{"gene":"DHX8","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":1991,"finding":"Yeast PRP22 (DHX8 ortholog) is required for release of spliced mRNA from the spliceosome after the second step of splicing. The protein shares extensive homology with PRP2 and PRP16, containing sequence elements characteristic of ATP-dependent RNA helicases and an RNA-binding motif.","method":"Genetic analysis, predicted protein sequence homology","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — foundational genetic study replicated and extended by multiple subsequent labs","pmids":["1992352"],"is_preprint":false},{"year":1994,"finding":"Human HRH1 (DHX8) is a functional homolog of yeast Prp22; it can partially rescue the temperature-sensitive phenotype of a yeast prp22 mutant. Unlike Prp22, HRH1 contains an RS domain that mediates interaction with SR protein family members in vitro and in the yeast two-hybrid system.","method":"Yeast complementation assay, in vitro binding, yeast two-hybrid","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — complementation rescue plus two orthogonal interaction assays, single lab","pmids":["7935475"],"is_preprint":false},{"year":1998,"finding":"Purified recombinant yeast Prp22 is an RNA-dependent ATPase and an ATP-dependent RNA helicase. It has two distinct functions: an ATP-independent role in the second catalytic (transesterification) step of splicing, and an ATP-requiring function in mRNA release from the spliceosome. The ATP-independent role in step 2 depends on the distance between the branchpoint and the 3' splice site.","method":"In vitro depletion/reconstitution splicing assay, purified recombinant protein ATPase and helicase assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified protein, multiple orthogonal assays, replicated by companion paper","pmids":["9524130"],"is_preprint":false},{"year":1998,"finding":"PRP22 unwinds RNA duplexes in a concentration- and ATP-dependent manner; ATP hydrolysis (not just binding) is required for RNA unwinding. Non-hydrolyzable ATP analogs did not substitute. Mutation of the putative ATP phosphate-binding motif eliminated both ATPase and RNA-unwinding activities. mRNA release from the spliceosome is an ATP-dependent process.","method":"RNA duplex unwinding assay, ATPase assay, site-directed mutagenesis of ATP-binding motif, non-hydrolyzable analog competition","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical reconstitution with mutagenesis, confirms findings of companion paper 9524130","pmids":["9582286"],"is_preprint":false},{"year":2002,"finding":"Prp22 motif III (SAT) mutations uncouple ATP hydrolysis from spliceosome disassembly, demonstrating that ATP hydrolysis is necessary but not sufficient for mRNA release. Intragenic suppressor mutations that restore RNA unwinding activity also restore mRNA release, linking helicase activity to the release function.","method":"Intragenic suppressor genetics, in vitro mRNA release assay, ATPase and helicase assays on purified mutant proteins","journal":"Genetics","confidence":"High","confidence_rationale":"Tier 1 / Strong — combined genetic suppressor analysis with in vitro biochemical characterization of multiple mutant proteins","pmids":["11861548"],"is_preprint":false},{"year":2002,"finding":"Lethal ATPase-defective mutations in Prp22 motifs II (D603A, E604A) and VI (Q804A, R808A) abolish mRNA release from the spliceosome but retain ATP-independent activity promoting step 2 transesterification. These mutant proteins bind spliceosomes and block wild-type Prp22 function in trans (dominant-negative effect).","method":"Site-directed mutagenesis, in vitro splicing assay, ATPase assay, spliceosome binding assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic mutagenesis with multiple orthogonal in vitro assays","pmids":["11856747"],"is_preprint":false},{"year":2003,"finding":"Residues in Prp22 motifs IV (F697) and V (T757, I764, T765) are critical for RNA unwinding and mRNA release. T757 and T765 couple ATP hydrolysis to RNA cofactor. A missense mutation in PRP8 (R1753K) suppresses helicase-deficient prp22 mutations, indicating that Prp22 disrupts an RNA/protein or RNA/RNA interaction normally stabilized by Prp8.","method":"Systematic mutagenesis, in vitro mRNA release assay, ATPase and helicase assays, extragenic suppressor screen","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutational analysis of 16 residues combined with genetic epistasis via suppressor screen","pmids":["14688266"],"is_preprint":false},{"year":2005,"finding":"Prp22 preferentially binds single-stranded RNA (~20-fold higher affinity than ssDNA or duplexes); optimal ATPase activity requires RNA ≥20 nt. Inclusion of hydrolyzable ATP reduces RNA binding affinity 3-4-fold. Prp22 unwinds RNA duplexes with 3'→5' directionality and can hydrolyze all common NTPs and dNTPs with comparable efficiency.","method":"Nitrocellulose filter binding assay, RNA-stimulated