{"gene":"DHX38","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":1991,"finding":"PRP16 (DHX38) is an RNA-dependent ATPase that interacts transiently with the spliceosome and is required specifically for the second catalytic step of pre-mRNA splicing in vitro; ATP binding and/or hydrolysis by PRP16 is concomitant with its release from the spliceosome.","method":"Protein purification, in vitro ATPase assay, in vitro splicing complementation assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — purified protein with direct in vitro ATPase and splicing activity demonstrated; seminal study replicated many times by independent labs","pmids":["1825134"],"is_preprint":false},{"year":1992,"finding":"PRP16 promotes a conformational change in the spliceosome that protects the 3' splice site from oligo-directed RNase H cleavage; this structural rearrangement requires ATP hydrolysis (ATP-γS, a competitive inhibitor, blocks both ATPase activity and 3' splice site protection). PRP16 can hydrolyze all NTPs and dNTPs, linking the nucleotide requirement of step 2 to PRP16.","method":"In vitro splicing assay, RNase H protection assay, ATPase activity with NTP analogs","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal biochemical assays; replicated by subsequent structural/mechanistic studies","pmids":["1464325"],"is_preprint":false},{"year":1993,"finding":"Prp16 ATPase activity governs a discard pathway for aberrantly branched lariat intermediates; suppressor alleles of PRP16 that allow splicing of branch-site mutants all map to the RNA-dependent ATPase region and show reduced ATPase activity in vitro, indicating that slowing ATP hydrolysis gives aberrant intermediates more time to proceed through the productive rather than the discard branch.","method":"Genetic suppressor screen, purification of mutant proteins, in vitro ATPase assay, in vivo steady-state splicing intermediate analysis","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (genetics, purified protein biochemistry, in vivo RNA analysis); replicated and expanded by subsequent studies","pmids":["8324826"],"is_preprint":false},{"year":1994,"finding":"Genetic interactions in yeast between Prp16 and U6 snRNA (single nucleotide deletions upstream of the 5' splice site-interacting sequence) and between Prp16 and U2-U6 helix I suppress prp16 cold-sensitive alleles, providing genetic evidence that U6 and U2 snRNAs are functional RNA ligands for Prp16's ATPase-driven remodeling activity.","method":"Genetic suppressor screen with mutagenized U6 snRNA library, site-directed mutagenesis of U2-U6 helix I, overexpression dominance analysis","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with multiple alleles; single lab, but several orthogonal genetic approaches","pmids":["8088513"],"is_preprint":false},{"year":1995,"finding":"SLU7 protein and the novel activity SSF1 are required together with PRP16 to promote the second catalytic step of splicing; using differential ATP requirements, SLU7 was shown to act after PRP16 in the splicing pathway.","method":"Glycerol gradient sedimentation to isolate PRP16-depleted spliceosomes, functional complementation with purified proteins/fractions, differential ATP requirement assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis established biochemically using depleted spliceosomes and purified factors; directly ordered pathway; replicated in subsequent studies","pmids":["7664739"],"is_preprint":false},{"year":1998,"finding":"Prp16 possesses ATP-dependent RNA unwinding (helicase) activity in vitro that is independent of sequence in either strand; the prp16-1 mutation near the ATP-binding motif abolishes both RNA-dependent ATPase and RNA unwinding activities.","method":"In vitro RNA duplex unwinding assay with purified protein, ATPase assay, mutant protein analysis","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct biochemical reconstitution of helicase activity with mutagenesis validation; single lab but rigorous in vitro assays","pmids":["9550699"],"is_preprint":false},{"year":1998,"finding":"The non-conserved N-terminal domain of Prp16 is essential for viability, required for nuclear localization, and mediates spliceosome binding specifically at the step of Prp16 function; this domain can interact in trans with the catalytic domain to allow complementation, indicating it targets the sequence-nonspecific helicase activity to the correct substrate.","method":"Deletion analysis, in vivo complementation, nuclear localization microscopy, spliceosome-binding assay, trans-complementation","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal in vivo and biochemical methods in single lab; functional domain mapped with complementation and localization assays","pmids":["9769096"],"is_preprint":false},{"year":1998,"finding":"Alanine-scanning mutagenesis of Prp16 defined essential residues: Gly-378, Lys-379, Thr-380 in motif I (GETGSGKT); Asp-473 and Glu-474 in the DEAH motif II; and Gln-685, Arg-686, Gly-688, Arg-689, Arg-692 in motif VI are all required for biological activity; the N-terminal 204 amino acids and C-terminal 100 residues are dispensable for in vivo function.","method":"Alanine-scanning mutagenesis, in vivo complementation of null strain, deletion analysis","journal":"Genetics","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — systematic mutagenesis across conserved motifs with in vivo viability readout; comprehensive structure-function map","pmids":["9611193"],"is_preprint":false},{"year":1998,"finding":"Human DHX38 (hPrp16) is required specifically for the second catalytic step of splicing: immunodepletion of hPrp16 from splicing extracts blocks step II, and activity is fully restored by recombinant hPrp16. hPrp16 associates with the spliceosome late in the splicing pathway. A chimeric yeast-human Prp16 protein rescues a yeast Prp16 knockout, demonstrating functional conservation.","method":"Immunodepletion from splicing extracts, recombinant protein complementation, spliceosome association assay, yeast knockout complementation with chimeric protein","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal depletion/add-back in human extracts plus cross-species complementation; multiple orthogonal methods","pmids":["9524131"],"is_preprint":false},{"year":2002,"finding":"Lethal Prp16 mutants in motifs I (G378A, K379A), II (D473A, E474A), and VI (Q685A, G688A, R689A, R692A) are defective for ATP hydrolysis and step 2 transesterification chemistry; these ATPase-defective mutants bind spliceosomes in vitro and block wild-type Prp16 function in trans (dominant-negative), establishing that ATP hydrolysis is mechanistically required for step 2 catalysis.","method":"Overexpression dominance assay in vivo, purification of recombinant mutant proteins, ATPase assay, in vitro splicing assay, spliceosome-binding assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — purified mutant proteins assayed biochemically plus in vivo dominant-negative analysis; multiple orthogonal methods","pmids":["11856747"],"is_preprint":false},{"year":2010,"finding":"Prp16 can associate with spliceosomes before 5' splice site cleavage and proofreads 5' splice site cleavage: when Prp16 is disabled, spliceosomes with an inactivated catalytic center can still catalyze 5' splice site cleavage (at reduced rate), but Prp16-mediated rejection is reversible, requiring the downstream discard ATPase Prp43 to complete discard.","method":"In vitro splicing assay with metal-ligand disruption at catalytic center, Prp16 inactivation, spliceosome association assay, genetic epistasis with Prp43","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple biochemical and genetic approaches; establishes new proofreading role at step 1 in addition to known step 2 role","pmids":["20705241"],"is_preprint":false},{"year":2010,"finding":"Prp16 promotes release of first-step factors Cwc25 and Yju2 from the spliceosome after lariat formation in a Prp16- and ATP-dependent manner, thereby enabling binding of Prp22, Prp18, and Slu7 to promote the second catalytic reaction; additionally, in the absence of ATP, Prp16 has an ATP-independent role in stabilizing Cwc25 binding to spliceosomes containing branch-point mutations to facilitate their splicing.","method":"In vitro splicing assay, spliceosome affinity purification, protein binding analysis, ATP-dependency experiments with branch-point mutant pre-mRNAs","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple biochemical assays in vitro; dual ATP-dependent and ATP-independent roles demonstrated with orthogonal methods","pmids":["21098140"],"is_preprint":false},{"year":2012,"finding":"NTR complex-mediated spliceosome disassembly is linked to Prp16 action: NTR can disassemble spliceosomes arrested specifically after ATP-dependent action of Prp16 (or