{"gene":"DDX43","run_date":"2026-04-28T17:46:02","timeline":{"discoveries":[{"year":2017,"finding":"DDX43 is an ATP-dependent dual RNA and DNA helicase containing a K-homology (KH) domain in its N-terminus that is required for full nucleic acid unwinding activity. It shows 5' to 3' polarity on RNA and 3' to 5' directionality on DNA. The KH domain is responsible for nucleic acid binding, and without it the C-terminal helicase domain alone shows no RNA unwinding and significantly reduced DNA unwinding activity. Single amino acid changes in the KH domain reduce both binding and unwinding activities.","method":"Recombinant protein purification, in vitro helicase assay, truncation mutations, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mutagenesis, multiple orthogonal methods in single study","pmids":["28468824"],"is_preprint":false},{"year":2020,"finding":"The KH domain of DDX43 preferentially binds pyrimidine-rich ssDNA and ssRNA (favoring TTGT/UUGU sequences), with the GXXG loop and an adjacent alanine residue critical for pyrimidine binding. The KH domain facilitates substrate specificity and unwinding processivity of the full-length DDX43 helicase, and DDX43 prefers substrates with TTGT/UUGU single-stranded tails.","method":"EMSA, NMR (15N-HSQC), SELEX, ChIP-seq, CLIP-seq, bioinformatics, mutational analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — multiple orthogonal methods including NMR structural validation and mutagenesis","pmids":["33199368"],"is_preprint":false},{"year":2012,"finding":"DDX43/HAGE is required for ABCB5+ malignant melanoma-initiating cell (MMIC)-dependent tumor growth. Knockdown of HAGE decreases NRAS protein expression with concomitant decrease in AKT and ERK pathway activation. HAGE promotes NRAS mRNA unwinding in vitro, and NRAS silenced by siRNA can be rescued by HAGE reintroduction, demonstrating that NRAS expression is dependent on HAGE helicase activity.","method":"shRNA knockdown, in vitro helicase/unwinding assay, siRNA rescue experiment, xenograft tumor transplantation in NOD/SCID mice","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal rescue experiment, in vitro unwinding assay, in vivo xenograft validation","pmids":["22393060"],"is_preprint":false},{"year":2014,"finding":"DDX43/HAGE prevents IFN-α-induced PML expression in ABCB5+ MMICs by promoting SOCS1 mRNA unwinding and protein expression, thereby inhibiting JAK-STAT pathway activation. Knockdown of HAGE decreases SOCS1 protein expression, activates JAK-STAT signaling, and increases PML expression. SOCS1 silenced by siRNA can be rescued by reintroduction of HAGE, confirming SOCS1 expression is dependent on HAGE helicase activity.","method":"shRNA knockdown, siRNA rescue, in vitro unwinding assay, stem cell proliferation assay, xenotransplantation assay","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 — rescue experiment confirms pathway dependence, in vitro mechanistic assay, in vivo validation","pmids":["24525737"],"is_preprint":false},{"year":2014,"finding":"DDX43 overexpression in MEK inhibitor-resistant uveal melanoma cells induces RAS protein levels (KRAS, HRAS, NRAS) and activates ERK and AKT pathways, mediating MEK inhibitor resistance. Depletion of DDX43 decreases RAS proteins and inhibits downstream signaling, while ectopic DDX43 expression in parental cells renders them resistant to MEK inhibition.","method":"shRNA/siRNA knockdown, ectopic overexpression, Western blot for RAS/ERK/AKT pathway activation, cell viability assays","journal":"Molecular cancer therapeutics","confidence":"Medium","confidence_rationale":"Tier 2 — clean gain- and loss-of-function with defined molecular pathway readouts in multiple cell lines","pmids":["24899684"],"is_preprint":false},{"year":2018,"finding":"DDX43 in CML promotes cell survival and tumorigenesis by arresting miR-186 (which normally targets DDX43) and releasing/upregulating lncRNA H19 through promoter demethylation. miR-186 targets DDX43 mRNA directly, and overexpression of miR-186 increases apoptosis. Silencing H19 inhibits cell survival.","method":"Overexpression and knockdown of DDX43, miRNA transfection, colony formation assay, apoptosis assay, methylation analysis","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — multiple functional readouts with defined molecular interactions, single lab","pmids":["29449695"],"is_preprint":false},{"year":2023,"finding":"DDX43 is an essential regulator of chromatin remodeling during spermiogenesis. Testis-specific Ddx43 knockout mice are infertile with defective histone-to-protamine replacement and post-meiotic chromatin condensation defects. Loss of ATP hydrolysis activity (ATPase-dead missense mutation) replicates the infertility phenotype, indicating dependence