{"gene":"DHX16","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2010,"finding":"DHX16 is required for human pre-mRNA splicing after formation of the pre-catalytic spliceosome (complex B); anti-DHX16 antiserum inhibited in vitro splicing and immunoprecipitated pre-mRNA, splicing intermediates, and spliceosomal snRNAs; helicase-domain mutants (G724N, S552L) accumulated unspliced transcripts and inhibited splicing in vitro when added to HeLa nuclear extracts.","method":"In vitro splicing assay with antibody inhibition and immunoprecipitation; dominant-negative helicase-domain mutants expressed in cells; nuclear extract complementation","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — multiple orthogonal methods (antibody inhibition, dominant-negative mutants, in vitro complementation) in a single focused study","pmids":["20423332"],"is_preprint":false},{"year":2010,"finding":"Expression of dominant-negative DHX16 mutant causes nuclear retention of unspliced pre-mRNAs; unspliced transcripts accumulate for many intron-containing genes (identified by genomic tiling microarray), are retained in the nucleus, and are not affected by blocking nonsense-mediated decay.","method":"Genomic tiling microarray; dominant-negative DHX16 mutant expression in human cells; nuclear/cytoplasmic fractionation; NMD inhibition","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genome-wide transcriptomic readout plus cellular fractionation, multiple orthogonal methods in one study","pmids":["20841358"],"is_preprint":false},{"year":2014,"finding":"GPKOW (human homolog of yeast Spp2) interacts directly with DHX16 via its G-patch domain and with RNA via its KOW1 domain; immunodepletion of GPKOW from HeLa nuclear extracts inactivates the spliceosome that still binds DHX16, and adding back recombinant GPKOW restores splicing; overexpression of GPKOW partially suppresses splicing defects caused by dominant-negative DHX16.","method":"Direct protein-protein interaction assay; immunodepletion/add-back in vitro splicing; in vivo overexpression rescue; domain mutation analysis","journal":"Bioscience reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal functional assays (depletion, add-back, in vivo rescue) plus domain mutation analysis in one study","pmids":["25296192"],"is_preprint":false},{"year":2022,"finding":"DHX16 acts as a pattern recognition receptor (PRR) that directly binds specific influenza viral RNA segments (those that undergo splicing) with high affinity via its RNA helicase motif; DHX16-dependent type I IFN production requires RIG-I and unanchored K48-polyubiquitin synthesized by TRIM6; silencing DHX16 in cells and in vivo diminishes IFN-I responses against influenza, Zika, and SARS-CoV-2.","method":"Affinity purification of unanchored poly-Ub from lung tissue; siRNA silencing in cells and in vivo; RNA binding assays; genetic requirement for RIG-I and TRIM6 established by knockdown/knockout; helicase-motif mutant analysis","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (pulldown, silencing in vivo, motif mutants, genetic epistasis with RIG-I and TRIM6) in one focused study","pmids":["35263596"],"is_preprint":false},{"year":2024,"finding":"Conditional knockout of Dhx16 in mouse hematopoietic cells causes depletion of HSCs, bone marrow failure, and lethality; mechanistically, Dhx16 loss causes intron 4 retention in Emg1 mRNA, reducing EMG1 protein, disrupting ribosome assembly, inducing nucleolar stress, and activating p53; overexpression of Emg1 in Dhx16-deficient HSCs partially restores ribosome assembly and HSC function.","method":"Conditional knockout mice; multi-omics (RNA-seq, proteomics); ribosome profiling; Emg1 overexpression rescue; p53 pathway activation assays","journal":"Leukemia","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined molecular mechanism, multi-omics, and genetic rescue in one study","pmids":["39333759"],"is_preprint":false},{"year":2011,"finding":"In zebrafish, maternal Dhx16 (encoded by the mission impossible locus) is required cell-autonomously for gastrulation cell movements and for activation of endodermal target genes downstream of casanova/sox32 but upstream of sox17 in the nodal signaling pathway; zygotic Dhx16 is also essential for embryonic viability.","method":"Forward genetic screen; positional cloning; genetic rescue; complementation analysis; cell transplantation; blastoderm explant assays","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — positional cloning with rescue and epistasis analysis, but pathway placement relies on genetic epistasis in a non-mammalian