Prp2/Prp22), but not before these ATPases act or upon their spliceosome binding; Prp16 and Slu7 (which both interact with Brr2) negatively impact Ntr2 binding to the spliceosome.","method":"Affinity purification of spliceosomes arrested at defined stages, NTR disassembly assay, Ntr2 spliceosome binding analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — well-defined biochemical stage-specific disassembly assays; multiple spliceosome arrest points tested","pmids":["23166295"],"is_preprint":false},{"year":2013,"finding":"Prp16 is required for destabilization of Yju2 and Cwc25 from the spliceosome after the first catalytic step; a truncated Yju2 (Yju2-N) with weak spliceosome affinity can support a low level of second-step splicing even in the absence of Prp16, suggesting that Prp16's role is specifically to displace stably-bound Yju2/Cwc25 to allow second-step factor binding.","method":"In vitro splicing complementation with truncated Yju2 fragments, spliceosome-binding assay, UV cross-linking to U2 snRNA","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro reconstitution with truncated proteins and UV cross-linking; single lab","pmids":["23438600"],"is_preprint":false},{"year":2014,"finding":"Prp16 destabilizes U2-U6 snRNA helix I during spliceosome remodeling between steps 1 and 2; the NTC protein Cwc2 stabilizes U2-U6 helix I, and a prp16-302 mutation stabilizes Cwc2 interactions with U6 snRNA while destabilizing Cwc2 interactions with pre-mRNA, indicating antagonistic functions between Cwc2 and Prp16 at the helix I/active site region.","method":"Genetic suppressor analysis, allele-specific epistasis, RNA-protein interaction assays (UV cross-linking), in vivo splicing assays","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and biochemical evidence from single lab; multiple allele combinations tested","pmids":["24848011"],"is_preprint":false},{"year":2014,"finding":"The C-terminal Sec63-2 domain of Brr2 modulates the ATPase activity of Prp16 in vitro by interfering with Prp16's ability to bind RNA; allele-specific genetic interactions between BRR2 and PRP16 mutations suppress or enhance growth defects, establishing a functional regulatory interaction between these two splicing helicases.","method":"In vitro ATPase assay with purified Brr2 Sec63-2 domain and Prp16, allele-specific genetic suppressor/enhancer analysis, physical interaction assay","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct in vitro ATPase modulation plus allele-specific genetic validation; two orthogonal methods","pmids":["25428373"],"is_preprint":false},{"year":2019,"finding":"DHX38 associates with satellite I noncoding RNA from the human centromere region in an interphase-specific manner; depletion of DHX38 causes defective chromosome segregation (similar to satellite I ncRNA knockdown) and impairs Aurora B function at mitosis, placing DHX38 in an ncRNP complex involved in mitotic regulation.","method":"RNA immunoprecipitation (RIP), siRNA knockdown, chromosome segregation assay, Aurora B functional analysis","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — RIP and knockdown phenotype; multiple functional readouts but single lab and no reconstitution","pmids":["31166646"],"is_preprint":false},{"year":2022,"finding":"DHX38 knockdown in human cells causes modulation of ~70 alternative splicing events and affects splicing of retina-specific genes FSCN2 and RHO; overexpression of DHX38 promotes usage of canonical and cryptic 5' splice sites in an HBB splicing reporter; the RP-linked G332D mutation modulates DHX38 splicing activity without detectably changing its spliceosome interaction profile.","method":"siRNA knockdown, RNA-seq splicing analysis, minigene splicing reporter assay, co-immunoprecipitation of spliceosomal factors","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — KD + RNA-seq + reporter assays; RP variant functional characterization; single lab, multiple orthogonal methods","pmids":["35385551"],"is_preprint":false},{"year":2022,"finding":"Dhx38 loss in zebrafish arrests erythro-myeloid progenitors (EMPs) and hematopoietic stem/progenitor cells in mitotic prometaphase with chromosome alignment defects; abnormal alternative splicing of genes related to chromosome segregation, microtubule cytoskeleton, cell cycle kinases, and DNA damage occurs in dhx38 mutants, and EMPs/HSPCs undergo p53-dependent apoptosis.","method":"CRISPR knockout zebrafish, cytological chromosome alignment analysis, RNA-seq alternative splicing analysis, p53 pathway analysis","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO model with defined mitotic and splicing phenotypes; single lab","pmids":["35929537"],"is_preprint":false},{"year":2023,"finding":"Prp16 has an ATP-independent role in promoting usage of aberrant 5' splice sites and mutated branch points: when a 5' splice site mutation is present or when Cwc24 is absent, Prp16 facilitates the branching reaction independently of ATP, and this function is mediated through the step-one factor Cwc25. Additionally, Prp16 prevents use of nearby cryptic branch sites while promoting mutated branch point usage.","method":"In vitro splicing assay with ATP analogs and mutant pre-mRNAs, deletion/depletion of Cwc24, Cwc25 interaction assays","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro reconstitution with multiple substrate/protein combinations; single lab, multiple orthogonal conditions","pmids":["37858289"],"is_preprint":false},{"year":2023,"finding":"DHX38 interacts with G3BP1 (Ras GTPase-activating protein-binding protein) as demonstrated by co-immunoprecipitation; DHX38 regulates G3BP1 expression, leading to activation of the MAPK/ERK signaling pathway and promoting EMT in NSCLC cells. Knockdown of G3BP1 reverses DHX38 overexpression-induced MAPK activation and EMT.","method":"Co-immunoprecipitation, LC-MS interactome, siRNA knockdown, ERK inhibitor (SCH772984) treatment, in vitro and in vivo tumor assays","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP plus knockdown rescue; multiple cell-based assays; single lab","pmids":["37931691"],"is_preprint":false},{"year":2023,"finding":"DHX38 directly interacts with RELL2 pre-mRNA (confirmed by RIP-qPCR) and regulates retention of intron 4 in RELL2 transcripts in gemcitabine-resistant pancreatic ductal adenocarcinoma cells; altered DHX38 expression causes corresponding changes in RELL2 intron 4 retention.","method":"RIP-qPCR, DHX38 knockdown/overexpression, RT-PCR for intron retention","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct RNA binding demonstrated by RIP; functional consequence of intron retention shown; single lab","pmids":["37506056"],"is_preprint":false},{"year":2023,"finding":"DHX38 deficiency in zebrafish and human cell lines causes significant accumulation of R-loops; DNA replication stress is the prerequisite for R-loop-induced DNA damage in DHX38 knockdown cells, establishing a DHX38/R-loop/replication stress/DNA damage regulatory axis in retinal progenitor cells.","method":"CRISPR dhx38 knockout zebrafish, R-loop immunofluorescence (S9.6 antibody), DHX38 siRNA knockdown in human cells, DNA damage markers","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO and cell-line KD with R-loop detection and DNA damage readouts; single lab, two experimental systems","pmids":["37867960"],"is_preprint":false},{"year":2024,"finding":"dhx38 knockout zebrafish display severe inner ear developmental defects (decrescent otocysts, absent semicircular canal protrusion, smaller otoliths) accompanied by DNA damage, p53-dependent apoptosis in inner ear cells, and abnormal alternative splicing of genes related to DNA damage repair and inner ear morphogenesis.","method":"CRISPR knockout zebrafish, bright-field morphology, in situ hybridization, immunofluorescence for apoptosis/DNA damage, RT-PCR alternative splicing analysis","journal":"Biomedicines","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO with defined morphological, apoptotic, and splicing phenotypes; single lab","pmids":["39857604"],"is_preprint":false},{"year":2025,"finding":"DHX38 knockdown significantly increases latent splice site (LSS) usage in a luminescence reporter and RNA-seq confirmed widespread LSS activation across hundreds of mRNAs, establishing DHX38 as a component of the nuclear SOS (suppression of splicing) quality control mechanism that prevents inappropriate use of latent splice sites.","method":"siRNA screen with luminescence reporter for LSS activation, RNA-seq after DHX38 knockdown","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint; single screen plus RNA-seq confirmation; no mechanistic reconstitution of the SOS pathway for DHX38 specifically","pmids":["bio_10.1101_2025.07.20.665773"],"is_preprint":true},{"year":2025,"finding":"YTHDF1 binds m6A-modified Dhx38 mRNA