{"gene":"DHX33","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2011,"finding":"DHX33 is a cell cycle-regulated nucleolar protein that associates with ribosomal DNA (rDNA) loci and interacts with the RNA Pol I transcription factor UBF. DHX33 knockdown decreased the association of Pol I with rDNA and caused a dramatic decrease in 47S rRNA synthesis. A DNA binding-defective mutant could not enhance rRNA synthesis, and an NTPase-defective mutant (K94R) acted as a dominant negative, inhibiting endogenous rRNA synthesis. DHX33 deficiency in primary human fibroblasts triggered a nucleolar p53 stress response and attenuated proliferation.","method":"Lentiviral RNAi screen, ChIP assay (rDNA association), Co-IP (UBF interaction), dominant-negative mutagenesis, rRNA synthesis assay, cell proliferation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (ChIP, Co-IP, mutagenesis, functional rRNA assay) in a single focused study, replicated in primary fibroblasts","pmids":["21930779"],"is_preprint":false},{"year":2013,"finding":"DHX33 is a cytosolic RNA sensor that activates the NLRP3 inflammasome. DHX33 binds dsRNA via its helicase C domain, interacts with NLRP3, and forms the inflammasome complex following RNA stimulation. shRNA knockdown of DHX33 blocked caspase-1 activation and IL-18/IL-1β secretion in human macrophages stimulated with poly I:C, reoviral RNA, or bacterial RNA.","method":"shRNA knockdown, Co-IP (DHX33-NLRP3 interaction), domain mapping (helicase C domain for dsRNA binding), caspase-1 activation assay, cytokine secretion assay","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, domain-mapping, functional loss-of-function with defined cytokine readout; replicated with multiple RNA stimuli","pmids":["23871209"],"is_preprint":false},{"year":2013,"finding":"DHX33 acts as a novel dsRNA sensor in myeloid dendritic cells (mDCs). The HELICc domain of DHX33 binds poly I:C. DHX33 interacts with IPS-1/MAVS via its HELICc region and the C-terminal domain of IPS-1, independently of RIG-I/MDA5. DHX33 knockdown blocked poly I:C- and reovirus-induced type I IFN production and activation of MAP kinases, NF-κB, and IRF3 in mDCs.","method":"shRNA knockdown, domain mapping (HELICc binding to poly I:C and IPS-1 C-terminus), Co-IP (DHX33–IPS-1 interaction), type I IFN production assay, MAP kinase/NF-κB/IRF3 activation assays","journal":"Cellular & molecular immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with domain mapping, functional KD with defined signaling readouts, single lab but multiple orthogonal methods","pmids":["24037184"],"is_preprint":false},{"year":2013,"finding":"DHX33 expression is exclusively controlled at the level of translation. ARF dramatically reduced polysome-associated DHX33 mRNAs, while RasV12 shifted DHX33 mRNAs to actively translating polysomes. RasV12-driven DHX33 translation was sensitive to PI3K, mTOR, and MAPK inhibitors. DHX33 knockdown abolished RasV12-induced rRNA transcription and protein translation and prevented in vitro and in vivo transforming properties of oncogenic Ras.","method":"Polysome fractionation, rRNA synthesis assay, pharmacological inhibition (PI3K, mTOR, MAPK), shRNA knockdown, in vitro/in vivo transformation assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — polysome fractionation with pharmacological epistasis, loss-of-function with in vivo readout, single lab with multiple orthogonal approaches","pmids":["23401854"],"is_preprint":false},{"year":2015,"finding":"DHX33 promotes mRNA translation initiation. DHX33 associates with ribosomal proteins, translation factors, and mRNAs in intact complexes. DHX33 reduction markedly reduced polyribosome formation and caused global inhibition of mRNA translation, rescued by wild-type but not helicase-defective DHX33. DHX33 loss caused accumulation of mRNA in 80S ribosome complexes, consistent with a stalling at initiation. DHX33 more preferentially promoted structured mRNA translation.","method":"RNA immunoprecipitation, mass spectrometry (interactome), polysome profiling, sucrose gradient sedimentation, helicase-dead mutant rescue experiment","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — polysome profiling with helicase-dead mutant rescue and MS interactome, single lab but multiple orthogonal methods","pmids":["26100019"],"is_preprint":false},{"year":2016,"finding":"DHX33 transcriptionally controls cell cycle genes (cyclins, E2F1, CDC, MCM genes) by physically associating with their promoters and controlling loading of active RNA polymerase II. DHX33 deficiency abrogated cell cycle progression and DNA replication and led to apoptosis. CRISPR-mediated knockout of DHX33 in zebrafish downregulated cyclin A2, cyclin B2, cyclin D1, cyclin E2, cdc6, cdc20, E2F1, and MCM complexes.","method":"ChIP assay (promoter association, RNA Pol II loading), shRNA knockdown (cell cycle, DNA replication, apoptosis readouts), CRISPR/Cas9 knockout in zebrafish","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP with functional KD/KO, replicated in multiple cell lines and in vivo zebrafish model","pmids":["27601587"],"is_preprint":false},{"year":2017,"finding":"c-Myc binds to the DHX33 upstream promoter region and stimulates its transcription, placing DHX33 as a critical downstream target of c-Myc. DHX33 promotes MMP9, MMP14, and PLAU transcription by directly binding to their promoters. Knockdown of DHX33 in c-Myc overexpressing cells significantly reduced cell proliferation, migration, anchorage-independent growth, and inhibited Myc-induced acute myeloid leukemia development.","method":"ChIP assay (c-Myc binding to DHX33 promoter; DHX33 binding to MMP9/MMP14/PLAU promoters), shRNA knockdown, in vitro/in vivo tumor assays","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP for both c-Myc→DHX33 and DHX33→target gene promoters, functional KD; single lab","pmids":["28498893"],"is_preprint":false},{"year":2017,"finding":"USP36 deubiquitinase reduces ubiquitination levels of DHX33 and increases its stability. Loss of USP36 destabilizes DHX33, leading to defects in ribosomal RNA synthesis and protein translation. USP36 knockout in mice causes preimplantation lethality (morula-to-blastocyst block), and shRNA reduction of DHX33 phenocopies USP36 loss in inducing apoptosis and cell cycle arrest.","method":"Usp36-knockout mouse model, O-propargyl-puromycin incorporation (protein synthesis), Northern blot (rRNA), ubiquitination assay, shRNA knockdown","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic mouse model, multiple biochemical readouts (ubiquitination, rRNA, protein synthesis), phenocopy by DHX33 KD","pmids":["29273634"],"is_preprint":false},{"year":2018,"finding":"Purified recombinant DHX33 protein possesses both RNA and DNA duplex unwinding (helicase) activity with an ATPase activity dependent on nucleic acid duplexes. ATPase activity is coupled to unwinding activity. Mutation of the key ATP-binding residue abolished DNA/RNA unwinding. Deletion of the RKK motif (involved in rDNA binding) reduced but did not abolish DNA unwinding.","method":"In vitro biochemical assay with purified recombinant protein: ATPase assay, helicase/unwinding assay, site-directed mutagenesis (ATP-binding site, RKK motif deletion)","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis, direct biochemical measurement; single lab","pmids":["29870660"],"is_preprint":false},{"year":2018,"finding":"DHX33 produces two protein isoforms from two in-frame start codons via leaky scanning during mRNA translation. Both isoforms are translated at equal efficiency. The shorter isoform has similar