{"gene":"DDX23","run_date":"2026-06-09T23:54:41","timeline":{"discoveries":[{"year":2008,"finding":"SRPK2 phosphorylates the RS domain of human PRP28/DDX23, and this phosphorylation is required for stable association of PRP28 with the U4/U6-U5 tri-snRNP and for tri-snRNP integration into the spliceosomal B complex. SRPK1 is predominantly associated with U1 snRNP, while SRPK2 associates with tri-snRNP. RNAi depletion of SRPK2 causes hypophosphorylation of PRP28 and destabilizes its tri-snRNP association.","method":"RNAi depletion in HeLa cells, immunodepletion and complementation assays in nuclear extract, mass spectrometry","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal immunodepletion/complementation in nuclear extract plus RNAi in cells, mechanistically replicated with multiple orthogonal approaches in a single rigorous study","pmids":["18425142"],"is_preprint":false},{"year":2017,"finding":"RNA polymerase II pausing initiates a signaling cascade in which SRPK2 phosphorylates DDX23, and phosphorylated DDX23 suppresses R-loops. In the absence of either SRPK2 or DDX23, R-loops accumulate and cause DNA double-strand breaks, revealing a role for DDX23 in maintaining genomic stability downstream of transcription dynamics.","method":"RNAi/siRNA knockdown of SRPK2 and DDX23 in human cells, R-loop immunofluorescence (S9.6 antibody), DNA damage markers (γH2AX), rescue experiments","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KD with defined cellular phenotypes (R-loops, DSBs), multiple orthogonal readouts in a single study, builds on established SRPK2-DDX23 phosphorylation axis","pmids":["28076779"],"is_preprint":false},{"year":1994,"finding":"Yeast Prp28 (ortholog of DDX23) is required for the first step of pre-mRNA splicing in vitro. Prp28 is not a stably associated snRNP protein. Purified Prp28 does not exhibit RNA helicase activity in strand displacement assays, suggesting additional factors are needed for its activation.","method":"Protein purification from S. cerevisiae, in vitro splicing assay, strand displacement assay","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of splicing requirement and direct biochemical helicase assay; foundational yeast ortholog study, independently replicated","pmids":["7520570"],"is_preprint":false},{"year":2016,"finding":"ATPase-deficient human Prp28/DDX23 blocks stable tri-snRNP association during B complex formation and stabilizes a novel assembly intermediate (pre-B complex) containing U1, U2, and loosely associated tri-snRNP, stalled before disruption of U1/5' splice-site base pairing. Disruption of the U1/5'ss interaction alone is insufficient to bypass the block, suggesting an additional function for hPrp28 ATPase activity at this stage.","method":"Dominant-negative ATPase-deficient hPrp28 mutant expression, spliceosomal complex purification and characterization, electron microscopy structural analysis, mass spectrometry","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — dominant-negative mutagenesis with structural characterization of intermediate complex plus biochemical fractionation, multiple orthogonal methods in a single rigorous study","pmids":["27377154"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of yeast Prp28 (ortholog of DDX23) reveals two RecA-like domains in a wide-open conformation. Prp28 has intrinsic RNA-dependent ATPase activity. Alanine scanning mutagenesis identified essential residues: Asp341/Glu342 (motif II) and Arg527/Arg530 (motif VI) for ATP binding, and Arg476 (motif Va) for RNA binding. Overexpression of ATP-site but not RNA-site defective mutants caused dominant-negative growth defects.","method":"X-ray crystallography (crystal structure), in vitro ATPase assay, alanine scanning mutagenesis, in vivo growth assays in S. cerevisiae","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure combined with in vitro enzymatic assay and systematic mutagenesis with functional in vivo validation","pmids":["25303995"],"is_preprint":false},{"year":2014,"finding":"The purified helicase domain of human Prp28/DDX23 binds ADP but not ATP and lacks detectable ATPase activity in isolation; however, within an assembled spliceosomal complex, hPrp28 gains ATP-binding activity. The crystal structure at 2.0 Å shows the helicase domain in a wide-open conformation with a closed P-loop that blocks γ-phosphate binding of ATP.","method":"X-ray crystallography (2.0 Å crystal structure of hPrp28ΔN), nucleotide-binding assays with purified protein and spliceosomal complex","journal":"Acta crystallographica. Section D, Biological crystallography","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with direct biochemical nucleotide-binding assays in isolated and complex-assembled conditions","pmids":["24914973"],"is_preprint":false},{"year":2015,"finding":"DDX23 promotes miR-21 biogenesis at the post-transcriptional level by facilitating primary-to-precursor (pri-to-pre) processing of miR-21 through direct interaction with the Drosha microprocessor. Mutagenesis demonstrated that the helicase activity of DDX23 is essential for this processing step.","method":"Co-immunoprecipitation, knockdown and overexpression in glioma cells, miRNA processing assays, helicase domain mutagenesis, xenograft mouse models","journal":"Brain : a journal of neurology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP with Drosha, functional mutagenesis of helicase domain, and in vivo xenograft validation; single lab but multiple orthogonal methods","pmids":["26121981"],"is_preprint":false},{"year":2015,"finding":"C. elegans DDX-23 (ortholog of DDX23) is required for primary miRNA processing: knockdown causes accumulation of pri-let-7 and reduction of mature let-7, lin-4, miR-48, miR-84, miR-241, and lsy-6, indicating DDX-23 acts at the level of Drosha-mediated pri-miRNA cleavage.","method":"RNAi knockdown in C. elegans, miRNA quantification (mature and primary forms), genetic sensitized background (let-7(mg279))","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean RNAi knockdown with direct molecular readout (pri-miRNA accumulation, mature miRNA decrease) across multiple miRNAs in a genetically tractable organism","pmids":["26601717"],"is_preprint":false},{"year":2013,"finding":"In yeast, Prp28 has an early ATP-independent function in commitment complex 2 (CC2) formation in addition to its known ATP-dependent role in spliceosome activation. Mutations in the N-terminal bromodomain-like region of Prp8 (U5 snRNP) suppress the cold-sensitive prp28-1 mutant at both the early CC2 assembly and later activation steps, placing Prp8 as a regulator of