on enzymatic activity. DDX43 regulates dynamic RNA regulatory processes in spermatids and targets the hub gene Elfn2, identified by eCLIP-seq.","method":"Testis-specific knockout mice, global knockout mice, ATPase-dead knock-in missense mutation, single-cell RNA-seq, eCLIP-seq, phenotypic analysis of spermatogenesis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 — genetic mouse model with ATPase-dead mutation, single-cell transcriptomics, eCLIP-seq, multiple orthogonal methods","pmids":["37120627"],"is_preprint":false},{"year":2019,"finding":"DDX43 exists as a homogeneous monomer in solution and preferentially binds single-stranded DNA or RNA substrates longer than 12 nt, with preference for guanosine. Full binding affinity requires physical connectivity of all domains (KH, linker, and helicase core); removal or disconnection of any domain reduces binding affinity approximately 10-fold.","method":"Recombinant protein purification from E. coli, EMSA, domain truncation and disconnection experiments","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro biochemical assays with mutagenesis/truncations, single lab","pmids":["31623828"],"is_preprint":false},{"year":2022,"finding":"DDX43 domain architecture comprises an N-terminal KH domain, a central flexible linker domain, and a C-terminal helicase core domain. The KH domain is required for ATPase and unwinding activity. DDX43 has documented roles in piRNA amplification, tumorigenesis, RAS signaling, and innate immunity.","method":"Biochemical structure-function review integrating EMSA, SELEX, ChIP-seq, CLIP-seq, NMR, and cellular studies","journal":"Methods (San Diego, Calif.)","confidence":"Medium","confidence_rationale":"Tier 1-2 — synthesis of multiple biochemical and structural methods, confirmatory review paper","pmids":["35257897"],"is_preprint":false},{"year":2025,"finding":"DDX43 inhibits HSV-2 replication in an interferon-independent manner. Overexpression of DDX43 inhibited HSV-2 replication while knockdown of endogenous DDX43 enhanced it, establishing DDX43 as a host restriction factor against HSV-2.","method":"Overexpression and siRNA knockdown in HeLa and ARPE-19 cells, viral replication assays, comparative transcriptomics/OPLS-DA","journal":"Viruses","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal gain- and loss-of-function with viral replication readout, interferon-independence confirmed","pmids":["41157636"],"is_preprint":false}],"current_model":"DDX43 (HAGE) is an ATP-dependent DEAD-box RNA/DNA helicase with an N-terminal KH domain that binds pyrimidine-rich sequences (favoring TTGT/UUGU), facilitates substrate specificity and unwinding processivity, and drives tumor progression by unwinding target mRNAs (including NRAS and SOCS1) to promote their translation; in the germline it is essential for spermiogenesis by regulating chromatin remodeling and histone-to-protamine replacement in an ATPase-dependent manner, and in innate immunity it restricts viral replication in an interferon-independent manner."},"narrative":{"teleology":[{"year":2012,"claim":"The first mechanistic link between DDX43 and a specific mRNA target was established when HAGE was shown to unwind NRAS mRNA, thereby controlling NRAS protein levels and downstream RAS–ERK–AKT signaling required for melanoma-initiating cell tumor growth.","evidence":"shRNA knockdown, siRNA rescue, in vitro unwinding assay, xenograft transplantation in NOD/SCID mice","pmids":["22393060"],"confidence":"High","gaps":["Structural basis for DDX43 recognition of NRAS mRNA was unknown","Whether DDX43 unwinds NRAS mRNA secondary structure or displaces bound proteins was not distinguished","Generalizability to other RAS family mRNAs was untested"]},{"year":2014,"claim":"DDX43's target repertoire was extended to SOCS1 mRNA, revealing how HAGE suppresses JAK–STAT signaling and interferon-induced PML expression in melanoma-initiating cells, and separately, how DDX43 overexpression drives MEK inhibitor resistance by upregulating multiple RAS proteins in uveal melanoma.","evidence":"shRNA/siRNA knockdown and rescue for SOCS1; ectopic overexpression and depletion with pathway readouts for RAS/ERK/AKT in MEK-resistant cells","pmids":["24525737","24899684"],"confidence":"High","gaps":["Direct binding of DDX43 to SOCS1 mRNA was inferred from unwinding assays but not mapped at nucleotide resolution","Whether DDX43 acts co-transcriptionally or post-transcriptionally on these targets was not resolved"]},{"year":2017,"claim":"Biochemical reconstitution revealed DDX43 as a dual RNA/DNA helicase with opposite polarity on the two substrates (5′→3′ on RNA, 3′→5′ on DNA), and identified the N-terminal KH domain as indispensable for nucleic acid binding and unwinding.","evidence":"Recombinant