cellular localization and functions to the full-length protein.","method":"Site-directed mutagenesis of AUG codons, expression in cell lines and mouse models, subcellular fractionation/localization assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis of start codons in cell lines and mouse models; single lab, two orthogonal systems","pmids":["29864424"],"is_preprint":false},{"year":2019,"finding":"A 54-kDa short DHX33 variant (DHX33-2, comprising the C-terminal helicase domain) preferentially localizes to the cytoplasm (unlike full-length DHX33-1 which is predominantly nuclear). DHX33-2 interacts with DDX3, eIF3, hnRNPs, and poly(A) binding protein, and binds a subset of mRNAs important in cell proliferation, stimulating their translation.","method":"Protein immunoprecipitation, RNA immunoprecipitation, RNA-seq, subcellular fractionation, translation assays","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP and RIP with RNA-seq and translation readouts; single lab","pmids":["30684270"],"is_preprint":false},{"year":2020,"finding":"DHX33 associates with gene promoters at CG-rich regions and recruits GADD45a (growth arrest and DNA damage protein 45a) together with AP-2β. DHX33 is required for GADD45a-mediated recruitment of TET methylcytosine dioxygenase (Tet1) to gene promoters, causing local DNA demethylation (reduced 5-hydroxymethylcytosine levels) and thereby activating transcription of a subset of genes. R-loop formation at GC-skew regions may serve as a guidance signal.","method":"ChIP assay (DHX33 promoter binding), Co-IP (DHX33–GADD45a–AP-2β interaction), 5-hmC measurement (DNA demethylation), shRNA knockdown, RNA Pol II loading assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — ChIP, Co-IP, and epigenetic (5-hmC) measurements with KD; multiple orthogonal methods in single lab","pmids":["32312884"],"is_preprint":false},{"year":2020,"finding":"DHX33 plays a critical role in the Warburg effect by transcriptionally controlling glycolytic genes (LDHA, PDK1, PKM2, ENO1, ENO2, HK1/2). DHX33 forms a complex with GADD45a and recruits it along with TET1 to glycolytic gene promoters, causing active DNA demethylation and enhanced histone H4 acetylation to promote their transcription.","method":"RNA-seq (transcriptome analysis), glycolysis activity assay, ChIP assay (promoter binding), Co-IP (DHX33–GADD45a–TET1 complex), DNA methylation/histone acetylation assays","journal":"Journal of cellular physiology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — RNA-seq, ChIP, Co-IP, and epigenetic assays; multiple orthogonal methods in single lab","pmids":["32617965"],"is_preprint":false},{"year":2022,"finding":"GSK-3β directly phosphorylates DHX33 at T482, triggering K48-linked ubiquitination-mediated proteasomal degradation of DHX33. K94 on the N-terminal region was identified as a major ubiquitination site. Cancer cells with frequent GSK-3β deactivation have elevated DHX33 stability as a result.","method":"In vitro kinase assay (GSK-3β phosphorylation of DHX33), site-directed mutagenesis (T482, K94), ubiquitination assay, protein stability assay in cancer cell lines and normal fibroblasts","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct phosphorylation and ubiquitination assays with mutagenesis; single lab","pmids":["36403931"],"is_preprint":false},{"year":2023,"finding":"Dhx33 plays an indispensable role in activation-induced upregulation of rDNA transcription in B cells. B-cell-specific deletion of Dhx33 impaired B-cell development, germinal center reactions, plasma cell differentiation, and antibody production. In the absence of Dhx33, activated B cells failed to upregulate 47S rRNA production and ribosome biogenesis, resulting in nucleolar stress, p53 accumulation, and cell death.","method":"CRISPR/Cas9-mediated functional analysis, conditional (B-cell-specific) knockout mouse model, rRNA synthesis assay, p53 immunoblot, flow cytometry (B-cell development/differentiation)","journal":"Cellular & molecular immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic in vivo mouse model with multiple orthogonal readouts (rRNA, p53, cell differentiation), replicated across B-cell developmental stages","pmids":["36631557"],"is_preprint":false},{"year":2023,"finding":"DHX33 binds to the promoters of HMGB family genes and regulates their transcription through promoter DNA demethylation in cancer cells. In RAS-driven lung tumorigenesis, DHX33 knockout debilitated RAS-induced nuclear HMGB overexpression.","method":"ChIP assay (DHX33 binding to HMGB promoters), DNA methylation assay, shRNA/KO in cancer cells and in vivo lung tumor model","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — ChIP and methylation assay with in vivo validation; single lab","pmids":["37543097"],"is_preprint":false},{"year":2023,"finding":"DHX33 mediates p53-null/mutant-driven upregulation of mevalonate pathway gene transcription. In p53 mutant/KrasG12D mice, DHX33 loss significantly debilitated upregulation of mevalonate pathway genes. DHX33 knockdown in human cancer cells inhibited mevalonate pathway gene transcription, placing DHX33 downstream of mutant p53 and Ras in this metabolic pathway.","method":"shRNA knockdown in cancer cells, genetic mouse model (p53 mutant + KrasG12D with DHX33 loss), RNA expression analysis","journal":"Biochimica et biophysica acta. General subjects","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in vivo mouse model and cell-based KD; single lab","pmids":["38143011"],"is_preprint":false},{"year":2024,"finding":"DHX33 promotes expression of lipid metabolism genes FADS1, FADS2, and SCD1, thereby sensitizing cancer cells to ferroptosis-mediated cell death. A DHX33 helicase inhibitor (KY386) kills cancer cells via the ferroptosis pathway.","method":"Small molecule inhibitor (KY386) treatment, gene expression analysis (FADS1/FADS2/SCD1), ferroptosis pathway assays, cell viability assays in vitro and in vivo","journal":"ACS omega","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pharmacological inhibitor with gene expression and cell death readouts; single lab, indirect mechanistic link","pmids":["38973855"],"is_preprint":false}],"current_model":"DHX33 is a multifunctional DEAH-box RNA/DNA helicase that operates in the nucleus as a cell cycle-regulated driver of rDNA transcription (interacting with UBF and RNA Pol I), as a transcriptional regulator that binds gene promoters—recruiting GADD45a/TET1 to cause local DNA demethylation and activate cell cycle, glycolytic, and other cancer-promoting genes—and in the cytoplasm as a dsRNA sensor that binds dsRNA via its HELICc domain and activates either the NLRP3 inflammasome (via direct interaction with NLRP3) or the IPS-1/MAVS type I IFN pathway depending on cell type; its ATPase-coupled helicase activity is essential for these functions, and its protein stability is regulated by GSK-3β-mediated phosphorylation at T482 (promoting ubiquitination/degradation) and by USP36-mediated deubiquitination (stabilization), while its translation is controlled by the PI3K/mTOR/MAPK axis downstream of oncogenic Ras."