model","pmids":["21396359"],"is_preprint":false},{"year":2025,"finding":"In Laodelphax striatellus (Hemiptera), DHX16 (an ATP-dependent RNA helicase and pre-mRNA splicing factor) interacts with LsFMD2315 to regulate female-specific splicing of doublesex (Lsdsx); RNAi depletion of DHX16 causes ectopic male-specific Lsdsx isoforms in females and ovipositor malformations; DHX16 does not interact directly with LsFMD-F, indicating LsFMD2315 acts as a scaffold.","method":"Co-immunoprecipitation in 293T cells; RNAi knockdown; RT-PCR for sex-specific splicing isoforms; phenotypic analysis","journal":"Insect science","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and RNAi in an invertebrate (potential ortholog context), single lab, limited mechanistic follow-up","pmids":["40329630"],"is_preprint":false}],"current_model":"DHX16 is a DEAH-box RNA helicase that functions as a core spliceosomal ATPase required for the transition from pre-catalytic spliceosome complex B to the first catalytic step of pre-mRNA splicing (analogous to yeast Prp2), physically interacts with the G-patch co-activator GPKOW to facilitate this step, and additionally acts as a pattern recognition receptor that directly binds specific viral RNAs to activate RIG-I-dependent type I interferon responses via unanchored K48-polyubiquitin and TRIM6; in hematopoietic stem cells, Dhx16 maintains ribosome assembly by ensuring correct splicing of Emg1 mRNA, linking its spliceosomal function to stem cell homeostasis through the p53 pathway."},"narrative":{"mechanistic_narrative":"DHX16 is a DEAH-box RNA helicase that functions as a core spliceosomal ATPase required for pre-mRNA splicing after assembly of the pre-catalytic spliceosome (complex B), where its activity drives entry into the catalytic phase of splicing [PMID:20423332, PMID:20841358]. Inhibition or dominant-negative mutation of its helicase domain blocks splicing in vitro and causes genome-wide nuclear retention of unspliced, intron-containing transcripts that are not cleared by nonsense-mediated decay [PMID:20423332, PMID:20841358]. The G-patch protein GPKOW binds DHX16 directly through its G-patch domain and RNA through its KOW1 domain, acting as an essential co-activator: GPKOW depletion inactivates DHX16-bound spliceosomes and recombinant GPKOW restores activity [PMID:25296192]. Beyond splicing, DHX16 acts as a viral RNA pattern recognition receptor, binding specific spliced influenza RNA segments via its helicase motif and triggering type I interferon responses that require RIG-I and TRIM6-synthesized unanchored K48-polyubiquitin, with broad antiviral relevance to influenza, Zika, and SARS-CoV-2 [PMID:35263596]. The physiological consequence of its splicing function is illustrated in hematopoietic stem cells, where Dhx16 loss causes Emg1 intron retention, impaired ribosome assembly, nucleolar stress, and p53 activation leading to stem cell depletion and bone marrow failure [PMID:39333759].","teleology":[{"year":2010,"claim":"Established that DHX16 acts at a defined late step of spliceosome activation rather than during early assembly, placing it at the transition into catalysis.","evidence":"In vitro splicing antibody inhibition, immunoprecipitation, and dominant-negative helicase mutants in HeLa nuclear extracts; genomic tiling microarray with nuclear/cytoplasmic fractionation","pmids":["20423332","20841358"],"confidence":"High","gaps":["No structural model of the DHX16-spliceosome interface","Direct RNA substrate specificity within the spliceosome not defined"]},{"year":2014,"claim":"Identified GPKOW as the direct G-patch co-activator that licenses DHX16 catalytic function, answering how the helicase is functionally coupled to the spliceosome and RNA.","evidence":"Direct protein-protein interaction assays, immunodepletion/add-back in vitro splicing, in vivo overexpression rescue, and domain mutation analysis","pmids":["25296192"],"confidence":"High","gaps":["Mechanism by which GPKOW stimulates DHX16 ATPase/helicase activity not resolved","No structure of the DHX16-GPKOW complex"]},{"year":2022,"claim":"Revealed a second, non-splicing role for DHX16 as a viral RNA sensor feeding into innate antiviral signaling, repurposing its RNA-binding capacity for pattern recognition.","evidence":"Unanchored poly-Ub affinity purification from lung, siRNA silencing in cells and in vivo, RNA binding assays, helicase-motif mutants, and genetic epistasis with RIG-I and TRIM6","pmids":["35263596"],"confidence":"High","gaps":["How DHX16 discriminates viral from cellular spliced RNAs is unclear","Molecular link between RNA binding and