both ATP-independent and ATP-dependent Prp28 functions.","method":"Genetic suppressor screen in S. cerevisiae, in vivo and in vitro spliceosome assembly assays, commitment complex analysis","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis by suppressor screen with biochemical spliceosome assembly assays, single lab","pmids":["24231520"],"is_preprint":false},{"year":2021,"finding":"Yeast Prp28 transiently interacts with the conserved 5' splice-site GU dinucleotide and makes splicing-dependent contacts with U1 snRNP protein U1C and tri-snRNP proteins Prp8, Brr2, and Snu114. Phosphorylated Npl3 (but not unphosphorylated Npl3) potentiates Prp28's ATPase activity, proposing Npl3 as a functional counterpart of the metazoan-specific phosphorylated N-terminal region of Prp28/DDX23.","method":"Co-immunoprecipitation, in vitro ATPase activity assay with phosphorylated vs. unphosphorylated Npl3, splicing-dependent crosslinking","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct in vitro ATPase stimulation assay with phospho-Npl3, Co-IP interactions with multiple spliceosomal proteins; single lab but multiple orthogonal methods","pmids":["34035302"],"is_preprint":false},{"year":2019,"finding":"Human DDX23 binds low-molecular-weight poly(I:C) (dsRNA) through its N-terminal region. Upon poly(I:C) or VSV stimulation, DDX23 translocates from the nucleus to the cytoplasm and forms complexes with TRIF or MAVS to initiate downstream NF-κB and IRF3 signaling. Knockdown of DDX23 enhances VSV replication and reduces NF-κB/IRF3 activation.","method":"Poly(I:C) pull-down assay, Co-immunoprecipitation with TRIF and MAVS, siRNA knockdown, viral replication assay, immunofluorescence for subcellular localization","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — pull-down for dsRNA binding, Co-IP for TRIF/MAVS interaction, KD with functional viral and signaling readouts; single lab but multiple approaches","pmids":["31620127"],"is_preprint":false},{"year":2020,"finding":"DDX23 interacts with FMDV IRES domains III and IV and suppresses IRES-dependent translation. DDX23 also interacts with FMDV 3C proteinase, which degrades DDX23 via the lysosomal pathway. Overexpression of DDX23 reduces FMDV replication, while knockdown/knockout increases it.","method":"Pull-down assay, Co-immunoprecipitation, siRNA knockdown, CRISPR knockout, overexpression, IRES-driven reporter translation assay, confocal microscopy","journal":"Viruses","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — direct binding assays combined with functional translation reporter and viral replication assays; single lab, multiple orthogonal methods","pmids":["33255534"],"is_preprint":false},{"year":2021,"finding":"DDX23 regulates mRNA processing of FOXM1 in ovarian cancer cells; DDX23 silencing reduces production of the FOXM1C oncogenic transcript and decreases FOXM1 protein. DDX23 transcription is directly activated by transcription factor E2F1, as shown by luciferase reporter and ChIP assays.","method":"Transcriptomic analysis of DDX23-silenced cells, ChIP assay, luciferase reporter assay, siRNA knockdown, rescue experiments","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — ChIP and reporter assay for E2F1 transcriptional activation, transcriptomics for splicing regulation, and rescue assays; single lab with multiple methods","pmids":["34966670"],"is_preprint":false},{"year":2023,"finding":"METTL3 methyltransferase directly m6A-modifies DDX23 mRNA in a YTHDF1-dependent manner, enhancing DDX23 expression. Elevated DDX23 activates PI3K/Akt signaling; DDX23 silencing suppresses PI3K/Akt activation and reduces pancreatic cancer cell malignancy and gemcitabine resistance.","method":"MeRIP-qPCR for m6A site identification, siRNA knockdown, overexpression rescue experiments, Western blotting for PI3K/Akt pathway components","journal":"Cell death & disease","confidence":"Low","confidence_rationale":"Tier 3 / Moderate — MeRIP-qPCR establishes METTL3 target, but PI3K/Akt pathway placement relies on knockdown phenotype without direct biochemical mechanism; single lab","pmids":["36977668"],"is_preprint":false},{"year":2024,"finding":"SRSF1 directly interacts with DDX23/PRP28; the interaction is mediated by the N-terminal RS-like domain of DDX23 and both RRM1 and the RS domain of SRSF1, as validated by bimolecular fluorescence complementation and in vitro binding assays. The RS-like region of DDX23's N-terminal domain is important for spliceosome incorporation, and larger N-terminal deletions alter DDX23's subnuclear localization.","method":"Proximity labeling (BioID) with mass spectrometry, bimolecular fluorescence complementation (BiFC), in vitro binding assays, deletion mutagenesis with subnuclear localization imaging","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal proximity labeling plus in vitro direct binding assay plus BiFC, with domain mutagenesis linking N-terminal RS-like domain to spliceosome incorporation and localization; multiple orthogonal methods in a single rigorous study","pmids":["38743621"],"is_preprint":false},{"year":2025,"finding":"Polymerase κ (Pol κ) physically interacts with DDX23 and recruits DDX23 to R-loop loci in chromatin to promote DDX23-mediated R-loop resolution. Individual ablation of either Pol κ or DDX23 augments R-loop accumulation in cells.","method":"Proximity labeling (BioID) and affinity pull-down followed by LC-MS/MS, co-immunoprecipitation, R-loop immunofluorescence (S9.6 antibody), individual KO/KD of Pol κ and DDX23","journal":"Analytical chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — two independent interaction approaches (proximity labeling + pull-down) with functional R-loop readout upon individual gene ablations; single lab","pmids":["41949541"],"is_preprint":false},{"year":2025,"finding":"LncPEDS1-AS interacts with splicing factor DDX23 to form a nuclear RNA-protein complex that facilitates splicing and maturation of PEDS1 pre-mRNA, thereby promoting resistance to lipid peroxidation in UTUC.","method":"RNA immunoprecipitation, co-immunoprecipitation, ASO-mediated knockdown with functional ROS/lipid peroxidation assays","journal":"Cell death & disease","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, interaction shown by RIP/Co-IP but mechanistic detail of DDX23's splicing role for PEDS1 is not directly dissected from the lncRNA's role","pmids":["41360761"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of Prp28 from thermophilic fungus Chaetomium thermophilum (ortholog of DDX23) determined at 3.2 Å resolution, confirming conservation of the two-RecA-domain open conformation