{"gene":"GPKOW","run_date":"2026-06-10T01:55:21","timeline":{"discoveries":[{"year":2014,"finding":"GPKOW directly interacts with the spliceosomal helicase DHX16/hPRP2 and with RNA. Immuno-depletion of GPKOW from HeLa nuclear extracts inactivates the spliceosome (which still retains bound DHX16), and splicing activity is restored by adding back recombinant GPKOW, establishing that GPKOW is essential for pre-mRNA splicing in vitro.","method":"Co-immunoprecipitation, RNA-binding assay, immuno-depletion from HeLa nuclear extracts with add-back of recombinant protein, in vitro splicing assay","journal":"Bioscience reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction shown by Co-IP, functional depletion-rescue experiment, multiple orthogonal methods in a single focused study","pmids":["25296192"],"is_preprint":false},{"year":2014,"finding":"The G-patch domain of GPKOW mediates its direct interaction with DHX16; mutations in this domain greatly diminish the GPKOW–DHX16 interaction. However, G-patch mutants retain splicing activity in vitro and can still suppress the DHX16 dominant-negative splicing defect in vivo, indicating that physical contact between GPKOW and DHX16 is not strictly required for functional activity.","method":"Site-directed mutagenesis of the G-patch domain, Co-immunoprecipitation, in vitro splicing assay, in vivo dominant-negative suppression assay","journal":"Bioscience reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis combined with in vitro functional assay and in vivo suppression assay in a single study","pmids":["25296192"],"is_preprint":false},{"year":2014,"finding":"Mutations in the KOW1 domain of GPKOW slightly reduce its RNA-binding activity and render the protein less functional both in vitro (splicing assay) and in vivo (suppression of DHX16 dominant-negative defect), indicating that KOW1-mediated RNA interaction contributes to GPKOW's splicing function.","method":"Site-directed mutagenesis of KOW1 domain, RNA-binding assay, in vitro splicing assay, in vivo splicing rescue assay","journal":"Bioscience reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis with orthogonal in vitro and in vivo functional readouts in a single focused study","pmids":["25296192"],"is_preprint":false},{"year":2014,"finding":"Overexpression of GPKOW in cells partially suppresses the splicing defect caused by expression of a dominant-negative DHX16 mutant, demonstrating a functional relationship between GPKOW and DHX16 in the spliceosome in vivo.","method":"In vivo overexpression rescue assay (dominant-negative DHX16 mutant cells), minigene splicing reporter","journal":"Bioscience reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean in vivo rescue experiment in a single lab with defined molecular readout","pmids":["25296192"],"is_preprint":false},{"year":2011,"finding":"GPKOW (also known as T54/MOS2 homolog) is a nuclear RNA-binding protein that is phosphorylated by the catalytic subunit of protein kinase A (PKA) at two sites in vitro. RNA immunoprecipitation combined with site-directed mutagenesis of the PKA phosphorylation sites showed that GPKOW binds RNA in vivo in a PKA-sensitive fashion, linking PKA signaling to GPKOW's RNA-binding activity.","method":"Yeast two-hybrid screen (identification of GPKOW as PKA Cβ2 interactor), in vitro PKA phosphorylation assay, site-directed mutagenesis of phosphorylation sites, RNA immunoprecipitation (RIP)","journal":"Journal of molecular signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus in vitro kinase assay plus RIP with mutagenesis; single lab, multiple orthogonal methods","pmids":["21880142"],"is_preprint":false},{"year":2011,"finding":"GPKOW was identified as a direct interaction partner of the PKA catalytic subunit Cβ2 through yeast two-hybrid screening, and the interaction was confirmed in a nuclear context, positioning GPKOW as a nuclear effector of PKA signaling.","method":"Yeast two-hybrid screen using PKA Cβ2 as bait","journal":"Journal of molecular signaling","confidence":"Low","confidence_rationale":"Tier 3 / Weak — yeast two-hybrid is a single method; full reciprocal validation not described in abstract","pmids":["21880142"],"is_preprint":false}],"current_model":"GPKOW is a nuclear RNA-binding protein that is an essential component of the human spliceosome: it directly binds pre-mRNA and the spliceosomal helicase DHX16/hPRP2 (via its G-patch domain), is required for pre-mRNA splicing in vitro, and functionally modulates DHX16 activity in vivo; its RNA-binding activity (mediated by the KOW1 domain) is regulated by phosphorylation by the PKA catalytic subunit."