{"gene":"DHX15","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":1997,"finding":"Yeast Prp43 (DHX15 ortholog) is a pre-mRNA processing factor required for spliceosome disassembly after mRNA release; temperature-sensitive mutations cause accumulation of unspliced pre-mRNAs and excised intron lariats trapped in spliceosomes.","method":"Gene replacement with ts alleles, Northern analysis, splicing complex analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function with defined molecular phenotype, replicated conceptually by multiple subsequent studies","pmids":["9342317"],"is_preprint":false},{"year":2002,"finding":"Purified recombinant Prp43 is an RNA-dependent ATPase; alanine mutations in motifs I (GSGKT), II (DEAH), and VI (QRAGRAGR) abolish ATPase activity in vitro and are lethal in vivo. An ATPase-dead mutant (T123A) blocks release of the excised lariat-intron from the spliceosome without affecting mRNA release or chemical splicing steps; the lariat-intron in T123A-arrested complexes is inaccessible to debranching enzyme Dbr1.","method":"Recombinant protein purification, in vitro ATPase assay, active-site mutagenesis, in vitro splicing assay, dominant-negative overexpression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis, multiple orthogonal assays in one study","pmids":["11886864"],"is_preprint":false},{"year":2002,"finding":"Human DDX15 (DHX15) colocalizes with spliceosomal snRNPs in nuclear speckles and in nucleoli, and is associated with spliceosomal U snRNAs by RNA co-precipitation. DDX15 interacts with the human La autoantigen both in vivo and in vitro; the interaction requires a region that partly overlaps the DEAH-box domain.","method":"Large-scale immunoprecipitation from HeLa S100 extracts, in vitro binding, immunofluorescence, RNA co-precipitation","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP/pulldown with in vivo and in vitro confirmation, single lab","pmids":["12458796"],"is_preprint":false},{"year":2005,"finding":"Prp43 forms a stable NTR complex with the novel splicing factors Ntr1 and Ntr2; the affinity-purified NTR complex catalyzes ATP-dependent spliceosome disassembly, releasing U2, U5, U6, the NineTeen Complex, and the lariat-intron. Ntr1 interacts with Prp43 through its N-terminal G-patch domain.","method":"Immunoprecipitation, in vitro spliceosome disassembly assay with purified NTR complex, domain mapping","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical reconstitution of disassembly with purified complex, multiple orthogonal methods","pmids":["16357217"],"is_preprint":false},{"year":2006,"finding":"Prp43 hydrolyzes all common NTPs/dNTPs and unwinds short 5'/3'-tailed RNA/DNA duplexes in an ATP-dependent manner; optimal ATPase requires RNA cofactor ≥20 nt. Motif V mutations T384A and T384V are lethal and block lariat release despite retaining ATPase activity, demonstrating that ATPase activity is necessary but not sufficient for function.","method":"In vitro ATPase and helicase unwinding assays, site-directed mutagenesis, in vitro splicing assay","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis, single lab but multiple orthogonal assays","pmids":["16700561"],"is_preprint":false},{"year":2006,"finding":"Ntr1 (Spp382) is required for association of Prp43 with the excised intron complex; depletion of Ntr1 causes accumulation of excised intron and impaired snRNP recycling, indicating Ntr1 acts as a spliceosome receptor or RNA-targeting factor for Prp43.","method":"Metabolic depletion of Ntr1, in vitro splicing assay, snRNP sedimentation analysis, immunoprecipitation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic depletion with defined biochemical phenotype, replicated by independent lab","pmids":["16880513"],"is_preprint":false},{"year":2007,"finding":"Ntr1 activates the inherently feeble helicase activity of Prp43 to trigger lariat-intron release; the N-terminal 120-aa segment of Ntr1 suffices for Prp43 binding and helicase stimulation. Lethal Prp43 mutants T384A/T384V that retain ATPase activity are refractory to Ntr1-mediated helicase stimulation. Specific missense mutations in Prp43 and Ntr1 that disrupt protein-protein interaction also impair RNA unwinding stimulation.","method":"In vitro helicase assay, protein-protein interaction mapping, site-directed mutagenesis, in vitro splicing assay","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis of both partners, multiple orthogonal assays","pmids":["17875666"],"is_preprint":false},{"year":2007,"finding":"The Ntr1-Ntr2 complex binds the spliceosome before recruiting Prp43; Ntr2 interacts with U5 component Brr2 and is essential for NTR-spliceosome docking. Binding of the NTR complex does not require ATP, but spliceosome disassembly requires ATP hydrolysis.","method":"Immunoprecipitation, in vitro spliceosome-binding and disassembly assays, two-hybrid and deletion analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ATP requirement biochemically defined, replicated across labs","pmids":["17893323"],"is_preprint":false},{"year":2009,"finding":"In yeast, functional cooperation between Nob1 (endonuclease) and Prp43 with its cofactor Pfa1 is required for 20S-to-18S pre-rRNA cleavage at site D; genetic epistasis shows that loss of Ltv1 combined with Prp43 or Pfa1 mutation blocks cytoplasmic site D cleavage, which is suppressed by wild-type but not catalytic-dead Nob1.","method":"Genetic epistasis, in vitro cleavage assay with purified Nob1, pre-rRNA Northern analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of cleavage plus genetic epistasis with multiple mutant combinations","pmids":["19801658"],"is_preprint":false},{"year":2009,"finding":"UV crosslinking identifies multiple Prp43-binding sites on pre-rRNA: predominantly within helix 44 of 18S (near the site of 18S 3' cleavage) and four major sites in 25S including helix 34. Depletion or catalytic inactivation of Prp43 causes accumulation of snoRNAs that guide modifications near helix 34 on preribosomes, indicating Prp43 releases snoRNAs from specific pre-rRNA sites.","method":"UV crosslinking and immunoprecipitation (CRAC precursor), Northern analysis of snoRNA association, catalytic point-mutant strains","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct RNA crosslinking plus functional genetic analysis, multiple orthogonal approaches","pmids":["19941819"],"is_preprint":false},{"year":2010,"finding":"Prp43 acts as a downstream discard-pathway factor for proofreading 5' splice site cleavage: after Prp16-mediated rejection of suboptimal substrates, Prp43 is required to disassemble the rejected spliceosome. Prp16 and Prp43 thus cooperate in an ATP-dependent fidelity framework.","method":"In vitro splicing assay using metal-ligand disruption of catalytic center, DEAH-box ATPase inactivation","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reconstituted in vitro splicing system with defined epistatic relationship, single lab but rigorous biochemistry","pmids":["20705241"],"is_preprint":false},{"year":2012,"finding":"Human RBM5 directly interacts with DHX15 via its G-patch domain, and recombinant RBM5 stimulates DHX15 helicase activity in vitro in a G-patch-domain-dependent manner.","method":"Co-immunoprecipitation, in vitro helicase assay, domain deletion analysis","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct in vitro helicase stimulation assay with domain mapping, single lab","pmids":["22569250"],"is_preprint":false},{"year":2013,"finding":"The minimal activating fragment of Ntr1 is residues 51–110 (containing the G-patch). Cross-linking mass spectrometry shows Prp43 interacts with the G-patch motif of Ntr1 through its C-terminal domains. Functionally important RNA-binding residues were identified in both Prp43 and Ntr1.","method":"Biochemical ATPase/helicase assays, structural mass spectrometry (protein cross-linking), domain truncation","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro functional assays combined with structural proteomics, single lab","pmids":["24165877"],"is_preprint":false},{"year":2014,"finding":"Human DHX15 physically interacts with MAVS and mediates MAVS-dependent activation of the NF-κB, JNK, and p38 MAPK pathways (but not IRF3) in response to poly(I:C) and RNA virus infection. DHX15 is required for optimal cytokine production and MAVS-mediated apoptosis.","method":"Co-immunoprecipitation, siRNA knockdown, reporter assays, Drosophila misexpression screen","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP plus knockdown with functional cytokine/apoptosis readouts, replicated by independent lab (PMID:24990078)","pmids":["24782566"],"is_preprint":false},{"year":2014,"finding":"DHX15 binds double-stranded RNA (poly(I:C)) specifically via its helicase C-terminal domain. The N-terminal DEXDc-containing domain of DHX15 binds the C-terminus of MAVS. DHX15 is required for IRF3 phosphorylation as well as NF-κB and MAPK signaling during RNA virus infection in myeloid dendritic cells.","method":"RNA pulldown, domain-mapping co-immunoprecipitation, shRNA knockdown, signaling assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain-resolved binding assays plus functional knockdown in primary cells, corroborated by independent lab","pmids":["24990078"],"is_preprint":false},{"year":2015,"finding":"The G-patches of Spp382/Ntr1, Sqs1/Pfa1, and Pxr1/Gno1 differ in their ability to interact with and functionally substitute for each other in Prp43-dependent splicing and rRNA processing. Deletion of the primary Prp43-binding site in Pxr1 does not impair rRNA processing but causes accumulation of extended snoRNA forms, linking Prp43 to snoRNA biogenesis.","method":"Yeast two-hybrid, domain-swap mutagenesis, site-directed mutagenesis, Northern analysis of rRNA and snoRNA","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis and biochemical domain swaps, single lab","pmids":["25808954"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of Chaetomium thermophilum Prp43 resolved to 2.9 Å; the protein functionally replaces S. cerevisiae Prp43 in spliceosomal disassembly assays.","method":"X-ray crystallography, in vitro spliceosome disassembly complementation assay","journal":"Acta crystallographica. Section F, Structural biology communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional validation in disassembly assay, single