unanchored K48-polyubiquitin recruitment not detailed"]},{"year":2024,"claim":"Connected DHX16 splicing fidelity to a physiological output by showing that loss disrupts Emg1 splicing, ribosome assembly, and HSC maintenance through nucleolar stress and p53.","evidence":"Conditional knockout mice, multi-omics including ribosome profiling, Emg1 overexpression rescue, and p53 pathway activation assays","pmids":["39333759"],"confidence":"High","gaps":["Whether other retained-intron transcripts contribute to the HSC phenotype is not established","Tissue-specificity of the Emg1 dependency not explored"]},{"year":2011,"claim":"Demonstrated an in vivo developmental requirement for Dhx16 in vertebrate gastrulation and endoderm specification, linking the splicing factor to embryonic patterning.","evidence":"Zebrafish forward genetic screen, positional cloning, genetic rescue, cell transplantation, and blastoderm explant epistasis analysis","pmids":["21396359"],"confidence":"Medium","gaps":["Whether the developmental defect reflects general splicing loss or specific target mis-splicing is undefined","Pathway placement relies on genetic epistasis in a non-mammalian model"]},{"year":2025,"claim":"Implicated DHX16 in regulated sex-specific alternative splicing via a scaffolding partner, extending its role to developmental splice-isoform control.","evidence":"Co-immunoprecipitation in 293T cells, RNAi knockdown, and RT-PCR for sex-specific doublesex isoforms in an insect","pmids":["40329630"],"confidence":"Low","gaps":["Single Co-IP without reciprocal validation in an invertebrate context","Direct role of DHX16 in dsx splice-site selection not biochemically demonstrated"]},{"year":null,"claim":"How DHX16 toggles between its core constitutive splicing role and its antiviral RNA-sensing role, and how each is regulated, remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No structural basis for substrate or partner selectivity","Regulation governing splicing versus PRR function unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[2,3]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[3]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[5]}],"complexes":["spliceosome"],"partners":["GPKOW","RIG-I","TRIM6"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O60231","full_name":"Pre-mRNA-splicing factor ATP-dependent RNA helicase DHX16","aliases":["ATP-dependent RNA helicase #3","DEAH-box protein 16"],"length_aa":1041,"mass_kda":119.3,"function":"Required for pre-mRNA splicing as a component of the spliceosome (PubMed:20423332, PubMed:20841358, PubMed:25296192, PubMed:29360106). Contributes to pre-mRNA splicing after spliceosome formation and prior to the first transesterification reaction. As a component of the minor spliceosome, involved in the splicing of U12-type introns in pre-mRNAs (Probable). Also plays a role in innate antiviral response by acting as a pattern recognition receptor sensing splicing signals in viral RNA (PubMed:35263596). Mechanistically, TRIM6 promotes the interaction between unanchored 'Lys-48'-polyubiquitin chains and DHX16, leading to DHX16 interaction with RIGI and ssRNA to amplify RIGI-dependent innate antiviral immune responses (PubMed:35263596)","subcellular_location":"Nucleus; Nucleus, nucleoplasm; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/O60231/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/DHX16","classification":"Common Essential","n_dependent_lines":1193,"n_total_lines":1208,"dependency_fraction":0.9875827814569537},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CAPZB","stoichiometry":0.2},{"gene":"CPSF6","stoichiometry":0.2},{"gene":"TOP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/DHX16","total_profiled":1310},"omim":[{"mim_id":"621554","title":"G-PATCH DOMAIN-CONTAINING PROTEIN 1; GPATCH1","url":"https://www.omim.org/entry/621554"},{"mim_id":"621530","title":"CWC25, SPLICEOSOME-ASSOCIATED PROTEIN; CWC25","url":"https://www.omim.org/entry/621530"},{"mim_id":"621401","title":"DEAH-BOX HELICASE 35; DHX35","url":"https://www.omim.org/entry/621401"},{"mim_id":"618733","title":"NEUROMUSCULAR OCULOAUDITORY SYNDROME; NMOAS","url":"https://www.omim.org/entry/618733"},{"mim_id":"610952","title":"CROOKED NECK PRE-mRNA SPLICING FACTOR-LIKE 1; CRNKL1","url":"https://www.omim.org/entry/610952"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DHX16"},"hgnc":{"alias_symbol":["DBP2","Prp2","PRPF2"],"prev_symbol":["DDX16"]},"alphafold":{"accession":"O60231","domains":[{"cath_id":"-","chopping":"5-70","consensus_level":"high","plddt":78.6708,"start":5,"end":70},{"cath_id":"3.40.50.300","chopping":"397-573","consensus_level":"high","plddt":90.3835,"start":397,"end":573},{"cath_id":"3.40.50.300","chopping":"580-757","consensus_level":"high","plddt":89.0692,"start":580,"end":757},{"cath_id":"1.10.10.2130","chopping":"760-807","consensus_level":"medium","plddt":88.2804,"start":760,"end":807},{"cath_id":"-","chopping":"819-1022","consensus_level":"medium","plddt":88.0139,"start":819,"end":1022}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O60231","model_url":"https://alphafold.ebi.ac.uk/files/AF-O60231-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O60231-F1-predicted_aligned_error_v6.png","plddt_mean":77.