protein purification, in vitro helicase assay with truncation and site-directed mutagenesis","pmids":["28468824"],"confidence":"High","gaps":["Why DDX43 displays opposite directionality on RNA versus DNA was mechanistically unexplained","Whether DNA helicase activity is physiologically relevant was unclear"]},{"year":2019,"claim":"Domain dissection showed DDX43 functions as a monomer and that physical connectivity of all three domains (KH, linker, helicase core) is required for high-affinity nucleic acid binding, explaining why isolated domains are insufficient.","evidence":"Recombinant protein purification, EMSA with domain truncation and disconnection constructs","pmids":["31623828"],"confidence":"Medium","gaps":["No high-resolution structure of the full-length protein to explain inter-domain communication","Role of the flexible linker beyond physical connectivity was not characterized"]},{"year":2020,"claim":"The KH domain's substrate specificity was defined: it preferentially recognizes pyrimidine-rich TTGT/UUGU motifs via its GXXG loop, conferring sequence-selective unwinding to the full-length helicase.","evidence":"SELEX, NMR (¹⁵N-HSQC), EMSA, ChIP-seq, CLIP-seq, mutational analysis","pmids":["33199368"],"confidence":"High","gaps":["Genome-wide identification of physiological RNA targets bearing UUGU motifs was not completed","Whether KH-mediated specificity explains selective unwinding of NRAS/SOCS1 mRNAs was not tested"]},{"year":2023,"claim":"A physiological in vivo role was established: DDX43 is essential for spermiogenesis, where its ATPase activity drives histone-to-protamine replacement; an ATPase-dead knock-in phenocopied full knockout infertility, and eCLIP identified Elfn2 as a direct RNA target in spermatids.","evidence":"Testis-specific and global Ddx43 knockout mice, ATPase-dead knock-in, single-cell RNA-seq, eCLIP-seq","pmids":["37120627"],"confidence":"High","gaps":["How DDX43 unwinding of target mRNAs like Elfn2 mechanistically triggers chromatin remodeling remains unknown","Whether DDX43 participates in piRNA amplification in vivo (as suggested by homology) was not directly tested"]},{"year":2025,"claim":"DDX43 was identified as a host antiviral factor that restricts HSV-2 replication independently of the interferon response, broadening its functional repertoire beyond oncogenesis and gametogenesis.","evidence":"Overexpression and siRNA knockdown in HeLa and ARPE-19 cells, viral replication assays","pmids":["41157636"],"confidence":"Medium","gaps":["The viral RNA or DNA target(s) of DDX43 during HSV-2 restriction were not identified","Whether antiviral activity depends on the KH domain or helicase activity specifically was not tested","Generalizability to other DNA or RNA viruses is unknown"]},{"year":null,"claim":"A full-length atomic structure of DDX43, genome-wide mapping of its physiological mRNA targets in somatic and germline cells, and the mechanism by which mRNA unwinding couples to translational activation or chromatin remodeling remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length crystal or cryo-EM structure exists","Transcriptome-wide target identification in cancer cells has not been performed","Mechanism linking mRNA unwinding to translational promotion is not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,6]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,1,7]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,1,7]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,2,3]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,3,4]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[6]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[9]}],"complexes":[],"partners":["NRAS","SOCS1","ELFN2"],"other_free_text":[]},"mechanistic_narrative":"DDX43 (HAGE) is an ATP-dependent DEAD-box helicase that couples an N-terminal K-homology (KH) domain to a C-terminal helicase core to unwind both RNA and DNA substrates, thereby regulating mRNA translation, chromatin remodeling, and antiviral defense. The KH domain binds pyrimidine-rich single-stranded nucleic acids (preferring TTGT/UUGU motifs) and is indispensable for full ATPase and unwinding activity; physical connectivity of all domains—KH, linker, and helicase core—is required for high-affinity substrate engagement [PMID:28468824, PMID:33199368, PMID:31623828]. In cancer cells, DDX43 unwinds NRAS and SOCS1 mRNAs to promote their translation, activating RAS–ERK–AKT signaling and suppressing JAK–STAT-mediated interferon responses, which collectively drive tumor-initiating cell survival and drug resistance [PMID:22393060, PMID:24525737, PMID:24899684]. In the male germline, DDX43 ATPase activity is essential for histone-to-protamine replacement during spermiogenesis—knockout or ATPase-dead knock-in mice are infertile with chromatin condensation defects—and DDX43 additionally functions as an interferon-independent host restriction factor against HSV-2 [PMID:37120627, PMID:41157636]."