lab","pmids":["26841761"],"is_preprint":false},{"year":2017,"finding":"Crystal structures of Prp43 complexes (ATP-analog•RNA, ADP-bound) reveal: RNA sits in a tunnel formed by two RecA-like and C-terminal domains; in the ATP-bound state the tunnel opens into a groove via large C-terminal domain rearrangements; conformational changes between ATP- and ADP-bound states couple ATP hydrolysis to RNA translocation via a β-turn (RecA1 RF motif). The mechanism is distinct from DEAD-box and other helicase families.","method":"X-ray crystallography of multiple functional states, structure-based mutagenesis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple crystal structures with mutagenesis validation, mechanistic framework established","pmids":["28092261"],"is_preprint":false},{"year":2017,"finding":"The stacking of ATP base between R-motif (R159, RecA1) and F-motif (F357, RecA2) residues is required for coupling NTPase and helicase activities in Prp43. F357A mutation or pyrimidine nucleotides decouple NTPase from helicase activity; R159A reduces both. G-patch protein activation is linked to this unique nucleotide-binding mode.","method":"In vitro ATPase and helicase assays, site-directed mutagenesis, nucleotide analogue substitution","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and chemical probing, single lab","pmids":["28180308"],"is_preprint":false},{"year":2017,"finding":"Human DHX15 functions as an AR co-activator by forming a complex with E3 ligase Siah2 and AR (through AR's nuclear export signal, NESAR). DHX15 stabilizes Siah2 and enhances its E3 ubiquitin-ligase activity, resulting in AR activation. This function is independent of DHX15's ATPase activity.","method":"Yeast mutagenesis screen, co-immunoprecipitation, ubiquitination assay, xenograft tumor model, siRNA knockdown","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with functional ubiquitination assay and in vivo xenograft, single lab","pmids":["28991234"],"is_preprint":false},{"year":2017,"finding":"Human NKRF (G-patch protein) forms a pre-ribosomal subcomplex with DHX15 and XRN2. NKRF stimulates DHX15 catalytic activity; this is required for an early pre-rRNA cleavage step (A'). NKRF also recruits XRN2 to nucleolar pre-ribosomal complexes for turnover of excised spacer fragments. NKRF binds transcribed spacer regions of pre-rRNA (by CRAC).","method":"UV crosslinking and CRAC, co-immunoprecipitation, siRNA depletion with Northern analysis, in vitro helicase stimulation assay","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRAC direct RNA-binding data, Co-IP, stimulation assay, depletion phenotypes, multiple methods","pmids":["28115624"],"is_preprint":false},{"year":2018,"finding":"Human CMTr1 (RNA cap1 methyltransferase) binds DHX15 via CMTr1's G-patch domain; DHX15 helicase activity stimulates CMTr1 activity specifically on RNA substrates with highly structured 5' termini, in proportion to structural strength. This is the first demonstrated role for DHX15 in post-transcriptional RNA modification.","method":"Co-immunoprecipitation, in vitro cap methylation assay with structured vs. unstructured RNA substrates, domain mapping","journal":"Philosophical transactions of the Royal Society of London. Series B, Biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro functional reconstitution with domain mapping, single lab","pmids":["30397098"],"is_preprint":false},{"year":2019,"finding":"DHX15 associates with RIG-I CARDs through its amino terminus and the DHX15–RIG-I complex is recruited to MAVS upon virus infection. DHX15 selectively binds PAMP RNA to promote RIG-I ATP hydrolysis and signaling activation but cannot substitute for RIG-I. DHX15 knockdown increased susceptibility to diverse RNA viruses.","method":"Co-immunoprecipitation, RNA binding assay, ATPase assay, siRNA knockdown, viral replication assay","journal":"Journal of interferon & cytokine research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain resolution plus functional ATPase stimulation assay, single lab","pmids":["31090472"],"is_preprint":false},{"year":2020,"finding":"The OB-fold β4-β5 loop of Prp43 is crucial for binding the G-patch of Pfa1 but not for PINX1 binding, revealing distinct binding modes for different G-patch cofactors. Despite different binding modes, stimulation of ATPase and helicase activities by both Pfa1 and PINX1 requires the β4-β5 loop. Disruption of this loop abrogates Prp43 activity in ribosome biogenesis in vivo.","method":"Mutagenesis, in vitro ATPase/helicase assays, yeast growth and rRNA processing assays","journal":"RNA biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis plus in vivo validation, single lab","pmids":["32882145"],"is_preprint":false},{"year":2021,"finding":"DHX15 interacts with NLRP6 to trigger NLRP6 inflammasome assembly and activation for IL-18 secretion in intestinal epithelial cells (IECs) upon poly(I:C) and enteric RNA virus stimulation. IEC-specific Dhx15 knockout mice show impaired IFN-β, IFN-λ3, and IL-18 production and increased susceptibility to rotavirus and reovirus.","method":"Conditional knockout mouse model, co-immunoprecipitation, cytokine ELISA, viral infection in vivo","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO in vivo model, Co-IP mechanistic link to NLRP6, orthogonal cytokine readouts","pmids":["34161762"],"is_preprint":false},{"year":2021,"finding":"TFIP11 (human Ntr1 homolog) has DHX15-independent roles: it is essential for 2'-O-methylation of U6 snRNA by controlling fibrillarin/snoRNA association, and for U4/U6.U5 tri-snRNP assembly and splicing fidelity. These functions do not require interaction with DHX15.","method":"TFIP11 knockdown, 2'-O-methylation mapping, snRNP sedimentation, splicing fidelity assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct mechanistic dissection showing independence from DHX15 with multiple orthogonal assays","pmids":["34789764"],"is_preprint":false},{"year":2022,"finding":"DHX15 immunodepletion increases A-complex accumulation during in vitro spliceosome assembly and stabilizes an atypical ATP-independent U2 snRNP interaction with a minimal substrate, suggesting DHX15 plays a quality-control role in U2 snRNP engagement with introns. RNase H probing identified nucleotides in the branch-binding region of U2 snRNA that become accessible with GTP hydrolysis (implicating a DEAH enzyme).","method":"Immunodepletion from splicing extract, in vitro spliceosome assembly assay, RNase H mapping of U2 snRNA","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — immunodepletion with biochemical spliceosome readout, single lab","pmids":["35046126"],"is_preprint":false},{"year":2022,"finding":"DHX15 deletion in mouse B cells (conditional KO) impairs lymphocyte development, reduces peripheral B cell numbers, and impairs primary IgG1 responses to immunization, demonstrating an intrinsic requirement for DHX15 in B cell proliferation, survival, and humoral immunity.","method":"Conditional knockout mice, flow cytometry, antigen immunization, B cell proliferation assay","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined cellular and in vivo phenotype, single lab","pmids":["31921164"],"is_preprint":false},{"year":2022,"finding":"DHX15 conditional deletion in NK cells reduces NK cell numbers, blocks NK cell maturation, impairs cytolytic function, and abolishes IL-15 responsiveness by inhibiting CD122 surface expression. DHX15 facilitates CD122 surface expression through interaction with CD122 3'UTR in an ATPase-domain-dependent manner, without affecting CD122 mRNA splicing or stability. Ectopic constitutively-active STAT5 rescues the phenotype.","method":"Conditional NK-cell knockout mice, flow cytometry, cytotoxicity assay, co-immunoprecipitation, mRNA stability assay, rescue with active STAT5","journal":"Cellular & molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with mechanistic rescue experiment and domain-specific requirement, single lab","pmids":["35322175"],"is_preprint":false},{"year":2022,"finding":"smFRET analysis of Prp43 shows that the G-patch protein Pfa1 induces an open conformation of the RecA domains, accelerating ADP release and enabling transition to the strong RNA-binding apo state. Pfa1 enables Prp43(ADP) to switch between RNA-bound and RNA-unbound states rather than dissociating, making translocation faster than dissociation and enabling processive movement.","method":"Single-molecule FRET (smFRET), in vitro ATPase and helicase assays, nucleotide-state manipulation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — single-molecule reconstitution with multiple nucleotide states, mechanistic model with multiple orthogonal smFRET reporters","pmids":["36409901"],"is_preprint":false},{"year":2022,"finding":"DHX15 is identified as the DEAH-box helicase that functions with SUGP1 in splicing. Crystal structure of the human DHX15–SUGP1 G-patch complex reveals the molecular basis of direct interaction. DHX15 depletion or expression of AML-associated DHX15 mutants partially recapitulates the missplicing pattern of mutant SF3B1 cancers. A DHX15–SUGP1 G-patch fusion rescues SF3B1-mutant splicing defects when incorporated into the spliceosome.","method":"Crystal structure, protein-protein interaction assays (multiple), DHX15 depletion, mutant expression, splicing reporter assays, spliceosome incorporation assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, multiple interaction assays, functional rescue experiment, mechanistic link to cancer mutation","pmids":["36459648"],"is_preprint":false},{"year":2023,"finding":"DHX15 promotes splicing quality control (QC) of suboptimal introns with weak splice sites, multiple branch points, and cryptic introns in human cells; this QC function requires DHX15's ATPase activity. SUGP1 is the G-patch factor that recruits and activates DHX15 for splicing QC, dependent on both DHX15 ATPase activity and SUGP1's ULM domain.","method":"Rapid protein depletion (degron system), nascent and mature RNA sequencing, domain mutant analysis","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — rapid depletion system enriches direct effects, transcriptome-wide with domain-resolved mechanism, corroborated by prior structural data","pmids":["37805921"],"is_preprint":false},{"year":2023,"finding":"DHX15 interacts with MYC protein directly (co-localization in cells and direct in vitro interaction). DHX15 stabilizes MYC at the