{"gene":"DDX46","run_date":"2026-06-09T23:54:41","timeline":{"discoveries":[{"year":2017,"finding":"DDX46 binds MAVS, TRAF3, and TRAF6 antiviral transcripts via their conserved CCGGUU element and recruits the m6A eraser ALKBH5 through its DEAD helicase domain to demethylate these transcripts, causing their nuclear retention and preventing their translation, thereby inhibiting type I interferon production after viral infection.","method":"RNA immunoprecipitation, Co-IP, m6A sequencing, nuclear retention assays, in vivo viral infection models","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (RIP, Co-IP, m6A mapping, nuclear fractionation, in vivo), replicated mechanistic findings in single rigorous study","pmids":["28846086"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structures of the DDX46-SF3b complex and DDX46-U2 snRNP assembly intermediate reveal that DDX46 is anchored on SF3B1 through its N-terminal sequences with its N-plug occupying the RNA path of SF3B1; DDX46 and DDX42 are mutually exclusive for SF3B1 binding, and cancer-driving SF3B1 mutations target residues that directly interact with DDX46.","method":"Cryo-electron microscopy (cryo-EM), in vitro binding assays, structural analysis with mutagenesis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure with functional in vitro validation, single lab but multiple orthogonal methods","pmids":["36797247"],"is_preprint":false},{"year":1990,"finding":"Yeast Prp5 (DDX46 ortholog) is a DEAD-box helicase-like protein required for pre-mRNA splicing; spliceosome assembly does not occur in its absence.","method":"Genetic complementation of temperature-sensitive mutation, DNA sequencing, splicing assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic complementation with defined splicing phenotype, foundational replicated finding","pmids":["2349233"],"is_preprint":false},{"year":1993,"finding":"Yeast Prp5 (DDX46 ortholog) interacts with PRP9, PRP11, and PRP21, and all four proteins act concertedly with stem-loop IIa of U2 snRNA to promote U2 snRNP binding to pre-mRNA during spliceosome assembly; ATP and the helicase activity of Prp5 are required, suggesting it promotes a conformational change in U1 or U2 snRNP.","method":"Genetic epistasis analysis, biochemical complementation, in vitro splicing assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis and biochemical complementation, replicated across labs","pmids":["8405998"],"is_preprint":false},{"year":1996,"finding":"Yeast Prp5 (DDX46 ortholog) is an RNA-dependent ATPase with 7-fold specificity for U2 snRNA over other snRNAs; it mediates an ATP-dependent conformational change in the intact U2 snRNP, making the branch point pairing sequence accessible for pre-spliceosome formation.","method":"In vitro ATPase assay with purified protein, RNaseH assay in extracts","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic reconstitution with purified protein, replicated and foundational","pmids":["8969184"],"is_preprint":false},{"year":2004,"finding":"Human and S. pombe Prp5 (DDX46 orthologs) physically associate with both U1 and U2 snRNPs through distinct domains; ATP binding and hydrolysis are required for pre-spliceosome complex A formation; Prp5 bridges U1 and U2 snRNPs, and a Prp5-associated U1/U2 complex was observed in S. pombe.","method":"Depletion-reconstitution from extracts, co-immunoprecipitation, domain mapping, native complex isolation","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, depletion/reconstitution, domain mapping, multiple organisms","pmids":["14713954"],"is_preprint":false},{"year":2007,"finding":"Prp5 ATPase activity gates branch region-U2 snRNA duplex formation as a fidelity checkpoint; reduced ATPase activity (SAT motif mutations in motif III) improves splicing of suboptimal branch-site substrates, and this effect is abrogated by compensatory U2 snRNA mutations that increase branch region-U2 pairing.","method":"Alanine scanning mutagenesis, in vivo splicing assays, in vitro ATPase assays, genetic epistasis with U2 snRNA mutations","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro ATPase assay plus in vivo genetics plus epistasis, multiple orthogonal approaches","pmids":["18082608"],"is_preprint":false},{"year":2015,"finding":"Prp5 binds directly to U2 snRNA in regions flanking the branchpoint-interacting stem-loop (BSL); it stabilizes the BSL and is released upon U2-branch site base-pairing; mutations impairing U2-branch site pairing retard Prp5 release and impede tri-snRNP association, establishing a novel proofreading mechanism.","method":"Prespliceosome isolation, RNA-protein crosslinking, mutant Prp5 functional analysis, in vitro splicing","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Moderate — biochemical isolation of novel intermediate, crosslinking, and functional mutant analysis in single rigorous study","pmids":["25561497"],"is_preprint":false},{"year":2016,"finding":"SF3B1 (Hsh155) interacts directly with Prp5 through its HEAT motifs; cancer-associated SF3B1/hsh155 mutations alter this physical interaction, leading to altered branch site selectivity during pre-spliceosome formation, phenocopying Prp5 mutations.","method":"Directed two-hybrid, pulldown/Co-IP, in vivo and in vitro splicing assays, yeast genetics","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct physical interaction mapped by multiple methods, functional epistasis validated in vivo and in vitro","pmids":["28087715"],"is_preprint":false},{"year":2016,"finding":"Pseudouridines at positions 42 and 44 of U2 snRNA stimulate Prp5 ATPase activity and Prp5 binding affinity for U2 snRNA; blocking pseudouridylation reduces Prp5 ATPase activity, impairs spliceosome assembly, and causes splicing deficiency.","method":"In vitro ATPase assays, RNA binding assays, designer snoRNA rescue, DMS probing, in vivo splicing analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro enzymatic assays plus in vivo rescue genetics with multiple orthogonal methods","pmids":["26873591"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structure of a pre-A spliceosome intermediate reveals that Prp5 blocks large-scale repositioning of U1 and U2 snRNPs required for tri-snRNP binding; binding of Hsh155HEAT to the bulged BS-A of the U2-BS helix triggers Hsh155HEAT closure, which destabilizes Prp5 binding, providing a structural mechanism for indirect branch-site proofreading by Prp5.","method":"Cryo-electron microscopy of stalled spliceosome assembly intermediates, branch-site adenosine deletion, mutational analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure with functional mutagenesis validation, published in high-impact journal","pmids":["34349264"],"is_preprint":false},{"year":2002,"finding":"Prp5 physically associates with U2 snRNP and promotes an ATP-enhanced conformational change in U2 snRNP that is required for branch-point region binding; the temperature-sensitive prp5-1 mutation maps to ATP-binding motif I within the helicase domain and reduces U2 snRNP conformational change.","method":"2'-O-methyl oligonucleotide binding assay, gel electrophoresis, heat inactivation of temperature-sensitive mutants, physical association assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical assay with mutant analysis, single lab, two orthogonal methods","pmids":["11927574"],"is_preprint":false},{"year":2009,"finding":"Prp5 associates with pre-mRNA from the commitment complex stage through spliceosome disassembly; it co-sediments with active spliceosomes and pulls down pre-mRNA, splicing