},"narrative":{"mechanistic_narrative":"GPKOW is a nuclear RNA-binding protein and an essential component of the human spliceosome required for pre-mRNA splicing [PMID:25296192]. It directly binds both pre-mRNA and the spliceosomal DEAH-box helicase DHX16/hPRP2, and immuno-depletion of GPKOW inactivates the spliceosome while DHX16 remains bound, with splicing activity restored by recombinant GPKOW add-back [PMID:25296192]. The physical GPKOW–DHX16 contact is mediated by the G-patch domain, yet G-patch mutants that lose this interaction retain splicing activity and still suppress a DHX16 dominant-negative defect, indicating GPKOW functionally modulates DHX16 in vivo independently of stable physical contact [PMID:25296192]. RNA engagement through the KOW1 domain contributes directly to splicing function, as KOW1 mutations reduce both RNA binding and activity in vitro and in vivo [PMID:25296192]. GPKOW's RNA-binding activity is regulated by phosphorylation: it is a direct partner and substrate of the PKA catalytic subunit Cβ2, which phosphorylates it at two sites, and its RNA binding in cells is PKA-sensitive, linking PKA signaling to GPKOW function [PMID:21880142].","teleology":[{"year":2011,"claim":"Whether GPKOW is connected to a signaling pathway was unknown; identifying it as a PKA substrate established a regulatory link between PKA signaling and its RNA-binding activity.","evidence":"Yeast two-hybrid with PKA Cβ2 bait, in vitro PKA kinase assay, phosphosite mutagenesis, and RNA immunoprecipitation","pmids":["21880142"],"confidence":"Medium","gaps":["PKA Cβ2 interaction rests on yeast two-hybrid without described reciprocal validation","Functional consequence of phosphorylation on splicing not tested","Physiological signals driving this phosphorylation not defined"]},{"year":2014,"claim":"Whether GPKOW has a role in splicing was unknown; depletion-rescue established it as an essential spliceosomal factor that directly binds pre-mRNA and the helicase DHX16.","evidence":"Co-IP, RNA-binding assay, immuno-depletion from HeLa nuclear extracts with recombinant add-back, in vitro splicing assay","pmids":["25296192"],"confidence":"High","gaps":["Spliceosomal assembly step at which GPKOW acts not pinpointed","Structural basis of pre-mRNA recognition not resolved"]},{"year":2014,"claim":"Whether the G-patch–DHX16 physical contact is required for function was unknown; G-patch mutagenesis showed the interaction is dispensable for splicing activity, dissociating physical binding from functional modulation.","evidence":"G-patch site-directed mutagenesis, Co-IP, in vitro splicing assay, in vivo dominant-negative DHX16 suppression assay","pmids":["25296192"],"confidence":"High","gaps":["Mechanism by which GPKOW modulates DHX16 without stable contact unexplained","Whether transient or alternative contacts substitute for the G-patch not addressed"]},{"year":2014,"claim":"The contribution of RNA binding to GPKOW function was unknown; KOW1 mutagenesis tied reduced RNA binding to reduced splicing activity, establishing KOW1-mediated RNA interaction as functionally relevant.","evidence":"KOW1 site-directed mutagenesis, RNA-binding assay, in vitro splicing assay, in vivo rescue assay","pmids":["25296192"],"confidence":"High","gaps":["RNA sequence/structure specificity of KOW1 binding undefined","Effect was modest, leaving other RNA-contacting regions unidentified"]},{"year":null,"claim":"How PKA phosphorylation, KOW1 RNA binding, and DHX16 modulation are integrated to control splicing in cells remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of GPKOW within an assembled spliceosome","Functional impact of PKA phosphorylation on splicing output untested","Genome-wide RNA targets and physiological splicing program of GPKOW uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,2,4]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,4]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0]}],"complexes":["spliceosome"],"partners":["DHX16","PRKACB"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q92917","full_name":"G-patch domain and KOW motifs-containing protein","aliases":["G-patch domain-containing protein 5","Protein MOS2 homolog","Protein T54"],"length_aa":476,"mass_kda":52.2,"function":"RNA-binding protein involved in pre-mRNA splicing. As a component of the minor spliceosome, involved in the splicing of U12-type introns in pre-mRNAs (Probable)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q92917/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/GPKOW","classification":"Common Essential","n_dependent_lines":1187,"n_total_lines":1208,"dependency_fraction":0.9826158940397351},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/GPKOW","total_profiled":1310},"omim":[{"mim_id":"602463","title":"DIHYDROPYRIMIDINASE-LIKE 2; DPYSL2","url":"https://www.omim.org/entry/602463"},{"mim_id":"306990","title":"MICROHYDRANENCEPHALY, X-LINKED; MHACX","url":"https://www.omim.org/entry/306990"},{"mim_id":"301003","title":"G-PATCH DOMAIN AND KOW MOTIFS; GPKOW","url":"https://www.omim.org/entry/301003"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/GPKOW"},"hgnc":{"alias_symbol":["T54","GPATC5","GPATCH5","Spp2","Mos2"],"prev_symbol":[]},"alphafold":{"accession":"Q92917","domains":[{"cath_id":"2.30.30.30","chopping":"246-308","consensus_level":"high","plddt":83.2575,"start":246,"end":308},{"cath_id":"2.30.30.140","chopping":"357-414","consensus_level":"medium","plddt":93.3003,"start":357,"end":414},{"cath_id":"2.30.30.30","chopping":"416-471","consensus_level":"medium","plddt":92.2023,"start":416,"end":471}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q92917","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q92917-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q92917-F1-predicted_aligned_error_v6.png","plddt_mean":69.