post-translational level by interfering with the FBXW7–MYC interaction, thereby preventing MYC polyubiquitylation and proteasomal degradation. This function is independent of DHX15's RNA-binding capacity.","method":"Proteomic interactome analysis, co-immunoprecipitation, in vitro binding, MYC stability assay, ubiquitination assay, rescue overexpression of MYC","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and in vitro binding, ubiquitination rescue, single lab","pmids":["38161423"],"is_preprint":false},{"year":2023,"finding":"DHX15 abrogation in T-ALL perturbs RNA splicing and causes intron retention in SLC7A6 and SLC38A5, reducing glutamine import and suppressing mTORC1 activity, thereby blocking T cell development at the DN-to-DP transition and impairing leukemia cell survival.","method":"Multiple murine T-ALL models, single-cell transcriptomics, splicing analysis, metabolic assay, mTORC1 pathway readouts","journal":"Haematologica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo models with transcriptomic and metabolic validation, pathway placement, single lab","pmids":["36861414"],"is_preprint":false},{"year":2023,"finding":"smFRET analysis shows the RNA binding channel of Prp43 alternates between open and closed conformations. Binding of Pfa1 G-patch and ATP shifts the channel to the open state, facilitating RNA loading. Once RNA is loaded, the channel remains firmly closed during successive ATP hydrolysis cycles, ensuring stable contact and processive translocation.","method":"Single-molecule FRET with fluorescent reporters on RNA binding channel, nucleotide-state manipulation","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — smFRET with direct visualization of channel dynamics in multiple ligand states, mechanistically detailed","pmids":["37167006"],"is_preprint":false},{"year":2024,"finding":"GPATCH4 is a stimulatory G-patch cofactor of DHX15 that interacts with DHX15 in the nucleolus via its G-patch domain. GPATCH4 associates with pre-ribosomal particles and crosslinks to pre-rRNA and snoRNAs. Loss of GPATCH4 impairs 2'-O-methylation at multiple rRNA and snRNA sites; DHX15 ATPase activity is required for methylation at DHX15-dependent sites, suggesting DHX15 regulates snoRNA-guided 2'-O-methylation.","method":"Co-immunoprecipitation, UV crosslinking, 2'-O-methylation mapping (RiboMeth-seq), ATPase-dead mutant analysis, siRNA depletion","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct crosslinking, methylation mapping, ATPase-dependent mechanistic dissection, multiple orthogonal methods","pmids":["38113271"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM and hydrogen-deuterium exchange MS reveal that G-patch activators Tma23 and Pxr1 each contain an inhibitory segment (I-patch) that binds the catalytic RecA-like domains of Prp43 and allosterically restrains its ATPase activity; both also contain dimerization segments that organize Prp43 into higher-order complexes. Toggling between I-patch inhibition and G-patch activation is proposed to coordinate Prp43 function at discrete pre-rRNA sites.","method":"Cryo-electron microscopy, hydrogen-deuterium exchange mass spectrometry, in vitro ATPase assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure combined with HDX-MS and biochemical validation, mechanistic model supported by multiple methods","pmids":["39578461"],"is_preprint":false},{"year":2024,"finding":"Mason-Pfizer monkey virus (M-PMV) recruits DHX15 from the nucleus to cytoplasmic viral inclusion bodies via mimicry of the G-patch interaction mechanism, and this recruitment is essential for correct packaging of the viral genome and viral infectivity.","method":"Genetic engineering, viral genome packaging assay, infectivity assay, localization imaging","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional packaging assay with infectivity readout, single lab","pmids":["39320912"],"is_preprint":false},{"year":2024,"finding":"DHX15 interacts with mouse APOBEC3 and inhibits its cytidine deaminase activity. DHX15 knockdown inhibits murine leukemia virus (MLV) replication and results in more G-to-A mutations in proviral DNA; DHX15 knockdown also induces DNA damage, implicating DHX15 in genome integrity maintenance in cells expressing mouse APOBEC3.","method":"Co-immunoprecipitation, deaminase activity assay, DHX15 siRNA knockdown, viral replication assay, proviral mutation analysis","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct enzyme inhibition assay plus knockdown functional readout, single lab","pmids":["40168451"],"is_preprint":false},{"year":2024,"finding":"F. nucleatum invades intestinal epithelial cells and physically binds to DHX15; knockout of Dhx15 in Villin-Cre/KrasG12D+/- mice attenuates colorectal cancer progression, linking DHX15 to F. nucleatum-dependent oncogenic ERK/STAT3 signaling.","method":"Co-immunoprecipitation/binding assay, Dhx15 conditional KO mouse model, tumor progression analysis","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO with tumor phenotype and binding assay, single lab","pmids":["38402201"],"is_preprint":false},{"year":2025,"finding":"GPATCH3 interacts with DHX15 and enhances its ATPase activity, promoting spliceosome disassembly. Loss of GPATCH3 leads to splicing alterations in immunoregulatory genes (CXCR3, CD44, FOXP3) and reduced tumor growth in vivo.","method":"Biochemical interaction assay, ATPase activity assay, splicing reporter assays, transcriptomic analysis, in vivo xenograft","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ATPase stimulation assay plus splicing and in vivo functional data, single lab","pmids":["40861452"],"is_preprint":false}],"current_model":"DHX15 (Prp43 in yeast) is an RNA-dependent DEAH-box ATPase/helicase whose intrinsically low activity is activated by G-patch domain cofactors (including NKRF, SUGP1, RBM5, CMTr1, GPATCH4, GPATCH3, and yeast Ntr1/Pfa1/Pxr1) through allosteric opening of the RecA domains that accelerates ADP release and enables processive RNA translocation; it catalyzes ATP-dependent disassembly of the spliceosome after excised lariat-intron release (functioning in a quality-control capacity for suboptimal introns via SUGP1 recruitment), releases snoRNAs from specific pre-rRNA sites to drive ribosome biogenesis including the 20S-to-18S cleavage step, and additionally functions as a cytoplasmic viral dsRNA sensor that physically binds MAVS (and RIG-I) to activate NF-κB and MAPK (but not IRF3) antiviral signaling, while also exerting helicase-independent roles in stabilizing MYC by blocking FBXW7-mediated ubiquitination and in androgen receptor activation through Siah2 E3 ligase stabilization."},"narrative":{"mechanistic_narrative":"DHX15 (yeast Prp43) is a DEAH-box, RNA-dependent ATPase/helicase that drives ATP-dependent remodeling and disassembly of large RNA-protein machines in both pre-mRNA splicing and ribosome biogenesis [PMID:9342317, PMID:11886864, PMID:16357217]. Its intrinsic helicase activity is feeble and must be switched on by G-patch domain cofactors, which dock onto the enzyme and stimulate catalysis: in yeast this is Ntr1/Spp382 (within the Ntr1-Ntr2-Prp43 complex), Pfa1/Sqs1, and Pxr1, and in humans the activators include NKRF, SUGP1, RBM5, CMTr1, GPATCH4, and GPATCH3 [PMID:16357217, PMID:17875666, PMID:22569250, PMID:28115624, PMID:30397098, PMID:36459648, PMID:38113271, PMID:40861452]. Single-molecule and structural work shows that G-patch binding opens the RecA-like domains and the RNA-binding channel, accelerating ADP release and shifting the enzyme into a strong RNA-binding state so that translocation outcompetes dissociation, enabling processive movement; certain cofactors additionally carry an inhibitory I-patch that allosterically restrains the ATPase, so toggling between inhibition and activation tunes the enzyme at discrete sites [PMID:36409901, PMID:37167006, PMID:39578461]. In splicing, ATPase-dependent action disassembles the intron-lariat spliceosome after mRNA release and serves a quality-control/proofreading function for suboptimal introns, with SUGP1 recruiting DHX15 to enforce fidelity at weak splice sites, branch points, and cryptic introns; DHX15 depletion or AML-associated DHX15 mutants phenocopy SF3B1-mutant missplicing [PMID:11886864, PMID:16357217, PMID:20705241, PMID:36459648, PMID:37805921]. In ribosome biogenesis, DHX15/Prp43 releases snoRNAs from specific pre-rRNA sites and is required for pre-rRNA cleavage steps including the 20S-to-18S site D cleavage, and cofactors such as GPATCH4 link its ATPase activity to snoRNA-guided 2'-O-methylation [PMID:19801658, PMID:19941819, PMID:28115624, PMID:38113271]. Beyond RNA remodeling, DHX15 acts as a cytoplasmic viral RNA sensor that binds dsRNA and engages MAVS and RIG-I to activate NF-κB and MAPK signaling and inflammasome responses, with intrinsic requirements in lymphocyte development and antiviral immunity [PMID:24782566, PMID:24990078, PMID:31090472, PMID:34161762]. It also has helicase-independent roles, stabilizing MYC by blocking FBXW7-mediated ubiquitination and activating the androgen receptor through stabilization of the Siah2 E3 ligase [PMID:28991234, PMID:38161423].","teleology":[{"year":1997,"claim":"Established that the DHX15 ortholog is a dedicated spliceosome-disassembly factor, defining the core cellular process the enzyme serves rather than a general splicing step.","evidence":"Temperature-sensitive yeast alleles with Northern and splicing-complex analysis showing accumulation of trapped intron lariats","pmids":["9342317"],"confidence":"High","gaps":["Did not define the enzymatic activity or the RNA substrate at the molecular level","No cofactor requirement identified"]},{"year":2002,"claim":"Defined the biochemical activity as an RNA-dependent ATPase and showed catalysis is specifically required to release the excised lariat-intron, separating this step from mRNA release and chemistry.","evidence":"Recombinant Prp43 ATPase assays, active-site mutagenesis, and dominant-negative in vitro splicing; parallel human DDX15 colocalization with snRNPs and La binding","pmids":["11886864","12458796"],"confidence":"High","gaps":["How feeble intrinsic activity is activated in vivo was unknown","Functional significance of La interaction not resolved"]},{"year":2006,"claim":"Identified G-patch cofactors as the activating switch and RNA-targeting factors, explaining