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DHX16","jax_strain_url":"https://www.jax.org/strain/search?query=DHX16"},"sequence":{"accession":"O60231","fasta_url":"https://rest.uniprot.org/uniprotkb/O60231.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O60231/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O60231"}},"corpus_meta":[{"pmid":"30506978","id":"PMC_30506978","title":"The DDX5/Dbp2 subfamily of DEAD-box RNA helicases.","date":"2018","source":"Wiley interdisciplinary reviews. 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England)","url":"https://pubmed.ncbi.nlm.nih.gov/30855040","citation_count":29,"is_preprint":false},{"pmid":"34302476","id":"PMC_34302476","title":"The RGG domain in the C-terminus of the DEAD box helicases Dbp2 and Ded1 is necessary for G-quadruplex destabilization.","date":"2021","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/34302476","citation_count":27,"is_preprint":false},{"pmid":"20841358","id":"PMC_20841358","title":"Nuclear retention of unspliced pre-mRNAs by mutant DHX16/hPRP2, a spliceosomal DEAH-box protein.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20841358","citation_count":23,"is_preprint":false},{"pmid":"25296192","id":"PMC_25296192","title":"GPKOW is essential for pre-mRNA splicing in vitro and suppresses splicing defect caused by dominant-negative DHX16 mutation in vivo.","date":"2014","source":"Bioscience 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anti-DHX16 antiserum inhibited in vitro splicing and immunoprecipitated pre-mRNA, splicing intermediates, and spliceosomal snRNAs; helicase-domain mutants (G724N, S552L) accumulated unspliced transcripts and inhibited splicing in vitro when added to HeLa nuclear extracts.\",\n      \"method\": \"In vitro splicing assay with antibody inhibition and immunoprecipitation; dominant-negative helicase-domain mutants expressed in cells; nuclear extract complementation\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — multiple orthogonal methods (antibody inhibition, dominant-negative mutants, in vitro complementation) in a single focused study\",\n      \"pmids\": [\"20423332\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Expression of dominant-negative DHX16 mutant causes nuclear retention of unspliced pre-mRNAs; unspliced transcripts accumulate for many intron-containing genes (identified by genomic tiling microarray), are retained in the nucleus, and are not affected by blocking nonsense-mediated decay.\",\n      \"method\": \"Genomic tiling microarray; dominant-negative DHX16 mutant expression in human cells; nuclear/cytoplasmic fractionation; NMD inhibition\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide transcriptomic readout plus cellular fractionation, multiple orthogonal methods in one study\",\n      \"pmids\": [\"20841358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GPKOW (human homolog of yeast Spp2) interacts directly with DHX16 via its G-patch domain and with RNA via its KOW1 domain; immunodepletion of GPKOW from HeLa nuclear extracts inactivates the spliceosome that still binds DHX16, and adding back recombinant GPKOW restores splicing; overexpression of GPKOW partially suppresses splicing defects caused by dominant-negative DHX16.\",\n      \"method\": \"Direct protein-protein interaction assay; immunodepletion/add-back in vitro splicing; in vivo overexpression rescue; domain mutation analysis\",\n      \"journal\": \"Bioscience reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal functional assays (depletion, add-back, in vivo rescue) plus domain mutation analysis in one study\",\n      \"pmids\": [\"25296192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DHX16 acts as a pattern recognition receptor (PRR) that directly binds specific influenza viral RNA segments (those that undergo splicing) with high affinity via its RNA helicase motif; DHX16-dependent type I IFN production requires RIG-I and unanchored K48-polyubiquitin synthesized by TRIM6; silencing DHX16 in cells and in vivo diminishes IFN-I responses against influenza, Zika, and SARS-CoV-2.