seen in yeast and human Prp28 structures.","method":"X-ray crystallography at 3.2 Å resolution","journal":"Acta crystallographica. Section F, Structural biology communications","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — crystal structure of ortholog; structural information only, no functional mutagenesis validation in this paper","pmids":["27139834"],"is_preprint":false}],"current_model":"DDX23/PRP28 is a DEAD-box RNA helicase and core component of the U4/U6-U5 tri-snRNP that uses SRPK2-dependent phosphorylation of its N-terminal RS-like domain for stable tri-snRNP association and integration into the pre-catalytic spliceosomal B complex, where its ATPase activity—stimulated by phosphorylated Npl3 (yeast) or regulated by SRPK2 (human)—disrupts U1 snRNP binding at the 5' splice site; additionally, DDX23 interacts with SRSF1 and other SR proteins via its RS-like domain, participates in primary miRNA processing through the Drosha microprocessor, resolves R-loops (a function reinforced by Pol κ recruitment), and acts as a cytoplasmic dsRNA sensor that translocates from nucleus to cytoplasm to engage TRIF or MAVS during antiviral signaling."},"narrative":{"mechanistic_narrative":"DDX23 (yeast Prp28) is a DEAD-box RNA helicase that functions at the heart of pre-mRNA splicing as a core component of the U4/U6-U5 tri-snRNP, where its ATPase activity drives the transition from a pre-catalytic pre-B intermediate to the activated spliceosome by remodeling the U1 snRNP/5' splice-site interaction [PMID:27377154, PMID:34035302]. Its enzymatic core comprises two RecA-like domains that adopt a wide-open conformation; the human helicase domain is intrinsically catalytically inert in isolation and acquires ATP-binding activity only upon spliceosomal assembly, while the yeast enzyme has measurable RNA-dependent ATPase activity [PMID:25303995, PMID:24914973]. Stable tri-snRNP association and integration into the B complex depend on SRPK2-mediated phosphorylation of the metazoan-specific N-terminal RS-like domain [PMID:18425142], a region that also mediates direct binding to SRSF1 and is required for spliceosome incorporation and proper subnuclear localization [PMID:38743621]; in yeast, phosphorylated Npl3 serves as the functional counterpart that potentiates Prp28 ATPase activity [PMID:34035302]. Beyond canonical splicing, DDX23 maintains genomic stability by resolving transcription-associated R-loops downstream of the SRPK2 phosphorylation axis and through recruitment by Polymerase κ to R-loop loci [PMID:28076779, PMID:41949541], and it promotes Drosha-dependent primary miRNA processing in a manner requiring its helicase activity [PMID:26121981, PMID:26601717]. DDX23 has additionally been implicated in antiviral defense as a cytoplasmic dsRNA sensor engaging TRIF/MAVS [PMID:31620127] and in cancer-associated splicing programs [PMID:34966670], though these activities are less fully resolved in the available corpus.","teleology":[{"year":1994,"claim":"Established that the DDX23 ortholog Prp28 is functionally required for the first catalytic step of splicing, setting the question of what activates a helicase that appears inactive alone.","evidence":"Protein purification and in vitro splicing/strand-displacement assays in S. cerevisiae","pmids":["7520570"],"confidence":"High","gaps":["No helicase activity detected for purified protein, implying missing activating factors","Did not define the RNA substrate remodeled during splicing"]},{"year":2008,"claim":"Answered how the metazoan helicase is integrated into the spliceosome by identifying SRPK2 phosphorylation of the RS domain as the requirement for stable tri-snRNP association and B-complex integration.","evidence":"RNAi depletion, immunodepletion/complementation in HeLa nuclear extract, mass spectrometry","pmids":["18425142"],"confidence":"High","gaps":["Did not resolve the structural consequence of phosphorylation on tri-snRNP binding","Phosphosite mapping on DDX23 not detailed"]},{"year":2013,"claim":"Revealed that Prp28 has an early ATP-independent role in commitment complex formation beyond its ATP-dependent activation function, with Prp8 regulating both activities.","evidence":"Genetic suppressor screen and spliceosome assembly assays in S. cerevisiae","pmids":["24231520"],"confidence":"Medium","gaps":["Molecular basis of the ATP-independent CC2 function unresolved","Human relevance of the early function not tested"]},{"year":2014,"claim":"Defined the catalytic architecture: crystal structures showed two RecA domains in an open conformation, and biochemistry distinguished active yeast ATPase from a human domain that is inert in isolation but ATP-competent within the assembled spliceosome.","evidence":"X-ray crystallography of yeast and human Prp28, ATPase and nucleotide-binding assays, alanine-scanning mutagenesis","pmids":["25303995","24914973"],"confidence":"High","gaps":["Structural state of DDX23 within an intact spliceosome not captured","Mechanism that switches the human domain from ADP- to ATP-binding within complex unknown"]},{"year":2015,"claim":"Extended DDX23 function beyond splicing by showing it promotes Drosha-dependent primary miRNA processing in a helicase-activity-dependent manner, conserved from worms to human glioma.","evidence":"Co-IP with Drosha, helicase-domain mutagenesis, knockdown/overexpression and xenografts; C. elegans RNAi with pri-/mature miRNA quantification","pmids":["26121981","26601717"],"confidence":"Medium","gaps":["Substrate hand-off between DDX23 and the microprocessor not biochemically reconstituted","Whether splicing and miRNA functions use the same molecular activity unclear"]},{"year":2016,"claim":"Characterized the pre-catalytic transition by showing an ATPase-deficient human Prp28 stalls a pre-B complex with loosely associated tri-snRNP before U1/5'ss disruption, indicating an ATPase function beyond merely displacing U1.","evidence":"Dominant-negative ATPase mutant, spliceosomal complex purification, electron microscopy, mass spectrometry; ortholog crystal structure confirmation","pmids":["27377154","27139834"],"confidence":"High","gaps":["The additional ATPase-dependent step beyond U1 displacement not defined","Direct RNA target of the remodeling not identified"]},{"year":2017,"claim":"Connected the SRPK2-DDX23 phosphorylation axis to genome maintenance, showing phosphorylated DDX23 suppresses R-loops and prevents double-strand breaks downstream of Pol II pausing.","evidence":"siRNA knockdown of SRPK2/DDX23, S9.6 R-loop immunofluorescence, γH2AX, rescue experiments","pmids":["28076779"],"confidence":"High","gaps":["Whether