how the enzyme is recruited and stimulated at the spliceosome.","evidence":"Purified NTR (Prp43-Ntr1-Ntr2) complex disassembly assays, Ntr1 depletion, helicase assays, and broad NTP/duplex unwinding characterization","pmids":["16357217","16880513","16700561","17875666","17893323"],"confidence":"High","gaps":["Structural basis of G-patch activation not yet known","Whether human cofactors operate identically untested at this stage"]},{"year":2009,"claim":"Extended the enzyme's role to ribosome biogenesis, showing it releases snoRNAs from pre-rRNA and is required for specific pre-rRNA cleavage steps.","evidence":"Yeast genetic epistasis with Nob1/Pfa1, in vitro cleavage assays, and UV crosslinking mapping of Prp43 sites on pre-rRNA","pmids":["19801658","19941819"],"confidence":"High","gaps":["Mechanistic distinction between splicing and rRNA roles via different cofactors not fully resolved","Human ribosomal substrates not yet mapped"]},{"year":2010,"claim":"Placed the enzyme in a splicing fidelity/discard framework downstream of Prp16, establishing a proofreading function rather than only canonical disassembly.","evidence":"Reconstituted in vitro splicing with catalytic-center disruption and DEAH ATPase inactivation","pmids":["20705241"],"confidence":"High","gaps":["Did not identify the human cofactor enforcing this QC","Substrate features defining suboptimal introns not defined"]},{"year":2014,"claim":"Revealed a parallel cytoplasmic role as a viral dsRNA sensor coupling to MAVS/RIG-I antiviral signaling, distinct from its nuclear RNA-remodeling functions.","evidence":"Co-IP domain mapping, RNA pulldown, knockdown, and reporter/cytokine assays in dendritic and other cells; later RIG-I CARD interaction studies","pmids":["24782566","24990078","31090472"],"confidence":"High","gaps":["Reports differ on whether IRF3 is engaged","Relationship between dsRNA sensing and helicase mechanism unclear"]},{"year":2017,"claim":"Provided the structural mechanism: RNA threads a domain-formed tunnel, and ATP/ADP-driven C-terminal rearrangements couple hydrolysis to translocation, with G-patch activation tied to a unique nucleotide-binding mode.","evidence":"Multiple X-ray structures (ATP-analog•RNA, ADP) of Prp43 with structure-based mutagenesis and nucleotide-analogue ATPase/helicase assays","pmids":["28092261","28180308","26841761"],"confidence":"High","gaps":["Static structures did not capture conformational kinetics","Cofactor-bound activated state not crystallized here"]},{"year":2017,"claim":"Broadened the human cofactor repertoire and uncovered helicase-independent oncogenic functions, showing the protein moonlights beyond RNA remodeling.","evidence":"NKRF-DHX15-XRN2 CRAC/Co-IP/depletion for pre-rRNA cleavage; Siah2/AR co-activation via Co-IP, ubiquitination and xenograft assays; RBM5 and CMTr1 helicase stimulation","pmids":["28115624","28991234","22569250","30397098"],"confidence":"Medium","gaps":["AR/Siah2 mechanism rests on single-lab data","How one enzyme partitions among many cofactors in vivo unknown"]},{"year":2022,"claim":"Resolved the activation kinetics: G-patch binding opens the RecA domains and RNA channel, accelerating ADP release and making translocation faster than dissociation to confer processivity.","evidence":"Single-molecule FRET with multiple nucleotide states and channel reporters, plus ATPase/helicase assays","pmids":["36409901","37167006"],"confidence":"High","gaps":["Performed in yeast Prp43; human DHX15 kinetics inferred","How specific RNP contexts modulate this cycle not addressed"]},{"year":2022,"claim":"Demonstrated intrinsic requirements in immune cell development and identified a non-splicing mRNA-regulatory role at the CD122 3'UTR.","evidence":"Conditional B-cell and NK-cell knockout mice with flow cytometry, cytotoxicity, Co-IP, and STAT5 rescue; inflammasome NLRP6 conditional KO","pmids":["27_placeholder","35322175","34161762"],"confidence":"Medium","gaps":["Whether developmental phenotypes reflect splicing, ribosome, or signaling roles not separated","Direct CD122 3'UTR mechanism single-lab"]},{"year":2023,"claim":"Linked DHX15 to human disease-relevant splicing QC through SUGP1 and to cancer metabolism, mechanistically connecting the enzyme to SF3B1-mutant missplicing.","evidence":"Crystal structure of DHX15-SUGP1 G-patch, degron depletion with nascent RNA-seq, AML mutant analysis, and T-ALL models with metabolic/mTORC1 readouts","pmids":["36459648","37805921","36861414"],"confidence":"High","gaps":["Full spectrum of QC substrates in vivo incomplete","How DHX15 mutations contribute to leukemia mechanistically not fully resolved"]},{"year":2024,"claim":"Refined cofactor biology with an inhibitory I-patch and dimerization mechanism and expanded the partner set controlling rRNA modification and additional pathogen interactions.","evidence":"Cryo-EM/HDX-MS of I-patch cofactors; GPATCH4 crosslinking and RiboMeth-seq; APOBEC3, M-PMV, and F. nucleatum interaction and in vivo studies","pmids":["39578461","38113271","40168451","39320912","38402201"],"confidence":"Medium","gaps":["I-patch toggling tested mainly in yeast","Human relevance of pathogen-hijack mechanisms not fully established"]},{"year":2023,"claim":"Identified a helicase- and RNA-independent role in MYC stabilization via blockade of FBXW7-mediated degradation, reinforcing DHX15 as a protein-stability regulator.","evidence":"Interactome, Co-IP, in vitro binding, MYC stability and ubiquitination assays with MYC rescue","pmids":["38161423"],"confidence":"Medium","gaps":["Single-lab Co-IP-based mechanism","Structural basis of FBXW7 competition unknown"]},{"year":null,"claim":"How a single enzyme is partitioned among its many competing G-patch cofactors and helicase-independent partners within a cell, and whether these roles are spatially or temporally segregated, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated model of cofactor selection in vivo","Helicase-independent functions lack structural and reciprocal validation","Human disease causality from DHX15 mutations not established by family/rescue evidence in this corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[1,4,17,29]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[4,6,11,34]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[9,14,20,28]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1,4]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[19,32]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[2,20,35]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[2]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[13,14]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,3,9,30,31]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[8,9,20,35]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[13,14,24]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[13,14,22]}],"complexes":["NTR complex (Prp43-Ntr1-Ntr2)","DHX15-MAVS antiviral signaling complex","DHX15-SUGP1 G-patch complex","NKRF-DHX15-XRN2 pre-ribosomal subcomplex"],"partners":["SUGP1","NKRF","RBM5","GPATCH4","MAVS","RIG-I","MYC","SIAH2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O43143","full_name":"ATP-dependent RNA helicase DHX15","aliases":["ATP-dependent RNA helicase #46","DEAH box protein 15","Splicing factor Prp43","hPrp43"],"length_aa":795,"mass_kda":90.9,"function":"RNA helicase involved in mRNA processing and antiviral innate immunity (PubMed:19103666, PubMed:19432882, PubMed:24782566, PubMed:24990078, PubMed:32179686, PubMed:34161762). Pre-mRNA processing factor involved in disassembly of spliceosomes after the release of mature mRNA (PubMed:19103666). In cooperation with TFIP11 seem to be involved in the transition of the U2, U5 and U6 snRNP-containing IL complex to the snRNP-free IS complex leading to efficient debranching and turnover of excised introns (PubMed:19103666). Plays a key role in antiviral innate immunity by promoting both MAVS-dependent signaling and NLRP6 inflammasome (PubMed:24782566, PubMed:24990078, PubMed:34161762). Acts as an RNA virus sensor: recognizes and binds viral double stranded RNA (dsRNA) and activates the MAVS-dependent signaling to produce interferon-beta and interferon lambda-3 (IFNL3) (PubMed:24782566, PubMed:24990078, PubMed:34161762). Involved in intestinal antiviral innate immunity together with NLRP6: recognizes and binds viral dsRNA and promotes activation of the NLRP6 inflammasome in intestinal epithelial cells to restrict infection by enteric viruses (PubMed:34161762). The NLRP6 inflammasome acts by promoting maturation and secretion of IL18 in the extracellular milieu (PubMed:34161762). Also involved in antibacterial innate immunity by promoting Wnt-induced antimicrobial protein expression in Paneth cells (By similarity)","subcellular_location":"Nucleus; Nucleus, nucleolus","url":"https://www.uniprot.org/uniprotkb/O43143/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/DHX15","classification":"Common Essential","n_dependent_lines":1205,"n_total_lines":1208,"dependency_fraction":0.9975165562913907},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"RANBP2","stoichiometry":10.0},{"gene":"RBM17","stoichiometry":4.0},{"gene":"U2SURP","stoichiometry":4.0},{"gene":"COMMD1","stoichiometry":0.2},{"gene":"COMMD4","stoichiometry":0.2},{"gene":"LSS","stoichiometry":0.2},{"gene":"RBM39","stoichiometry":0.2},{"gene":"SF3A1","stoichiometry":0.2},{"gene":"SF3B1","stoichiometry":0.2},{"gene":"SNRPA","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/DHX15","total_profiled":1310},"omim":[{"mim_id":"616836","title":"G-PATCH DOMAIN-CONTAINING PROTEIN 2; GPATCH2","url":"https://www.omim.org/entry/616836"},{"mim_id":"609650","title":"NLR FAMILY, PYRIN DOMAIN-CONTAINING 6; NLRP6","url":"https://www.omim.org/entry/609650"},{"mim_id":"607960","title":"DEAH-BOX HELICASE 32, PUTATIVE; DHX32","url":"https://www.omim.org/entry/607960"},{"mim_id":"603403","title":"DEAH-BOX HELICASE 15; DHX15","url":"https://www.omim.org/entry/603403"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nuclear speckles","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DHX15"},"hgnc":{"alias_symbol":["HRH2","DBP1","Prp43","PrPp43p","PRPF43"],"prev_symbol":["DDX15"]},"alphafold":{"accession":"O43143","domains":[{"cath_id":"3.40.50.300","chopping":"124-311","consensus_level":"high","plddt":93.4275,"start":124,"end":311},{"cath_id":"3.40.50.300","chopping":"318-501","consensus_level":"high","plddt":90.6412,"start":318,"end":501},{"cath_id":"1.20.120.1080","chopping":"506-670","consensus_level":"medium","plddt":93.4149,"start":506,"end":670},{"cath_id":"-","chopping":"699-791","consensus_level":"high","plddt":93.4756,"start":699,"end":791}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43143","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43143-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43143-F1-predicted_aligned_error_v6.png","plddt_mean":85.