},"narrative":{"mechanistic_narrative":"DHX33 is a DEAH-box helicase with intrinsic ATP-dependent RNA and DNA duplex-unwinding activity that operates as a multifunctional driver of ribosome biogenesis, transcription, and innate immune sensing, with its ATPase-coupled helicase activity essential across these roles [PMID:29870660, PMID:21930779]. In the nucleolus it is a cell cycle-regulated activator of RNA Pol I transcription, associating with rDNA loci and the transcription factor UBF to drive 47S rRNA synthesis; its loss triggers a nucleolar p53 stress response and arrests proliferation [PMID:21930779], a requirement made physiologically concrete in activated B cells where Dhx33 is indispensable for activation-induced rDNA transcription, germinal center reactions, and antibody production [PMID:36631557]. Beyond rRNA, DHX33 binds gene promoters at CG-rich regions and recruits a GADD45a/AP-2β complex together with TET1 to drive local DNA demethylation and transcriptional activation of cell cycle, glycolytic (Warburg), HMGB, and other target genes, thereby controlling RNA Pol II loading [PMID:27601587, PMID:32312884, PMID:32617965]. DHX33 also promotes cap- and structure-dependent mRNA translation initiation, associating with ribosomal proteins, translation factors, and mRNAs, with a cytoplasmic short isoform partnering DDX3, eIF3, hnRNPs, and PABP [PMID:26100019, PMID:30684270]. In the cytoplasm DHX33 functions as a dsRNA sensor that binds dsRNA through its HELICc domain and either nucleates the NLRP3 inflammasome through direct NLRP3 interaction in macrophages or engages IPS-1/MAVS to drive type I IFN in dendritic cells [PMID:23871209, PMID:24037184]. DHX33 sits at an oncogenic nexus: it is a c-Myc transcriptional target and a translationally controlled effector of oncogenic Ras via the PI3K/mTOR/MAPK axis [PMID:28498893, PMID:23401854], and its abundance is set post-translationally by GSK-3β phosphorylation at T482 promoting K48-ubiquitination/degradation and by USP36-mediated deubiquitination promoting stability [PMID:36403931, PMID:29273634].","teleology":[{"year":2011,"claim":"Established DHX33's first defined cellular role by showing it is a nucleolar, cell cycle-regulated activator of Pol I-driven rRNA synthesis rather than an uncharacterized helicase.","evidence":"RNAi, ChIP at rDNA, Co-IP with UBF, dominant-negative NTPase mutant, and rRNA synthesis/proliferation assays in primary fibroblasts","pmids":["21930779"],"confidence":"High","gaps":["Did not resolve how DHX33 is recruited to rDNA versus other genomic loci","Catalytic activity was inferred from mutants, not measured directly"]},{"year":2013,"claim":"Revealed an unexpected cytoplasmic innate-immune function, defining DHX33 as a dsRNA sensor that bifurcates into inflammasome and IFN signaling depending on cell type.","evidence":"shRNA knockdown, reciprocal Co-IP and domain mapping (HELICc for dsRNA/partner binding) with NLRP3 in macrophages and IPS-1/MAVS in dendritic cells; caspase-1, cytokine, and type I IFN readouts","pmids":["23871209","24037184"],"confidence":"High","gaps":["How a single helicase chooses between NLRP3 and MAVS pathways is unresolved","Relationship between nuclear and cytoplasmic pools not addressed"]},{"year":2013,"claim":"Connected DHX33 to oncogenic signaling by showing its expression is gated at translation downstream of Ras through PI3K/mTOR/MAPK, making it a required effector of Ras transformation.","evidence":"Polysome fractionation, pharmacological epistasis (PI3K/mTOR/MAPK inhibitors), shRNA, and in vitro/in vivo transformation assays","pmids":["23401854"],"confidence":"High","gaps":["The specific cis-elements directing translational control of DHX33 mRNA were not mapped"]},{"year":2015,"claim":"Expanded DHX33's function from rRNA synthesis to global mRNA translation initiation, showing it resolves structured mRNAs to permit polysome assembly.","evidence":"RNA-IP, MS interactome, polysome profiling and sucrose gradients with helicase-dead rescue","pmids":["26100019"],"confidence":"High","gaps":["Which step of initiation DHX33 acts at mechanistically was not defined beyond 80S accumulation"]},{"year":2016,"claim":"Demonstrated DHX33 acts as a direct transcriptional regulator of cell cycle genes via promoter binding and Pol II loading, linking it to proliferation and DNA replication in vivo.","evidence":"ChIP for promoter association and Pol II loading, shRNA phenotyping, and CRISPR knockout in zebrafish","pmids":["27601587"],"confidence":"High","gaps":["How DHX33 is targeted to specific gene promoters was not yet known"]},{"year":2017,"claim":"Placed DHX33 in the c-Myc oncogenic circuit and identified pro-invasive transcriptional targets, while a separate study defined USP36 as the deubiquitinase stabilizing DHX33.","evidence":"ChIP for c-Myc→DHX33 and DHX33→MMP9/MMP14/PLAU promoters with tumor assays; Usp36-knockout mouse, ubiquitination/rRNA/protein-synthesis readouts with DHX33-KD phenocopy","pmids":["28498893","29273634"],"confidence":"High","gaps":["The E3 ligase opposing USP36 was not identified in this work","Direct DHX33–USP36 binding interface not mapped"]},{"year":2018,"claim":"Provided direct biochemical proof that DHX33 is a bona fide ATP-dependent RNA/DNA helicase and characterized isoform generation by leaky scanning.","evidence":"In vitro ATPase and unwinding assays with purified recombinant protein and ATP-binding/RKK-motif mutants; AUG-codon mutagenesis defining two equally translated isoforms","pmids":["29870660","29864424"],"confidence":"High","gaps":["No structural model of the helicase or duplex-engagement mechanism","Physiological substrate specificity in cells not resolved by in vitro assays"]},{"year":2019,"claim":"Defined a cytoplasmic short isoform (DHX33-2) with a distinct translation-promoting interactome, distinguishing it from the predominantly nuclear full-length protein.","evidence":"Protein-IP, RIP with RNA-seq, subcellular fractionation and translation assays identifying DDX3/eIF3/hnRNP/PABP partners","pmids":["30684270"],"confidence":"Medium","gaps":["Functional non-redundancy between isoforms not established by genetic separation","Single lab, no reciprocal validation of all partners"]},{"year":2020,"claim":"Uncovered the mechanism by which DHX33 activates target genes—recruiting a GADD45a/AP-2β/TET1 demethylation machinery to CG-rich promoters—and applied it to glycolytic gene control underlying the Warburg effect.","evidence":"ChIP, Co-IP of DHX33–GADD45a–AP-2β/TET1, 5-hmC and histone acetylation measurements, RNA-seq and glycolysis assays with knockdown","pmids":["32312884","32617965"],"confidence":"High","gaps":["The proposed R-loop guidance signal for promoter targeting was not directly demonstrated","Whether helicase activity is required for demethylation recruitment not isolated"]},{"year":2023,"claim":"Demonstrated a genetic in vivo requirement for Dhx33 in B-cell rRNA upregulation and immunity, and extended the demethylation-driven transcriptional program to HMGB and mevalonate-pathway genes downstream of Ras and mutant p53.","evidence":"B-cell conditional knockout mouse with rRNA/p53/differentiation readouts; ChIP/methylation assays and p53-mutant/KrasG12D mouse epistasis","pmids":["36631557","37543097","38143011"],"confidence":"High","gaps":["Mechanism coupling oncogenic genotype to DHX33 target selection not defined","HMGB/mevalonate links are Medium-confidence single-lab studies"]},{"year":2022,"claim":"Established post-translational control of DHX33 abundance through GSK-3β phosphorylation at T482 driving K48-ubiquitination, explaining elevated DHX33 stability in cancers with GSK-3β inactivation.","evidence":"In vitro kinase assay, T482/K94 mutagenesis, ubiquitination and protein-stability assays in cancer cells and fibroblasts","pmids":["36403931"],"confidence":"Medium","gaps":["The responsible E3 ligase was not identified","Single lab; in vivo relevance of the T482 phospho-degron not tested"]},{"year":2024,"claim":"Linked DHX33 to lipid metabolism and ferroptosis sensitivity and provided a candidate small-molecule helicase