DDX23 unwinds R-loops directly or via splicing not separated","Genomic loci of DDX23-dependent R-loop resolution not mapped"]},{"year":2019,"claim":"Proposed a non-splicing antiviral role in which DDX23 binds cytoplasmic dsRNA and engages TRIF/MAVS to drive NF-κB/IRF3 signaling, restricting viral replication.","evidence":"Poly(I:C) pull-down, Co-IP with TRIF/MAVS, siRNA knockdown, VSV replication and signaling readouts, localization imaging","pmids":["31620127"],"confidence":"Medium","gaps":["Nuclear-to-cytoplasmic translocation trigger not mechanistically defined","Single-lab finding without reciprocal validation of TRIF/MAVS complexes"]},{"year":2021,"claim":"Refined the activation mechanism by mapping Prp28 contacts to the 5'ss GU and to U1C/Prp8/Brr2/Snu114, and identified phospho-Npl3 as the yeast functional counterpart of the metazoan phospho-N-terminus that stimulates ATPase activity.","evidence":"Co-IP, splicing-dependent crosslinking, in vitro ATPase assays with phospho- vs unphospho-Npl3","pmids":["34035302"],"confidence":"Medium","gaps":["Structural basis of Npl3-mediated ATPase stimulation unresolved","Whether human SRPK2-DDX23 and yeast Npl3-Prp28 are mechanistically equivalent not directly tested"]},{"year":2024,"claim":"Established a direct SR-protein partnership, defining the DDX23 N-terminal RS-like domain and SRSF1 RRM1/RS domain as the binding interface and linking this region to spliceosome incorporation and subnuclear localization.","evidence":"BioID proximity labeling/MS, BiFC, in vitro binding assays, deletion mutagenesis with localization imaging","pmids":["38743621"],"confidence":"High","gaps":["Functional consequence of the SRSF1 interaction for specific splicing events not defined","Phosphorylation dependence of the SRSF1 interaction not tested"]},{"year":2025,"claim":"Identified Polymerase κ as a recruiter of DDX23 to chromatin R-loop loci, providing a targeting mechanism for its R-loop resolution activity.","evidence":"BioID and affinity pull-down with LC-MS/MS, Co-IP, S9.6 R-loop immunofluorescence, individual KO/KD","pmids":["41949541"],"confidence":"Medium","gaps":["Whether DDX23 enzymatically unwinds R-loops at Pol κ sites not shown directly","Single-lab study"]},{"year":null,"claim":"How DDX23 partitions among its splicing, R-loop, miRNA-processing, and antiviral activities—and whether a single helicase/ATPase activity underlies all of them—remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking the distinct functions to defined RNA substrates","Structure of DDX23 within an active spliceosome or R-loop complex not determined","Regulatory switch governing nuclear vs cytoplasmic functions unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[2,3,4,6]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[9,10,16]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[3,4,5]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[2,4]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[10,14]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[10]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,3,6]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[1,15]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[10]}],"complexes":["U4/U6-U5 tri-snRNP","spliceosome (pre-B/B complex)","Drosha microprocessor"],"partners":["SRPK2","SRSF1","DROSHA","PRP8","BRR2","SNU114","TRIF","MAVS"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BUQ8","full_name":"Probable ATP-dependent RNA helicase DDX23","aliases":["100 kDa U5 snRNP-specific protein","DEAD box protein 23","PRP28 homolog","U5-100kD"],"length_aa":820,"mass_kda":95.6,"function":"Involved in pre-mRNA splicing and its phosphorylated form (by SRPK2) is required for spliceosomal B complex formation (PubMed:18425142). Independently of its spliceosome formation function, required for the suppression of incorrect R-loops formed during transcription; R-loops are composed of a DNA:RNA hybrid and the associated non-template single-stranded DNA (PubMed:28076779)","subcellular_location":"Nucleus; Chromosome","url":"https://www.uniprot.org/uniprotkb/Q9BUQ8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/DDX23","classification":"Common Essential","n_dependent_lines":1188,"n_total_lines":1208,"dependency_fraction":0.9834437086092715},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PRPF8","stoichiometry":10.0},{"gene":"RBM42","stoichiometry":10.0},{"gene":"CD2BP2","stoichiometry":4.0},{"gene":"EFTUD2","stoichiometry":4.0},{"gene":"PRPF4B","stoichiometry":4.0},{"gene":"SNRNP40","stoichiometry":4.0},{"gene":"CPSF6","stoichiometry":0.2},{"gene":"DDX21","stoichiometry":0.2},{"gene":"RBM39","stoichiometry":0.2},{"gene":"RBM6","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/DDX23","total_profiled":1310},"omim":[{"mim_id":"612172","title":"DEAD-BOX HELICASE 23; DDX23","url":"https://www.omim.org/entry/612172"},{"mim_id":"602980","title":"SRSF PROTEIN KINASE 2; SRPK2","url":"https://www.omim.org/entry/602980"},{"mim_id":"601664","title":"SMALL NUCLEAR RIBONUCLEOPROTEIN, 200-KD; SNRNP200","url":"https://www.omim.org/entry/601664"},{"mim_id":"180740","title":"SMALL NUCLEAR RIBONUCLEOPROTEIN, U1 SUBUNIT, 70-KD; SNRNP70","url":"https://www.omim.org/entry/180740"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nucleoli","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DDX23"},"hgnc":{"alias_symbol":["Prp28","U5-100KD","PRPF28","SNRNP100"],"prev_symbol":[]},"alphafold":{"accession":"Q9BUQ8","domains":[{"cath_id":"3.40.50.300","chopping":"378-628","consensus_level":"high","plddt":91.5369,"start":378,"end":628},{"cath_id":"3.40.50.300","chopping":"634-800","consensus_level":"high","plddt":86.5441,"start":634,"end":800}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BUQ8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BUQ8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BUQ8-F1-predicted_aligned_error_v6.png","plddt_mean":77.