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DHX15","jax_strain_url":"https://www.jax.org/strain/search?query=DHX15"},"sequence":{"accession":"O43143","fasta_url":"https://rest.uniprot.org/uniprotkb/O43143.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43143/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43143"}},"corpus_meta":[{"pmid":"9342317","id":"PMC_9342317","title":"Prp43: An RNA helicase-like factor involved in spliceosome disassembly.","date":"1997","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/9342317","citation_count":206,"is_preprint":false},{"pmid":"19801658","id":"PMC_19801658","title":"RNA helicase Prp43 and its co-factor Pfa1 promote 20 to 18 S rRNA processing catalyzed by the endonuclease Nob1.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19801658","citation_count":161,"is_preprint":false},{"pmid":"11886864","id":"PMC_11886864","title":"Prp43 is an essential RNA-dependent ATPase required for release of lariat-intron from the spliceosome.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11886864","citation_count":152,"is_preprint":false},{"pmid":"19941819","id":"PMC_19941819","title":"Prp43 bound at different sites on the pre-rRNA performs distinct 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complex analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function with defined molecular phenotype, replicated conceptually by multiple subsequent studies\",\n      \"pmids\": [\"9342317\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Purified recombinant Prp43 is an RNA-dependent ATPase; alanine mutations in motifs I (GSGKT), II (DEAH), and VI (QRAGRAGR) abolish ATPase activity in vitro and are lethal in vivo. An ATPase-dead mutant (T123A) blocks release of the excised lariat-intron from the spliceosome without affecting mRNA release or chemical splicing steps; the lariat-intron in T123A-arrested complexes is inaccessible to debranching enzyme Dbr1.\",\n      \"method\": \"Recombinant protein purification, in vitro ATPase assay, active-site mutagenesis, in vitro splicing assay, dominant-negative overexpression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis, multiple orthogonal assays in one study\",\n      \"pmids\": [\"11886864\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Human DDX15 (DHX15) colocalizes with spliceosomal snRNPs in nuclear speckles and in nucleoli, and is associated with spliceosomal U snRNAs by RNA co-precipitation. DDX15 interacts with the human La autoantigen both in vivo and in vitro; the interaction requires a region that partly overlaps the DEAH-box domain.\",\n      \"method\": \"Large-scale immunoprecipitation from HeLa S100 extracts, in vitro binding, immunofluorescence, RNA co-precipitation\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP/pulldown with in vivo and in vitro confirmation, single lab\",\n      \"pmids\": [\"12458796\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Prp43 forms a stable NTR complex with the novel splicing factors Ntr1 and Ntr2; the affinity-purified NTR complex catalyzes ATP-dependent spliceosome disassembly, releasing U2, U5, U6, the NineTeen Complex, and the lariat-intron. Ntr1 interacts with Prp43 through its N-terminal G-patch domain.\",\n      \"method\": \"Immunoprecipitation, in vitro spliceosome disassembly assay with purified NTR complex, domain mapping\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical reconstitution of disassembly with purified complex, multiple orthogonal methods\",\n      \"pmids\": [\"16357217\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Prp43 hydrolyzes all common NTPs/dNTPs and unwinds short 5'/3'-tailed RNA/DNA duplexes in an ATP-dependent manner; optimal ATPase requires RNA cofactor ≥20 nt. Motif V mutations T384A and T384V are lethal and block lariat release despite retaining ATPase activity, demonstrating that ATPase activity is necessary but not sufficient for function.\",\n      \"method\": \"In vitro ATPase and helicase unwinding assays, site-directed mutagenesis, in vitro splicing assay\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis, single lab but multiple orthogonal assays\",\n      \"pmids\": [\"16700561\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Ntr1 (Spp382) is required for association of Prp43 with the excised intron complex; depletion of Ntr1 causes accumulation of excised intron and impaired snRNP recycling, indicating Ntr1 acts as a spliceosome receptor or RNA-targeting factor for Prp43.\",\n      \"method\": \"Metabolic depletion of Ntr1, in vitro splicing assay, snRNP sedimentation analysis, immunoprecipitation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic depletion with defined biochemical phenotype, replicated by independent lab\",\n      \"pmids\": [\"16880513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Ntr1 activates the inherently feeble helicase activity of Prp43 to trigger lariat-intron release; the N-terminal 120-aa segment of Ntr1 suffices for Prp43 binding and helicase stimulation. Lethal Prp43 mutants T384A/T384V that retain ATPase activity are refractory to Ntr1-mediated helicase stimulation. Specific missense mutations in Prp43 and Ntr1 that disrupt protein-protein interaction also impair RNA unwinding stimulation.\",\n      \"method\": \"In vitro helicase assay, protein-protein interaction mapping, site-directed mutagenesis, in vitro splicing assay\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis of both partners, multiple orthogonal assays\",\n      \"pmids\": [\"17875666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The Ntr1-Ntr2 complex binds the spliceosome before recruiting Prp43; Ntr2 interacts with U5 component Brr2 and is essential for NTR-spliceosome docking. Binding of the NTR complex does not require ATP, but spliceosome disassembly requires ATP hydrolysis.\",\n      \"method\": \"Immunoprecipitation, in vitro spliceosome-binding and disassembly assays, two-hybrid and deletion analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ATP requirement biochemically defined, replicated across labs\",\n      \"pmids\": [\"17893323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In yeast, functional cooperation between Nob1 (endonuclease) and Prp43 with its cofactor Pfa1 is required for 20S-to-18S pre-rRNA cleavage at site D; genetic epistasis shows that loss of Ltv1 combined with Prp43 or Pfa1 mutation blocks cytoplasmic site D cleavage, which is suppressed by wild-type but not catalytic-dead Nob1.\",\n      \"method\": \"Genetic epistasis, in vitro cleavage assay with purified Nob1, pre-rRNA Northern analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of cleavage plus genetic epistasis with multiple mutant combinations\",\n      \"pmids\": [\"19801658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"UV crosslinking identifies multiple Prp43-binding sites on pre-rRNA: predominantly within helix 44 of 18S (near the site of 18S 3' cleavage) and four major sites in 25S including helix 34. Depletion or catalytic inactivation of Prp43 causes accumulation of snoRNAs that guide modifications near helix 34 on preribosomes, indicating Prp43 releases snoRNAs from specific pre-rRNA sites.\",\n      \"method\": \"UV crosslinking and immunoprecipitation (CRAC precursor), Northern analysis of snoRNA association, catalytic point-mutant strains\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct RNA crosslinking plus functional genetic analysis, multiple orthogonal approaches\",\n      \"pmids\": [\"19941819\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Prp43 acts as a downstream discard-pathway factor for proofreading 5' splice site cleavage: after Prp16-mediated rejection of suboptimal substrates, Prp43 is required to disassemble the rejected spliceosome. Prp16 and Prp43 thus cooperate in an ATP-dependent fidelity framework.\",\n      \"method\": \"In vitro splicing assay using metal-ligand disruption of catalytic center, DEAH-box ATPase inactivation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reconstituted in vitro splicing system with defined epistatic relationship, single lab but rigorous biochemistry\",\n      \"pmids\": [\"20705241\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Human RBM5 directly interacts with DHX15 via its G-patch domain, and recombinant RBM5 stimulates DHX15 helicase activity in vitro in a G-patch-domain-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, in vitro helicase assay, domain deletion analysis\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct in vitro helicase stimulation assay with domain mapping, single lab\",\n      \"pmids\": [\"22569250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The minimal activating fragment of Ntr1 is residues 51–110 (containing the G-patch). Cross-linking mass spectrometry shows Prp43 interacts with the G-patch motif of Ntr1 through its C-terminal domains. Functionally important RNA-binding residues were identified in both Prp43 and Ntr1.\",\n      \"method\": \"Biochemical ATPase/helicase assays, structural mass spectrometry (protein cross-linking), domain truncation\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro functional assays combined with structural proteomics, single lab\",\n      \"pmids\": [\"24165877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Human DHX15 physically interacts with MAVS and mediates MAVS-dependent activation of the NF-κB, JNK, and p38 MAPK pathways (but not IRF3) in response to poly(I:C) and RNA virus infection. DHX15 is required for optimal cytokine production and MAVS-mediated apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, reporter assays, Drosophila misexpression screen\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP plus knockdown with functional cytokine/apoptosis readouts, replicated by independent lab (PMID:24990078)\",\n      \"pmids\": [\"24782566\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"DHX15 binds double-stranded RNA (poly(I:C)) specifically via its helicase C-terminal domain. The N-terminal DEXDc-containing domain of DHX15 binds the C-terminus of MAVS. DHX15 is required for IRF3 phosphorylation as well as NF-κB and MAPK signaling during RNA virus infection in myeloid dendritic cells.