\",\n      \"method\": \"Affinity purification of unanchored poly-Ub from lung tissue; siRNA silencing in cells and in vivo; RNA binding assays; genetic requirement for RIG-I and TRIM6 established by knockdown/knockout; helicase-motif mutant analysis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (pulldown, silencing in vivo, motif mutants, genetic epistasis with RIG-I and TRIM6) in one focused study\",\n      \"pmids\": [\"35263596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Conditional knockout of Dhx16 in mouse hematopoietic cells causes depletion of HSCs, bone marrow failure, and lethality; mechanistically, Dhx16 loss causes intron 4 retention in Emg1 mRNA, reducing EMG1 protein, disrupting ribosome assembly, inducing nucleolar stress, and activating p53; overexpression of Emg1 in Dhx16-deficient HSCs partially restores ribosome assembly and HSC function.\",\n      \"method\": \"Conditional knockout mice; multi-omics (RNA-seq, proteomics); ribosome profiling; Emg1 overexpression rescue; p53 pathway activation assays\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined molecular mechanism, multi-omics, and genetic rescue in one study\",\n      \"pmids\": [\"39333759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In zebrafish, maternal Dhx16 (encoded by the mission impossible locus) is required cell-autonomously for gastrulation cell movements and for activation of endodermal target genes downstream of casanova/sox32 but upstream of sox17 in the nodal signaling pathway; zygotic Dhx16 is also essential for embryonic viability.\",\n      \"method\": \"Forward genetic screen; positional cloning; genetic rescue; complementation analysis; cell transplantation; blastoderm explant assays\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — positional cloning with rescue and epistasis analysis, but pathway placement relies on genetic epistasis in a non-mammalian model\",\n      \"pmids\": [\"21396359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In Laodelphax striatellus (Hemiptera), DHX16 (an ATP-dependent RNA helicase and pre-mRNA splicing factor) interacts with LsFMD2315 to regulate female-specific splicing of doublesex (Lsdsx); RNAi depletion of DHX16 causes ectopic male-specific Lsdsx isoforms in females and ovipositor malformations; DHX16 does not interact directly with LsFMD-F, indicating LsFMD2315 acts as a scaffold.\",\n      \"method\": \"Co-immunoprecipitation in 293T cells; RNAi knockdown; RT-PCR for sex-specific splicing isoforms; phenotypic analysis\",\n      \"journal\": \"Insect science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and RNAi in an invertebrate (potential ortholog context), single lab, limited mechanistic follow-up\",\n      \"pmids\": [\"40329630\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DHX16 is a DEAH-box RNA helicase that functions as a core spliceosomal ATPase required for the transition from pre-catalytic spliceosome complex B to the first catalytic step of pre-mRNA splicing (analogous to yeast Prp2), physically interacts with the G-patch co-activator GPKOW to facilitate this step, and additionally acts as a pattern recognition receptor that directly binds specific viral RNAs to activate RIG-I-dependent type I interferon responses via unanchored K48-polyubiquitin and TRIM6; in hematopoietic stem cells, Dhx16 maintains ribosome assembly by ensuring correct splicing of Emg1 mRNA, linking its spliceosomal function to stem cell homeostasis through the p53 pathway.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DHX16 is a DEAH-box RNA helicase that functions as a core spliceosomal ATPase required for pre-mRNA splicing after assembly of the pre-catalytic spliceosome (complex B), where its activity drives entry into the catalytic phase of splicing [#0, #1]. Inhibition or dominant-negative mutation of its helicase domain blocks splicing in vitro and causes genome-wide nuclear retention of unspliced, intron-containing transcripts that are not cleared by nonsense-mediated decay [#0, #1]. The G-patch protein GPKOW binds DHX16 directly through its G-patch domain and RNA through its KOW1 domain, acting as an essential co-activator: GPKOW depletion inactivates DHX16-bound spliceosomes and recombinant GPKOW restores activity [#2]. Beyond splicing, DHX16 acts as a viral RNA pattern recognition receptor, binding specific spliced influenza RNA segments via its helicase motif and triggering type I interferon responses that require RIG-I and TRIM6-synthesized unanchored K48-polyubiquitin, with broad antiviral relevance to influenza, Zika, and SARS-CoV-2 [#3]. The physiological consequence of its splicing function is illustrated in hematopoietic stem cells, where Dhx16 loss causes Emg1 intron retention, impaired ribosome assembly, nucleolar stress, and p53 activation leading to stem cell depletion and bone marrow failure [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Established that DHX16 acts at a defined late step of spliceosome activation rather than during early assembly, placing it at the transition into catalysis.