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DDX23","jax_strain_url":"https://www.jax.org/strain/search?query=DDX23"},"sequence":{"accession":"Q9BUQ8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BUQ8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BUQ8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BUQ8"}},"corpus_meta":[{"pmid":"18425142","id":"PMC_18425142","title":"Phosphorylation of human PRP28 by SRPK2 is required for integration of the U4/U6-U5 tri-snRNP into the spliceosome.","date":"2008","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/18425142","citation_count":114,"is_preprint":false},{"pmid":"28076779","id":"PMC_28076779","title":"Transcription Dynamics Prevent RNA-Mediated Genomic Instability through SRPK2-Dependent DDX23 Phosphorylation.","date":"2017","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/28076779","citation_count":95,"is_preprint":false},{"pmid":"26121981","id":"PMC_26121981","title":"DEAD-box RNA helicase DDX23 modulates glioma malignancy via elevating miR-21 biogenesis.","date":"2015","source":"Brain : a journal of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/26121981","citation_count":66,"is_preprint":false},{"pmid":"7520570","id":"PMC_7520570","title":"PRP28, a 'DEAD-box' protein, is required for the first step of mRNA splicing in vitro.","date":"1994","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/7520570","citation_count":66,"is_preprint":false},{"pmid":"27377154","id":"PMC_27377154","title":"A spliceosome intermediate with loosely associated tri-snRNP accumulates in the absence of Prp28 ATPase activity.","date":"2016","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/27377154","citation_count":66,"is_preprint":false},{"pmid":"36977668","id":"PMC_36977668","title":"METTL3 enhances pancreatic ductal adenocarcinoma progression and gemcitabine resistance through modifying DDX23 mRNA N6 adenosine methylation.","date":"2023","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/36977668","citation_count":40,"is_preprint":false},{"pmid":"31620127","id":"PMC_31620127","title":"DDX23, an Evolutionary Conserved dsRNA Sensor, Participates in Innate Antiviral Responses by Pairing With TRIF or MAVS.","date":"2019","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/31620127","citation_count":36,"is_preprint":false},{"pmid":"18729217","id":"PMC_18729217","title":"The Caenorhabditis elegans DDX-23, a homolog of yeast splicing factor PRP28, is required for the sperm-oocyte switch and differentiation of various cell types.","date":"2008","source":"Developmental dynamics : an official publication of the American Association of Anatomists","url":"https://pubmed.ncbi.nlm.nih.gov/18729217","citation_count":31,"is_preprint":false},{"pmid":"25303995","id":"PMC_25303995","title":"Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28.","date":"2014","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/25303995","citation_count":22,"is_preprint":false},{"pmid":"24914973","id":"PMC_24914973","title":"Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28.","date":"2014","source":"Acta crystallographica. 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SRPK1 is predominantly associated with U1 snRNP, while SRPK2 associates with tri-snRNP. RNAi depletion of SRPK2 causes hypophosphorylation of PRP28 and destabilizes its tri-snRNP association.\",\n      \"method\": \"RNAi depletion in HeLa cells, immunodepletion and complementation assays in nuclear extract, mass spectrometry\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal immunodepletion/complementation in nuclear extract plus RNAi in cells, mechanistically replicated with multiple orthogonal approaches in a single rigorous study\",\n      \"pmids\": [\"18425142\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RNA polymerase II pausing initiates a signaling cascade in which SRPK2 phosphorylates DDX23, and phosphorylated DDX23 suppresses R-loops. In the absence of either SRPK2 or DDX23, R-loops accumulate and cause DNA double-strand breaks, revealing a role for DDX23 in maintaining genomic stability downstream of transcription dynamics.\",\n      \"method\": \"RNAi/siRNA knockdown of SRPK2 and DDX23 in human cells, R-loop immunofluorescence (S9.6 antibody), DNA damage markers (γH2AX), rescue experiments\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with defined cellular phenotypes (R-loops, DSBs), multiple orthogonal readouts in a single study, builds on established SRPK2-DDX23 phosphorylation axis\",\n      \"pmids\": [\"28076779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Yeast Prp28 (ortholog of DDX23) is required for the first step of pre-mRNA splicing in vitro. Prp28 is not a stably associated snRNP protein. Purified Prp28 does not exhibit RNA helicase activity in strand displacement assays, suggesting additional factors are needed for its activation.\",\n      \"method\": \"Protein purification from S. cerevisiae, in vitro splicing assay, strand displacement assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of splicing requirement and direct biochemical helicase assay; foundational yeast ortholog study, independently replicated\",\n      \"pmids\": [\"7520570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ATPase-deficient human Prp28/DDX23 blocks stable tri-snRNP association during B complex formation and stabilizes a novel assembly intermediate (pre-B complex) containing U1, U2, and loosely associated tri-snRNP, stalled before disruption of U1/5' splice-site base pairing. Disruption of the U1/5'ss interaction alone is insufficient to bypass the block, suggesting an additional function for hPrp28 ATPase activity at this stage.\",\n      \"method\": \"Dominant-negative ATPase-deficient hPrp28 mutant expression, spliceosomal complex purification and characterization, electron microscopy structural analysis, mass spectrometry\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — dominant-negative mutagenesis with structural characterization of intermediate complex plus biochemical fractionation, multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"27377154\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of yeast Prp28 (ortholog of DDX23) reveals two RecA-like domains in a wide-open conformation. Prp28 has intrinsic RNA-dependent ATPase activity. Alanine scanning mutagenesis identified essential residues: Asp341/Glu342 (motif II) and Arg527/Arg530 (motif VI) for ATP binding, and Arg476 (motif Va) for RNA binding. Overexpression of ATP-site but not RNA-site defective mutants caused dominant-negative growth defects.\",\n      \"method\": \"X-ray crystallography (crystal structure), in vitro ATPase assay, alanine scanning mutagenesis, in vivo growth assays in S. cerevisiae\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure combined with in vitro enzymatic assay and systematic mutagenesis with functional in vivo validation\",\n      \"pmids\": [\"25303995\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The purified helicase domain of human Prp28/DDX23 binds ADP but not ATP and lacks detectable ATPase activity in isolation; however, within an assembled spliceosomal complex, hPrp28 gains ATP-binding activity. The crystal structure at 2.0 Å shows the helicase domain in a wide-open conformation with a closed P-loop that blocks γ-phosphate binding of ATP.