at the coding sequence (CDS) and enhances its translational efficiency without altering mRNA levels, as demonstrated by MeRIP-seq and RIP-seq in mouse retina; loss of Ythdf1 reduces Dhx38 protein levels and contributes to retinal degeneration.","method":"MeRIP-seq, RIP-seq, Ythdf1 knockout mouse, polysome/translation efficiency assay, single-cell RNA-seq","journal":"Zoological research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — integrative sequencing plus KO model; YTHDF1 as m6A reader for DHX38 mRNA established by two orthogonal sequencing methods; single lab","pmids":["40116022"],"is_preprint":false}],"current_model":"DHX38 (PRP16) is a DEAH-box RNA-dependent ATPase and RNA helicase that functions primarily as a spliceosomal remodeling factor: it binds the spliceosome transiently after the first catalytic step (lariat formation), uses ATP hydrolysis to drive a conformational rearrangement that releases first-step factors Cwc25 and Yju2 and destabilizes U2-U6 helix I, thereby enabling binding of second-step factors (Prp22, Prp18, Slu7) and 3' splice site cleavage/exon ligation; it also proofreads both the first and second catalytic steps by directing aberrant intermediates to a Prp43-dependent discard pathway, can act ATP-independently to promote aberrant splice site usage via Cwc25, associates with satellite I ncRNA to support mitotic chromosome segregation, prevents R-loop accumulation to safeguard genome integrity, and interacts with G3BP1 to activate MAPK signaling in cancer contexts, with its mRNA translation regulated by the m6A reader YTHDF1."},"narrative":{"mechanistic_narrative":"DHX38 (PRP16) is a DEAH-box RNA-dependent ATPase and RNA helicase that acts as a spliceosomal remodeling factor governing the transition between the two catalytic steps of pre-mRNA splicing [PMID:1825134, PMID:9550699]. It associates transiently with the spliceosome late in the pathway, where ATP binding/hydrolysis is coupled to its release and to a conformational rearrangement that protects the 3' splice site and licenses the second transesterification step [PMID:1825134, PMID:1464325]; ATPase-deficient motif mutants bind spliceosomes but block step 2 in a dominant-negative manner, establishing that catalysis depends on its NTP hydrolysis [PMID:11856747]. Mechanistically, after lariat formation DHX38 destabilizes the first-step factors Cwc25 and Yju2 and disrupts U2-U6 helix I, thereby enabling recruitment of second-step factors Prp22, Prp18, and Slu7 to drive exon ligation [PMID:21098140, PMID:23438600, PMID:24848011]. Its sequence-nonspecific helicase activity is targeted to the correct substrate by a non-conserved N-terminal domain and is regulated through functional interaction with the helicase Brr2 [PMID:9769096, PMID:25428373]. Beyond promoting forward catalysis, DHX38 proofreads both step 1 and step 2, routing aberrant intermediates to a Prp43-dependent discard pathway, and slowing its ATPase rate permits aberrant intermediates to proceed productively [PMID:8324826, PMID:20705241]; it also possesses an ATP-independent activity that promotes usage of aberrant 5' splice sites and mutated branch points via the step-one factor Cwc25 [PMID:21098140, PMID:37858289]. In human and zebrafish systems DHX38 controls alternative splicing and splice-site fidelity, and its loss arrests cells in mitotic prometaphase, causes R-loop accumulation and replication-stress-driven DNA damage, and triggers p53-dependent apoptosis in developing retina, inner ear, and hematopoietic progenitors [PMID:35385551, PMID:35929537, PMID:37867960, PMID:39857604]. DHX38 additionally associates with centromeric satellite I ncRNA to support chromosome segregation and Aurora B function [PMID:31166646], interacts with G3BP1 to activate MAPK/ERK signaling and EMT in cancer [PMID:37931691], and its own mRNA translation is enhanced by the m6A reader YTHDF1 [PMID:40116022].","teleology":[{"year":1991,"claim":"Established DHX38/PRP16 as an RNA-dependent ATPase that transiently engages the spliceosome and is specifically required for the second catalytic step, defining the first functional foothold for the protein.","evidence":"Protein purification with in vitro ATPase and splicing complementation assays","pmids":["1825134"],"confidence":"High","gaps":["Did not resolve the molecular substrate of remodeling","Helicase activity not yet demonstrated"]},{"year":1992,"claim":"Linked ATP hydrolysis to a defined conformational rearrangement of the spliceosome, showing the energy of NTP hydrolysis drives a structural change that protects the 3' splice site for step 2.","evidence":"In vitro splicing with RNase H protection and ATPase assays using NTP analogs","pmids":["1464325"],"confidence":"High","gaps":["Identity of the rearranged RNA elements not defined","Helicase mechanism not yet shown"]},{"year":1993,"claim":"Revealed a kinetic proofreading function whereby PRP16 ATPase activity discards aberrantly branched intermediates, explaining how splicing fidelity is enforced.","evidence":"Genetic suppressor screen with purified mutant proteins and in vivo intermediate analysis in yeast","pmids":["8324826"],"confidence":"High","gaps":["Downstream discard machinery not identified","Direct coupling of ATPase rate to discard not reconstituted"]},{"year":1994,"claim":"Identified U6 and U2-U6 helix I snRNAs as functional RNA ligands of PRP16's remodeling activity, pointing to the catalytic RNA core as its target.","evidence":"Genetic suppressor screens and site-directed mutagenesis of snRNAs in yeast","pmids":["8088513"],"confidence":"Medium","gaps":["Genetic interaction does not prove direct physical contact","Mechanism of helix I destabilization unresolved at this stage"]},{"year":1995,"claim":"Ordered the second-step pathway by showing SLU7 and SSF1 act downstream of PRP16, building a sequential factor map for exon ligation.","evidence":"Glycerol gradient isolation of PRP16-depleted spliceosomes and functional complementation with differential ATP requirements","pmids":["7664739"],"confidence":"High","gaps":["Physical handoff between PRP16 and second-step factors not visualized","SSF1 molecular identity not fully defined"]},{"year":1998,"claim":"Demonstrated intrinsic ATP-dependent RNA unwinding activity and mapped the structure-function determinants, defining DHX38 as a bona fide helicase whose catalytic motifs and substrate-targeting N-terminus are separable.","evidence":"In vitro RNA duplex unwinding, alanine-scanning and deletion mutagenesis, complementation and localization assays in yeast","pmids":["9550699","9611193","9769096"],"confidence":"High","gaps":["How the N-terminal domain recognizes the correct spliceosome state not defined","In vivo unwinding substrate not directly captured"]},{"year":1998,"claim":"Extended PRP16 function to humans, showing hPrp16 is required for step 2 and is functionally conserved across species.","evidence":"Immunodepletion/add-back in human extracts and cross-species chimera complementation in yeast","pmids":["9524131"],"confidence":"High","gaps":["Human-specific cofactors not identified","Structural basis of conservation not addressed"]},{"year":2002,"claim":"Established that ATP hydrolysis itself, not merely ATP binding, is mechanistically required for step 2 chemistry, via dominant-negative ATPase-defective mutants.","evidence":"In vivo dominance assay plus purified mutant ATPase, splicing, and spliceosome-binding assays","pmids":["11856747"],"confidence":"High","gaps":["Conformational consequence of hydrolysis not structurally resolved","Coupling of motor cycle to factor release not yet shown"]},{"year":2010,"claim":"Expanded proofreading to step 1 (5' splice site cleavage) and showed Prp16-mediated rejection is reversible and requires Prp43 to complete discard, integrating DHX38 into a two-step fidelity surveillance system.","evidence":"In vitro splicing with catalytic-center disruption, Prp16 inactivation, and genetic epistasis with Prp43","pmids":["20705241"],"confidence":"High","gaps":["Mechanism of Prp16-to-Prp43 handoff not defined","Structural state of rejected intermediates unknown"]},{"year":2013,"claim":"Defined the substrate of remodeling as the stably bound first-step factors Cwc25 and Yju2, whose displacement by Prp16 (and destabilization of U2-U6 helix I) enables second-step factor binding.","evidence":"In vitro splicing with truncated Yju2, spliceosome-binding and UV cross-linking assays; genetic and cross-linking analysis of helix I","pmids":["21098140","23438600","24848011"],"confidence":"Medium","gaps":["Direct structural snapshot of factor displacement not obtained","Antagonism with Cwc2 inferred genetically/biochemically only"]},{"year":2014,"claim":"Identified Brr2 as a regulator of Prp16 ATPase activity, and connected Prp16 action to NTR-mediated spliceosome