"method\": \"Mutagenesis of oligomerization domains, functional assays in HeLa cells for mRNA export, stress granule formation, and translation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — structure-function mutagenesis with multiple functional readouts, single lab\",\n      \"pmids\": [\"32981894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Disruption of Gle1 nucleocytoplasmic shuttling causes nuclear accumulation of specific mRNAs with elongated 3'-UTRs due to changes in transcription state (not altered nuclear export); shuttling inhibition results in increased DDX1 nucleoplasmic localization, decreased DDX1 interaction with Gle1 and the pre-mRNA cleavage factor CstF-64, and increased nuclear R-loop signal; Gle1 colocalizes with R-loops, revealing a nuclear role in coordinating DDX1 function in transcription termination.\",\n      \"method\": \"Peptide-mediated shuttling disruption, RNA sequencing, immunofluorescence, co-immunoprecipitation, R-loop detection\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — multiple orthogonal approaches but mechanistic link to DDX1/transcription termination based partly on correlative evidence\",\n      \"pmids\": [\"32755435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The RNA-binding E3 ubiquitin ligase MKRN2 physically interacts with GLE1; MKRN2 knockdown or knockout in zebrafish partially rescues retinal developmental defects caused by GLE1 depletion, indicating MKRN2 is epistatic to GLE1; MKRN2 knockdown enhances nuclear export of MKRN2-associated mRNAs.\",\n      \"method\": \"Affinity-purification mass spectrometry, zebrafish morpholino knockdown and CRISPR knockout with epistasis analysis, ribonomic (RIP) assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — AP-MS interaction confirmed by zebrafish genetic epistasis with specific developmental phenotype readout\",\n      \"pmids\": [\"32460013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Gle1 binding to Dbp5 is required for Dbp5 to direct tRNA export in vivo; tRNA (or dsRNA) does not itself activate Dbp5 ATPase activity but acts synergistically with Gle1 to fully activate Dbp5; Dbp5 is recruited to tRNA independently of canonical mRNA export factors Los1, Msn5, or Mex67.\",\n      \"method\": \"Co-immunoprecipitation (in vivo), Dbp5 dominant-negative overexpression epistasis, in vitro ATPase assays with tRNA\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro biochemistry plus in vivo co-IP and genetic epistasis, single lab\",\n      \"pmids\": [\"38189406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Phosphorylation of Gle1 at threonine 102 (T102) serves as a priming event for further phosphorylation in Gle1's N-terminal low complexity cluster; basally phosphorylated (pT102) Gle1 is predominantly nuclear with punctate distribution; under sodium arsenite stress, pT102-Gle1 shifts to cytoplasm; pT102-Gle1 co-immunoprecipitates with DDX1 in a phosphatase-sensitive manner, linking phosphorylation to Gle1's role in regulating DDX1 during transcription termination.\",\n      \"method\": \"In vitro kinase assays, phospho-specific antibody immunofluorescence microscopy, co-immunoprecipitation\",\n      \"journal\": \"Advances in biological regulation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro kinase assays plus phospho-specific antibody immunofluorescence and co-IP, single lab\",\n      \"pmids\": [\"37801910\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Crystal structure of N-terminally truncated Gle1 from Debaryomyces hansenii (DhGle1ΔN) was determined at 1.5 Å resolution, revealing 13 α-helices and a RecA-like fold; comparison with homolog structures in complexes identifies distinct secondary structural elements in α1, α3, α4, and α8 that may relate to Dbp5 interaction.\",\n      \"method\": \"X-ray crystallography at 1.5 Å resolution\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 for structure — high-resolution crystal structure, but limited functional validation beyond structural comparison\",\n      \"pmids\": [\"40004126\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"GLE1 is a conserved, multifunctional regulator of DEAD-box RNA helicases that localizes to the cytoplasmic face of the nuclear pore complex (NPC) via interactions with Nup155, hCG1, and Nup42; at the NPC, IP6-bound Gle1 activates the DEAD-box ATPase Dbp5/DDX19B by thermodynamically coupling ATP binding to Gle1 binding and accelerating rate-limiting phosphate release, thereby driving mRNP remodeling and directional mRNA (and tRNA) export; Gle1 also regulates translation initiation (by