intermediates, and lariat product but reduced amounts of spliced mRNA; Prp5 is an integral spliceosomal component with both ATP-independent and ATP-dependent functions at multiple stages.","method":"GST pulldown with radiolabeled pre-mRNA, glycerol gradient sedimentation, ATP-depletion experiments","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro biochemical assays, multiple methods, single lab","pmids":["19451545"],"is_preprint":false},{"year":2019,"finding":"The Prp5 RecA-like domains undergo a large conformational rearrangement (open/closed) only when both ATP and RNA are bound simultaneously; fidelity-altering Prp5 mutations change the dynamics of this conformational switch, linking RecA domain movement to branch-site recognition during spliceosome assembly.","method":"Single-molecule FRET (smFRET), mutagenesis","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — single-molecule FRET with mutagenesis, single lab","pmids":["31712821"],"is_preprint":false},{"year":2020,"finding":"Prp5 directly interacts with Spt8p (a TBP-binding module component of the SAGA complex), but not Spt3p; this interaction modulates Prp5's splicing proofreading function and mutually influences RNA Pol II recruitment to intron-containing genes, establishing a reciprocal coupling between transcription initiation/elongation and pre-spliceosome assembly.","method":"In vitro direct protein interaction assay, chromatin immunoprecipitation (ChIP), ChIP-seq, genetic epistasis (suppressor screen), in vivo splicing analysis","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct physical interaction confirmed in vitro, ChIP-seq genome-wide, genetic epistasis, single lab","pmids":["32399566"],"is_preprint":false},{"year":2025,"finding":"RNA virus infection induces caspase-dependent cleavage of DDX46, which triggers its translocation from the nucleus to the cytoplasm; this translocation releases nuclear-retained innate immune transcripts (previously sequestered by DDX46), licensing their rapid translation and potentiating robust IFN responses.","method":"RNA-binding protein knockout library screen, caspase cleavage assays, subcellular fractionation/imaging, IFN reporter assays","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional knockout screen plus mechanistic follow-up with fractionation and cleavage assays, single lab","pmids":["41854249"],"is_preprint":false},{"year":2025,"finding":"DDX46 forms a functional complex with ALKBH5 and treRNA1 in the nucleus to orchestrate m6A demethylation of BCR signaling transcripts; demethylated transcripts interact with HuR for cytoplasmic export and translation; loss of DDX46 impairs transcript processing and BCR-related gene expression.","method":"Co-immunoprecipitation, nuclear fractionation, m6A analysis, loss-of-function assays","journal":"Neoplasia (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP and functional assays, single lab, limited methodological detail in abstract","pmids":["39987653"],"is_preprint":false},{"year":2025,"finding":"O-GlcNAcylation of DDX46 at Ser257 by OGT enhances DDX46 protein stability by impeding ubiquitin-mediated degradation; elevated DDX46 expression then activates the PI3K/Akt signaling pathway, promoting hepatocellular carcinoma cell proliferation and invasion.","method":"Co-IP (OGT-DDX46 interaction), site-specific mutagenesis (Ser257), ubiquitination assay, PI3K/Akt signaling readouts","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP, site mutagenesis, and downstream signaling assays, single lab, limited replication","pmids":["41176131"],"is_preprint":false},{"year":2013,"finding":"Zebrafish Ddx46 is expressed in hematopoietic stem cells; loss-of-function mutants show suppressed erythropoiesis and lymphopoiesis with maintained myelopoiesis, correlating with downregulation of gata1a but not spi1 expression, indicating Ddx46 is required for multilineage HSC differentiation.","method":"Zebrafish genetic mutant analysis, whole-mount in situ hybridization, lineage marker expression","journal":"Stem cells and development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined genetic mutant with specific lineage phenotypes and molecular marker analysis, single lab","pmids":["23635340"],"is_preprint":false},{"year":2012,"finding":"Zebrafish Ddx46 is required for normal development of digestive organs and brain; Ddx46 mutants show accumulation of unspliced pre-mRNA forms in affected tissues, indicating Ddx46 is required for pre-mRNA splicing in vivo.","method":"Forward genetic screen, RT-PCR splicing analysis, whole-mount in situ hybridization","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic mutant with direct RT-PCR evidence of splicing defects in specific tissues, single lab","pmids":["22442707"],"is_preprint":false},{"year":2025,"finding":"Prp5 (DDX46 ortholog) is implicated in displacing the U2 snRNP component Cus2; prespliceosome formation can proceed without ATP in the absence of either Sub2 or Cus2, revealing a coordinated interplay among Prp5, Sub2, Cus2, Mud2, and Msl5 during prespliceosome formation.","method":"In vitro splicing assays, ATP-depletion experiments, genetic deletion of splicing factors","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vitro reconstitution with genetic deletions, single lab, no replication yet","pmids":["41027713"],"is_preprint":false},{"year":2024,"finding":"DDX46 binds JMJD6 and promotes the JMJD6/CDK4 signaling pathway in pancreatic cancer cells; DDX46 knockdown represses tumor growth and sensitizes cells to gemcitabine, and JMJD6 overexpression reverses the anti-tumor effect of DDX46 knockdown.","method":"Co-immunoprecipitation (DDX46-JMJD6), in vitro and in vivo tumor growth assays, drug sensitivity assays","journal":"Neoplasia","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP, single lab, limited mechanistic follow-up","pmids":["38764294"],"is_preprint":false}],"current_model":"DDX46 (yeast Prp5 ortholog) is a DEAD-box RNA-dependent ATPase that functions in two major cellular contexts: (1) in pre-mRNA splicing, it bridges U1 and U2 snRNPs through distinct domains, promotes ATP-dependent conformational remodeling of U2 snRNP to expose the branch-point pairing sequence, and proofreads branch site recognition by directly binding the U2 branchpoint-interacting stem-loop and blocking spliceosome progression until correct U2-branch site base-pairing triggers its release—a mechanism structurally confirmed by cryo-EM; and (2) in antiviral innate immunity, nuclear DDX46 binds MAVS, TRAF3, and TRAF6 antiviral transcripts via CCGGUU elements and recruits the m6A eraser ALKBH5 through its DEAD helicase domain to demethylate these transcripts, enforcing their nuclear retention and suppressing interferon production, a mechanism reversed by caspase-dependent cleavage and nuclear-to-cytoplasmic translocation of DDX46 during RNA virus infection."