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=GPKOW","jax_strain_url":"https://www.jax.org/strain/search?query=GPKOW"},"sequence":{"accession":"Q92917","fasta_url":"https://rest.uniprot.org/uniprotkb/Q92917.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q92917/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q92917"}},"corpus_meta":[{"pmid":"24588742","id":"PMC_24588742","title":"MoS₂ 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Immuno-depletion of GPKOW from HeLa nuclear extracts inactivates the spliceosome (which still retains bound DHX16), and splicing activity is restored by adding back recombinant GPKOW, establishing that GPKOW is essential for pre-mRNA splicing in vitro.\",\n      \"method\": \"Co-immunoprecipitation, RNA-binding assay, immuno-depletion from HeLa nuclear extracts with add-back of recombinant protein, in vitro splicing assay\",\n      \"journal\": \"Bioscience reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interaction shown by Co-IP, functional depletion-rescue experiment, multiple orthogonal methods in a single focused study\",\n      \"pmids\": [\"25296192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The G-patch domain of GPKOW mediates its direct interaction with DHX16; mutations in this domain greatly diminish the GPKOW–DHX16 interaction. However, G-patch mutants retain splicing activity in vitro and can still suppress the DHX16 dominant-negative splicing defect in vivo, indicating that physical contact between GPKOW and DHX16 is not strictly required for functional activity.\",\n      \"method\": \"Site-directed mutagenesis of the G-patch domain, Co-immunoprecipitation, in vitro splicing assay, in vivo dominant-negative suppression assay\",\n      \"journal\": \"Bioscience reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis combined with in vitro functional assay and in vivo suppression assay in a single study\",\n      \"pmids\": [\"25296192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Mutations in the KOW1 domain of GPKOW slightly reduce its RNA-binding activity and render the protein less functional both in vitro (splicing assay) and in vivo (suppression of DHX16 dominant-negative defect), indicating that KOW1-mediated RNA interaction contributes to GPKOW's splicing function.\",\n      \"method\": \"Site-directed mutagenesis of KOW1 domain, RNA-binding assay, in vitro splicing assay, in vivo splicing rescue assay\",\n      \"journal\": \"Bioscience reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis with orthogonal in vitro and in vivo functional readouts in a single focused study\",\n      \"pmids\": [\"25296192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Overexpression of GPKOW in cells partially suppresses the splicing defect caused by expression of a dominant-negative DHX16 mutant, demonstrating a functional relationship between GPKOW and DHX16 in the spliceosome in vivo.\",\n      \"method\": \"In vivo overexpression rescue assay (dominant-negative DHX16 mutant cells), minigene splicing reporter\",\n      \"journal\": \"Bioscience reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean in vivo rescue experiment in a single lab with defined molecular readout\",\n      \"pmids\": [\"25296192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"GPKOW (also known as T54/MOS2 homolog) is a nuclear RNA-binding protein that is phosphorylated by the catalytic subunit of protein kinase A (PKA) at two sites in vitro. RNA immunoprecipitation combined with site-directed mutagenesis of the PKA phosphorylation sites showed that GPKOW binds RNA in vivo in a PKA-sensitive fashion, linking PKA signaling to GPKOW's RNA-binding activity.\",\n      \"method\": \"Yeast two-hybrid screen (identification of GPKOW as PKA Cβ2 interactor), in vitro PKA phosphorylation assay, site-directed mutagenesis of phosphorylation sites, RNA immunoprecipitation (RIP)\",\n      \"journal\": \"Journal of molecular signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus in vitro kinase assay plus RIP with mutagenesis; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"21880142\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"GPKOW was identified as a direct interaction partner of the PKA catalytic subunit Cβ2 through yeast two-hybrid screening, and the interaction was confirmed in a nuclear context, positioning GPKOW as a nuclear effector of PKA signaling.