inhibitor.","evidence":"KY386 inhibitor treatment, FADS1/FADS2/SCD1 expression and ferroptosis/viability assays in vitro and in vivo","pmids":["38973855"],"confidence":"Low","gaps":["Indirect mechanistic link; inhibitor specificity for DHX33 not rigorously established","Single lab, no genetic confirmation of the ferroptosis axis"]},{"year":null,"claim":"How DHX33 partitions between its nuclear (rDNA/promoter), translational, and cytoplasmic immune-sensing functions, and what determines its promoter and substrate selectivity, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of DHX33 or its complexes","Signals controlling subcellular partitioning of isoforms unknown","Rules for promoter/mRNA target selection undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[8,0]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[1,2,4,8]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,8,11]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[8,4]},{"term_id":"GO:0140097","term_label":"catalytic activity, acting on DNA","supporting_discovery_ids":[8]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[8]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[5,6,11,12]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[1,2]},{"term_id":"GO:0045182","term_label":"translation regulator activity","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[0,14]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[5,11,9]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,2,10]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,5,11,12]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,4]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[4]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[5]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,2,14]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[12,16,17]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[11,12]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,6,16]}],"complexes":["NLRP3 inflammasome"],"partners":["UBF","NLRP3","MAVS","GADD45A","TET1","TFAP2B","DDX3X","USP36"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9H6R0","full_name":"ATP-dependent RNA helicase DHX33","aliases":["DEAH box protein 33"],"length_aa":707,"mass_kda":78.9,"function":"Implicated in nucleolar organization, ribosome biogenesis, protein synthesis and cytoplasmic dsRNA sensing (By similarity) (PubMed:21930779, PubMed:23871209, PubMed:26100019). Stimulates RNA polymerase I transcription of the 47S precursor rRNA. Associates with ribosomal DNA (rDNA) loci where it is involved in POLR1A recruitment (PubMed:21930779). In the cytoplasm, promotes elongation-competent 80S ribosome assembly at the late stage of mRNA translation initiation (PubMed:26100019). Senses cytosolic dsRNA mediating NLRP3 inflammasome formation in macrophages and type I interferon production in myeloid dendritic cells (PubMed:23871209). Required for NLRP3 activation induced by viral dsRNA and bacterial RNA (PubMed:23871209). In dendritic cells, required for induction of type I interferon production induced by cytoplasmic dsRNA via the activation of MAPK and NF-kappa-B signaling pathways (By similarity)","subcellular_location":"Nucleus, nucleolus; Nucleus, nucleoplasm; Cytoplasm; Nucleus; Inflammasome","url":"https://www.uniprot.org/uniprotkb/Q9H6R0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/DHX33","classification":"Common Essential","n_dependent_lines":1166,"n_total_lines":1208,"dependency_fraction":0.9652317880794702},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"USP36","stoichiometry":4.0},{"gene":"FKBP5","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/DHX33","total_profiled":1310},"omim":[{"mim_id":"614405","title":"DEAH-BOX HELICASE 33; DHX33","url":"https://www.omim.org/entry/614405"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoli","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DHX33"},"hgnc":{"alias_symbol":["FLJ21972","DKFZp762F2011"],"prev_symbol":["DDX33"]},"alphafold":{"accession":"Q9H6R0","domains":[{"cath_id":"3.40.50.300","chopping":"64-252","consensus_level":"high","plddt":90.3928,"start":64,"end":252},{"cath_id":"3.40.50.300","chopping":"259-434","consensus_level":"high","plddt":91.0686,"start":259,"end":434},{"cath_id":"1.10.10","chopping":"439-500","consensus_level":"medium","plddt":89.2418,"start":439,"end":500},{"cath_id":"-","chopping":"505-707","consensus_level":"medium","plddt":90.5547,"start":505,"end":707}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H6R0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H6R0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H6R0-F1-predicted_aligned_error_v6.png","plddt_mean":85.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DHX33","jax_strain_url":"https://www.jax.org/strain/search?query=DHX33"},"sequence":{"accession":"Q9H6R0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H6R0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H6R0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H6R0"}},"corpus_meta":[{"pmid":"23871209","id":"PMC_23871209","title":"The DHX33 RNA helicase senses cytosolic RNA and activates the NLRP3 inflammasome.","date":"2013","source":"Immunity","url":"https://pubmed.ncbi.nlm.nih.gov/23871209","citation_count":174,"is_preprint":false},{"pmid":"21930779","id":"PMC_21930779","title":"Identification of DHX33 as a mediator of rRNA synthesis and cell growth.","date":"2011","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/21930779","citation_count":55,"is_preprint":false},{"pmid":"24037184","id":"PMC_24037184","title":"The interaction between the helicase DHX33 and IPS-1 as a novel pathway to sense double-stranded RNA and RNA viruses in myeloid dendritic cells.","date":"2013","source":"Cellular & molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/24037184","citation_count":49,"is_preprint":false},{"pmid":"29273634","id":"PMC_29273634","title":"Loss of the deubiquitinase USP36 destabilizes the RNA helicase DHX33 and causes preimplantation lethality in mice.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29273634","citation_count":38,"is_preprint":false},{"pmid":"27693040","id":"PMC_27693040","title":"miR-634 exhibits anti-tumor activities toward hepatocellular carcinoma via Rab1A and DHX33.","date":"2016","source":"Molecular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/27693040","citation_count":35,"is_preprint":false},{"pmid":"26100019","id":"PMC_26100019","title":"The DHX33 RNA Helicase Promotes mRNA Translation Initiation.","date":"2015","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/26100019","citation_count":30,"is_preprint":false},{"pmid":"28498893","id":"PMC_28498893","title":"Role of DHX33 in c-Myc-induced cancers.","date":"2017","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/28498893","citation_count":29,"is_preprint":false},{"pmid":"23401854","id":"PMC_23401854","title":"P19ARF and RasV¹² offer opposing regulation of DHX33 translation to dictate tumor cell fate.","date":"2013","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/23401854","citation_count":24,"is_preprint":false},{"pmid":"27601587","id":"PMC_27601587","title":"DHX33 Transcriptionally Controls Genes Involved in the Cell Cycle.","date":"2016","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/27601587","citation_count":24,"is_preprint":false},{"pmid":"30552990","id":"PMC_30552990","title":"The RNA helicase DHX33 is required for cancer cell proliferation in human glioblastoma and confers resistance to PI3K/mTOR