inhibiting Ded1/DDX3 ATPase and interacting with eIF3), translation termination (in complex with termination factors), stress granule dynamics (controlled by MAPK/GSK3-mediated phosphorylation), and transcription termination (by coordinating nuclear DDX1 function at 3'-end processing complexes), with Gle1 oligomerization via its coiled-coil domain required specifically for mRNA export but not all translation functions, and human disease mutations (LCCS1, LAAHD, ALS) disrupting NPC localization, oligomerization, or IP6-dependent Dbp5 activation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"GLE1 is a conserved regulator of DEAD-box RNA helicases that couples nuclear pore complex (NPC) function to mRNA export, translation, and transcription termination. At the cytoplasmic face of the NPC, where it is anchored via interactions with Nup155, hCG1, and Nup42, IP6-bound Gle1 activates Dbp5/DDX19B by thermodynamically coupling its own binding to ATP loading on Dbp5 (>150-fold increase in ATP affinity) and accelerating rate-limiting phosphate release ~20-fold, driving iterative mRNP remodeling for directional mRNA and tRNA export [PMID:16783363, PMID:35286399, PMID:38189406]. Gle1 also inhibits the DEAD-box helicase Ded1/DDX3 in an IP6-independent manner to modulate translation initiation, interacts with eIF3 and translation termination factors for broader translational control, and coordinates DDX1 function in nuclear transcription termination and 3′-end processing [PMID:21949122, PMID:18724935, PMID:32755435]. Gle1 oligomerization via its coiled-coil domain is specifically required for mRNA export and stress granule dynamics—the latter regulated by MAPK/GSK3-mediated phosphorylation—and disease-causing mutations in LCCS1/LAAHD disrupt oligomer architecture and NPC localization [PMID:24243016, PMID:30429220].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Establishing that hGle1 localizes to the NPC via Nup155 binding and shuttles between nucleus and cytoplasm answered how Gle1 is positioned to function in mRNA export.\",\n      \"evidence\": \"FRAP, microinjection, cell-permeable peptide competition in HeLa cells; yeast two-hybrid and in vitro pulldowns for Nup155 interaction\",\n      \"pmids\": [\"12668658\", \"14645504\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Gle1-Nup155 interface not resolved\", \"Whether shuttling is cargo-associated or autonomous was unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identification of the hGle1B–hCG1–Nup155 heterotrimeric complex at the NPC cytoplasmic face defined the molecular platform through which Gle1 is tethered for mRNA export.\",\n      \"evidence\": \"In vitro reconstitution of trimeric complex, siRNA depletion of hCG1 with mRNA export readout\",\n      \"pmids\": [\"16000379\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the complex at the intact NPC not determined\", \"Contribution of Nup42 to human NPC tethering not yet tested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrating that Gle1 and IP6 together stimulate Dbp5 ATPase activity revealed the enzymatic mechanism by which Gle1 drives directional mRNA export at the NPC.\",\n      \"evidence\": \"In vitro ATPase kinetic assays and genetic epistasis, independently replicated by two labs\",\n      \"pmids\": [\"16783363\", \"16783364\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise kinetic mechanism of stimulation (binding vs. catalysis) not yet dissected\", \"Whether Gle1-IP6 activates other DEAD-box proteins was unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Discovery that Gle1 functions in both translation termination (IP6-dependent, with termination factors) and translation initiation (via eIF3, IP6-independent) expanded Gle1's role beyond mRNA export to cytoplasmic gene expression control.\",\n      \"evidence\": \"Co-immunoprecipitation with termination factors and eIF3, genetic interactions with nip1-1, in vivo translation assays in S. cerevisiae\",\n      \"pmids\": [\"18724935\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct target helicase for translation termination role not identified\", \"Mechanism of eIF3 interaction not structurally resolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Crystal structure of Dbp5 C-terminal domain mapped the Gle1-interaction surface and showed that charge-complementary residues are essential for Gle1-stimulated ATPase activity and viability.