\",\n      \"method\": \"X-ray crystallography (2.0 Å crystal structure of hPrp28ΔN), nucleotide-binding assays with purified protein and spliceosomal complex\",\n      \"journal\": \"Acta crystallographica. Section D, Biological crystallography\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with direct biochemical nucleotide-binding assays in isolated and complex-assembled conditions\",\n      \"pmids\": [\"24914973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"DDX23 promotes miR-21 biogenesis at the post-transcriptional level by facilitating primary-to-precursor (pri-to-pre) processing of miR-21 through direct interaction with the Drosha microprocessor. Mutagenesis demonstrated that the helicase activity of DDX23 is essential for this processing step.\",\n      \"method\": \"Co-immunoprecipitation, knockdown and overexpression in glioma cells, miRNA processing assays, helicase domain mutagenesis, xenograft mouse models\",\n      \"journal\": \"Brain : a journal of neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP with Drosha, functional mutagenesis of helicase domain, and in vivo xenograft validation; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"26121981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"C. elegans DDX-23 (ortholog of DDX23) is required for primary miRNA processing: knockdown causes accumulation of pri-let-7 and reduction of mature let-7, lin-4, miR-48, miR-84, miR-241, and lsy-6, indicating DDX-23 acts at the level of Drosha-mediated pri-miRNA cleavage.\",\n      \"method\": \"RNAi knockdown in C. elegans, miRNA quantification (mature and primary forms), genetic sensitized background (let-7(mg279))\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean RNAi knockdown with direct molecular readout (pri-miRNA accumulation, mature miRNA decrease) across multiple miRNAs in a genetically tractable organism\",\n      \"pmids\": [\"26601717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In yeast, Prp28 has an early ATP-independent function in commitment complex 2 (CC2) formation in addition to its known ATP-dependent role in spliceosome activation. Mutations in the N-terminal bromodomain-like region of Prp8 (U5 snRNP) suppress the cold-sensitive prp28-1 mutant at both the early CC2 assembly and later activation steps, placing Prp8 as a regulator of both ATP-independent and ATP-dependent Prp28 functions.\",\n      \"method\": \"Genetic suppressor screen in S. cerevisiae, in vivo and in vitro spliceosome assembly assays, commitment complex analysis\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis by suppressor screen with biochemical spliceosome assembly assays, single lab\",\n      \"pmids\": [\"24231520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Yeast Prp28 transiently interacts with the conserved 5' splice-site GU dinucleotide and makes splicing-dependent contacts with U1 snRNP protein U1C and tri-snRNP proteins Prp8, Brr2, and Snu114. Phosphorylated Npl3 (but not unphosphorylated Npl3) potentiates Prp28's ATPase activity, proposing Npl3 as a functional counterpart of the metazoan-specific phosphorylated N-terminal region of Prp28/DDX23.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ATPase activity assay with phosphorylated vs. unphosphorylated Npl3, splicing-dependent crosslinking\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct in vitro ATPase stimulation assay with phospho-Npl3, Co-IP interactions with multiple spliceosomal proteins; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"34035302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Human DDX23 binds low-molecular-weight poly(I:C) (dsRNA) through its N-terminal region. Upon poly(I:C) or VSV stimulation, DDX23 translocates from the nucleus to the cytoplasm and forms complexes with TRIF or MAVS to initiate downstream NF-κB and IRF3 signaling. Knockdown of DDX23 enhances VSV replication and reduces NF-κB/IRF3 activation.\",\n      \"method\": \"Poly(I:C) pull-down assay, Co-immunoprecipitation with TRIF and MAVS, siRNA knockdown, viral replication assay, immunofluorescence for subcellular localization\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — pull-down for dsRNA binding, Co-IP for TRIF/MAVS interaction, KD with functional viral and signaling readouts; single lab but multiple approaches\",\n      \"pmids\": [\"31620127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"DDX23 interacts with FMDV IRES domains III and IV and suppresses IRES-dependent translation. DDX23 also interacts with FMDV 3C proteinase, which degrades DDX23 via the lysosomal pathway. Overexpression of DDX23 reduces FMDV replication, while knockdown/knockout increases it.\",\n      \"method\": \"Pull-down assay, Co-immunoprecipitation, siRNA knockdown, CRISPR knockout, overexpression, IRES-driven reporter translation assay, confocal microscopy\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — direct binding assays combined with functional translation reporter and viral replication assays; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"33255534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DDX23 regulates mRNA processing of FOXM1 in ovarian cancer cells; DDX23 silencing reduces production of the FOXM1C oncogenic transcript and decreases FOXM1 protein. DDX23 transcription is directly activated by transcription factor E2F1, as shown by luciferase reporter and ChIP assays.\",\n      \"method\": \"Transcriptomic analysis of DDX23-silenced cells, ChIP assay, luciferase reporter assay, siRNA knockdown, rescue experiments\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — ChIP and reporter assay for E2F1 transcriptional activation, transcriptomics for splicing regulation, and rescue assays; single lab with multiple methods\",\n      \"pmids\": [\"34966670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"METTL3 methyltransferase directly m6A-modifies DDX23 mRNA in a YTHDF1-dependent manner, enhancing DDX23 expression. Elevated DDX23 activates PI3K/Akt signaling; DDX23 silencing suppresses PI3K/Akt activation and reduces pancreatic cancer cell malignancy and gemcitabine resistance.\",\n      \"method\": \"MeRIP-qPCR for m6A site identification, siRNA knockdown, overexpression rescue experiments, Western blotting for PI3K/Akt pathway components\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — MeRIP-qPCR establishes METTL3 target, but PI3K/Akt pathway placement relies on knockdown phenotype without direct biochemical mechanism; single lab\",\n      \"pmids\": [\"36977668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SRSF1 directly interacts with DDX23/PRP28; the interaction is mediated by the N-terminal RS-like domain of DDX23 and both RRM1 and the RS domain of SRSF1, as validated by bimolecular fluorescence complementation and in vitro binding assays. The RS-like region of DDX23's N-terminal domain is important for spliceosome incorporation, and larger N-terminal deletions alter DDX23's subnuclear localization.