inhibition.","date":"2018","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/30552990","citation_count":24,"is_preprint":false},{"pmid":"32004669","id":"PMC_32004669","title":"DHX33 promotes colon cancer development downstream of Wnt signaling.","date":"2020","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/32004669","citation_count":18,"is_preprint":false},{"pmid":"32717723","id":"PMC_32717723","title":"Circular RNA DHX33 promotes malignant behavior in ccRCC by targeting miR-489-3p/MEK1 axis.","date":"2020","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/32717723","citation_count":17,"is_preprint":false},{"pmid":"33843021","id":"PMC_33843021","title":"Long non-coding RNA HOTAIR promotes hepatocellular carcinoma progression by regulating miR-526b-3p/DHX33 axis.","date":"2021","source":"Genes & genomics","url":"https://pubmed.ncbi.nlm.nih.gov/33843021","citation_count":17,"is_preprint":false},{"pmid":"32617965","id":"PMC_32617965","title":"Function of DHX33 in promoting Warburg effect via regulation of glycolytic genes.","date":"2020","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/32617965","citation_count":16,"is_preprint":false},{"pmid":"32767810","id":"PMC_32767810","title":"Targeting RNA helicase DHX33 blocks Ras-driven lung tumorigenesis in vivo.","date":"2020","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/32767810","citation_count":13,"is_preprint":false},{"pmid":"36631557","id":"PMC_36631557","title":"Dhx33 promotes B-cell growth and proliferation by controlling activation-induced rRNA upregulation.","date":"2023","source":"Cellular & molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/36631557","citation_count":12,"is_preprint":false},{"pmid":"32312884","id":"PMC_32312884","title":"DHX33 Recruits Gadd45a To Cause DNA Demethylation and Regulates a Subset of Gene Transcription.","date":"2020","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/32312884","citation_count":10,"is_preprint":false},{"pmid":"29870660","id":"PMC_29870660","title":"Recombinant DHX33 Protein Possesses Dual DNA/RNA Helicase Activity.","date":"2018","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29870660","citation_count":8,"is_preprint":false},{"pmid":"23890068","id":"PMC_23890068","title":"RNA helicase DHX33 puts a new twist on NLRP3 inflammasome activation.","date":"2013","source":"Immunity","url":"https://pubmed.ncbi.nlm.nih.gov/23890068","citation_count":7,"is_preprint":false},{"pmid":"36403931","id":"PMC_36403931","title":"GSK-3β phosphorylation of DHX33 leads to its ubiquitination mediated protein degradation.","date":"2022","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/36403931","citation_count":4,"is_preprint":false},{"pmid":"37838340","id":"PMC_37838340","title":"Development of small molecule inhibitors targeting RNA helicase DHX33 as anti-cancer agents.","date":"2023","source":"Bioorganic & medicinal chemistry letters","url":"https://pubmed.ncbi.nlm.nih.gov/37838340","citation_count":3,"is_preprint":false},{"pmid":"38143011","id":"PMC_38143011","title":"DHX33 mediates p53 to regulate mevalonate pathway gene transcription in human cancers.","date":"2023","source":"Biochimica et biophysica acta. General subjects","url":"https://pubmed.ncbi.nlm.nih.gov/38143011","citation_count":2,"is_preprint":false},{"pmid":"29864424","id":"PMC_29864424","title":"Alternative translation initiation from two in-frame start codons in DHX33 gene.","date":"2018","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/29864424","citation_count":2,"is_preprint":false},{"pmid":"37543097","id":"PMC_37543097","title":"RNA helicase DHX33 regulates HMGB family genes in human cancer cells.","date":"2023","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/37543097","citation_count":1,"is_preprint":false},{"pmid":"30684270","id":"PMC_30684270","title":"A 54-kDa short variant of DHX33 functions in regulating mRNA translation.","date":"2019","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/30684270","citation_count":1,"is_preprint":false},{"pmid":"38973855","id":"PMC_38973855","title":"An RNA Helicase DHX33 Inhibitor Shows Broad Anticancer Activity via Inducing Ferroptosis in Cancer Cells.","date":"2024","source":"ACS omega","url":"https://pubmed.ncbi.nlm.nih.gov/38973855","citation_count":0,"is_preprint":false},{"pmid":"42243442","id":"PMC_42243442","title":"DHX33 inhibitors induced transcriptional changes for a subset of genes in cancer cells.","date":"2026","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/42243442","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12074,"output_tokens":5096,"usd":0.056331,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13445,"output_tokens":4771,"usd":0.09325,"stage2_stop_reason":"end_turn"},"total_usd":0.149581,"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\": 2011,\n      \"finding\": \"DHX33 is a cell cycle-regulated nucleolar protein that associates with ribosomal DNA (rDNA) loci and interacts with the RNA Pol I transcription factor UBF. DHX33 knockdown decreased the association of Pol I with rDNA and caused a dramatic decrease in 47S rRNA synthesis. A DNA binding-defective mutant could not enhance rRNA synthesis, and an NTPase-defective mutant (K94R) acted as a dominant negative, inhibiting endogenous rRNA synthesis. DHX33 deficiency in primary human fibroblasts triggered a nucleolar p53 stress response and attenuated proliferation.\",\n      \"method\": \"Lentiviral RNAi screen, ChIP assay (rDNA association), Co-IP (UBF interaction), dominant-negative mutagenesis, rRNA synthesis assay, cell proliferation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (ChIP, Co-IP, mutagenesis, functional rRNA assay) in a single focused study, replicated in primary fibroblasts\",\n      \"pmids\": [\"21930779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"DHX33 is a cytosolic RNA sensor that activates the NLRP3 inflammasome. DHX33 binds dsRNA via its helicase C domain, interacts with NLRP3, and forms the inflammasome complex following RNA stimulation. shRNA knockdown of DHX33 blocked caspase-1 activation and IL-18/IL-1β secretion in human macrophages stimulated with poly I:C, reoviral RNA, or bacterial RNA.\",\n      \"method\": \"shRNA knockdown, Co-IP (DHX33-NLRP3 interaction), domain mapping (helicase C domain for dsRNA binding), caspase-1 activation assay, cytokine secretion assay\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, domain-mapping, functional loss-of-function with defined cytokine readout; replicated with multiple RNA stimuli\",\n      \"pmids\": [\"23871209\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"DHX33 acts as a novel dsRNA sensor in myeloid dendritic cells (mDCs). The HELICc domain of DHX33 binds poly I:C. DHX33 interacts with IPS-1/MAVS via its HELICc region and the C-terminal domain of IPS-1, independently of RIG-I/MDA5. DHX33 knockdown blocked poly I:C- and reovirus-induced type I IFN production and activation of MAP kinases, NF-κB, and IRF3 in mDCs.\",\n      \"method\": \"shRNA knockdown, domain mapping (HELICc binding to poly I:C and IPS-1 C-terminus), Co-IP (DHX33–IPS-1 interaction), type I IFN production assay, MAP kinase/NF-κB/IRF3 activation assays\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with domain mapping, functional KD with defined signaling readouts, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"24037184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"DHX33 expression is exclusively controlled at the level of translation. ARF dramatically reduced polysome-associated DHX33 mRNAs, while RasV12 shifted DHX33 mRNAs to actively translating polysomes. RasV12-driven DHX33 translation was sensitive to PI3K, mTOR, and MAPK inhibitors. DHX33 knockdown abolished RasV12-induced rRNA transcription and protein translation and prevented in vitro and in vivo transforming properties of oncogenic Ras.