disassembly, embedding DHX38 in a helicase regulatory network.","evidence":"In vitro ATPase modulation with Brr2 Sec63-2 domain, allele-specific genetics, and stage-specific NTR disassembly assays","pmids":["25428373","23166295"],"confidence":"High","gaps":["Physiological trigger for Brr2 regulation of Prp16 unclear","Structural basis of Brr2-Prp16 interplay not resolved"]},{"year":2019,"claim":"Revealed a splicing-independent role: DHX38 binds centromeric satellite I ncRNA and supports chromosome segregation and Aurora B function, broadening its cellular reach to mitosis.","evidence":"RNA immunoprecipitation, siRNA knockdown, chromosome segregation and Aurora B functional assays in human cells","pmids":["31166646"],"confidence":"Medium","gaps":["Direct vs splicing-mediated contribution to segregation not separated","No reconstitution of the ncRNP complex"]},{"year":2023,"claim":"Connected DHX38 to genome integrity by showing its loss causes R-loop accumulation and replication-stress-driven DNA damage, defining a DHX38/R-loop/replication-stress axis.","evidence":"CRISPR knockout zebrafish and siRNA knockdown in human cells with R-loop (S9.6) immunostaining and DNA damage markers","pmids":["37867960"],"confidence":"Medium","gaps":["Whether R-loop control is direct or a downstream consequence of splicing defects unresolved","No biochemical demonstration of R-loop resolution by DHX38"]},{"year":2023,"claim":"Documented cancer-context roles in which DHX38 interacts with G3BP1 to activate MAPK/ERK signaling and EMT, and regulates intron retention of specific transcripts in drug-resistant tumors.","evidence":"Co-IP/LC-MS, knockdown rescue, ERK inhibition, RIP-qPCR and intron-retention RT-PCR in cancer cell lines and tumor assays","pmids":["37931691","37506056"],"confidence":"Medium","gaps":["Mechanism linking splicing activity to G3BP1/MAPK not defined","Single-lab co-IP without reciprocal structural validation"]},{"year":2022,"claim":"Tied DHX38 function to development and splice-site fidelity in vertebrates, where loss arrests progenitors in prometaphase, perturbs alternative splicing, and provoked retina/ear/hematopoietic defects with p53-dependent apoptosis.","evidence":"siRNA knockdown with RNA-seq and minigene reporters in human cells; CRISPR knockout zebrafish with cytological, splicing, and p53 analyses","pmids":["35385551","35929537","39857604"],"confidence":"Medium","gaps":["Causal chain from splicing changes to mitotic arrest not fully resolved","RP-linked variant mechanism not mapped to a defined biochemical defect"]},{"year":2025,"claim":"Identified upstream control of DHX38 abundance, with the m6A reader YTHDF1 enhancing Dhx38 mRNA translation, linking epitranscriptomic regulation to DHX38 protein levels and retinal health.","evidence":"MeRIP-seq, RIP-seq, polysome assays, and Ythdf1 knockout mouse retina","pmids":["40116022"],"confidence":"Medium","gaps":["Direct functional rescue of retinal phenotype by restoring DHX38 not shown","Tissue specificity of this regulation not defined"]},{"year":null,"claim":"How DHX38's ATP-driven motor cycle is mechanistically partitioned between forward step-2 catalysis, kinetic proofreading/discard, splicing-independent R-loop suppression, and chromosome segregation remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model coupling the helicase cycle to factor release in human spliceosomes","Whether mitotic and genome-integrity roles are direct or splicing-dependent is unknown","No reconstitution of the proposed nuclear SOS latent-splice-site surveillance role for DHX38"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,1,5,9]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[5]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[3,16,21]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,8,11,17]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[16,18]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[22,23]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[20]}],"complexes":["spliceosome"],"partners":["SLU7","BRR2","G3BP1","CWC25","YJU2","YTHDF1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q92620","full_name":"Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP16","aliases":["ATP-dependent RNA helicase DHX38","DEAH box protein 38"],"length_aa":1227,"mass_kda":140.5,"function":"Probable ATP-binding RNA helicase (Probable). Involved in pre-mRNA splicing as component of the spliceosome (PubMed:29301961, PubMed:9524131)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q92620/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/DHX38","classification":"Common Essential","n_dependent_lines":640,"n_total_lines":1208,"dependency_fraction":0.5298013245033113},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CD2BP2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/DHX38","total_profiled":1310},"omim":[{"mim_id":"618220","title":"RETINITIS PIGMENTOSA 84; RP84","url":"https://www.omim.org/entry/618220"},{"mim_id":"605584","title":"DEAH-BOX HELICASE 38; DHX38","url":"https://www.omim.org/entry/605584"},{"mim_id":"268000","title":"RETINITIS PIGMENTOSA; RP","url":"https://www.omim.org/entry/268000"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DHX38"},"hgnc":{"alias_symbol":["Prp16","KIAA0224","hPrp16","PRPF16"],"prev_symbol":["DDX38"]},"alphafold":{"accession":"Q92620","domains":[{"cath_id":"3.40.50.300","chopping":"440-462_710-888","consensus_level":"high","plddt":86.0566,"start":440,"end":888},{"cath_id":"3.40.50.300","chopping":"506-704","consensus_level":"high","plddt":84.3536,"start":506,"end":704},{"cath_id":"-","chopping":"950-1153","consensus_level":"medium","plddt":85.6789,"start":950,"end":1153}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q92620","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q92620-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q92620-F1-predicted_aligned_error_v6.png","plddt_mean":68.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DHX38","jax_strain_url":"https://www.jax.org/strain/search?query=DHX38"},"sequence":{"accession":"Q92620","fasta_url":"https://rest.uniprot.org/uniprotkb/Q92620.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q92620/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q92620"}},"corpus_meta":[{"pmid":"1825134","id":"PMC_1825134","title":"PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome.","date":"1991","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/1825134","citation_count":301,"is_preprint":false},{"pmid":"1464325","id":"PMC_1464325","title":"A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis.","date":"1992","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/1464325","citation_count":185,"is_preprint":false},{"pmid":"8324826","id":"PMC_8324826","title":"A mechanism to enhance mRNA splicing fidelity: the RNA-dependent ATPase Prp16 governs usage of a discard pathway for aberrant lariat intermediates.","date":"1993","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/8324826","citation_count":162,"is_preprint":false},{"pmid":"7664739","id":"PMC_7664739","title":"SLU7 and a novel activity, SSF1, act during the PRP16-dependent step of yeast pre-mRNA splicing.","date":"1995","source":"The EMBO 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signalling","url":"https://pubmed.ncbi.nlm.nih.gov/37931691","citation_count":14,"is_preprint":false},{"pmid":"24848011","id":"PMC_24848011","title":"Remodeling of U2-U6 snRNA helix I during pre-mRNA splicing by Prp16 and the NineTeen Complex protein Cwc2.","date":"2014","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/24848011","citation_count":13,"is_preprint":false},{"pmid":"37858289","id":"PMC_37858289","title":"An ATP-independent role for Prp16 in promoting aberrant splicing.","date":"2023","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/37858289","citation_count":12,"is_preprint":false},{"pmid":"25428373","id":"PMC_25428373","title":"Brr2p carboxy-terminal Sec63 domain modulates Prp16 splicing RNA helicase.","date":"2014","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/25428373","citation_count":12,"is_preprint":false},{"pmid":"8203164","id":"PMC_8203164","title":"The complete sequence of an 18,002 bp segment of Saccharomyces cerevisiae chromosome XI contains the HBS1, MRP-L20 and PRP16 genes, and six new open reading frames.","date":"1994","source":"Yeast (Chichester, England)","url":"https://pubmed.ncbi.nlm.nih.gov/8203164","citation_count":12,"is_preprint":false},{"pmid":"35385551","id":"PMC_35385551","title":"Retinitis pigmentosa-linked mutation in DHX38 modulates its splicing activity.","date":"2022","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/35385551","citation_count":11,"is_preprint":false},{"pmid":"35929537","id":"PMC_35929537","title":"Dhx38 is required for the maintenance and differentiation