\",\n      \"method\": \"Proximity labeling (BioID) with mass spectrometry, bimolecular fluorescence complementation (BiFC), in vitro binding assays, deletion mutagenesis with subnuclear localization imaging\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal proximity labeling plus in vitro direct binding assay plus BiFC, with domain mutagenesis linking N-terminal RS-like domain to spliceosome incorporation and localization; multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"38743621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Polymerase κ (Pol κ) physically interacts with DDX23 and recruits DDX23 to R-loop loci in chromatin to promote DDX23-mediated R-loop resolution. Individual ablation of either Pol κ or DDX23 augments R-loop accumulation in cells.\",\n      \"method\": \"Proximity labeling (BioID) and affinity pull-down followed by LC-MS/MS, co-immunoprecipitation, R-loop immunofluorescence (S9.6 antibody), individual KO/KD of Pol κ and DDX23\",\n      \"journal\": \"Analytical chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — two independent interaction approaches (proximity labeling + pull-down) with functional R-loop readout upon individual gene ablations; single lab\",\n      \"pmids\": [\"41949541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"LncPEDS1-AS interacts with splicing factor DDX23 to form a nuclear RNA-protein complex that facilitates splicing and maturation of PEDS1 pre-mRNA, thereby promoting resistance to lipid peroxidation in UTUC.\",\n      \"method\": \"RNA immunoprecipitation, co-immunoprecipitation, ASO-mediated knockdown with functional ROS/lipid peroxidation assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, interaction shown by RIP/Co-IP but mechanistic detail of DDX23's splicing role for PEDS1 is not directly dissected from the lncRNA's role\",\n      \"pmids\": [\"41360761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of Prp28 from thermophilic fungus Chaetomium thermophilum (ortholog of DDX23) determined at 3.2 Å resolution, confirming conservation of the two-RecA-domain open conformation seen in yeast and human Prp28 structures.\",\n      \"method\": \"X-ray crystallography at 3.2 Å resolution\",\n      \"journal\": \"Acta crystallographica. Section F, Structural biology communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — crystal structure of ortholog; structural information only, no functional mutagenesis validation in this paper\",\n      \"pmids\": [\"27139834\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DDX23/PRP28 is a DEAD-box RNA helicase and core component of the U4/U6-U5 tri-snRNP that uses SRPK2-dependent phosphorylation of its N-terminal RS-like domain for stable tri-snRNP association and integration into the pre-catalytic spliceosomal B complex, where its ATPase activity—stimulated by phosphorylated Npl3 (yeast) or regulated by SRPK2 (human)—disrupts U1 snRNP binding at the 5' splice site; additionally, DDX23 interacts with SRSF1 and other SR proteins via its RS-like domain, participates in primary miRNA processing through the Drosha microprocessor, resolves R-loops (a function reinforced by Pol κ recruitment), and acts as a cytoplasmic dsRNA sensor that translocates from nucleus to cytoplasm to engage TRIF or MAVS during antiviral signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DDX23 (yeast Prp28) is a DEAD-box RNA helicase that functions at the heart of pre-mRNA splicing as a core component of the U4/U6-U5 tri-snRNP, where its ATPase activity drives the transition from a pre-catalytic pre-B intermediate to the activated spliceosome by remodeling the U1 snRNP/5' splice-site interaction [#3, #9]. Its enzymatic core comprises two RecA-like domains that adopt a wide-open conformation; the human helicase domain is intrinsically catalytically inert in isolation and acquires ATP-binding activity only upon spliceosomal assembly, while the yeast enzyme has measurable RNA-dependent ATPase activity [#4, #5]. Stable tri-snRNP association and integration into the B complex depend on SRPK2-mediated phosphorylation of the metazoan-specific N-terminal RS-like domain [#0], a region that also mediates direct binding to SRSF1 and is required for spliceosome incorporation and proper subnuclear localization [#14]; in yeast, phosphorylated Npl3 serves as the functional counterpart that potentiates Prp28 ATPase activity [#9]. Beyond canonical splicing, DDX23 maintains genomic stability by resolving transcription-associated R-loops downstream of the SRPK2 phosphorylation axis and through recruitment by Polymerase \\u03ba to R-loop loci [#1, #15], and it promotes Drosha-dependent primary miRNA processing in a manner requiring its helicase activity [#6, #7]. DDX23 has additionally been implicated in antiviral defense as a cytoplasmic dsRNA sensor engaging TRIF/MAVS [#10] and in cancer-associated splicing programs [#12], though these activities are less fully resolved in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Established that the DDX23 ortholog Prp28 is functionally required for the first catalytic step of splicing, setting the question of what activates a helicase that appears inactive alone.\",\n      \"evidence\": \"Protein purification and in vitro splicing/strand-displacement assays in S. cerevisiae\",\n      \"pmids\": [\"7520570\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No helicase activity detected for purified protein, implying missing activating factors\", \"Did not define the RNA substrate remodeled during splicing\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Answered how the metazoan helicase is integrated into the spliceosome by identifying SRPK2 phosphorylation of the RS domain as the requirement for stable tri-snRNP association and B-complex integration.\",\n      \"evidence\": \"RNAi depletion, immunodepletion/complementation in HeLa nuclear extract, mass spectrometry\",\n      \"pmids\": [\"18425142\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural consequence of phosphorylation on tri-snRNP binding\", \"Phosphosite mapping on DDX23 not detailed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Revealed that Prp28 has an early ATP-independent role in commitment complex formation beyond its ATP-dependent activation function, with Prp8 regulating both activities.