\",\n      \"method\": \"Polysome fractionation, rRNA synthesis assay, pharmacological inhibition (PI3K, mTOR, MAPK), shRNA knockdown, in vitro/in vivo transformation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — polysome fractionation with pharmacological epistasis, loss-of-function with in vivo readout, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"23401854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"DHX33 promotes mRNA translation initiation. DHX33 associates with ribosomal proteins, translation factors, and mRNAs in intact complexes. DHX33 reduction markedly reduced polyribosome formation and caused global inhibition of mRNA translation, rescued by wild-type but not helicase-defective DHX33. DHX33 loss caused accumulation of mRNA in 80S ribosome complexes, consistent with a stalling at initiation. DHX33 more preferentially promoted structured mRNA translation.\",\n      \"method\": \"RNA immunoprecipitation, mass spectrometry (interactome), polysome profiling, sucrose gradient sedimentation, helicase-dead mutant rescue experiment\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — polysome profiling with helicase-dead mutant rescue and MS interactome, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"26100019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"DHX33 transcriptionally controls cell cycle genes (cyclins, E2F1, CDC, MCM genes) by physically associating with their promoters and controlling loading of active RNA polymerase II. DHX33 deficiency abrogated cell cycle progression and DNA replication and led to apoptosis. CRISPR-mediated knockout of DHX33 in zebrafish downregulated cyclin A2, cyclin B2, cyclin D1, cyclin E2, cdc6, cdc20, E2F1, and MCM complexes.\",\n      \"method\": \"ChIP assay (promoter association, RNA Pol II loading), shRNA knockdown (cell cycle, DNA replication, apoptosis readouts), CRISPR/Cas9 knockout in zebrafish\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP with functional KD/KO, replicated in multiple cell lines and in vivo zebrafish model\",\n      \"pmids\": [\"27601587\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"c-Myc binds to the DHX33 upstream promoter region and stimulates its transcription, placing DHX33 as a critical downstream target of c-Myc. DHX33 promotes MMP9, MMP14, and PLAU transcription by directly binding to their promoters. Knockdown of DHX33 in c-Myc overexpressing cells significantly reduced cell proliferation, migration, anchorage-independent growth, and inhibited Myc-induced acute myeloid leukemia development.\",\n      \"method\": \"ChIP assay (c-Myc binding to DHX33 promoter; DHX33 binding to MMP9/MMP14/PLAU promoters), shRNA knockdown, in vitro/in vivo tumor assays\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP for both c-Myc→DHX33 and DHX33→target gene promoters, functional KD; single lab\",\n      \"pmids\": [\"28498893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"USP36 deubiquitinase reduces ubiquitination levels of DHX33 and increases its stability. Loss of USP36 destabilizes DHX33, leading to defects in ribosomal RNA synthesis and protein translation. USP36 knockout in mice causes preimplantation lethality (morula-to-blastocyst block), and shRNA reduction of DHX33 phenocopies USP36 loss in inducing apoptosis and cell cycle arrest.\",\n      \"method\": \"Usp36-knockout mouse model, O-propargyl-puromycin incorporation (protein synthesis), Northern blot (rRNA), ubiquitination assay, shRNA knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic mouse model, multiple biochemical readouts (ubiquitination, rRNA, protein synthesis), phenocopy by DHX33 KD\",\n      \"pmids\": [\"29273634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Purified recombinant DHX33 protein possesses both RNA and DNA duplex unwinding (helicase) activity with an ATPase activity dependent on nucleic acid duplexes. ATPase activity is coupled to unwinding activity. Mutation of the key ATP-binding residue abolished DNA/RNA unwinding. Deletion of the RKK motif (involved in rDNA binding) reduced but did not abolish DNA unwinding.\",\n      \"method\": \"In vitro biochemical assay with purified recombinant protein: ATPase assay, helicase/unwinding assay, site-directed mutagenesis (ATP-binding site, RKK motif deletion)\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis, direct biochemical measurement; single lab\",\n      \"pmids\": [\"29870660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DHX33 produces two protein isoforms from two in-frame start codons via leaky scanning during mRNA translation. Both isoforms are translated at equal efficiency. The shorter isoform has similar cellular localization and functions to the full-length protein.\",\n      \"method\": \"Site-directed mutagenesis of AUG codons, expression in cell lines and mouse models, subcellular fractionation/localization assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis of start codons in cell lines and mouse models; single lab, two orthogonal systems\",\n      \"pmids\": [\"29864424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"A 54-kDa short DHX33 variant (DHX33-2, comprising the C-terminal helicase domain) preferentially localizes to the cytoplasm (unlike full-length DHX33-1 which is predominantly nuclear). DHX33-2 interacts with DDX3, eIF3, hnRNPs, and poly(A) binding protein, and binds a subset of mRNAs important in cell proliferation, stimulating their translation.\",\n      \"method\": \"Protein immunoprecipitation, RNA immunoprecipitation, RNA-seq, subcellular fractionation, translation assays\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP and RIP with RNA-seq and translation readouts; single lab\",\n      \"pmids\": [\"30684270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"DHX33 associates with gene promoters at CG-rich regions and recruits GADD45a (growth arrest and DNA damage protein 45a) together with AP-2β. DHX33 is required for GADD45a-mediated recruitment of TET methylcytosine dioxygenase (Tet1) to gene promoters, causing local DNA demethylation (reduced 5-hydroxymethylcytosine levels) and thereby activating transcription of a subset of genes. R-loop formation at GC-skew regions may serve as a guidance signal.\",\n      \"method\": \"ChIP assay (DHX33 promoter binding), Co-IP (DHX33–GADD45a–AP-2β interaction), 5-hmC measurement (DNA demethylation), shRNA knockdown, RNA Pol II loading assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP, Co-IP, and epigenetic (5-hmC) measurements with KD; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"32312884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"DHX33 plays a critical role in the Warburg effect by transcriptionally controlling glycolytic genes (LDHA, PDK1, PKM2, ENO1, ENO2, HK1/2). DHX33 forms a complex with GADD45a and recruits it along with TET1 to glycolytic gene promoters, causing active DNA demethylation and enhanced histone H4 acetylation to promote their transcription.\",\n      \"method\": \"RNA-seq (transcriptome analysis), glycolysis activity assay, ChIP assay (promoter binding), Co-IP (DHX33–GADD45a–TET1 complex), DNA methylation/histone acetylation assays\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA-seq, ChIP, Co-IP, and epigenetic assays; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"32617965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"GSK-3β directly phosphorylates DHX33 at T482, triggering K48-linked ubiquitination-mediated proteasomal degradation of DHX33. K94 on the N-terminal region was identified as a major ubiquitination site. Cancer cells with frequent GSK-3β deactivation have elevated DHX33 stability as a result.