\",\n      \"evidence\": \"X-ray crystallography at 1.8 Å, structure-guided mutagenesis with in vitro and in vivo validation\",\n      \"pmids\": [\"19805289\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full Gle1-Dbp5 co-crystal structure not obtained\", \"Conformational changes upon Gle1 binding not visualized\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Mapping the IP6 binding pocket on Gle1 and showing that IP6-binding mutants phenocopy IP6-depleted cells established Gle1 as the primary effector of IP6 signaling in both mRNA export and translation termination.\",\n      \"evidence\": \"Structure-guided Gle1 point mutants tested in vitro (ATPase stimulation) and in vivo (export and translation assays)\",\n      \"pmids\": [\"20371601\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether IP6 binding induces a conformational change in Gle1 was not determined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Reconstitution of the Dbp5 catalytic cycle showed that Gle1-IP6 stimulates ATP binding and primes RNA loading while Nup159 modulates nucleotide state, explaining how a single Dbp5 molecule performs multiple remodeling cycles at the NPC.\",\n      \"evidence\": \"In vitro ATPase reconstitution, Dbp5 mutant bypass analysis, FRAP dynamics, dominant-negative competition\",\n      \"pmids\": [\"21576266\", \"21576265\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise role of Nup159 in ADP release versus Gle1 dissociation debated\", \"Real-time single-molecule visualization of the cycle not achieved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Finding that Gle1 inhibits Ded1 ATPase activity (IP6-independently) revealed that Gle1 can both activate and inhibit distinct DEAD-box helicases, with opposing effects tuning translation initiation.\",\n      \"evidence\": \"In vitro ATPase inhibition assays, co-IP, genetic suppression of ded1-120 by gle1-4, in vitro translation\",\n      \"pmids\": [\"21949122\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for differential activation (Dbp5) versus inhibition (Ded1) unknown\", \"Whether inhibition occurs on polysomes or free mRNPs not resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstration that Gle1 forms disk-shaped oligomers via its coiled-coil domain and that the LCCS1 FinMajor mutation disrupts oligomerization and specifically impairs mRNA export (but not translation) linked a human disease mechanism to a discrete structural feature of Gle1.\",\n      \"evidence\": \"Electron microscopy of oligomeric particles, in vitro self-association assays, in vivo dissection of export versus translation in FinMajor mutants\",\n      \"pmids\": [\"24243016\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Oligomer stoichiometry and arrangement within the NPC not resolved\", \"Whether other LAAHD/ALS mutations also disrupt oligomerization not systematically tested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Reconstitution of the Nup42-Gle1-Dbp5 trimeric complex and demonstration that Nup42 and IP6 non-additively enhance Gle1-mediated Dbp5 activation clarified how NPC-tethered Gle1 achieves maximal helicase stimulation in situ.\",\n      \"evidence\": \"In vitro trimeric complex reconstitution, ATPase stimulation, structure-function mutagenesis in yeast and human cells\",\n      \"pmids\": [\"28869701\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cryo-EM or crystal structure of the intact trimeric complex not obtained\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showing that Gle1 inhibits Ded1 by reducing its RNA affinity defined the molecular mechanism through which Gle1 gates Ded1-mediated translation repression.\",\n      \"evidence\": \"RNA binding assays with Ded1 mutants, in vitro translation, genetic suppressor analysis\",\n      \"pmids\": [\"28784717\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding interface between Gle1 and Ded1 not mapped\", \"In vivo transcriptome-wide impact on Ded1 targets not profiled\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Quantitative kinetic analysis revised the Dbp5 catalytic cycle: Gle1 slows ADP release rather than Nup159 accelerating it, and Nup159 instead weakens Gle1-Dbp5(ADP) interaction to promote Gle1 release, redefining the roles of both cofactors.\",\n      \"evidence\": \"Solution-based kinetic and thermodynamic nucleotide binding assays\",\n      \"pmids\": [\"29782832\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this revised model holds under NPC-proximal crowding conditions not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Discovery that MAPK (ERK/JNK) and GSK3 phosphorylate Gle1A's N-terminal domain during stress, modulating self-association, DDX3 inhibition, and stress granule dynamics, revealed a signaling input that toggles Gle1's cytoplasmic functions.