of erythro-myeloid progenitors and hematopoietic stem cells by alternative splicing.","date":"2022","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/35929537","citation_count":11,"is_preprint":false},{"pmid":"37506056","id":"PMC_37506056","title":"DHX38 restricts chemoresistance by regulating the alternative pre-mRNA splicing of RELL2 in pancreatic ductal adenocarcinoma.","date":"2023","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/37506056","citation_count":9,"is_preprint":false},{"pmid":"30810475","id":"PMC_30810475","title":"Early splicing functions of fission yeast Prp16 and its unexpected requirement for gene Silencing is governed by intronic features.","date":"2019","source":"RNA biology","url":"https://pubmed.ncbi.nlm.nih.gov/30810475","citation_count":9,"is_preprint":false},{"pmid":"34965029","id":"PMC_34965029","title":"The splicing factor DHX38/PRP16 is required for ovarian clear cell carcinoma tumorigenesis, as revealed by a CRISPR-Cas9 screen.","date":"2022","source":"FEBS open bio","url":"https://pubmed.ncbi.nlm.nih.gov/34965029","citation_count":8,"is_preprint":false},{"pmid":"31166646","id":"PMC_31166646","title":"DEAH box RNA helicase DHX38 associates with satellite I noncoding RNA involved in chromosome segregation.","date":"2019","source":"Genes to cells : devoted to molecular & 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reader YTHDF1 in retinal visual function by regulating TULP1 and DHX38 translation.","date":"2025","source":"Zoological research","url":"https://pubmed.ncbi.nlm.nih.gov/40116022","citation_count":2,"is_preprint":false},{"pmid":"36662708","id":"PMC_36662708","title":"Correction: Retinitis pigmentosa-linked mutation in DHX38 modulates its splicing activity.","date":"2023","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/36662708","citation_count":1,"is_preprint":false},{"pmid":"35719279","id":"PMC_35719279","title":"A Novel Missense Variant C.2571 (P.Ala857=) of the DHX38 Gene in a Saudi Family Causes an Autosomal Recessive Retinitis Pigmentosa.","date":"2022","source":"Middle East African journal of ophthalmology","url":"https://pubmed.ncbi.nlm.nih.gov/35719279","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.07.20.665773","title":"Identification of RNA binding proteins that mediate a quality control mechanism of splicing","date":"2025-07-23","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.20.665773","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.12.05.626984","title":"Prp16 enables efficient splicing of introns with diverse exonic consensus elements in the short-intron rich  <i>Cryptococcus neoformans</i>  transcriptome","date":"2024-12-05","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.05.626984","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17519,"output_tokens":6558,"usd":0.075464,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15441,"output_tokens":5055,"usd":0.10179,"stage2_stop_reason":"end_turn"},"total_usd":0.177254,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1991,\n      \"finding\": \"PRP16 (DHX38) is an RNA-dependent ATPase that interacts transiently with the spliceosome and is required specifically for the second catalytic step of pre-mRNA splicing in vitro; ATP binding and/or hydrolysis by PRP16 is concomitant with its release from the spliceosome.\",\n      \"method\": \"Protein purification, in vitro ATPase assay, in vitro splicing complementation assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — purified protein with direct in vitro ATPase and splicing activity demonstrated; seminal study replicated many times by independent labs\",\n      \"pmids\": [\"1825134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"PRP16 promotes a conformational change in the spliceosome that protects the 3' splice site from oligo-directed RNase H cleavage; this structural rearrangement requires ATP hydrolysis (ATP-γS, a competitive inhibitor, blocks both ATPase activity and 3' splice site protection). PRP16 can hydrolyze all NTPs and dNTPs, linking the nucleotide requirement of step 2 to PRP16.\",\n      \"method\": \"In vitro splicing assay, RNase H protection assay, ATPase activity with NTP analogs\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal biochemical assays; replicated by subsequent structural/mechanistic studies\",\n      \"pmids\": [\"1464325\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Prp16 ATPase activity governs a discard pathway for aberrantly branched lariat intermediates; suppressor alleles of PRP16 that allow splicing of branch-site mutants all map to the RNA-dependent ATPase region and show reduced ATPase activity in vitro, indicating that slowing ATP hydrolysis gives aberrant intermediates more time to proceed through the productive rather than the discard branch.\",\n      \"method\": \"Genetic suppressor screen, purification of mutant proteins, in vitro ATPase assay, in vivo steady-state splicing intermediate analysis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (genetics, purified protein biochemistry, in vivo RNA analysis); replicated and expanded by subsequent studies\",\n      \"pmids\": [\"8324826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Genetic interactions in yeast between Prp16 and U6 snRNA (single nucleotide deletions upstream of the 5' splice site-interacting sequence) and between Prp16 and U2-U6 helix I suppress prp16 cold-sensitive alleles, providing genetic evidence that U6 and U2 snRNAs are functional RNA ligands for Prp16's ATPase-driven remodeling activity.\",\n      \"method\": \"Genetic suppressor screen with mutagenized U6 snRNA library, site-directed mutagenesis of U2-U6 helix I, overexpression dominance analysis\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with multiple alleles; single lab, but several orthogonal genetic approaches\",\n      \"pmids\": [\"8088513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"SLU7 protein and the novel activity SSF1 are required together with PRP16 to promote the second catalytic step of splicing; using differential ATP requirements, SLU7 was shown to act after PRP16 in the splicing pathway.\",\n      \"method\": \"Glycerol gradient sedimentation to isolate PRP16-depleted spliceosomes, functional complementation with purified proteins/fractions, differential ATP requirement assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis established biochemically using depleted spliceosomes and purified factors; directly ordered pathway; replicated in subsequent studies\",\n      \"pmids\": [\"7664739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Prp16 possesses ATP-dependent RNA unwinding (helicase) activity in vitro that is independent of sequence in either strand; the prp16-1 mutation near the ATP-binding motif abolishes both RNA-dependent ATPase and RNA unwinding activities.\",\n      \"method\": \"In vitro RNA duplex unwinding assay with purified protein, ATPase assay, mutant protein analysis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct biochemical reconstitution of helicase activity with mutagenesis validation; single lab but rigorous in vitro assays\",\n      \"pmids\": [\"9550699\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The non-conserved N-terminal domain of Prp16 is essential for viability, required for nuclear localization, and mediates spliceosome binding specifically at the step of Prp16 function; this domain can interact in trans with the catalytic domain to allow complementation, indicating it targets the sequence-nonspecific helicase activity to the correct substrate.\",\n      \"method\": \"Deletion analysis, in vivo complementation, nuclear localization microscopy, spliceosome-binding assay, trans-complementation\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal in vivo and biochemical methods in single lab; functional domain mapped with complementation and localization assays\",\n      \"pmids\": [\"9769096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Alanine-scanning mutagenesis of Prp16 defined essential residues: Gly-378, Lys-379, Thr-380 in motif I (GETGSGKT); Asp-473 and Glu-474 in the DEAH motif II; and Gln-685, Arg-686, Gly-688, Arg-689, Arg-692 in motif VI are all required for biological activity; the N-terminal 204 amino acids and C-terminal 100 residues are dispensable for in vivo function.\",\n      \"method\": \"Alanine-scanning mutagenesis, in vivo complementation of null strain, deletion analysis\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — systematic mutagenesis across conserved motifs with in vivo viability readout; comprehensive structure-function map\",\n      \"pmids\": [\"9611193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Human DHX38 (hPrp16) is required specifically for the second catalytic step of splicing: immunodepletion of hPrp16 from splicing extracts blocks step II, and activity is fully restored by recombinant hPrp16. hPrp16 associates with the spliceosome late in the splicing pathway. A chimeric yeast-human Prp16 protein rescues a yeast Prp16 knockout, demonstrating functional conservation.