\",\n      \"method\": \"Yeast two-hybrid screen using PKA Cβ2 as bait\",\n      \"journal\": \"Journal of molecular signaling\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — yeast two-hybrid is a single method; full reciprocal validation not described in abstract\",\n      \"pmids\": [\"21880142\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"GPKOW is a nuclear RNA-binding protein that is an essential component of the human spliceosome: it directly binds pre-mRNA and the spliceosomal helicase DHX16/hPRP2 (via its G-patch domain), is required for pre-mRNA splicing in vitro, and functionally modulates DHX16 activity in vivo; its RNA-binding activity (mediated by the KOW1 domain) is regulated by phosphorylation by the PKA catalytic subunit.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GPKOW is a nuclear RNA-binding protein and an essential component of the human spliceosome required for pre-mRNA splicing [#0]. It directly binds both pre-mRNA and the spliceosomal DEAH-box helicase DHX16/hPRP2, and immuno-depletion of GPKOW inactivates the spliceosome while DHX16 remains bound, with splicing activity restored by recombinant GPKOW add-back [#0]. The physical GPKOW\\u2013DHX16 contact is mediated by the G-patch domain, yet G-patch mutants that lose this interaction retain splicing activity and still suppress a DHX16 dominant-negative defect, indicating GPKOW functionally modulates DHX16 in vivo independently of stable physical contact [#1, #3]. RNA engagement through the KOW1 domain contributes directly to splicing function, as KOW1 mutations reduce both RNA binding and activity in vitro and in vivo [#2]. GPKOW's RNA-binding activity is regulated by phosphorylation: it is a direct partner and substrate of the PKA catalytic subunit C\\u03b22, which phosphorylates it at two sites, and its RNA binding in cells is PKA-sensitive, linking PKA signaling to GPKOW function [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Whether GPKOW is connected to a signaling pathway was unknown; identifying it as a PKA substrate established a regulatory link between PKA signaling and its RNA-binding activity.\",\n      \"evidence\": \"Yeast two-hybrid with PKA C\\u03b22 bait, in vitro PKA kinase assay, phosphosite mutagenesis, and RNA immunoprecipitation\",\n      \"pmids\": [\"21880142\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"PKA C\\u03b22 interaction rests on yeast two-hybrid without described reciprocal validation\",\n        \"Functional consequence of phosphorylation on splicing not tested\",\n        \"Physiological signals driving this phosphorylation not defined\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Whether GPKOW has a role in splicing was unknown; depletion-rescue established it as an essential spliceosomal factor that directly binds pre-mRNA and the helicase DHX16.\",\n      \"evidence\": \"Co-IP, RNA-binding assay, immuno-depletion from HeLa nuclear extracts with recombinant add-back, in vitro splicing assay\",\n      \"pmids\": [\"25296192\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Spliceosomal assembly step at which GPKOW acts not pinpointed\",\n        \"Structural basis of pre-mRNA recognition not resolved\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Whether the G-patch\\u2013DHX16 physical contact is required for function was unknown; G-patch mutagenesis showed the interaction is dispensable for splicing activity, dissociating physical binding from functional modulation.\",\n      \"evidence\": \"G-patch site-directed mutagenesis, Co-IP, in vitro splicing assay, in vivo dominant-negative DHX16 suppression assay\",\n      \"pmids\": [\"25296192\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism by which GPKOW modulates DHX16 without stable contact unexplained\",\n        \"Whether transient or alternative contacts substitute for the G-patch not addressed\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The contribution of RNA binding to GPKOW function was unknown; KOW1 mutagenesis tied reduced RNA binding to reduced splicing activity, establishing KOW1-mediated RNA interaction as functionally relevant.\",\n      \"evidence\": \"KOW1 site-directed mutagenesis, RNA-binding assay, in vitro splicing assay, in vivo rescue assay\",\n      \"pmids\": [\"25296192\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"RNA sequence/structure specificity of KOW1 binding undefined\",\n        \"Effect was modest, leaving other RNA-contacting regions unidentified\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PKA phosphorylation, KOW1 RNA binding, and DHX16 modulation are integrated to control splicing in cells remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No structural model of GPKOW within an assembled spliceosome\",\n        \"Functional impact of PKA phosphorylation on splicing output untested\",\n        \"Genome-wide RNA targets and physiological splicing program of GPKOW uncharacterized\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [\"spliceosome\"],\n    \"partners\": [\"DHX16\", \"PRKACB\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}