\",\n      \"evidence\": \"In vitro splicing antibody inhibition, immunoprecipitation, and dominant-negative helicase mutants in HeLa nuclear extracts; genomic tiling microarray with nuclear/cytoplasmic fractionation\",\n      \"pmids\": [\"20423332\", \"20841358\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural model of the DHX16-spliceosome interface\", \"Direct RNA substrate specificity within the spliceosome not defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified GPKOW as the direct G-patch co-activator that licenses DHX16 catalytic function, answering how the helicase is functionally coupled to the spliceosome and RNA.\",\n      \"evidence\": \"Direct protein-protein interaction assays, immunodepletion/add-back in vitro splicing, in vivo overexpression rescue, and domain mutation analysis\",\n      \"pmids\": [\"25296192\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which GPKOW stimulates DHX16 ATPase/helicase activity not resolved\", \"No structure of the DHX16-GPKOW complex\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed a second, non-splicing role for DHX16 as a viral RNA sensor feeding into innate antiviral signaling, repurposing its RNA-binding capacity for pattern recognition.\",\n      \"evidence\": \"Unanchored poly-Ub affinity purification from lung, siRNA silencing in cells and in vivo, RNA binding assays, helicase-motif mutants, and genetic epistasis with RIG-I and TRIM6\",\n      \"pmids\": [\"35263596\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How DHX16 discriminates viral from cellular spliced RNAs is unclear\", \"Molecular link between RNA binding and unanchored K48-polyubiquitin recruitment not detailed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected DHX16 splicing fidelity to a physiological output by showing that loss disrupts Emg1 splicing, ribosome assembly, and HSC maintenance through nucleolar stress and p53.\",\n      \"evidence\": \"Conditional knockout mice, multi-omics including ribosome profiling, Emg1 overexpression rescue, and p53 pathway activation assays\",\n      \"pmids\": [\"39333759\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other retained-intron transcripts contribute to the HSC phenotype is not established\", \"Tissue-specificity of the Emg1 dependency not explored\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated an in vivo developmental requirement for Dhx16 in vertebrate gastrulation and endoderm specification, linking the splicing factor to embryonic patterning.\",\n      \"evidence\": \"Zebrafish forward genetic screen, positional cloning, genetic rescue, cell transplantation, and blastoderm explant epistasis analysis\",\n      \"pmids\": [\"21396359\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the developmental defect reflects general splicing loss or specific target mis-splicing is undefined\", \"Pathway placement relies on genetic epistasis in a non-mammalian model\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated DHX16 in regulated sex-specific alternative splicing via a scaffolding partner, extending its role to developmental splice-isoform control.\",\n      \"evidence\": \"Co-immunoprecipitation in 293T cells, RNAi knockdown, and RT-PCR for sex-specific doublesex isoforms in an insect\",\n      \"pmids\": [\"40329630\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP without reciprocal validation in an invertebrate context\", \"Direct role of DHX16 in dsx splice-site selection not biochemically demonstrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How DHX16 toggles between its core constitutive splicing role and its antiviral RNA-sensing role, and how each is regulated, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural basis for substrate or partner selectivity\", \"Regulation governing splicing versus PRR function unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [\"spliceosome\"],\n    \"partners\": [\"GPKOW\", \"RIG-I\", \"TRIM6\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}