\",\n      \"method\": \"Immunodepletion from splicing extracts, recombinant protein complementation, spliceosome association assay, yeast knockout complementation with chimeric protein\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal depletion/add-back in human extracts plus cross-species complementation; multiple orthogonal methods\",\n      \"pmids\": [\"9524131\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Lethal Prp16 mutants in motifs I (G378A, K379A), II (D473A, E474A), and VI (Q685A, G688A, R689A, R692A) are defective for ATP hydrolysis and step 2 transesterification chemistry; these ATPase-defective mutants bind spliceosomes in vitro and block wild-type Prp16 function in trans (dominant-negative), establishing that ATP hydrolysis is mechanistically required for step 2 catalysis.\",\n      \"method\": \"Overexpression dominance assay in vivo, purification of recombinant mutant proteins, ATPase assay, in vitro splicing assay, spliceosome-binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — purified mutant proteins assayed biochemically plus in vivo dominant-negative analysis; multiple orthogonal methods\",\n      \"pmids\": [\"11856747\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Prp16 can associate with spliceosomes before 5' splice site cleavage and proofreads 5' splice site cleavage: when Prp16 is disabled, spliceosomes with an inactivated catalytic center can still catalyze 5' splice site cleavage (at reduced rate), but Prp16-mediated rejection is reversible, requiring the downstream discard ATPase Prp43 to complete discard.\",\n      \"method\": \"In vitro splicing assay with metal-ligand disruption at catalytic center, Prp16 inactivation, spliceosome association assay, genetic epistasis with Prp43\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple biochemical and genetic approaches; establishes new proofreading role at step 1 in addition to known step 2 role\",\n      \"pmids\": [\"20705241\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Prp16 promotes release of first-step factors Cwc25 and Yju2 from the spliceosome after lariat formation in a Prp16- and ATP-dependent manner, thereby enabling binding of Prp22, Prp18, and Slu7 to promote the second catalytic reaction; additionally, in the absence of ATP, Prp16 has an ATP-independent role in stabilizing Cwc25 binding to spliceosomes containing branch-point mutations to facilitate their splicing.\",\n      \"method\": \"In vitro splicing assay, spliceosome affinity purification, protein binding analysis, ATP-dependency experiments with branch-point mutant pre-mRNAs\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical assays in vitro; dual ATP-dependent and ATP-independent roles demonstrated with orthogonal methods\",\n      \"pmids\": [\"21098140\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"NTR complex-mediated spliceosome disassembly is linked to Prp16 action: NTR can disassemble spliceosomes arrested specifically after ATP-dependent action of Prp16 (or Prp2/Prp22), but not before these ATPases act or upon their spliceosome binding; Prp16 and Slu7 (which both interact with Brr2) negatively impact Ntr2 binding to the spliceosome.\",\n      \"method\": \"Affinity purification of spliceosomes arrested at defined stages, NTR disassembly assay, Ntr2 spliceosome binding analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — well-defined biochemical stage-specific disassembly assays; multiple spliceosome arrest points tested\",\n      \"pmids\": [\"23166295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Prp16 is required for destabilization of Yju2 and Cwc25 from the spliceosome after the first catalytic step; a truncated Yju2 (Yju2-N) with weak spliceosome affinity can support a low level of second-step splicing even in the absence of Prp16, suggesting that Prp16's role is specifically to displace stably-bound Yju2/Cwc25 to allow second-step factor binding.\",\n      \"method\": \"In vitro splicing complementation with truncated Yju2 fragments, spliceosome-binding assay, UV cross-linking to U2 snRNA\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro reconstitution with truncated proteins and UV cross-linking; single lab\",\n      \"pmids\": [\"23438600\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Prp16 destabilizes U2-U6 snRNA helix I during spliceosome remodeling between steps 1 and 2; the NTC protein Cwc2 stabilizes U2-U6 helix I, and a prp16-302 mutation stabilizes Cwc2 interactions with U6 snRNA while destabilizing Cwc2 interactions with pre-mRNA, indicating antagonistic functions between Cwc2 and Prp16 at the helix I/active site region.\",\n      \"method\": \"Genetic suppressor analysis, allele-specific epistasis, RNA-protein interaction assays (UV cross-linking), in vivo splicing assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and biochemical evidence from single lab; multiple allele combinations tested\",\n      \"pmids\": [\"24848011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The C-terminal Sec63-2 domain of Brr2 modulates the ATPase activity of Prp16 in vitro by interfering with Prp16's ability to bind RNA; allele-specific genetic interactions between BRR2 and PRP16 mutations suppress or enhance growth defects, establishing a functional regulatory interaction between these two splicing helicases.\",\n      \"method\": \"In vitro ATPase assay with purified Brr2 Sec63-2 domain and Prp16, allele-specific genetic suppressor/enhancer analysis, physical interaction assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct in vitro ATPase modulation plus allele-specific genetic validation; two orthogonal methods\",\n      \"pmids\": [\"25428373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"DHX38 associates with satellite I noncoding RNA from the human centromere region in an interphase-specific manner; depletion of DHX38 causes defective chromosome segregation (similar to satellite I ncRNA knockdown) and impairs Aurora B function at mitosis, placing DHX38 in an ncRNP complex involved in mitotic regulation.\",\n      \"method\": \"RNA immunoprecipitation (RIP), siRNA knockdown, chromosome segregation assay, Aurora B functional analysis\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — RIP and knockdown phenotype; multiple functional readouts but single lab and no reconstitution\",\n      \"pmids\": [\"31166646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DHX38 knockdown in human cells causes modulation of ~70 alternative splicing events and affects splicing of retina-specific genes FSCN2 and RHO; overexpression of DHX38 promotes usage of canonical and cryptic 5' splice sites in an HBB splicing reporter; the RP-linked G332D mutation modulates DHX38 splicing activity without detectably changing its spliceosome interaction profile.\",\n      \"method\": \"siRNA knockdown, RNA-seq splicing analysis, minigene splicing reporter assay, co-immunoprecipitation of spliceosomal factors\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — KD + RNA-seq + reporter assays; RP variant functional characterization; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"35385551\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Dhx38 loss in zebrafish arrests erythro-myeloid progenitors (EMPs) and hematopoietic stem/progenitor cells in mitotic prometaphase with chromosome alignment defects; abnormal alternative splicing of genes related to chromosome segregation, microtubule cytoskeleton, cell cycle kinases, and DNA damage occurs in dhx38 mutants, and EMPs/HSPCs undergo p53-dependent apoptosis.\",\n      \"method\": \"CRISPR knockout zebrafish, cytological chromosome alignment analysis, RNA-seq alternative splicing analysis, p53 pathway analysis\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO model with defined mitotic and splicing phenotypes; single lab\",\n      \"pmids\": [\"35929537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Prp16 has an ATP-independent role in promoting usage of aberrant 5' splice sites and mutated branch points: when a 5' splice site mutation is present or when Cwc24 is absent, Prp16 facilitates the branching reaction independently of ATP, and this function is mediated through the step-one factor Cwc25. Additionally, Prp16 prevents use of nearby cryptic branch sites while promoting mutated branch point usage.