\",\n      \"method\": \"In vitro kinase assay (GSK-3β phosphorylation of DHX33), site-directed mutagenesis (T482, K94), ubiquitination assay, protein stability assay in cancer cell lines and normal fibroblasts\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct phosphorylation and ubiquitination assays with mutagenesis; single lab\",\n      \"pmids\": [\"36403931\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Dhx33 plays an indispensable role in activation-induced upregulation of rDNA transcription in B cells. B-cell-specific deletion of Dhx33 impaired B-cell development, germinal center reactions, plasma cell differentiation, and antibody production. In the absence of Dhx33, activated B cells failed to upregulate 47S rRNA production and ribosome biogenesis, resulting in nucleolar stress, p53 accumulation, and cell death.\",\n      \"method\": \"CRISPR/Cas9-mediated functional analysis, conditional (B-cell-specific) knockout mouse model, rRNA synthesis assay, p53 immunoblot, flow cytometry (B-cell development/differentiation)\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic in vivo mouse model with multiple orthogonal readouts (rRNA, p53, cell differentiation), replicated across B-cell developmental stages\",\n      \"pmids\": [\"36631557\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"DHX33 binds to the promoters of HMGB family genes and regulates their transcription through promoter DNA demethylation in cancer cells. In RAS-driven lung tumorigenesis, DHX33 knockout debilitated RAS-induced nuclear HMGB overexpression.\",\n      \"method\": \"ChIP assay (DHX33 binding to HMGB promoters), DNA methylation assay, shRNA/KO in cancer cells and in vivo lung tumor model\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — ChIP and methylation assay with in vivo validation; single lab\",\n      \"pmids\": [\"37543097\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"DHX33 mediates p53-null/mutant-driven upregulation of mevalonate pathway gene transcription. In p53 mutant/KrasG12D mice, DHX33 loss significantly debilitated upregulation of mevalonate pathway genes. DHX33 knockdown in human cancer cells inhibited mevalonate pathway gene transcription, placing DHX33 downstream of mutant p53 and Ras in this metabolic pathway.\",\n      \"method\": \"shRNA knockdown in cancer cells, genetic mouse model (p53 mutant + KrasG12D with DHX33 loss), RNA expression analysis\",\n      \"journal\": \"Biochimica et biophysica acta. General subjects\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in vivo mouse model and cell-based KD; single lab\",\n      \"pmids\": [\"38143011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DHX33 promotes expression of lipid metabolism genes FADS1, FADS2, and SCD1, thereby sensitizing cancer cells to ferroptosis-mediated cell death. A DHX33 helicase inhibitor (KY386) kills cancer cells via the ferroptosis pathway.\",\n      \"method\": \"Small molecule inhibitor (KY386) treatment, gene expression analysis (FADS1/FADS2/SCD1), ferroptosis pathway assays, cell viability assays in vitro and in vivo\",\n      \"journal\": \"ACS omega\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pharmacological inhibitor with gene expression and cell death readouts; single lab, indirect mechanistic link\",\n      \"pmids\": [\"38973855\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DHX33 is a multifunctional DEAH-box RNA/DNA helicase that operates in the nucleus as a cell cycle-regulated driver of rDNA transcription (interacting with UBF and RNA Pol I), as a transcriptional regulator that binds gene promoters—recruiting GADD45a/TET1 to cause local DNA demethylation and activate cell cycle, glycolytic, and other cancer-promoting genes—and in the cytoplasm as a dsRNA sensor that binds dsRNA via its HELICc domain and activates either the NLRP3 inflammasome (via direct interaction with NLRP3) or the IPS-1/MAVS type I IFN pathway depending on cell type; its ATPase-coupled helicase activity is essential for these functions, and its protein stability is regulated by GSK-3β-mediated phosphorylation at T482 (promoting ubiquitination/degradation) and by USP36-mediated deubiquitination (stabilization), while its translation is controlled by the PI3K/mTOR/MAPK axis downstream of oncogenic Ras.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DHX33 is a DEAH-box helicase with intrinsic ATP-dependent RNA and DNA duplex-unwinding activity that operates as a multifunctional driver of ribosome biogenesis, transcription, and innate immune sensing, with its ATPase-coupled helicase activity essential across these roles [#8, #0]. In the nucleolus it is a cell cycle-regulated activator of RNA Pol I transcription, associating with rDNA loci and the transcription factor UBF to drive 47S rRNA synthesis; its loss triggers a nucleolar p53 stress response and arrests proliferation [#0], a requirement made physiologically concrete in activated B cells where Dhx33 is indispensable for activation-induced rDNA transcription, germinal center reactions, and antibody production [#14]. Beyond rRNA, DHX33 binds gene promoters at CG-rich regions and recruits a GADD45a/AP-2β complex together with TET1 to drive local DNA demethylation and transcriptional activation of cell cycle, glycolytic (Warburg), HMGB, and other target genes, thereby controlling RNA Pol II loading [#5, #11, #12]. DHX33 also promotes cap- and structure-dependent mRNA translation initiation, associating with ribosomal proteins, translation factors, and mRNAs, with a cytoplasmic short isoform partnering DDX3, eIF3, hnRNPs, and PABP [#4, #10]. In the cytoplasm DHX33 functions as a dsRNA sensor that binds dsRNA through its HELICc domain and either nucleates the NLRP3 inflammasome through direct NLRP3 interaction in macrophages or engages IPS-1/MAVS to drive type I IFN in dendritic cells [#1, #2]. DHX33 sits at an oncogenic nexus: it is a c-Myc transcriptional target and a translationally controlled effector of oncogenic Ras via the PI3K/mTOR/MAPK axis [#6, #3], and its abundance is set post-translationally by GSK-3β phosphorylation at T482 promoting K48-ubiquitination/degradation and by USP36-mediated deubiquitination promoting stability [#13, #7].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Established DHX33's first defined cellular role by showing it is a nucleolar, cell cycle-regulated activator of Pol I-driven rRNA synthesis rather than an uncharacterized helicase.\",\n      \"evidence\": \"RNAi, ChIP at rDNA, Co-IP with UBF, dominant-negative NTPase mutant, and rRNA synthesis/proliferation assays in primary fibroblasts\",\n      \"pmids\": [\"21930779\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how DHX33 is recruited to rDNA versus other genomic loci\", \"Catalytic activity was inferred from mutants, not measured directly\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Revealed an unexpected cytoplasmic innate-immune function, defining DHX33 as a dsRNA sensor that bifurcates into inflammasome and IFN signaling depending on cell type.