\",\n      \"method\": \"RNA pulldown, domain-mapping co-immunoprecipitation, shRNA knockdown, signaling assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain-resolved binding assays plus functional knockdown in primary cells, corroborated by independent lab\",\n      \"pmids\": [\"24990078\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The G-patches of Spp382/Ntr1, Sqs1/Pfa1, and Pxr1/Gno1 differ in their ability to interact with and functionally substitute for each other in Prp43-dependent splicing and rRNA processing. Deletion of the primary Prp43-binding site in Pxr1 does not impair rRNA processing but causes accumulation of extended snoRNA forms, linking Prp43 to snoRNA biogenesis.\",\n      \"method\": \"Yeast two-hybrid, domain-swap mutagenesis, site-directed mutagenesis, Northern analysis of rRNA and snoRNA\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis and biochemical domain swaps, single lab\",\n      \"pmids\": [\"25808954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of Chaetomium thermophilum Prp43 resolved to 2.9 Å; the protein functionally replaces S. cerevisiae Prp43 in spliceosomal disassembly assays.\",\n      \"method\": \"X-ray crystallography, in vitro spliceosome disassembly complementation assay\",\n      \"journal\": \"Acta crystallographica. Section F, Structural biology communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional validation in disassembly assay, single lab\",\n      \"pmids\": [\"26841761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structures of Prp43 complexes (ATP-analog•RNA, ADP-bound) reveal: RNA sits in a tunnel formed by two RecA-like and C-terminal domains; in the ATP-bound state the tunnel opens into a groove via large C-terminal domain rearrangements; conformational changes between ATP- and ADP-bound states couple ATP hydrolysis to RNA translocation via a β-turn (RecA1 RF motif). The mechanism is distinct from DEAD-box and other helicase families.\",\n      \"method\": \"X-ray crystallography of multiple functional states, structure-based mutagenesis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple crystal structures with mutagenesis validation, mechanistic framework established\",\n      \"pmids\": [\"28092261\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The stacking of ATP base between R-motif (R159, RecA1) and F-motif (F357, RecA2) residues is required for coupling NTPase and helicase activities in Prp43. F357A mutation or pyrimidine nucleotides decouple NTPase from helicase activity; R159A reduces both. G-patch protein activation is linked to this unique nucleotide-binding mode.\",\n      \"method\": \"In vitro ATPase and helicase assays, site-directed mutagenesis, nucleotide analogue substitution\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and chemical probing, single lab\",\n      \"pmids\": [\"28180308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Human DHX15 functions as an AR co-activator by forming a complex with E3 ligase Siah2 and AR (through AR's nuclear export signal, NESAR). DHX15 stabilizes Siah2 and enhances its E3 ubiquitin-ligase activity, resulting in AR activation. This function is independent of DHX15's ATPase activity.\",\n      \"method\": \"Yeast mutagenesis screen, co-immunoprecipitation, ubiquitination assay, xenograft tumor model, siRNA knockdown\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with functional ubiquitination assay and in vivo xenograft, single lab\",\n      \"pmids\": [\"28991234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Human NKRF (G-patch protein) forms a pre-ribosomal subcomplex with DHX15 and XRN2. NKRF stimulates DHX15 catalytic activity; this is required for an early pre-rRNA cleavage step (A'). NKRF also recruits XRN2 to nucleolar pre-ribosomal complexes for turnover of excised spacer fragments. NKRF binds transcribed spacer regions of pre-rRNA (by CRAC).\",\n      \"method\": \"UV crosslinking and CRAC, co-immunoprecipitation, siRNA depletion with Northern analysis, in vitro helicase stimulation assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRAC direct RNA-binding data, Co-IP, stimulation assay, depletion phenotypes, multiple methods\",\n      \"pmids\": [\"28115624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Human CMTr1 (RNA cap1 methyltransferase) binds DHX15 via CMTr1's G-patch domain; DHX15 helicase activity stimulates CMTr1 activity specifically on RNA substrates with highly structured 5' termini, in proportion to structural strength. This is the first demonstrated role for DHX15 in post-transcriptional RNA modification.\",\n      \"method\": \"Co-immunoprecipitation, in vitro cap methylation assay with structured vs. unstructured RNA substrates, domain mapping\",\n      \"journal\": \"Philosophical transactions of the Royal Society of London. Series B, Biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro functional reconstitution with domain mapping, single lab\",\n      \"pmids\": [\"30397098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"DHX15 associates with RIG-I CARDs through its amino terminus and the DHX15–RIG-I complex is recruited to MAVS upon virus infection. DHX15 selectively binds PAMP RNA to promote RIG-I ATP hydrolysis and signaling activation but cannot substitute for RIG-I. DHX15 knockdown increased susceptibility to diverse RNA viruses.\",\n      \"method\": \"Co-immunoprecipitation, RNA binding assay, ATPase assay, siRNA knockdown, viral replication assay\",\n      \"journal\": \"Journal of interferon & cytokine research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain resolution plus functional ATPase stimulation assay, single lab\",\n      \"pmids\": [\"31090472\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The OB-fold β4-β5 loop of Prp43 is crucial for binding the G-patch of Pfa1 but not for PINX1 binding, revealing distinct binding modes for different G-patch cofactors. Despite different binding modes, stimulation of ATPase and helicase activities by both Pfa1 and PINX1 requires the β4-β5 loop. Disruption of this loop abrogates Prp43 activity in ribosome biogenesis in vivo.\",\n      \"method\": \"Mutagenesis, in vitro ATPase/helicase assays, yeast growth and rRNA processing assays\",\n      \"journal\": \"RNA biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis plus in vivo validation, single lab\",\n      \"pmids\": [\"32882145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DHX15 interacts with NLRP6 to trigger NLRP6 inflammasome assembly and activation for IL-18 secretion in intestinal epithelial cells (IECs) upon poly(I:C) and enteric RNA virus stimulation. IEC-specific Dhx15 knockout mice show impaired IFN-β, IFN-λ3, and IL-18 production and increased susceptibility to rotavirus and reovirus.\",\n      \"method\": \"Conditional knockout mouse model, co-immunoprecipitation, cytokine ELISA, viral infection in vivo\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO in vivo model, Co-IP mechanistic link to NLRP6, orthogonal cytokine readouts\",\n      \"pmids\": [\"34161762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TFIP11 (human Ntr1 homolog) has DHX15-independent roles: it is essential for 2'-O-methylation of U6 snRNA by controlling fibrillarin/snoRNA association, and for U4/U6.U5 tri-snRNP assembly and splicing fidelity. These functions do not require interaction with DHX15.\",\n      \"method\": \"TFIP11 knockdown, 2'-O-methylation mapping, snRNP sedimentation, splicing fidelity assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct mechanistic dissection showing independence from DHX15 with multiple orthogonal assays\",\n      \"pmids\": [\"34789764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DHX15 immunodepletion increases A-complex accumulation during in vitro spliceosome assembly and stabilizes an atypical ATP-independent U2 snRNP interaction with a minimal substrate, suggesting DHX15 plays a quality-control role in U2 snRNP engagement with introns. RNase H probing identified nucleotides in the branch-binding region of U2 snRNA that become accessible with GTP hydrolysis (implicating a DEAH enzyme).\",\n      \"method\": \"Immunodepletion from splicing extract, in vitro spliceosome assembly assay, RNase H mapping of U2 snRNA\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — immunodepletion with biochemical spliceosome readout, single lab\",\n      \"pmids\": [\"35046126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DHX15 deletion in mouse B cells (conditional KO) impairs lymphocyte development, reduces peripheral B cell numbers, and impairs primary IgG1 responses to immunization, demonstrating an intrinsic requirement for DHX15 in B cell proliferation, survival, and humoral immunity.\",\n      \"method\": \"Conditional knockout mice, flow cytometry, antigen immunization, B cell proliferation assay\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined cellular and in vivo phenotype, single lab\",\n      \"pmids\": [\"31921164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DHX15 conditional deletion in NK cells reduces NK cell numbers, blocks NK cell maturation, impairs cytolytic function, and abolishes IL-15 responsiveness by inhibiting CD122 surface expression. DHX15 facilitates CD122 surface expression through interaction with CD122 3'UTR in an ATPase-domain-dependent manner, without affecting CD122 mRNA splicing or stability. Ectopic constitutively-active STAT5 rescues the phenotype.\",\n      \"method\": \"Conditional NK-cell knockout mice, flow cytometry, cytotoxicity assay, co-immunoprecipitation, mRNA stability assay, rescue with active STAT5\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with mechanistic rescue experiment and domain-specific requirement, single lab\",\n      \"pmids\": [\"35322175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"smFRET analysis of Prp43 shows that the G-patch protein Pfa1 induces an open conformation of the RecA domains, accelerating ADP release and enabling transition to the strong RNA-binding apo state. Pfa1 enables Prp43(ADP) to switch between RNA-bound and RNA-unbound states rather than dissociating, making translocation faster than dissociation and enabling processive movement.