\",\n      \"evidence\": \"Genetic suppressor screen and spliceosome assembly assays in S. cerevisiae\",\n      \"pmids\": [\"24231520\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of the ATP-independent CC2 function unresolved\", \"Human relevance of the early function not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined the catalytic architecture: crystal structures showed two RecA domains in an open conformation, and biochemistry distinguished active yeast ATPase from a human domain that is inert in isolation but ATP-competent within the assembled spliceosome.\",\n      \"evidence\": \"X-ray crystallography of yeast and human Prp28, ATPase and nucleotide-binding assays, alanine-scanning mutagenesis\",\n      \"pmids\": [\"25303995\", \"24914973\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural state of DDX23 within an intact spliceosome not captured\", \"Mechanism that switches the human domain from ADP- to ATP-binding within complex unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extended DDX23 function beyond splicing by showing it promotes Drosha-dependent primary miRNA processing in a helicase-activity-dependent manner, conserved from worms to human glioma.\",\n      \"evidence\": \"Co-IP with Drosha, helicase-domain mutagenesis, knockdown/overexpression and xenografts; C. elegans RNAi with pri-/mature miRNA quantification\",\n      \"pmids\": [\"26121981\", \"26601717\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate hand-off between DDX23 and the microprocessor not biochemically reconstituted\", \"Whether splicing and miRNA functions use the same molecular activity unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Characterized the pre-catalytic transition by showing an ATPase-deficient human Prp28 stalls a pre-B complex with loosely associated tri-snRNP before U1/5'ss disruption, indicating an ATPase function beyond merely displacing U1.\",\n      \"evidence\": \"Dominant-negative ATPase mutant, spliceosomal complex purification, electron microscopy, mass spectrometry; ortholog crystal structure confirmation\",\n      \"pmids\": [\"27377154\", \"27139834\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The additional ATPase-dependent step beyond U1 displacement not defined\", \"Direct RNA target of the remodeling not identified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected the SRPK2-DDX23 phosphorylation axis to genome maintenance, showing phosphorylated DDX23 suppresses R-loops and prevents double-strand breaks downstream of Pol II pausing.\",\n      \"evidence\": \"siRNA knockdown of SRPK2/DDX23, S9.6 R-loop immunofluorescence, \\u03b3H2AX, rescue experiments\",\n      \"pmids\": [\"28076779\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether DDX23 unwinds R-loops directly or via splicing not separated\", \"Genomic loci of DDX23-dependent R-loop resolution not mapped\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Proposed a non-splicing antiviral role in which DDX23 binds cytoplasmic dsRNA and engages TRIF/MAVS to drive NF-\\u03baB/IRF3 signaling, restricting viral replication.\",\n      \"evidence\": \"Poly(I:C) pull-down, Co-IP with TRIF/MAVS, siRNA knockdown, VSV replication and signaling readouts, localization imaging\",\n      \"pmids\": [\"31620127\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Nuclear-to-cytoplasmic translocation trigger not mechanistically defined\", \"Single-lab finding without reciprocal validation of TRIF/MAVS complexes\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Refined the activation mechanism by mapping Prp28 contacts to the 5'ss GU and to U1C/Prp8/Brr2/Snu114, and identified phospho-Npl3 as the yeast functional counterpart of the metazoan phospho-N-terminus that stimulates ATPase activity.\",\n      \"evidence\": \"Co-IP, splicing-dependent crosslinking, in vitro ATPase assays with phospho- vs unphospho-Npl3\",\n      \"pmids\": [\"34035302\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of Npl3-mediated ATPase stimulation unresolved\", \"Whether human SRPK2-DDX23 and yeast Npl3-Prp28 are mechanistically equivalent not directly tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established a direct SR-protein partnership, defining the DDX23 N-terminal RS-like domain and SRSF1 RRM1/RS domain as the binding interface and linking this region to spliceosome incorporation and subnuclear localization.\",\n      \"evidence\": \"BioID proximity labeling/MS, BiFC, in vitro binding assays, deletion mutagenesis with localization imaging\",\n      \"pmids\": [\"38743621\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of the SRSF1 interaction for specific splicing events not defined\", \"Phosphorylation dependence of the SRSF1 interaction not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified Polymerase \\u03ba as a recruiter of DDX23 to chromatin R-loop loci, providing a targeting mechanism for its R-loop resolution activity.\",\n      \"evidence\": \"BioID and affinity pull-down with LC-MS/MS, Co-IP, S9.6 R-loop immunofluorescence, individual KO/KD\",\n      \"pmids\": [\"41949541\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether DDX23 enzymatically unwinds R-loops at Pol \\u03ba sites not shown directly\", \"Single-lab study\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How DDX23 partitions among its splicing, R-loop, miRNA-processing, and antiviral activities—and whether a single helicase/ATPase activity underlies all of them—remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking the distinct functions to defined RNA substrates\", \"Structure of DDX23 within an active spliceosome or R-loop complex not determined\", \"Regulatory switch governing nuclear vs cytoplasmic functions unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [2, 3, 4, 6]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [9, 10, 16]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [3, 4, 5]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [10, 14]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 3, 6]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [1, 15]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"complexes\": [\n      \"U4/U6-U5 tri-snRNP\",\n      \"spliceosome (pre-B/B complex)\",\n      \"Drosha microprocessor\"\n    ],\n    \"partners\": [\n      \"SRPK2\",\n      \"SRSF1\",\n      \"DROSHA\",\n      \"PRP8\",\n      \"BRR2\",\n      \"SNU114\",\n      \"TRIF\",\n      \"MAVS\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}