\",\n      \"method\": \"In vitro splicing assay with ATP analogs and mutant pre-mRNAs, deletion/depletion of Cwc24, Cwc25 interaction assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro reconstitution with multiple substrate/protein combinations; single lab, multiple orthogonal conditions\",\n      \"pmids\": [\"37858289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"DHX38 interacts with G3BP1 (Ras GTPase-activating protein-binding protein) as demonstrated by co-immunoprecipitation; DHX38 regulates G3BP1 expression, leading to activation of the MAPK/ERK signaling pathway and promoting EMT in NSCLC cells. Knockdown of G3BP1 reverses DHX38 overexpression-induced MAPK activation and EMT.\",\n      \"method\": \"Co-immunoprecipitation, LC-MS interactome, siRNA knockdown, ERK inhibitor (SCH772984) treatment, in vitro and in vivo tumor assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP plus knockdown rescue; multiple cell-based assays; single lab\",\n      \"pmids\": [\"37931691\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"DHX38 directly interacts with RELL2 pre-mRNA (confirmed by RIP-qPCR) and regulates retention of intron 4 in RELL2 transcripts in gemcitabine-resistant pancreatic ductal adenocarcinoma cells; altered DHX38 expression causes corresponding changes in RELL2 intron 4 retention.\",\n      \"method\": \"RIP-qPCR, DHX38 knockdown/overexpression, RT-PCR for intron retention\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct RNA binding demonstrated by RIP; functional consequence of intron retention shown; single lab\",\n      \"pmids\": [\"37506056\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"DHX38 deficiency in zebrafish and human cell lines causes significant accumulation of R-loops; DNA replication stress is the prerequisite for R-loop-induced DNA damage in DHX38 knockdown cells, establishing a DHX38/R-loop/replication stress/DNA damage regulatory axis in retinal progenitor cells.\",\n      \"method\": \"CRISPR dhx38 knockout zebrafish, R-loop immunofluorescence (S9.6 antibody), DHX38 siRNA knockdown in human cells, DNA damage markers\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO and cell-line KD with R-loop detection and DNA damage readouts; single lab, two experimental systems\",\n      \"pmids\": [\"37867960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"dhx38 knockout zebrafish display severe inner ear developmental defects (decrescent otocysts, absent semicircular canal protrusion, smaller otoliths) accompanied by DNA damage, p53-dependent apoptosis in inner ear cells, and abnormal alternative splicing of genes related to DNA damage repair and inner ear morphogenesis.\",\n      \"method\": \"CRISPR knockout zebrafish, bright-field morphology, in situ hybridization, immunofluorescence for apoptosis/DNA damage, RT-PCR alternative splicing analysis\",\n      \"journal\": \"Biomedicines\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO with defined morphological, apoptotic, and splicing phenotypes; single lab\",\n      \"pmids\": [\"39857604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DHX38 knockdown significantly increases latent splice site (LSS) usage in a luminescence reporter and RNA-seq confirmed widespread LSS activation across hundreds of mRNAs, establishing DHX38 as a component of the nuclear SOS (suppression of splicing) quality control mechanism that prevents inappropriate use of latent splice sites.\",\n      \"method\": \"siRNA screen with luminescence reporter for LSS activation, RNA-seq after DHX38 knockdown\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint; single screen plus RNA-seq confirmation; no mechanistic reconstitution of the SOS pathway for DHX38 specifically\",\n      \"pmids\": [\"bio_10.1101_2025.07.20.665773\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"YTHDF1 binds m6A-modified Dhx38 mRNA at the coding sequence (CDS) and enhances its translational efficiency without altering mRNA levels, as demonstrated by MeRIP-seq and RIP-seq in mouse retina; loss of Ythdf1 reduces Dhx38 protein levels and contributes to retinal degeneration.\",\n      \"method\": \"MeRIP-seq, RIP-seq, Ythdf1 knockout mouse, polysome/translation efficiency assay, single-cell RNA-seq\",\n      \"journal\": \"Zoological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — integrative sequencing plus KO model; YTHDF1 as m6A reader for DHX38 mRNA established by two orthogonal sequencing methods; single lab\",\n      \"pmids\": [\"40116022\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DHX38 (PRP16) is a DEAH-box RNA-dependent ATPase and RNA helicase that functions primarily as a spliceosomal remodeling factor: it binds the spliceosome transiently after the first catalytic step (lariat formation), uses ATP hydrolysis to drive a conformational rearrangement that releases first-step factors Cwc25 and Yju2 and destabilizes U2-U6 helix I, thereby enabling binding of second-step factors (Prp22, Prp18, Slu7) and 3' splice site cleavage/exon ligation; it also proofreads both the first and second catalytic steps by directing aberrant intermediates to a Prp43-dependent discard pathway, can act ATP-independently to promote aberrant splice site usage via Cwc25, associates with satellite I ncRNA to support mitotic chromosome segregation, prevents R-loop accumulation to safeguard genome integrity, and interacts with G3BP1 to activate MAPK signaling in cancer contexts, with its mRNA translation regulated by the m6A reader YTHDF1.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DHX38 (PRP16) is a DEAH-box RNA-dependent ATPase and RNA helicase that acts as a spliceosomal remodeling factor governing the transition between the two catalytic steps of pre-mRNA splicing [#0, #5]. It associates transiently with the spliceosome late in the pathway, where ATP binding/hydrolysis is coupled to its release and to a conformational rearrangement that protects the 3' splice site and licenses the second transesterification step [#0, #1]; ATPase-deficient motif mutants bind spliceosomes but block step 2 in a dominant-negative manner, establishing that catalysis depends on its NTP hydrolysis [#9]. Mechanistically, after lariat formation DHX38 destabilizes the first-step factors Cwc25 and Yju2 and disrupts U2-U6 helix I, thereby enabling recruitment of second-step factors Prp22, Prp18, and Slu7 to drive exon ligation [#11, #13, #14]. Its sequence-nonspecific helicase activity is targeted to the correct substrate by a non-conserved N-terminal domain and is regulated through functional interaction with the helicase Brr2 [#6, #15]. Beyond promoting forward catalysis, DHX38 proofreads both step 1 and step 2, routing aberrant intermediates to a Prp43-dependent discard pathway, and slowing its ATPase rate permits aberrant intermediates to proceed productively [#2, #10]; it also possesses an ATP-independent activity that promotes usage of aberrant 5' splice sites and mutated branch points via the step-one factor Cwc25 [#11, #19]. In human and zebrafish systems DHX38 controls alternative splicing and splice-site fidelity, and its loss arrests cells in mitotic prometaphase, causes R-loop accumulation and replication-stress-driven DNA damage, and triggers p53-dependent apoptosis in developing retina, inner ear, and hematopoietic progenitors [#17, #18, #22, #23]. DHX38 additionally associates with centromeric satellite I ncRNA to support chromosome segregation and Aurora B function [#16], interacts with G3BP1 to activate MAPK/ERK signaling and EMT in cancer [#20], and its own mRNA translation is enhanced by the m6A reader YTHDF1 [#25].\",\n  \"teleology\": [\n    {\n      \"year\": 1991,\n      \"claim\": \"Established DHX38/PRP16 as an RNA-dependent ATPase that transiently engages the spliceosome and is specifically required for the second catalytic step, defining the first functional foothold for the protein.\",\n      \"evidence\": \"Protein purification with in vitro ATPase and splicing complementation assays\",\n      \"pmids\": [\"1825134\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the molecular substrate of remodeling\", \"Helicase activity not yet demonstrated\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Linked ATP hydrolysis to a defined conformational rearrangement of the spliceosome, showing the energy of NTP hydrolysis drives a structural change that protects the 3' splice site for step 2.\",\n      \"evidence\": \"In vitro splicing with RNase H protection and ATPase assays using NTP analogs\",\n      \"pmids\": [\"1464325\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the rearranged RNA elements not defined\", \"Helicase mechanism not yet shown\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Revealed a kinetic proofreading function whereby PRP16 ATPase activity discards aberrantly branched intermediates, explaining how splicing fidelity is enforced.