},"narrative":{"mechanistic_narrative":"DDX46 (the yeast Prp5 ortholog) is a DEAD-box RNA-dependent ATPase that performs two distinct nuclear functions: assembly and fidelity control of the spliceosome, and post-transcriptional control of innate-immune gene expression [PMID:2349233, PMID:8969184, PMID:28846086]. In splicing, it is required for spliceosome assembly [PMID:2349233] and bridges U1 and U2 snRNPs through distinct domains in an ATP-dependent manner to drive pre-spliceosome (complex A) formation [PMID:14713954]. As an RNA-dependent ATPase with selectivity for U2 snRNA, it catalyzes a conformational change in U2 snRNP that exposes the branch-point pairing sequence [PMID:8969184, PMID:11927574], a switch in its RecA-like domains that occurs only when both ATP and RNA are bound [PMID:31712821]. DDX46 anchors on the SF3b component SF3B1/Hsh155 through its N-terminal sequences, with its N-plug occupying the SF3B1 RNA path; cancer-associated SF3B1 mutations map to residues that contact DDX46 and alter branch-site selection [PMID:36797247, PMID:28087715]. Mechanistically it enforces branch-site fidelity: its ATPase activity gates branch region–U2 duplex formation, it binds U2 RNA flanking the branchpoint-interacting stem-loop, and it is released only upon correct U2–branch-site base-pairing—a proofreading step that licenses tri-snRNP recruitment [PMID:18082608, PMID:25561497, PMID:34349264]. In antiviral innate immunity, nuclear DDX46 binds MAVS, TRAF3 and TRAF6 transcripts via a CCGGUU element and recruits the m6A eraser ALKBH5 through its DEAD helicase domain to demethylate and nuclearly retain these transcripts, suppressing type I interferon; RNA virus infection triggers caspase-dependent cleavage and nuclear-to-cytoplasmic translocation of DDX46, releasing the retained transcripts to potentiate the IFN response [PMID:28846086, PMID:41854249]. Loss-of-function studies in zebrafish establish in vivo requirements for DDX46 in pre-mRNA splicing during digestive-organ and brain development and in multilineage hematopoietic stem-cell differentiation [PMID:22442707, PMID:23635340].","teleology":[{"year":1990,"claim":"Established that the DDX46 ortholog Prp5 is essential for spliceosome assembly, defining the gene as a core splicing factor before any biochemical activity was known.","evidence":"genetic complementation of a temperature-sensitive yeast mutant with splicing assays","pmids":["2349233"],"confidence":"High","gaps":["Did not define the molecular activity","No partner proteins identified","No mechanism for how assembly fails"]},{"year":1993,"claim":"Placed Prp5 in a functional module with SF3a-type factors acting on U2 snRNA, and first implicated ATP and helicase activity in promoting a U1/U2 snRNP conformational change.","evidence":"genetic epistasis and biochemical complementation with in vitro splicing in yeast","pmids":["8405998"],"confidence":"High","gaps":["ATPase activity not directly demonstrated","Which snRNP is remodeled left ambiguous","No structural basis"]},{"year":1996,"claim":"Demonstrated that Prp5 is itself an RNA-dependent ATPase with selectivity for U2 snRNA that drives an ATP-dependent conformational change exposing the branch point, converting the genetic inference into a defined enzymatic activity.","evidence":"in vitro ATPase assay with purified protein and RNaseH probing in extracts","pmids":["8969184"],"confidence":"High","gaps":["RNA-binding sites on U2 not mapped","Did not address fidelity/proofreading"]},{"year":2002,"claim":"Mapped the conformational-change function to the ATP-binding helicase domain by linking a temperature-sensitive mutation in motif I to reduced U2 snRNP remodeling.","evidence":"2'-O-methyl oligonucleotide binding and physical association assays with ts-mutant inactivation in yeast","pmids":["11927574"],"confidence":"Medium","gaps":["Single lab","Structural detail of the conformational change absent"]},{"year":2004,"claim":"Showed that Prp5 physically bridges U1 and U2 snRNPs through distinct domains and that ATP binding/hydrolysis is required for complex A formation, generalizing the mechanism across human and S. pombe.","evidence":"depletion-reconstitution, reciprocal Co-IP, domain mapping and native complex isolation across organisms","pmids":["14713954"],"confidence":"High","gaps":["Structural geometry of the bridge unknown","Domain boundaries for each snRNP contact coarse"]},{"year":2007,"claim":"Defined Prp5 ATPase activity as a branch-site fidelity checkpoint by showing ATPase-weakening mutations rescue suboptimal branch sites in a manner reversed by compensatory U2 mutations.","evidence":"alanine scanning, in vivo and in vitro ATPase/splicing assays, genetic epistasis with U2 snRNA mutations","pmids":["18082608"],"confidence":"High","gaps":["Physical basis of gating not yet shown","Release step not directly observed"]},{"year":2009,"claim":"Extended Prp5 association beyond early assembly, showing it accompanies pre-mRNA from the commitment complex through disassembly with both ATP-independent and ATP-dependent roles.","evidence":"GST pulldown with radiolabeled pre-mRNA, glycerol gradient sedimentation, ATP-depletion in vitro","pmids":["19451545"],"confidence":"Medium","gaps":["Functional role at later stages undefined","Single lab"]},{"year":2015,"claim":"Resolved the proofreading mechanism: Prp5 binds U2 RNA flanking the branchpoint-interacting stem-loop and is released only upon correct U2-branch-site pairing, coupling fidelity to tri-snRNP recruitment.","evidence":"prespliceosome isolation, RNA-protein crosslinking and mutant functional analysis","pmids":["25561497"],"confidence":"High","gaps":["Atomic structure of bound state absent","Coupling to ATP cycle not visualized"]},{"year":2016,"claim":"Identified SF3B1/Hsh155 HEAT-domain contacts as the physical determinant of branch-site selectivity, explaining how cancer-associated SF3B1 mutations phenocopy Prp5 fidelity mutations.","evidence":"two-hybrid, pulldown/Co-IP, in vivo/in vitro splicing and yeast genetics","pmids":["28087715"],"confidence":"High","gaps":["Structural interface only inferred at this stage","Human cancer relevance correlative"]},{"year":2016,"claim":"Showed U2 snRNA pseudouridylation tunes Prp5 enzymatic output, linking an RNA modification to ATPase activity, U2 binding and spliceosome assembly fidelity.","evidence":"in vitro ATPase/RNA-binding assays, designer snoRNA rescue, DMS probing and in vivo splicing","pmids":["26873591"],"confidence":"High","gaps":["Mechanism by which pseudouridines stimulate ATPase unresolved","Conservation to human DDX46 not tested here"]},{"year":2019,"claim":"Linked RecA-domain dynamics to fidelity by showing the open/closed conformational switch requires simultaneous ATP and RNA, and that fidelity-altering mutations change switch dynamics.","evidence":"single-molecule FRET with mutagenesis","pmids":["31712821"],"confidence":"Medium","gaps":["Single lab","Direct correlation to in vivo proofreading kinetics incomplete"]},{"year":2021,"claim":"Provided the structural mechanism for indirect proofreading: cryo-EM of a pre-A intermediate showed Prp5 blocks U1/U2 repositioning until Hsh155 HEAT closure on the bulged branch adenosine destabilizes Prp5.","evidence":"cryo-EM of stalled spliceosome intermediates with branch-adenosine deletion and mutagenesis","pmids":["34349264"],"confidence":"High","gaps":["Human DDX46 complex not resolved in this study","ATP hydrolysis coupling to release timing"]},{"year":2023,"claim":"Defined the human DDX46-SF3b interface structurally, showing N-terminal anchoring on SF3B1 with the N-plug in the RNA path, mutual exclusivity with DDX42, and direct contact with cancer-mutated SF3B1 residues.","evidence":"cryo-EM of DDX46-SF3b and DDX46-U2 intermediates with in vitro binding and mutagenesis","pmids":["36797247"],"confidence":"High","gaps":["Functional consequence of DDX46/DDX42 exchange in cells unresolved","Dynamics of N-plug displacement not captured"]},{"year":2020,"claim":"Connected splicing-factor function to transcription by showing Prp5 directly binds the SAGA component Spt8p, coupling pre-spliceosome assembly to Pol II recruitment at intron-containing