},"prefetch_data":{"uniprot":{"accession":"Q9NXZ2","full_name":"Probable ATP-dependent RNA helicase DDX43","aliases":["Cancer/testis antigen 13","CT13","DEAD box protein 43","DEAD box protein HAGE","Helical antigen"],"length_aa":648,"mass_kda":72.8,"function":"","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q9NXZ2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DDX43","classification":"Not Classified","n_dependent_lines":20,"n_total_lines":1208,"dependency_fraction":0.016556291390728478},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/DDX43","total_profiled":1310},"omim":[{"mim_id":"606286","title":"DEAD-BOX HELICASE 43; DDX43","url":"https://www.omim.org/entry/606286"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"placenta","ntpm":4.6},{"tissue":"testis","ntpm":18.3}],"url":"https://www.proteinatlas.org/search/DDX43"},"hgnc":{"alias_symbol":["HAGE","DKFZp434H2114","CT13"],"prev_symbol":[]},"alphafold":{"accession":"Q9NXZ2","domains":[{"cath_id":"3.30.1370.10","chopping":"71-132","consensus_level":"high","plddt":86.8187,"start":71,"end":132},{"cath_id":"3.40.50.300","chopping":"221-308_319-451","consensus_level":"medium","plddt":92.0247,"start":221,"end":451},{"cath_id":"3.40.50.300","chopping":"462-634","consensus_level":"high","plddt":89.0299,"start":462,"end":634}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NXZ2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NXZ2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NXZ2-F1-predicted_aligned_error_v6.png","plddt_mean":79.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DDX43","jax_strain_url":"https://www.jax.org/strain/search?query=DDX43"},"sequence":{"accession":"Q9NXZ2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NXZ2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NXZ2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NXZ2"}},"corpus_meta":[{"pmid":"12399967","id":"PMC_12399967","title":"Frequent expression of HAGE in presentation chronic myeloid leukaemias.","date":"2002","source":"Leukemia","url":"https://pubmed.ncbi.nlm.nih.gov/12399967","citation_count":68,"is_preprint":false},{"pmid":"17296563","id":"PMC_17296563","title":"Epigenetic regulation of human cancer/testis antigen gene, HAGE, in chronic myeloid leukemia.","date":"2007","source":"Haematologica","url":"https://pubmed.ncbi.nlm.nih.gov/17296563","citation_count":44,"is_preprint":false},{"pmid":"20058853","id":"PMC_20058853","title":"HAGE, a cancer/testis antigen expressed at the protein level in a variety of cancers.","date":"2010","source":"Cancer immunity","url":"https://pubmed.ncbi.nlm.nih.gov/20058853","citation_count":36,"is_preprint":false},{"pmid":"37120627","id":"PMC_37120627","title":"Single-cell RNA-seq uncovers dynamic processes orchestrated by RNA-binding protein DDX43 in chromatin remodeling during spermiogenesis.","date":"2023","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/37120627","citation_count":35,"is_preprint":false},{"pmid":"28468824","id":"PMC_28468824","title":"The DEAD-box protein DDX43 (HAGE) is a dual RNA-DNA helicase and has a K-homology domain required for full nucleic acid unwinding activity.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28468824","citation_count":33,"is_preprint":false},{"pmid":"22393060","id":"PMC_22393060","title":"The helicase HAGE expressed by malignant melanoma-initiating cells is required for tumor cell proliferation in vivo.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22393060","citation_count":32,"is_preprint":false},{"pmid":"22527039","id":"PMC_22527039","title":"Hydroxybutyrate prevents protein aggregation in the halotolerant bacterium Pseudomonas sp. 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It shows 5' to 3' polarity on RNA and 3' to 5' directionality on DNA. The KH domain is responsible for nucleic acid binding, and without it the C-terminal helicase domain alone shows no RNA unwinding and significantly reduced DNA unwinding activity. Single amino acid changes in the KH domain reduce both binding and unwinding activities.\",\n      \"method\": \"Recombinant protein purification, in vitro helicase assay, truncation mutations, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutagenesis, multiple orthogonal methods in single study\",\n      \"pmids\": [\"28468824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The KH domain of DDX43 preferentially binds pyrimidine-rich ssDNA and ssRNA (favoring TTGT/UUGU sequences), with the GXXG loop and an adjacent alanine residue critical for pyrimidine binding. The KH domain facilitates substrate specificity and unwinding processivity of the full-length DDX43 helicase, and DDX43 prefers substrates with TTGT/UUGU single-stranded tails.