\",\n      \"evidence\": \"Mass spectrometry of phosphorylation sites, in vitro kinase assays, phosphomimetic/phosphodeficient mutant SG phenotypes\",\n      \"pmids\": [\"30429220\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphatase(s) responsible for dephosphorylation not identified\", \"Whether phosphorylation also affects mRNA export function not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identification of two independent self-association regions (coiled-coil and a 10-aa aggregation-prone motif), both required for mRNA export and stress granule formation but dispensable for basal translation, dissected which Gle1 functions require oligomerization.\",\n      \"evidence\": \"Mutagenesis of oligomerization domains with functional assays in HeLa cells for export, SG formation, and translation\",\n      \"pmids\": [\"32981894\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of each domain to oligomer architecture not structurally resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealing that Gle1 shuttling disruption causes nuclear mRNA accumulation through altered transcription state (not export block), via reduced DDX1 interaction with CstF-64 and increased R-loops, established a nuclear role for Gle1 in transcription termination and 3′-end processing.\",\n      \"evidence\": \"Peptide-mediated shuttling inhibition, RNA-seq, co-IP of DDX1/CstF-64, R-loop immunofluorescence\",\n      \"pmids\": [\"32755435\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct enzymatic activity of Gle1 on DDX1 not biochemically demonstrated\", \"Genome-wide sites of Gle1-dependent transcription termination not mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Definitive kinetic dissection showed Gle1 activates Dbp5 by two synergistic mechanisms—coupling Gle1 binding to ATP binding (>100-fold selectivity) and accelerating Pi release ~20-fold—providing the most complete mechanistic picture of Gle1-dependent helicase activation.\",\n      \"evidence\": \"Stopped-flow and steady-state fluorescence-based nucleotide binding and ATPase assays\",\n      \"pmids\": [\"35286399\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether these kinetic parameters change when Dbp5 is RNA-loaded at the NPC not tested\", \"Structural basis for preferential ATP-state binding not visualized\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrating that Gle1-Dbp5 interaction is required for tRNA export and that tRNA synergizes with Gle1 to activate Dbp5 expanded Gle1's role beyond mRNA to a broader RNA export regulator.\",\n      \"evidence\": \"In vivo co-IP, dominant-negative Dbp5 overexpression epistasis, in vitro ATPase assays with tRNA substrates\",\n      \"pmids\": [\"38189406\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Gle1-Dbp5 acts on tRNA at the NPC or in the nucleoplasm not resolved\", \"Specific tRNA species affected not profiled\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A high-resolution structure of the full Gle1-Dbp5 complex (ideally with IP6, Nup42, and RNA) is needed to explain how Gle1 differentially activates Dbp5 while inhibiting Ded1/DDX3, and how disease mutations structurally disrupt these interactions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No co-crystal or cryo-EM structure of the full Gle1-Dbp5-Nup42-IP6 complex\", \"Structural basis for Gle1 activation versus inhibition of different DEAD-box helicases unknown\", \"How Gle1 oligomeric architecture integrates into the intact NPC scaffold not visualized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 6, 16, 17, 18]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 9, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [10, 11]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [15, 20]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [21, 24]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [19]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 1, 4, 14, 23]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 6, 18]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 10, 14]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [21]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [15, 20]}\n    ],\n    \"complexes\": [\n      \"Nup155–hCG1–Gle1B NPC subcomplex\",\n      \"Nup42–Gle1–Dbp5 trimeric complex\",\n      \"Gle1–Gfd1–Nab2 export complex\"\n    ],\n    \"partners\": [\n      \"DBP5\",\n      \"DDX19B\",\n      \"DDX3\",\n      \"DDX1\",\n      \"NUP155\",\n      \"NUP42\",\n      \"NUPL2\",\n      \"MKRN2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}