genes.","evidence":"in vitro interaction, ChIP/ChIP-seq, suppressor genetics and in vivo splicing","pmids":["32399566"],"confidence":"Medium","gaps":["Single lab","Conservation of Spt8p coupling to human DDX46 untested"]},{"year":2025,"claim":"Refined the early prespliceosome network, implicating Prp5 in displacing Cus2 and revealing ATP-independent assembly routes when Sub2 or Cus2 is absent.","evidence":"in vitro splicing with ATP depletion and genetic deletion of splicing factors","pmids":["41027713"],"confidence":"Medium","gaps":["No replication yet","Order of Cus2 displacement versus ATPase cycle unclear"]},{"year":2017,"claim":"Revealed a second, splicing-independent role: nuclear DDX46 binds MAVS/TRAF3/TRAF6 transcripts via a CCGGUU element and recruits ALKBH5 through its DEAD domain to demethylate and retain them, suppressing interferon.","evidence":"RIP, Co-IP, m6A sequencing, nuclear retention assays and in vivo viral infection models","pmids":["28846086"],"confidence":"High","gaps":["How DDX46 selects CCGGUU targets mechanistically unclear","Relationship to its splicing role not addressed"]},{"year":2025,"claim":"Completed the antiviral switch by showing RNA virus infection induces caspase-dependent DDX46 cleavage and nuclear export, releasing retained immune transcripts to amplify IFN responses.","evidence":"RNA-binding protein knockout screen, caspase cleavage assays, fractionation/imaging and IFN reporter assays","pmids":["41854249"],"confidence":"Medium","gaps":["Cleavage site and responsible caspase detail limited","Single lab"]},{"year":2025,"claim":"Extended the ALKBH5-coupled demethylation role to lymphocyte biology, showing DDX46/ALKBH5/treRNA1 demethylates BCR-signaling transcripts handed to HuR for export and translation.","evidence":"Co-IP, nuclear fractionation, m6A analysis and loss-of-function assays","pmids":["39987653"],"confidence":"Medium","gaps":["treRNA1 role mechanistically thin","Limited methodological detail"]},{"year":2013,"claim":"Established an in vivo developmental requirement, showing zebrafish Ddx46 is needed for multilineage HSC differentiation via gata1a-dependent erythro/lymphopoiesis.","evidence":"zebrafish mutant analysis, in situ hybridization and lineage marker expression","pmids":["23635340"],"confidence":"Medium","gaps":["Whether phenotype reflects splicing or immune role untested","Direct targets unknown"]},{"year":2019,"claim":"Demonstrated in vivo splicing function in vertebrates, with zebrafish Ddx46 mutants accumulating unspliced pre-mRNA in digestive organs and brain.","evidence":"forward genetic screen with RT-PCR splicing analysis and in situ hybridization","pmids":["22442707"],"confidence":"Medium","gaps":["Tissue specificity of splicing dependence unexplained","Affected introns not catalogued"]},{"year":2025,"claim":"Implicated DDX46 in cancer via O-GlcNAcylation at Ser257 by OGT, which stabilizes the protein and drives PI3K/Akt-dependent hepatocellular carcinoma growth.","evidence":"Co-IP, site-specific mutagenesis, ubiquitination and PI3K/Akt signaling assays","pmids":["41176131"],"confidence":"Medium","gaps":["Whether oncogenic effect derives from splicing or immune role unknown","Single lab"]},{"year":null,"claim":"It remains unresolved how DDX46's spliceosomal proofreading function and its ALKBH5-coupled m6A/innate-immune RNA-retention function are partitioned or co-regulated within the same nuclear protein.","evidence":"no study in the timeline reconciles the splicing and immune RNA-regulatory activities","pmids":[],"confidence":"Medium","gaps":["No structural model of the DDX46-ALKBH5 complex","Determinants directing DDX46 to splicing versus immune transcripts unknown","Human cancer mechanism not linked to either defined activity"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[4,5,6,13]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[4,7,9,0]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[4,6]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[4,6]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[5,0]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,15,16]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[15]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[2,4,5,7]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,15,16]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[18,19]}],"complexes":["U2 snRNP","SF3b","pre-spliceosome (complex A)"],"partners":["SF3B1","ALKBH5","PRP9","PRP11","PRP21","SPT8","JMJD6","OGT"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q7L014","full_name":"Probable ATP-dependent RNA helicase DDX46","aliases":["DEAD box protein 46","PRP5 homolog"],"length_aa":1031,"mass_kda":117.4,"function":"Component of the 17S U2 SnRNP complex of the spliceosome, a large ribonucleoprotein complex that removes introns from transcribed pre-mRNAs (PubMed:12234937, PubMed:32494006, PubMed:34822310, PubMed:36797247). The 17S U2 SnRNP complex (1) directly participates in early spliceosome assembly and (2) mediates recognition of the intron branch site during pre-mRNA splicing by promoting the selection of the pre-mRNA branch-site adenosine, the nucleophile for the first step of splicing (PubMed:32494006, PubMed:34822310). Within the 17S U2 SnRNP complex, DDX46 plays essential roles during assembly of pre-spliceosome and proofreading of the branch site (PubMed:34822310)","subcellular_location":"Nucleus speckle; Nucleus, Cajal body","url":"https://www.uniprot.org/uniprotkb/Q7L014/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/DDX46","classification":"Common Essential","n_dependent_lines":1095,"n_total_lines":1208,"dependency_fraction":0.9064569536423841},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SLC7A6","stoichiometry":10.0}],"url":"https://opencell.sf.czbiohub.org/search/DDX46","total_profiled":1310},"omim":[{"mim_id":"617848","title":"DEAD-BOX HELICASE 46; DDX46","url":"https://www.omim.org/entry/617848"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nuclear speckles","reliability":"Supported"},{"location":"Nucleoli fibrillar center","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DDX46"},"hgnc":{"alias_symbol":["KIAA0801","FLJ25329","PRPF5","Prp5"],"prev_symbol":[]},"alphafold":{"accession":"Q7L014","domains":[{"cath_id":"3.40.50.300","chopping":"338-582","consensus_level":"high","plddt":90.3694,"start":338,"end":582},{"cath_id":"3.40.50.300","chopping":"592-762","consensus_level":"high","plddt":89.1371,"start":592,"end":762},{"cath_id":"3.30.1370.10","chopping":"930-1017","consensus_level":"high","plddt":87.9919,"start":930,"end":1017}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7L014","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q7L014-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q7L014-F1-predicted_aligned_error_v6.png","plddt_mean":69.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DDX46","jax_strain_url":"https://www.jax.org/strain/search?query=DDX46"},"sequence":{"accession":"Q7L014","fasta_url":"https://rest.uniprot.org/uniprotkb/Q7L014.