\",\n      \"method\": \"EMSA, NMR (15N-HSQC), SELEX, ChIP-seq, CLIP-seq, bioinformatics, mutational analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple orthogonal methods including NMR structural validation and mutagenesis\",\n      \"pmids\": [\"33199368\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"DDX43/HAGE is required for ABCB5+ malignant melanoma-initiating cell (MMIC)-dependent tumor growth. Knockdown of HAGE decreases NRAS protein expression with concomitant decrease in AKT and ERK pathway activation. HAGE promotes NRAS mRNA unwinding in vitro, and NRAS silenced by siRNA can be rescued by HAGE reintroduction, demonstrating that NRAS expression is dependent on HAGE helicase activity.\",\n      \"method\": \"shRNA knockdown, in vitro helicase/unwinding assay, siRNA rescue experiment, xenograft tumor transplantation in NOD/SCID mice\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal rescue experiment, in vitro unwinding assay, in vivo xenograft validation\",\n      \"pmids\": [\"22393060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"DDX43/HAGE prevents IFN-α-induced PML expression in ABCB5+ MMICs by promoting SOCS1 mRNA unwinding and protein expression, thereby inhibiting JAK-STAT pathway activation. Knockdown of HAGE decreases SOCS1 protein expression, activates JAK-STAT signaling, and increases PML expression. SOCS1 silenced by siRNA can be rescued by reintroduction of HAGE, confirming SOCS1 expression is dependent on HAGE helicase activity.\",\n      \"method\": \"shRNA knockdown, siRNA rescue, in vitro unwinding assay, stem cell proliferation assay, xenotransplantation assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — rescue experiment confirms pathway dependence, in vitro mechanistic assay, in vivo validation\",\n      \"pmids\": [\"24525737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"DDX43 overexpression in MEK inhibitor-resistant uveal melanoma cells induces RAS protein levels (KRAS, HRAS, NRAS) and activates ERK and AKT pathways, mediating MEK inhibitor resistance. Depletion of DDX43 decreases RAS proteins and inhibits downstream signaling, while ectopic DDX43 expression in parental cells renders them resistant to MEK inhibition.\",\n      \"method\": \"shRNA/siRNA knockdown, ectopic overexpression, Western blot for RAS/ERK/AKT pathway activation, cell viability assays\",\n      \"journal\": \"Molecular cancer therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean gain- and loss-of-function with defined molecular pathway readouts in multiple cell lines\",\n      \"pmids\": [\"24899684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DDX43 in CML promotes cell survival and tumorigenesis by arresting miR-186 (which normally targets DDX43) and releasing/upregulating lncRNA H19 through promoter demethylation. miR-186 targets DDX43 mRNA directly, and overexpression of miR-186 increases apoptosis. Silencing H19 inhibits cell survival.\",\n      \"method\": \"Overexpression and knockdown of DDX43, miRNA transfection, colony formation assay, apoptosis assay, methylation analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional readouts with defined molecular interactions, single lab\",\n      \"pmids\": [\"29449695\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"DDX43 is an essential regulator of chromatin remodeling during spermiogenesis. Testis-specific Ddx43 knockout mice are infertile with defective histone-to-protamine replacement and post-meiotic chromatin condensation defects. Loss of ATP hydrolysis activity (ATPase-dead missense mutation) replicates the infertility phenotype, indicating dependence on enzymatic activity. DDX43 regulates dynamic RNA regulatory processes in spermatids and targets the hub gene Elfn2, identified by eCLIP-seq.\",\n      \"method\": \"Testis-specific knockout mice, global knockout mice, ATPase-dead knock-in missense mutation, single-cell RNA-seq, eCLIP-seq, phenotypic analysis of spermatogenesis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — genetic mouse model with ATPase-dead mutation, single-cell transcriptomics, eCLIP-seq, multiple orthogonal methods\",\n      \"pmids\": [\"37120627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"DDX43 exists as a homogeneous monomer in solution and preferentially binds single-stranded DNA or RNA substrates longer than 12 nt, with preference for guanosine. Full binding affinity requires physical connectivity of all domains (KH, linker, and helicase core); removal or disconnection of any domain reduces binding affinity approximately 10-fold.