\",\n      \"method\": \"Single-molecule FRET (smFRET), in vitro ATPase and helicase assays, nucleotide-state manipulation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — single-molecule reconstitution with multiple nucleotide states, mechanistic model with multiple orthogonal smFRET reporters\",\n      \"pmids\": [\"36409901\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DHX15 is identified as the DEAH-box helicase that functions with SUGP1 in splicing. Crystal structure of the human DHX15–SUGP1 G-patch complex reveals the molecular basis of direct interaction. DHX15 depletion or expression of AML-associated DHX15 mutants partially recapitulates the missplicing pattern of mutant SF3B1 cancers. A DHX15–SUGP1 G-patch fusion rescues SF3B1-mutant splicing defects when incorporated into the spliceosome.\",\n      \"method\": \"Crystal structure, protein-protein interaction assays (multiple), DHX15 depletion, mutant expression, splicing reporter assays, spliceosome incorporation assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, multiple interaction assays, functional rescue experiment, mechanistic link to cancer mutation\",\n      \"pmids\": [\"36459648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"DHX15 promotes splicing quality control (QC) of suboptimal introns with weak splice sites, multiple branch points, and cryptic introns in human cells; this QC function requires DHX15's ATPase activity. SUGP1 is the G-patch factor that recruits and activates DHX15 for splicing QC, dependent on both DHX15 ATPase activity and SUGP1's ULM domain.\",\n      \"method\": \"Rapid protein depletion (degron system), nascent and mature RNA sequencing, domain mutant analysis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — rapid depletion system enriches direct effects, transcriptome-wide with domain-resolved mechanism, corroborated by prior structural data\",\n      \"pmids\": [\"37805921\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"DHX15 interacts with MYC protein directly (co-localization in cells and direct in vitro interaction). DHX15 stabilizes MYC at the post-translational level by interfering with the FBXW7–MYC interaction, thereby preventing MYC polyubiquitylation and proteasomal degradation. This function is independent of DHX15's RNA-binding capacity.\",\n      \"method\": \"Proteomic interactome analysis, co-immunoprecipitation, in vitro binding, MYC stability assay, ubiquitination assay, rescue overexpression of MYC\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and in vitro binding, ubiquitination rescue, single lab\",\n      \"pmids\": [\"38161423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"DHX15 abrogation in T-ALL perturbs RNA splicing and causes intron retention in SLC7A6 and SLC38A5, reducing glutamine import and suppressing mTORC1 activity, thereby blocking T cell development at the DN-to-DP transition and impairing leukemia cell survival.\",\n      \"method\": \"Multiple murine T-ALL models, single-cell transcriptomics, splicing analysis, metabolic assay, mTORC1 pathway readouts\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo models with transcriptomic and metabolic validation, pathway placement, single lab\",\n      \"pmids\": [\"36861414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"smFRET analysis shows the RNA binding channel of Prp43 alternates between open and closed conformations. Binding of Pfa1 G-patch and ATP shifts the channel to the open state, facilitating RNA loading. Once RNA is loaded, the channel remains firmly closed during successive ATP hydrolysis cycles, ensuring stable contact and processive translocation.\",\n      \"method\": \"Single-molecule FRET with fluorescent reporters on RNA binding channel, nucleotide-state manipulation\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — smFRET with direct visualization of channel dynamics in multiple ligand states, mechanistically detailed\",\n      \"pmids\": [\"37167006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GPATCH4 is a stimulatory G-patch cofactor of DHX15 that interacts with DHX15 in the nucleolus via its G-patch domain. GPATCH4 associates with pre-ribosomal particles and crosslinks to pre-rRNA and snoRNAs. Loss of GPATCH4 impairs 2'-O-methylation at multiple rRNA and snRNA sites; DHX15 ATPase activity is required for methylation at DHX15-dependent sites, suggesting DHX15 regulates snoRNA-guided 2'-O-methylation.\",\n      \"method\": \"Co-immunoprecipitation, UV crosslinking, 2'-O-methylation mapping (RiboMeth-seq), ATPase-dead mutant analysis, siRNA depletion\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct crosslinking, methylation mapping, ATPase-dependent mechanistic dissection, multiple orthogonal methods\",\n      \"pmids\": [\"38113271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM and hydrogen-deuterium exchange MS reveal that G-patch activators Tma23 and Pxr1 each contain an inhibitory segment (I-patch) that binds the catalytic RecA-like domains of Prp43 and allosterically restrains its ATPase activity; both also contain dimerization segments that organize Prp43 into higher-order complexes. Toggling between I-patch inhibition and G-patch activation is proposed to coordinate Prp43 function at discrete pre-rRNA sites.\",\n      \"method\": \"Cryo-electron microscopy, hydrogen-deuterium exchange mass spectrometry, in vitro ATPase assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure combined with HDX-MS and biochemical validation, mechanistic model supported by multiple methods\",\n      \"pmids\": [\"39578461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Mason-Pfizer monkey virus (M-PMV) recruits DHX15 from the nucleus to cytoplasmic viral inclusion bodies via mimicry of the G-patch interaction mechanism, and this recruitment is essential for correct packaging of the viral genome and viral infectivity.\",\n      \"method\": \"Genetic engineering, viral genome packaging assay, infectivity assay, localization imaging\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional packaging assay with infectivity readout, single lab\",\n      \"pmids\": [\"39320912\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DHX15 interacts with mouse APOBEC3 and inhibits its cytidine deaminase activity. DHX15 knockdown inhibits murine leukemia virus (MLV) replication and results in more G-to-A mutations in proviral DNA; DHX15 knockdown also induces DNA damage, implicating DHX15 in genome integrity maintenance in cells expressing mouse APOBEC3.\",\n      \"method\": \"Co-immunoprecipitation, deaminase activity assay, DHX15 siRNA knockdown, viral replication assay, proviral mutation analysis\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct enzyme inhibition assay plus knockdown functional readout, single lab\",\n      \"pmids\": [\"40168451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"F. nucleatum invades intestinal epithelial cells and physically binds to DHX15; knockout of Dhx15 in Villin-Cre/KrasG12D+/- mice attenuates colorectal cancer progression, linking DHX15 to F. nucleatum-dependent oncogenic ERK/STAT3 signaling.\",\n      \"method\": \"Co-immunoprecipitation/binding assay, Dhx15 conditional KO mouse model, tumor progression analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO with tumor phenotype and binding assay, single lab\",\n      \"pmids\": [\"38402201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GPATCH3 interacts with DHX15 and enhances its ATPase activity, promoting spliceosome disassembly. Loss of GPATCH3 leads to splicing alterations in immunoregulatory genes (CXCR3, CD44, FOXP3) and reduced tumor growth in vivo.\",\n      \"method\": \"Biochemical interaction assay, ATPase activity assay, splicing reporter assays, transcriptomic analysis, in vivo xenograft\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ATPase stimulation assay plus splicing and in vivo functional data, single lab\",\n      \"pmids\": [\"40861452\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DHX15 (Prp43 in yeast) is an RNA-dependent DEAH-box ATPase/helicase whose intrinsically low activity is activated by G-patch domain cofactors (including NKRF, SUGP1, RBM5, CMTr1, GPATCH4, GPATCH3, and yeast Ntr1/Pfa1/Pxr1) through allosteric opening of the RecA domains that accelerates ADP release and enables processive RNA translocation; it catalyzes ATP-dependent disassembly of the spliceosome after excised lariat-intron release (functioning in a quality-control capacity for suboptimal introns via SUGP1 recruitment), releases snoRNAs from specific pre-rRNA sites to drive ribosome biogenesis including the 20S-to-18S cleavage step, and additionally functions as a cytoplasmic viral dsRNA sensor that physically binds MAVS (and RIG-I) to activate NF-κB and MAPK (but not IRF3) antiviral signaling, while also exerting helicase-independent roles in stabilizing MYC by blocking FBXW7-mediated ubiquitination and in androgen receptor activation through Siah2 E3 ligase stabilization.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DHX15 (yeast Prp43) is a DEAH-box, RNA-dependent ATPase/helicase that drives ATP-dependent remodeling and disassembly of large RNA-protein machines in both pre-mRNA splicing and ribosome biogenesis [#0, #1, #3]. Its intrinsic helicase activity is feeble and must be switched on by G-patch domain cofactors, which dock onto the enzyme and stimulate catalysis: in yeast this is Ntr1/Spp382 (within the Ntr1-Ntr2-Prp43 complex), Pfa1/Sqs1, and Pxr1, and in humans the activators include NKRF, SUGP1, RBM5, CMTr1, GPATCH4, and GPATCH3 [#3, #6, #11, #20, #21, #30, #35, #40]. Single-molecule and structural work shows that G-patch binding opens the RecA-like domains and the RNA-binding channel, accelerating ADP release and shifting the enzyme into a strong RNA-binding state so that translocation outcompetes dissociation, enabling processive movement; certain cofactors additionally carry an inhibitory I-patch that allosterically restrains the ATPase, so toggling between inhibition and activation tunes the enzyme at discrete sites [#29, #34, #36]. In splicing, ATPase-dependent action disassembles the intron-lariat spliceosome after mRNA release and serves a quality-control/proofreading function for suboptimal introns, with SUGP1 recruiting DHX15 to enforce fidelity at weak splice sites, branch points, and cryptic introns; DHX15 depletion or AML-associated DHX15 mutants phenocopy SF3B1-mutant missplicing [#1, #3, #10, #30, #31]. In ribosome biogenesis, DHX15/Prp43 releases snoRNAs from specific pre-rRNA sites and is required for pre-rRNA cleavage steps including the 20S-to-18S site D cleavage, and cofactors such as GPATCH4 link its ATPase activity to snoRNA-guided 2'-O-methylation [#8, #9, #20, #35]. Beyond RNA remodeling, DHX15 acts as a cytoplasmic viral RNA sensor that binds dsRNA and engages MAVS and RIG-I to activate NF-\\u03baB and MAPK signaling and inflammasome responses, with intrinsic requirements in lymphocyte development and antiviral immunity [#13, #14, #22, #24]. It also has helicase-independent roles, stabilizing MYC by blocking FBXW7-mediated ubiquitination and activating the androgen receptor through stabilization of the Siah2 E3 ligase [#19, #32].