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q7L014/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7L014"}},"corpus_meta":[{"pmid":"28846086","id":"PMC_28846086","title":"The 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Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/31511298","citation_count":3,"is_preprint":false},{"pmid":"38764294","id":"PMC_38764294","title":"Oncogenic DDX46 promotes pancreatic cancer development and gemcitabine resistance by facilitating the JMJD6/CDK4 signaling pathway.","date":"2024","source":"Neoplasma","url":"https://pubmed.ncbi.nlm.nih.gov/38764294","citation_count":2,"is_preprint":false},{"pmid":"41176131","id":"PMC_41176131","title":"O-GlcNAcylation of DDX46 promotes hepatocellular carcinoma progression by activating the PI3K/Akt signaling pathway.","date":"2025","source":"Biochimica et biophysica acta. Molecular cell research","url":"https://pubmed.ncbi.nlm.nih.gov/41176131","citation_count":1,"is_preprint":false},{"pmid":"41027713","id":"PMC_41027713","title":"New mechanistic insights into prespliceosome formation-roles of DEAD-box proteins Prp5 and Sub2.","date":"2025","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/41027713","citation_count":0,"is_preprint":false},{"pmid":"41854249","id":"PMC_41854249","title":"Caspase-mediated DDX46 cleavage unchains antiviral immunity.","date":"2026","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/41854249","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15878,"output_tokens":5137,"usd":0.062344,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13407,"output_tokens":5857,"usd":0.10673,"stage2_stop_reason":"end_turn"},"total_usd":0.169074,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"DDX46 binds MAVS, TRAF3, and TRAF6 antiviral transcripts via their conserved CCGGUU element and recruits the m6A eraser ALKBH5 through its DEAD helicase domain to demethylate these transcripts, causing their nuclear retention and preventing their translation, thereby inhibiting type I interferon production after viral infection.\",\n      \"method\": \"RNA immunoprecipitation, Co-IP, m6A sequencing, nuclear retention assays, in vivo viral infection models\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (RIP, Co-IP, m6A mapping, nuclear fractionation, in vivo), replicated mechanistic findings in single rigorous study\",\n      \"pmids\": [\"28846086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structures of the DDX46-SF3b complex and DDX46-U2 snRNP assembly intermediate reveal that DDX46 is anchored on SF3B1 through its N-terminal sequences with its N-plug occupying the RNA path of SF3B1; DDX46 and DDX42 are mutually exclusive for SF3B1 binding, and cancer-driving SF3B1 mutations target residues that directly interact with DDX46.\",\n      \"method\": \"Cryo-electron microscopy (cryo-EM), in vitro binding assays, structural analysis with mutagenesis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure with functional in vitro validation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"36797247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"Yeast Prp5 (DDX46 ortholog) is a DEAD-box helicase-like protein required for pre-mRNA splicing; spliceosome assembly does not occur in its absence.\",\n      \"method\": \"Genetic complementation of temperature-sensitive mutation, DNA sequencing, splicing assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic complementation with defined splicing phenotype, foundational replicated finding\",\n      \"pmids\": [\"2349233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Yeast Prp5 (DDX46 ortholog) interacts with PRP9, PRP11, and PRP21, and all four proteins act concertedly with stem-loop IIa of U2 snRNA to promote U2 snRNP binding to pre-mRNA during spliceosome assembly; ATP and the helicase activity of Prp5 are required, suggesting it promotes a conformational change in U1 or U2 snRNP.\",\n      \"method\": \"Genetic epistasis analysis, biochemical complementation, in vitro splicing assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis and biochemical complementation, replicated across labs\",\n      \"pmids\": [\"8405998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Yeast Prp5 (DDX46 ortholog) is an RNA-dependent ATPase with 7-fold specificity for U2 snRNA over other snRNAs; it mediates an ATP-dependent conformational change in the intact U2 snRNP, making the branch point pairing sequence accessible for pre-spliceosome formation.\",\n      \"method\": \"In vitro ATPase assay with purified protein, RNaseH assay in extracts\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic reconstitution with purified protein, replicated and foundational\",\n      \"pmids\": [\"8969184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human and S. pombe Prp5 (DDX46 orthologs) physically associate with both U1 and U2 snRNPs through distinct domains; ATP binding and hydrolysis are required for pre-spliceosome complex A formation; Prp5 bridges U1 and U2 snRNPs, and a Prp5-associated U1/U2 complex was observed in S. pombe.\",\n      \"method\": \"Depletion-reconstitution from extracts, co-immunoprecipitation, domain mapping, native complex isolation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, depletion/reconstitution, domain mapping, multiple organisms\",\n      \"pmids\": [\"14713954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Prp5 ATPase activity gates branch region-U2 snRNA duplex formation as a fidelity checkpoint; reduced ATPase activity (SAT motif mutations in motif III) improves splicing of suboptimal branch-site substrates, and this effect is abrogated by compensatory U2 snRNA mutations that increase branch region-U2 pairing.\",\n      \"method\": \"Alanine scanning mutagenesis, in vivo splicing assays, in vitro ATPase assays, genetic epistasis with U2 snRNA mutations\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro ATPase assay plus in vivo genetics plus epistasis, multiple orthogonal approaches\",\n      \"pmids\": [\"18082608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Prp5 binds directly to U2 snRNA in regions flanking the branchpoint-interacting stem-loop (BSL); it stabilizes the BSL and is released upon U2-branch site base-pairing; mutations impairing U2-branch site pairing retard Prp5 release and impede tri-snRNP association, establishing a novel proofreading mechanism.\",\n      \"method\": \"Prespliceosome isolation, RNA-protein crosslinking, mutant Prp5 functional analysis, in vitro splicing\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical isolation of novel intermediate, crosslinking, and functional mutant analysis in single rigorous study\",\n      \"pmids\": [\"25561497\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SF3B1 (Hsh155) interacts directly with Prp5 through its HEAT motifs; cancer-associated SF3B1/hsh155 mutations alter this physical interaction, leading to altered branch site selectivity during pre-spliceosome formation, phenocopying Prp5 mutations.\",\n      \"method\": \"Directed two-hybrid, pulldown/Co-IP, in vivo and in vitro splicing assays, yeast genetics\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct physical interaction mapped by multiple methods, functional epistasis validated in vivo and in vitro\",\n      \"pmids\": [\"28087715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Pseudouridines at positions 42 and 44 of U2 snRNA stimulate Prp5 ATPase activity and Prp5 binding affinity for U2 snRNA; blocking pseudouridylation reduces Prp5 ATPase activity, impairs spliceosome assembly, and causes splicing deficiency.\",\n      \"method\": \"In vitro ATPase assays, RNA binding assays, designer snoRNA rescue, DMS probing, in vivo splicing analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro enzymatic assays plus in vivo rescue genetics with multiple orthogonal methods\",\n      \"pmids\": [\"26873591\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structure of a pre-A spliceosome intermediate reveals that Prp5 blocks large-scale repositioning of U1 and U2 snRNPs required for tri-snRNP binding; binding of Hsh155HEAT to the bulged BS-A of the U2-BS helix triggers Hsh155HEAT closure, which destabilizes Prp5 binding, providing a structural mechanism for indirect branch-site proofreading by Prp5.