\",\n      \"method\": \"Recombinant protein purification from E. coli, EMSA, domain truncation and disconnection experiments\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro biochemical assays with mutagenesis/truncations, single lab\",\n      \"pmids\": [\"31623828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DDX43 domain architecture comprises an N-terminal KH domain, a central flexible linker domain, and a C-terminal helicase core domain. The KH domain is required for ATPase and unwinding activity. DDX43 has documented roles in piRNA amplification, tumorigenesis, RAS signaling, and innate immunity.\",\n      \"method\": \"Biochemical structure-function review integrating EMSA, SELEX, ChIP-seq, CLIP-seq, NMR, and cellular studies\",\n      \"journal\": \"Methods (San Diego, Calif.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — synthesis of multiple biochemical and structural methods, confirmatory review paper\",\n      \"pmids\": [\"35257897\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DDX43 inhibits HSV-2 replication in an interferon-independent manner. Overexpression of DDX43 inhibited HSV-2 replication while knockdown of endogenous DDX43 enhanced it, establishing DDX43 as a host restriction factor against HSV-2.\",\n      \"method\": \"Overexpression and siRNA knockdown in HeLa and ARPE-19 cells, viral replication assays, comparative transcriptomics/OPLS-DA\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal gain- and loss-of-function with viral replication readout, interferon-independence confirmed\",\n      \"pmids\": [\"41157636\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DDX43 (HAGE) is an ATP-dependent DEAD-box RNA/DNA helicase with an N-terminal KH domain that binds pyrimidine-rich sequences (favoring TTGT/UUGU), facilitates substrate specificity and unwinding processivity, and drives tumor progression by unwinding target mRNAs (including NRAS and SOCS1) to promote their translation; in the germline it is essential for spermiogenesis by regulating chromatin remodeling and histone-to-protamine replacement in an ATPase-dependent manner, and in innate immunity it restricts viral replication in an interferon-independent manner.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"DDX43 (HAGE) is an ATP-dependent DEAD-box helicase that couples an N-terminal K-homology (KH) domain to a C-terminal helicase core to unwind both RNA and DNA substrates, thereby regulating mRNA translation, chromatin remodeling, and antiviral defense. The KH domain binds pyrimidine-rich single-stranded nucleic acids (preferring TTGT/UUGU motifs) and is indispensable for full ATPase and unwinding activity; physical connectivity of all domains—KH, linker, and helicase core—is required for high-affinity substrate engagement [PMID:28468824, PMID:33199368, PMID:31623828]. In cancer cells, DDX43 unwinds NRAS and SOCS1 mRNAs to promote their translation, activating RAS–ERK–AKT signaling and suppressing JAK–STAT-mediated interferon responses, which collectively drive tumor-initiating cell survival and drug resistance [PMID:22393060, PMID:24525737, PMID:24899684]. In the male germline, DDX43 ATPase activity is essential for histone-to-protamine replacement during spermiogenesis—knockout or ATPase-dead knock-in mice are infertile with chromatin condensation defects—and DDX43 additionally functions as an interferon-independent host restriction factor against HSV-2 [PMID:37120627, PMID:41157636].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"The first mechanistic link between DDX43 and a specific mRNA target was established when HAGE was shown to unwind NRAS mRNA, thereby controlling NRAS protein levels and downstream RAS–ERK–AKT signaling required for melanoma-initiating cell tumor growth.\",\n      \"evidence\": \"shRNA knockdown, siRNA rescue, in vitro unwinding assay, xenograft transplantation in NOD/SCID mice\",\n      \"pmids\": [\"22393060\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for DDX43 recognition of NRAS mRNA was unknown\",\n        \"Whether DDX43 unwinds NRAS mRNA secondary structure or displaces bound proteins was not distinguished\",\n        \"Generalizability to other RAS family mRNAs was untested\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"DDX43's target repertoire was extended to SOCS1 mRNA, revealing how HAGE suppresses JAK–STAT signaling and interferon-induced PML expression in melanoma-initiating cells, and separately, how DDX43 overexpression drives MEK inhibitor resistance by upregulating multiple RAS proteins in uveal melanoma.