\",\n      \"evidence\": \"Genetic suppressor screen with purified mutant proteins and in vivo intermediate analysis in yeast\",\n      \"pmids\": [\"8324826\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream discard machinery not identified\", \"Direct coupling of ATPase rate to discard not reconstituted\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Identified U6 and U2-U6 helix I snRNAs as functional RNA ligands of PRP16's remodeling activity, pointing to the catalytic RNA core as its target.\",\n      \"evidence\": \"Genetic suppressor screens and site-directed mutagenesis of snRNAs in yeast\",\n      \"pmids\": [\"8088513\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Genetic interaction does not prove direct physical contact\", \"Mechanism of helix I destabilization unresolved at this stage\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Ordered the second-step pathway by showing SLU7 and SSF1 act downstream of PRP16, building a sequential factor map for exon ligation.\",\n      \"evidence\": \"Glycerol gradient isolation of PRP16-depleted spliceosomes and functional complementation with differential ATP requirements\",\n      \"pmids\": [\"7664739\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physical handoff between PRP16 and second-step factors not visualized\", \"SSF1 molecular identity not fully defined\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Demonstrated intrinsic ATP-dependent RNA unwinding activity and mapped the structure-function determinants, defining DHX38 as a bona fide helicase whose catalytic motifs and substrate-targeting N-terminus are separable.\",\n      \"evidence\": \"In vitro RNA duplex unwinding, alanine-scanning and deletion mutagenesis, complementation and localization assays in yeast\",\n      \"pmids\": [\"9550699\", \"9611193\", \"9769096\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the N-terminal domain recognizes the correct spliceosome state not defined\", \"In vivo unwinding substrate not directly captured\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Extended PRP16 function to humans, showing hPrp16 is required for step 2 and is functionally conserved across species.\",\n      \"evidence\": \"Immunodepletion/add-back in human extracts and cross-species chimera complementation in yeast\",\n      \"pmids\": [\"9524131\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Human-specific cofactors not identified\", \"Structural basis of conservation not addressed\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Established that ATP hydrolysis itself, not merely ATP binding, is mechanistically required for step 2 chemistry, via dominant-negative ATPase-defective mutants.\",\n      \"evidence\": \"In vivo dominance assay plus purified mutant ATPase, splicing, and spliceosome-binding assays\",\n      \"pmids\": [\"11856747\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational consequence of hydrolysis not structurally resolved\", \"Coupling of motor cycle to factor release not yet shown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Expanded proofreading to step 1 (5' splice site cleavage) and showed Prp16-mediated rejection is reversible and requires Prp43 to complete discard, integrating DHX38 into a two-step fidelity surveillance system.\",\n      \"evidence\": \"In vitro splicing with catalytic-center disruption, Prp16 inactivation, and genetic epistasis with Prp43\",\n      \"pmids\": [\"20705241\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of Prp16-to-Prp43 handoff not defined\", \"Structural state of rejected intermediates unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the substrate of remodeling as the stably bound first-step factors Cwc25 and Yju2, whose displacement by Prp16 (and destabilization of U2-U6 helix I) enables second-step factor binding.\",\n      \"evidence\": \"In vitro splicing with truncated Yju2, spliceosome-binding and UV cross-linking assays; genetic and cross-linking analysis of helix I\",\n      \"pmids\": [\"21098140\", \"23438600\", \"24848011\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct structural snapshot of factor displacement not obtained\", \"Antagonism with Cwc2 inferred genetically/biochemically only\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified Brr2 as a regulator of Prp16 ATPase activity, and connected Prp16 action to NTR-mediated spliceosome disassembly, embedding DHX38 in a helicase regulatory network.\",\n      \"evidence\": \"In vitro ATPase modulation with Brr2 Sec63-2 domain, allele-specific genetics, and stage-specific NTR disassembly assays\",\n      \"pmids\": [\"25428373\", \"23166295\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological trigger for Brr2 regulation of Prp16 unclear\", \"Structural basis of Brr2-Prp16 interplay not resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed a splicing-independent role: DHX38 binds centromeric satellite I ncRNA and supports chromosome segregation and Aurora B function, broadening its cellular reach to mitosis.\",\n      \"evidence\": \"RNA immunoprecipitation, siRNA knockdown, chromosome segregation and Aurora B functional assays in human cells\",\n      \"pmids\": [\"31166646\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs splicing-mediated contribution to segregation not separated\", \"No reconstitution of the ncRNP complex\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected DHX38 to genome integrity by showing its loss causes R-loop accumulation and replication-stress-driven DNA damage, defining a DHX38/R-loop/replication-stress axis.\",\n      \"evidence\": \"CRISPR knockout zebrafish and siRNA knockdown in human cells with R-loop (S9.6) immunostaining and DNA damage markers\",\n      \"pmids\": [\"37867960\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether R-loop control is direct or a downstream consequence of splicing defects unresolved\", \"No biochemical demonstration of R-loop resolution by DHX38\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Documented cancer-context roles in which DHX38 interacts with G3BP1 to activate MAPK/ERK signaling and EMT, and regulates intron retention of specific transcripts in drug-resistant tumors.\",\n      \"evidence\": \"Co-IP/LC-MS, knockdown rescue, ERK inhibition, RIP-qPCR and intron-retention RT-PCR in cancer cell lines and tumor assays\",\n      \"pmids\": [\"37931691\", \"37506056\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking splicing activity to G3BP1/MAPK not defined\", \"Single-lab co-IP without reciprocal structural validation\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Tied DHX38 function to development and splice-site fidelity in vertebrates, where loss arrests progenitors in prometaphase, perturbs alternative splicing, and provoked retina/ear/hematopoietic defects with p53-dependent apoptosis.\",\n      \"evidence\": \"siRNA knockdown with RNA-seq and minigene reporters in human cells; CRISPR knockout zebrafish with cytological, splicing, and p53 analyses\",\n      \"pmids\": [\"35385551\", \"35929537\", \"39857604\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal chain from splicing changes to mitotic arrest not fully resolved\", \"RP-linked variant mechanism not mapped to a defined biochemical defect\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified upstream control of DHX38 abundance, with the m6A reader YTHDF1 enhancing Dhx38 mRNA translation, linking epitranscriptomic regulation to DHX38 protein levels and retinal health.\",\n      \"evidence\": \"MeRIP-seq, RIP-seq, polysome assays, and Ythdf1 knockout mouse retina\",\n      \"pmids\": [\"40116022\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct functional rescue of retinal phenotype by restoring DHX38 not shown\", \"Tissue specificity of this regulation not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How DHX38's ATP-driven motor cycle is mechanistically partitioned between forward step-2 catalysis, kinetic proofreading/discard, splicing-independent R-loop suppression, and chromosome segregation remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model coupling the helicase cycle to factor release in human spliceosomes\", \"Whether mitotic and genome-integrity roles are direct or splicing-dependent is unknown\", \"No reconstitution of the proposed nuclear SOS latent-splice-site surveillance role for DHX38\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 1, 5, 9]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [3, 16, 21]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 8, 11, 17]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [16, 18]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [22, 23]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [20]}\n    ],\n    \"complexes\": [\"spliceosome\"],\n    \"partners\": [\"SLU7\", \"BRR2\", \"G3BP1\", \"CWC25\", \"YJU2\", \"YTHDF1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}