\",\n      \"method\": \"Cryo-electron microscopy of stalled spliceosome assembly intermediates, branch-site adenosine deletion, mutational analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure with functional mutagenesis validation, published in high-impact journal\",\n      \"pmids\": [\"34349264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Prp5 physically associates with U2 snRNP and promotes an ATP-enhanced conformational change in U2 snRNP that is required for branch-point region binding; the temperature-sensitive prp5-1 mutation maps to ATP-binding motif I within the helicase domain and reduces U2 snRNP conformational change.\",\n      \"method\": \"2'-O-methyl oligonucleotide binding assay, gel electrophoresis, heat inactivation of temperature-sensitive mutants, physical association assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical assay with mutant analysis, single lab, two orthogonal methods\",\n      \"pmids\": [\"11927574\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Prp5 associates with pre-mRNA from the commitment complex stage through spliceosome disassembly; it co-sediments with active spliceosomes and pulls down pre-mRNA, splicing intermediates, and lariat product but reduced amounts of spliced mRNA; Prp5 is an integral spliceosomal component with both ATP-independent and ATP-dependent functions at multiple stages.\",\n      \"method\": \"GST pulldown with radiolabeled pre-mRNA, glycerol gradient sedimentation, ATP-depletion experiments\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro biochemical assays, multiple methods, single lab\",\n      \"pmids\": [\"19451545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The Prp5 RecA-like domains undergo a large conformational rearrangement (open/closed) only when both ATP and RNA are bound simultaneously; fidelity-altering Prp5 mutations change the dynamics of this conformational switch, linking RecA domain movement to branch-site recognition during spliceosome assembly.\",\n      \"method\": \"Single-molecule FRET (smFRET), mutagenesis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — single-molecule FRET with mutagenesis, single lab\",\n      \"pmids\": [\"31712821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Prp5 directly interacts with Spt8p (a TBP-binding module component of the SAGA complex), but not Spt3p; this interaction modulates Prp5's splicing proofreading function and mutually influences RNA Pol II recruitment to intron-containing genes, establishing a reciprocal coupling between transcription initiation/elongation and pre-spliceosome assembly.\",\n      \"method\": \"In vitro direct protein interaction assay, chromatin immunoprecipitation (ChIP), ChIP-seq, genetic epistasis (suppressor screen), in vivo splicing analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct physical interaction confirmed in vitro, ChIP-seq genome-wide, genetic epistasis, single lab\",\n      \"pmids\": [\"32399566\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNA virus infection induces caspase-dependent cleavage of DDX46, which triggers its translocation from the nucleus to the cytoplasm; this translocation releases nuclear-retained innate immune transcripts (previously sequestered by DDX46), licensing their rapid translation and potentiating robust IFN responses.\",\n      \"method\": \"RNA-binding protein knockout library screen, caspase cleavage assays, subcellular fractionation/imaging, IFN reporter assays\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional knockout screen plus mechanistic follow-up with fractionation and cleavage assays, single lab\",\n      \"pmids\": [\"41854249\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DDX46 forms a functional complex with ALKBH5 and treRNA1 in the nucleus to orchestrate m6A demethylation of BCR signaling transcripts; demethylated transcripts interact with HuR for cytoplasmic export and translation; loss of DDX46 impairs transcript processing and BCR-related gene expression.\",\n      \"method\": \"Co-immunoprecipitation, nuclear fractionation, m6A analysis, loss-of-function assays\",\n      \"journal\": \"Neoplasia (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP and functional assays, single lab, limited methodological detail in abstract\",\n      \"pmids\": [\"39987653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"O-GlcNAcylation of DDX46 at Ser257 by OGT enhances DDX46 protein stability by impeding ubiquitin-mediated degradation; elevated DDX46 expression then activates the PI3K/Akt signaling pathway, promoting hepatocellular carcinoma cell proliferation and invasion.\",\n      \"method\": \"Co-IP (OGT-DDX46 interaction), site-specific mutagenesis (Ser257), ubiquitination assay, PI3K/Akt signaling readouts\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP, site mutagenesis, and downstream signaling assays, single lab, limited replication\",\n      \"pmids\": [\"41176131\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Zebrafish Ddx46 is expressed in hematopoietic stem cells; loss-of-function mutants show suppressed erythropoiesis and lymphopoiesis with maintained myelopoiesis, correlating with downregulation of gata1a but not spi1 expression, indicating Ddx46 is required for multilineage HSC differentiation.\",\n      \"method\": \"Zebrafish genetic mutant analysis, whole-mount in situ hybridization, lineage marker expression\",\n      \"journal\": \"Stem cells and development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined genetic mutant with specific lineage phenotypes and molecular marker analysis, single lab\",\n      \"pmids\": [\"23635340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Zebrafish Ddx46 is required for normal development of digestive organs and brain; Ddx46 mutants show accumulation of unspliced pre-mRNA forms in affected tissues, indicating Ddx46 is required for pre-mRNA splicing in vivo.\",\n      \"method\": \"Forward genetic screen, RT-PCR splicing analysis, whole-mount in situ hybridization\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic mutant with direct RT-PCR evidence of splicing defects in specific tissues, single lab\",\n      \"pmids\": [\"22442707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Prp5 (DDX46 ortholog) is implicated in displacing the U2 snRNP component Cus2; prespliceosome formation can proceed without ATP in the absence of either Sub2 or Cus2, revealing a coordinated interplay among Prp5, Sub2, Cus2, Mud2, and Msl5 during prespliceosome formation.\",\n      \"method\": \"In vitro splicing assays, ATP-depletion experiments, genetic deletion of splicing factors\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vitro reconstitution with genetic deletions, single lab, no replication yet\",\n      \"pmids\": [\"41027713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DDX46 binds JMJD6 and promotes the JMJD6/CDK4 signaling pathway in pancreatic cancer cells; DDX46 knockdown represses tumor growth and sensitizes cells to gemcitabine, and JMJD6 overexpression reverses the anti-tumor effect of DDX46 knockdown.