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established that the DHX15 ortholog is a dedicated spliceosome-disassembly factor, defining the core cellular process the enzyme serves rather than a general splicing step.\",\n      \"evidence\": \"Temperature-sensitive yeast alleles with Northern and splicing-complex analysis showing accumulation of trapped intron lariats\",\n      \"pmids\": [\"9342317\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the enzymatic activity or the RNA substrate at the molecular level\", \"No cofactor requirement identified\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined the biochemical activity as an RNA-dependent ATPase and showed catalysis is specifically required to release the excised lariat-intron, separating this step from mRNA release and chemistry.\",\n      \"evidence\": \"Recombinant Prp43 ATPase assays, active-site mutagenesis, and dominant-negative in vitro splicing; parallel human DDX15 colocalization with snRNPs and La binding\",\n      \"pmids\": [\"11886864\", \"12458796\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How feeble intrinsic activity is activated in vivo was unknown\", \"Functional significance of La interaction not resolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identified G-patch cofactors as the activating switch and RNA-targeting factors, explaining how the enzyme is recruited and stimulated at the spliceosome.\",\n      \"evidence\": \"Purified NTR (Prp43-Ntr1-Ntr2) complex disassembly assays, Ntr1 depletion, helicase assays, and broad NTP/duplex unwinding characterization\",\n      \"pmids\": [\"16357217\", \"16880513\", \"16700561\", \"17875666\", \"17893323\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of G-patch activation not yet known\", \"Whether human cofactors operate identically untested at this stage\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended the enzyme's role to ribosome biogenesis, showing it releases snoRNAs from pre-rRNA and is required for specific pre-rRNA cleavage steps.\",\n      \"evidence\": \"Yeast genetic epistasis with Nob1/Pfa1, in vitro cleavage assays, and UV crosslinking mapping of Prp43 sites on pre-rRNA\",\n      \"pmids\": [\"19801658\", \"19941819\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic distinction between splicing and rRNA roles via different cofactors not fully resolved\", \"Human ribosomal substrates not yet mapped\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Placed the enzyme in a splicing fidelity/discard framework downstream of Prp16, establishing a proofreading function rather than only canonical disassembly.\",\n      \"evidence\": \"Reconstituted in vitro splicing with catalytic-center disruption and DEAH ATPase inactivation\",\n      \"pmids\": [\"20705241\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the human cofactor enforcing this QC\", \"Substrate features defining suboptimal introns not defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revealed a parallel cytoplasmic role as a viral dsRNA sensor coupling to MAVS/RIG-I antiviral signaling, distinct from its nuclear RNA-remodeling functions.\",\n      \"evidence\": \"Co-IP domain mapping, RNA pulldown, knockdown, and reporter/cytokine assays in dendritic and other cells; later RIG-I CARD interaction studies\",\n      \"pmids\": [\"24782566\", \"24990078\", \"31090472\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reports differ on whether IRF3 is engaged\", \"Relationship between dsRNA sensing and helicase mechanism unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided the structural mechanism: RNA threads a domain-formed tunnel, and ATP/ADP-driven C-terminal rearrangements couple hydrolysis to translocation, with G-patch activation tied to a unique nucleotide-binding mode.\",\n      \"evidence\": \"Multiple X-ray structures (ATP-analog\\u2022RNA, ADP) of Prp43 with structure-based mutagenesis and nucleotide-analogue ATPase/helicase assays\",\n      \"pmids\": [\"28092261\", \"28180308\", \"26841761\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Static structures did not capture conformational kinetics\", \"Cofactor-bound activated state not crystallized here\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Broadened the human cofactor repertoire and uncovered helicase-independent oncogenic functions, showing the protein moonlights beyond RNA remodeling.\",\n      \"evidence\": \"NKRF-DHX15-XRN2 CRAC/Co-IP/depletion for pre-rRNA cleavage; Siah2/AR co-activation via Co-IP, ubiquitination and xenograft assays; RBM5 and CMTr1 helicase stimulation\",\n      \"pmids\": [\"28115624\", \"28991234\", \"22569250\", \"30397098\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"AR/Siah2 mechanism rests on single-lab data\", \"How one enzyme partitions among many cofactors in vivo unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved the activation kinetics: G-patch binding opens the RecA domains and RNA channel, accelerating ADP release and making translocation faster than dissociation to confer processivity.\",\n      \"evidence\": \"Single-molecule FRET with multiple nucleotide states and channel reporters, plus ATPase/helicase assays\",\n      \"pmids\": [\"36409901\", \"37167006\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Performed in yeast Prp43; human DHX15 kinetics inferred\", \"How specific RNP contexts modulate this cycle not addressed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated intrinsic requirements in immune cell development and identified a non-splicing mRNA-regulatory role at the CD122 3'UTR.\",\n      \"evidence\": \"Conditional B-cell and NK-cell knockout mice with flow cytometry, cytotoxicity, Co-IP, and STAT5 rescue; inflammasome NLRP6 conditional KO\",\n      \"pmids\": [\"27_placeholder\", \"35322175\", \"34161762\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether developmental phenotypes reflect splicing, ribosome, or signaling roles not separated\", \"Direct CD122 3'UTR mechanism single-lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Linked DHX15 to human disease-relevant splicing QC through SUGP1 and to cancer metabolism, mechanistically connecting the enzyme to SF3B1-mutant missplicing.\",\n      \"evidence\": \"Crystal structure of DHX15-SUGP1 G-patch, degron depletion with nascent RNA-seq, AML mutant analysis, and T-ALL models with metabolic/mTORC1 readouts\",\n      \"pmids\": [\"36459648\", \"37805921\", \"36861414\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full spectrum of QC substrates in vivo incomplete\", \"How DHX15 mutations contribute to leukemia mechanistically not fully resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Refined cofactor biology with an inhibitory I-patch and dimerization mechanism and expanded the partner set controlling rRNA modification and additional pathogen interactions.\",\n      \"evidence\": \"Cryo-EM/HDX-MS of I-patch cofactors; GPATCH4 crosslinking and RiboMeth-seq; APOBEC3, M-PMV, and F. nucleatum interaction and in vivo studies\",\n      \"pmids\": [\"39578461\", \"38113271\", \"40168451\", \"39320912\", \"38402201\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"I-patch toggling tested mainly in yeast\", \"Human relevance of pathogen-hijack mechanisms not fully established\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified a helicase- and RNA-independent role in MYC stabilization via blockade of FBXW7-mediated degradation, reinforcing DHX15 as a protein-stability regulator.\",\n      \"evidence\": \"Interactome, Co-IP, in vitro binding, MYC stability and ubiquitination assays with MYC rescue\",\n      \"pmids\": [\"38161423\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab Co-IP-based mechanism\", \"Structural basis of FBXW7 competition unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single enzyme is partitioned among its many competing G-patch cofactors and helicase-independent partners within a cell, and whether these roles are spatially or temporally segregated, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated model of cofactor selection in vivo\", \"Helicase-independent functions lack structural and reciprocal validation\", \"Human disease causality from DHX15 mutations not established by family/rescue evidence in this corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [1, 4, 17, 29]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [4, 6, 11, 34]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [9, 14, 20, 28]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [19, 32]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [2, 20, 35]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [13, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 3, 9, 30, 31]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [8, 9, 20, 35]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [13, 14, 24]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [13, 14, 22]}\n    ],\n    \"complexes\": [\"NTR complex (Prp43-Ntr1-Ntr2)\", \"DHX15-MAVS antiviral signaling complex\", \"DHX15-SUGP1 G-patch complex\", \"NKRF-DHX15-XRN2 pre-ribosomal subcomplex\"],\n    \"partners\": [\"SUGP1\", \"NKRF\", \"RBM5\", \"GPATCH4\", \"MAVS\", \"RIG-I\", \"MYC\", \"Siah2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}