\",\n      \"evidence\": \"shRNA/siRNA knockdown and rescue for SOCS1; ectopic overexpression and depletion with pathway readouts for RAS/ERK/AKT in MEK-resistant cells\",\n      \"pmids\": [\"24525737\", \"24899684\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct binding of DDX43 to SOCS1 mRNA was inferred from unwinding assays but not mapped at nucleotide resolution\",\n        \"Whether DDX43 acts co-transcriptionally or post-transcriptionally on these targets was not resolved\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Biochemical reconstitution revealed DDX43 as a dual RNA/DNA helicase with opposite polarity on the two substrates (5′→3′ on RNA, 3′→5′ on DNA), and identified the N-terminal KH domain as indispensable for nucleic acid binding and unwinding.\",\n      \"evidence\": \"Recombinant protein purification, in vitro helicase assay with truncation and site-directed mutagenesis\",\n      \"pmids\": [\"28468824\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Why DDX43 displays opposite directionality on RNA versus DNA was mechanistically unexplained\",\n        \"Whether DNA helicase activity is physiologically relevant was unclear\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Domain dissection showed DDX43 functions as a monomer and that physical connectivity of all three domains (KH, linker, helicase core) is required for high-affinity nucleic acid binding, explaining why isolated domains are insufficient.\",\n      \"evidence\": \"Recombinant protein purification, EMSA with domain truncation and disconnection constructs\",\n      \"pmids\": [\"31623828\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No high-resolution structure of the full-length protein to explain inter-domain communication\",\n        \"Role of the flexible linker beyond physical connectivity was not characterized\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"The KH domain's substrate specificity was defined: it preferentially recognizes pyrimidine-rich TTGT/UUGU motifs via its GXXG loop, conferring sequence-selective unwinding to the full-length helicase.\",\n      \"evidence\": \"SELEX, NMR (¹⁵N-HSQC), EMSA, ChIP-seq, CLIP-seq, mutational analysis\",\n      \"pmids\": [\"33199368\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Genome-wide identification of physiological RNA targets bearing UUGU motifs was not completed\",\n        \"Whether KH-mediated specificity explains selective unwinding of NRAS/SOCS1 mRNAs was not tested\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A physiological in vivo role was established: DDX43 is essential for spermiogenesis, where its ATPase activity drives histone-to-protamine replacement; an ATPase-dead knock-in phenocopied full knockout infertility, and eCLIP identified Elfn2 as a direct RNA target in spermatids.\",\n      \"evidence\": \"Testis-specific and global Ddx43 knockout mice, ATPase-dead knock-in, single-cell RNA-seq, eCLIP-seq\",\n      \"pmids\": [\"37120627\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How DDX43 unwinding of target mRNAs like Elfn2 mechanistically triggers chromatin remodeling remains unknown\",\n        \"Whether DDX43 participates in piRNA amplification in vivo (as suggested by homology) was not directly tested\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"DDX43 was identified as a host antiviral factor that restricts HSV-2 replication independently of the interferon response, broadening its functional repertoire beyond oncogenesis and gametogenesis.\",\n      \"evidence\": \"Overexpression and siRNA knockdown in HeLa and ARPE-19 cells, viral replication assays\",\n      \"pmids\": [\"41157636\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"The viral RNA or DNA target(s) of DDX43 during HSV-2 restriction were not identified\",\n        \"Whether antiviral activity depends on the KH domain or helicase activity specifically was not tested\",\n        \"Generalizability to other DNA or RNA viruses is unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A full-length atomic structure of DDX43, genome-wide mapping of its physiological mRNA targets in somatic and germline cells, and the mechanism by which mRNA unwinding couples to translational activation or chromatin remodeling remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No full-length crystal or cryo-EM structure exists\",\n        \"Transcriptome-wide target identification in cancer cells has not been performed\",\n        \"Mechanism linking mRNA unwinding to translational promotion is not defined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 1, 7]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 1, 7]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 2, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3, 4]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"NRAS\",\n      \"SOCS1\",\n      \"ELFN2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}