\",\n      \"evidence\": \"shRNA knockdown, reciprocal Co-IP and domain mapping (HELICc for dsRNA/partner binding) with NLRP3 in macrophages and IPS-1/MAVS in dendritic cells; caspase-1, cytokine, and type I IFN readouts\",\n      \"pmids\": [\"23871209\", \"24037184\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a single helicase chooses between NLRP3 and MAVS pathways is unresolved\", \"Relationship between nuclear and cytoplasmic pools not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Connected DHX33 to oncogenic signaling by showing its expression is gated at translation downstream of Ras through PI3K/mTOR/MAPK, making it a required effector of Ras transformation.\",\n      \"evidence\": \"Polysome fractionation, pharmacological epistasis (PI3K/mTOR/MAPK inhibitors), shRNA, and in vitro/in vivo transformation assays\",\n      \"pmids\": [\"23401854\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The specific cis-elements directing translational control of DHX33 mRNA were not mapped\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Expanded DHX33's function from rRNA synthesis to global mRNA translation initiation, showing it resolves structured mRNAs to permit polysome assembly.\",\n      \"evidence\": \"RNA-IP, MS interactome, polysome profiling and sucrose gradients with helicase-dead rescue\",\n      \"pmids\": [\"26100019\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which step of initiation DHX33 acts at mechanistically was not defined beyond 80S accumulation\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated DHX33 acts as a direct transcriptional regulator of cell cycle genes via promoter binding and Pol II loading, linking it to proliferation and DNA replication in vivo.\",\n      \"evidence\": \"ChIP for promoter association and Pol II loading, shRNA phenotyping, and CRISPR knockout in zebrafish\",\n      \"pmids\": [\"27601587\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How DHX33 is targeted to specific gene promoters was not yet known\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Placed DHX33 in the c-Myc oncogenic circuit and identified pro-invasive transcriptional targets, while a separate study defined USP36 as the deubiquitinase stabilizing DHX33.\",\n      \"evidence\": \"ChIP for c-Myc→DHX33 and DHX33→MMP9/MMP14/PLAU promoters with tumor assays; Usp36-knockout mouse, ubiquitination/rRNA/protein-synthesis readouts with DHX33-KD phenocopy\",\n      \"pmids\": [\"28498893\", \"29273634\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The E3 ligase opposing USP36 was not identified in this work\", \"Direct DHX33–USP36 binding interface not mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Provided direct biochemical proof that DHX33 is a bona fide ATP-dependent RNA/DNA helicase and characterized isoform generation by leaky scanning.\",\n      \"evidence\": \"In vitro ATPase and unwinding assays with purified recombinant protein and ATP-binding/RKK-motif mutants; AUG-codon mutagenesis defining two equally translated isoforms\",\n      \"pmids\": [\"29870660\", \"29864424\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural model of the helicase or duplex-engagement mechanism\", \"Physiological substrate specificity in cells not resolved by in vitro assays\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined a cytoplasmic short isoform (DHX33-2) with a distinct translation-promoting interactome, distinguishing it from the predominantly nuclear full-length protein.\",\n      \"evidence\": \"Protein-IP, RIP with RNA-seq, subcellular fractionation and translation assays identifying DDX3/eIF3/hnRNP/PABP partners\",\n      \"pmids\": [\"30684270\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional non-redundancy between isoforms not established by genetic separation\", \"Single lab, no reciprocal validation of all partners\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Uncovered the mechanism by which DHX33 activates target genes—recruiting a GADD45a/AP-2β/TET1 demethylation machinery to CG-rich promoters—and applied it to glycolytic gene control underlying the Warburg effect.\",\n      \"evidence\": \"ChIP, Co-IP of DHX33–GADD45a–AP-2β/TET1, 5-hmC and histone acetylation measurements, RNA-seq and glycolysis assays with knockdown\",\n      \"pmids\": [\"32312884\", \"32617965\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The proposed R-loop guidance signal for promoter targeting was not directly demonstrated\", \"Whether helicase activity is required for demethylation recruitment not isolated\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated a genetic in vivo requirement for Dhx33 in B-cell rRNA upregulation and immunity, and extended the demethylation-driven transcriptional program to HMGB and mevalonate-pathway genes downstream of Ras and mutant p53.\",\n      \"evidence\": \"B-cell conditional knockout mouse with rRNA/p53/differentiation readouts; ChIP/methylation assays and p53-mutant/KrasG12D mouse epistasis\",\n      \"pmids\": [\"36631557\", \"37543097\", \"38143011\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism coupling oncogenic genotype to DHX33 target selection not defined\", \"HMGB/mevalonate links are Medium-confidence single-lab studies\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established post-translational control of DHX33 abundance through GSK-3β phosphorylation at T482 driving K48-ubiquitination, explaining elevated DHX33 stability in cancers with GSK-3β inactivation.\",\n      \"evidence\": \"In vitro kinase assay, T482/K94 mutagenesis, ubiquitination and protein-stability assays in cancer cells and fibroblasts\",\n      \"pmids\": [\"36403931\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The responsible E3 ligase was not identified\", \"Single lab; in vivo relevance of the T482 phospho-degron not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked DHX33 to lipid metabolism and ferroptosis sensitivity and provided a candidate small-molecule helicase inhibitor.\",\n      \"evidence\": \"KY386 inhibitor treatment, FADS1/FADS2/SCD1 expression and ferroptosis/viability assays in vitro and in vivo\",\n      \"pmids\": [\"38973855\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Indirect mechanistic link; inhibitor specificity for DHX33 not rigorously established\", \"Single lab, no genetic confirmation of the ferroptosis axis\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How DHX33 partitions between its nuclear (rDNA/promoter), translational, and cytoplasmic immune-sensing functions, and what determines its promoter and substrate selectivity, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of DHX33 or its complexes\", \"Signals controlling subcellular partitioning of isoforms unknown\", \"Rules for promoter/mRNA target selection undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [8, 0]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [1, 2, 4, 8]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 8, 11]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [8, 4]},\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [5, 6, 11, 12]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0045182\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [0, 14]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5, 11, 9]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 2, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 5, 11, 12]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 2, 14]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [12, 16, 17]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [11, 12]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 6, 16]}\n    ],\n    \"complexes\": [\"NLRP3 inflammasome\"],\n    \"partners\": [\"UBF\", \"NLRP3\", \"MAVS\", \"GADD45a\", \"TET1\", \"TFAP2B\", \"DDX3X\", \"USP36\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}