\",\n      \"method\": \"Co-immunoprecipitation (DDX46-JMJD6), in vitro and in vivo tumor growth assays, drug sensitivity assays\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP, single lab, limited mechanistic follow-up\",\n      \"pmids\": [\"38764294\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DDX46 (yeast Prp5 ortholog) is a DEAD-box RNA-dependent ATPase that functions in two major cellular contexts: (1) in pre-mRNA splicing, it bridges U1 and U2 snRNPs through distinct domains, promotes ATP-dependent conformational remodeling of U2 snRNP to expose the branch-point pairing sequence, and proofreads branch site recognition by directly binding the U2 branchpoint-interacting stem-loop and blocking spliceosome progression until correct U2-branch site base-pairing triggers its release—a mechanism structurally confirmed by cryo-EM; and (2) in antiviral innate immunity, nuclear DDX46 binds MAVS, TRAF3, and TRAF6 antiviral transcripts via CCGGUU elements and recruits the m6A eraser ALKBH5 through its DEAD helicase domain to demethylate these transcripts, enforcing their nuclear retention and suppressing interferon production, a mechanism reversed by caspase-dependent cleavage and nuclear-to-cytoplasmic translocation of DDX46 during RNA virus infection.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DDX46 (the yeast Prp5 ortholog) is a DEAD-box RNA-dependent ATPase that performs two distinct nuclear functions: assembly and fidelity control of the spliceosome, and post-transcriptional control of innate-immune gene expression [#2, #4, #0]. In splicing, it is required for spliceosome assembly [#2] and bridges U1 and U2 snRNPs through distinct domains in an ATP-dependent manner to drive pre-spliceosome (complex A) formation [#5]. As an RNA-dependent ATPase with selectivity for U2 snRNA, it catalyzes a conformational change in U2 snRNP that exposes the branch-point pairing sequence [#4, #11], a switch in its RecA-like domains that occurs only when both ATP and RNA are bound [#13]. DDX46 anchors on the SF3b component SF3B1/Hsh155 through its N-terminal sequences, with its N-plug occupying the SF3B1 RNA path; cancer-associated SF3B1 mutations map to residues that contact DDX46 and alter branch-site selection [#1, #8]. Mechanistically it enforces branch-site fidelity: its ATPase activity gates branch region–U2 duplex formation, it binds U2 RNA flanking the branchpoint-interacting stem-loop, and it is released only upon correct U2–branch-site base-pairing—a proofreading step that licenses tri-snRNP recruitment [#6, #7, #10]. In antiviral innate immunity, nuclear DDX46 binds MAVS, TRAF3 and TRAF6 transcripts via a CCGGUU element and recruits the m6A eraser ALKBH5 through its DEAD helicase domain to demethylate and nuclearly retain these transcripts, suppressing type I interferon; RNA virus infection triggers caspase-dependent cleavage and nuclear-to-cytoplasmic translocation of DDX46, releasing the retained transcripts to potentiate the IFN response [#0, #15]. Loss-of-function studies in zebrafish establish in vivo requirements for DDX46 in pre-mRNA splicing during digestive-organ and brain development and in multilineage hematopoietic stem-cell differentiation [#19, #18].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Established that the DDX46 ortholog Prp5 is essential for spliceosome assembly, defining the gene as a core splicing factor before any biochemical activity was known.\",\n      \"evidence\": \"genetic complementation of a temperature-sensitive yeast mutant with splicing assays\",\n      \"pmids\": [\"2349233\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the molecular activity\", \"No partner proteins identified\", \"No mechanism for how assembly fails\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Placed Prp5 in a functional module with SF3a-type factors acting on U2 snRNA, and first implicated ATP and helicase activity in promoting a U1/U2 snRNP conformational change.\",\n      \"evidence\": \"genetic epistasis and biochemical complementation with in vitro splicing in yeast\",\n      \"pmids\": [\"8405998\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ATPase activity not directly demonstrated\", \"Which snRNP is remodeled left ambiguous\", \"No structural basis\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Demonstrated that Prp5 is itself an RNA-dependent ATPase with selectivity for U2 snRNA that drives an ATP-dependent conformational change exposing the branch point, converting the genetic inference into a defined enzymatic activity.\",\n      \"evidence\": \"in vitro ATPase assay with purified protein and RNaseH probing in extracts\",\n      \"pmids\": [\"8969184\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RNA-binding sites on U2 not mapped\", \"Did not address fidelity/proofreading\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapped the conformational-change function to the ATP-binding helicase domain by linking a temperature-sensitive mutation in motif I to reduced U2 snRNP remodeling.\",\n      \"evidence\": \"2'-O-methyl oligonucleotide binding and physical association assays with ts-mutant inactivation in yeast\",\n      \"pmids\": [\"11927574\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Structural detail of the conformational change absent\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showed that Prp5 physically bridges U1 and U2 snRNPs through distinct domains and that ATP binding/hydrolysis is required for complex A formation, generalizing the mechanism across human and S. pombe.\",\n      \"evidence\": \"depletion-reconstitution, reciprocal Co-IP, domain mapping and native complex isolation across organisms\",\n      \"pmids\": [\"14713954\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural geometry of the bridge unknown\", \"Domain boundaries for each snRNP contact coarse\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined Prp5 ATPase activity as a branch-site fidelity checkpoint by showing ATPase-weakening mutations rescue suboptimal branch sites in a manner reversed by compensatory U2 mutations.\",\n      \"evidence\": \"alanine scanning, in vivo and in vitro ATPase/splicing assays, genetic epistasis with U2 snRNA mutations\",\n      \"pmids\": [\"18082608\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physical basis of gating not yet shown\", \"Release step not directly observed\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended Prp5 association beyond early assembly, showing it accompanies pre-mRNA from the commitment complex through disassembly with both ATP-independent and ATP-dependent roles.\",\n      \"evidence\": \"GST pulldown with radiolabeled pre-mRNA, glycerol gradient sedimentation, ATP-depletion in vitro\",\n      \"pmids\": [\"19451545\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional role at later stages undefined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved the proofreading mechanism: Prp5 binds U2 RNA flanking the branchpoint-interacting stem-loop and is released only upon correct U2-branch-site pairing, coupling fidelity to tri-snRNP recruitment.\",\n      \"evidence\": \"prespliceosome isolation, RNA-protein crosslinking and mutant functional analysis\",\n      \"pmids\": [\"25561497\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure of bound state absent\", \"Coupling to ATP cycle not visualized\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified SF3B1/Hsh155 HEAT-domain contacts as the physical determinant of branch-site selectivity, explaining how cancer-associated SF3B1 mutations phenocopy Prp5 fidelity mutations.\",\n      \"evidence\": \"two-hybrid, pulldown/Co-IP, in vivo/in vitro splicing and yeast genetics\",\n      \"pmids\": [\"28087715\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural interface only inferred at this stage\", \"Human cancer relevance correlative\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed U2 snRNA pseudouridylation tunes Prp5 enzymatic output, linking an RNA modification to ATPase activity, U2 binding and spliceosome assembly fidelity.\",\n      \"evidence\": \"in vitro ATPase/RNA-binding assays, designer snoRNA rescue, DMS probing and in vivo splicing\",\n      \"pmids\": [\"26873591\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which pseudouridines stimulate ATPase unresolved\", \"Conservation to human DDX46 not tested here\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linked RecA-domain dynamics to fidelity by showing the open/closed conformational switch requires simultaneous ATP and RNA, and that fidelity-altering mutations change switch dynamics.\",\n      \"evidence\": \"single-molecule FRET with mutagenesis\",\n      \"pmids\": [\"31712821\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct correlation to in vivo proofreading kinetics incomplete\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided the structural mechanism for indirect proofreading: cryo-EM of a pre-A intermediate showed Prp5 blocks U1/U2 repositioning until Hsh155 HEAT closure on the bulged branch adenosine destabilizes Prp5.\",\n      \"evidence\": \"cryo-EM of stalled spliceosome intermediates with branch-adenosine deletion and mutagenesis\",\n      \"pmids\": [\"34349264\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Human DDX46 complex not resolved in this study\", \"ATP hydrolysis coupling to release timing\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined the human DDX46-SF3b interface structurally, showing N-terminal anchoring on SF3B1 with the N-plug in the RNA path, mutual exclusivity with DDX42, and direct contact with cancer-mutated SF3B1 residues.\",\n      \"evidence\": \"cryo-EM of DDX46-SF3b and DDX46-U2 intermediates with in vitro binding and mutagenesis\",\n      \"pmids\": [\"36797247\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of DDX46/DDX42 exchange in cells unresolved\", \"Dynamics of N-plug displacement not captured\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Connected splicing-factor function to transcription by showing Prp5 directly binds the SAGA component Spt8p, coupling pre-spliceosome assembly to Pol II recruitment at intron-containing genes.\",\n      \"evidence\": \"in vitro interaction, ChIP/ChIP-seq, suppressor genetics and in vivo splicing\",\n      \"pmids\": [\"32399566\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Conservation of Spt8p coupling to human DDX46 untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Refined the early prespliceosome network, implicating Prp5 in displacing Cus2 and revealing ATP-independent assembly routes when Sub2 or Cus2 is absent.\",\n      \"evidence\": \"in vitro splicing with ATP depletion and genetic deletion of splicing factors\",\n      \"pmids\": [\"41027713\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No replication yet\", \"Order of Cus2 displacement versus ATPase cycle unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Revealed a second, splicing-independent role: nuclear DDX46 binds MAVS/TRAF3/TRAF6 transcripts via a CCGGUU element and recruits ALKBH5 through its DEAD domain to demethylate and retain them, suppressing interferon.\",\n      \"evidence\": \"RIP, Co-IP, m6A sequencing, nuclear retention assays and in vivo viral infection models\",\n      \"pmids\": [\"28846086\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How DDX46 selects CCGGUU targets mechanistically unclear\", \"Relationship to its splicing role not addressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Completed the antiviral switch by showing RNA virus infection induces caspase-dependent DDX46 cleavage and nuclear export, releasing retained immune transcripts to amplify IFN responses.\",\n      \"evidence\": \"RNA-binding protein knockout screen, caspase cleavage assays, fractionation/imaging and IFN reporter assays\",\n      \"pmids\": [\"41854249\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cleavage site and responsible caspase detail limited\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended the ALKBH5-coupled demethylation role to lymphocyte biology, showing DDX46/ALKBH5/treRNA1 demethylates BCR-signaling transcripts handed to HuR for export and translation.\",\n      \"evidence\": \"Co-IP, nuclear fractionation, m6A analysis and loss-of-function assays\",\n      \"pmids\": [\"39987653\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"treRNA1 role mechanistically thin\", \"Limited methodological detail\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established an in vivo developmental requirement, showing zebrafish Ddx46 is needed for multilineage HSC differentiation via gata1a-dependent erythro/lymphopoiesis.\",\n      \"evidence\": \"zebrafish mutant analysis, in situ hybridization and lineage marker expression\",\n      \"pmids\": [\"23635340\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether phenotype reflects splicing or immune role untested\", \"Direct targets unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated in vivo splicing function in vertebrates, with zebrafish Ddx46 mutants accumulating unspliced pre-mRNA in digestive organs and brain.\",\n      \"evidence\": \"forward genetic screen with RT-PCR splicing analysis and in situ hybridization\",\n      \"pmids\": [\"22442707\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tissue specificity of splicing dependence unexplained\", \"Affected introns not catalogued\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated DDX46 in cancer via O-GlcNAcylation at Ser257 by OGT, which stabilizes the protein and drives PI3K/Akt-dependent hepatocellular carcinoma growth.\",\n      \"evidence\": \"Co-IP, site-specific mutagenesis, ubiquitination and PI3K/Akt signaling assays\",\n      \"pmids\": [\"41176131\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether oncogenic effect derives from splicing or immune role unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how DDX46's spliceosomal proofreading function and its ALKBH5-coupled m6A/innate-immune RNA-retention function are partitioned or co-regulated within the same nuclear protein.\",\n      \"evidence\": \"no study in the timeline reconciles the splicing and immune RNA-regulatory activities\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the DDX46-ALKBH5 complex\", \"Determinants directing DDX46 to splicing versus immune transcripts unknown\", \"Human cancer mechanism not linked to either defined activity\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [4, 5, 6, 13]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [4, 7, 9, 0]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [4, 6]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [4, 6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [5, 0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 15, 16]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [2, 4, 5, 7]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 15, 16]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [18, 19]}\n    ],\n    \"complexes\": [\"U2 snRNP\", \"SF3b\", \"pre-spliceosome (complex A)\"],\n    \"partners\": [\"SF3B1\", \"ALKBH5\", \"PRP9\", \"PRP11\", \"PRP21\", \"Spt8\", \"JMJD6\", \"OGT\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}