ATPase assay, RNA duplex unwinding assay with directionality determination","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal quantitative biochemical assays with purified protein","pmids":["16008364"],"is_preprint":false},{"year":2008,"finding":"Prp22 interacts with the mRNA downstream of the exon-exon junction prior to mRNA release (detected by site-specific crosslinking and RNase H protection). A rearrangement accompanying the second transesterification step deposits Prp22 on the mRNA, and the 3'→5' helicase then disrupts mRNA/U5 snRNP contacts to liberate the mRNA. Spliceosome disassembly requires >13 ribonucleotides downstream of the 3' splice site.","method":"Site-specific crosslinking, RNase H protection assay, in vitro splicing assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct RNA-protein crosslinking and protection assays defining binding site and mechanistic model","pmids":["18570877"],"is_preprint":false},{"year":2009,"finding":"Prp45 (yeast ortholog of SNW1/SKIP) affects the stoichiometric association of Prp22 with the spliceosome. In prp45 mutant cells, Prp22 is underrepresented in Cwc2-associated spliceosomal complexes, and expression of Prp45(119-379) restores Prp22 partitioning and rescues splicing phenotypes, establishing a functional link between Prp45 and Prp22 recruitment.","method":"Affinity purification of spliceosomal complexes, in vivo splicing assays with reporter constructs, genetic rescue experiments","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — affinity purification plus genetic rescue, single lab","pmids":["19016306"],"is_preprint":false},{"year":2012,"finding":"Crystal structure of the C-terminal domain (CTD) of human Prp22 (DHX8) was determined by MAD. The CTD fold resembles yeast Prp43-CTD and shares structural similarity with winged-helix and ratchet domains of DNA helicase Hel308, suggesting analogous function in dsRNA binding/unwinding. The CTD has a significant impact on the ATPase activity of yPrp22 in vitro.","method":"X-ray crystallography (MAD phasing), in vitro ATPase assay of CTD deletion constructs, homology modeling","journal":"Biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional validation of CTD contribution to ATPase activity","pmids":["23096351"],"is_preprint":false},{"year":2012,"finding":"NTR complex (Prp43+Ntr1+Ntr2) can disassemble spliceosomes specifically arrested after the ATP-dependent action of Prp22 (or Prp2, Prp16), but not at steps before these ATPases act or upon their mere binding. This links spliceosome disassembly competence to prior Prp22 ATPase activity.","method":"Affinity purification of arrested spliceosome intermediates, in vitro disassembly assay with NTR complex","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro disassembly assay with defined arrested intermediates, single lab","pmids":["23166295"],"is_preprint":false},{"year":2012,"finding":"Loss-of-function of dhx8 in zebrafish (mmy mutant, truncation) causes splicing defects in many genes including hematopoietic genes, hematopoietic blockage, and cell division defects (disorganized mitotic spindles, multipolar spindles). DHX8 knockdown in HeLa cells confirmed the cell division defects.","method":"Positional cloning of ENU mutant, splicing analysis, DHX8 siRNA knockdown in HeLa cells with mitotic spindle imaging","journal":"Developmental dynamics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo vertebrate knockout combined with human cell knockdown confirming cell division phenotype","pmids":["22411201"],"is_preprint":false},{"year":2017,"finding":"Human cactin physically interacts with DHX8 and SRRM2 (detected by co-immunoprecipitation). Cactin depletion impairs efficient pre-mRNA splicing; the cactin-DHX8-SRRM2 complex is required for faithful splicing of specific pre-mRNAs including sororin, whose mis-splicing causes premature sister chromatid separation.","method":"Co-immunoprecipitation, siRNA depletion, RNA-seq splicing analysis, sister chromatid cohesion assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus functional depletion with splicing readout, single lab","pmids":["28062851"],"is_preprint":false},{"year":2019,"finding":"Crystal structures of human DHX8 helicase core (DHX8Δ547) bound to ADP and to poly(A)6 ssRNA reveal that RNA binding triggers ADP release through conformational changes in the DEAH-, P-loop, and hook-turn motifs. R620 and the hook-turn/hook-loop regions are required for helicase activity. The hook-turn acts as a gatekeeper regulating directional movement of the 3' RNA end through the RNA-binding channel. DHX8 has preferential in vitro binding for adenine-rich RNA.","method":"X-ray crystallography (ADP-bound and RNA-bound structures), ATPase assay, RNA-binding assay, site-directed mutagenesis of R620 and hook-turn","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — two crystal structures with mutagenesis and multiple biochemical assays characterizing mechanism of ADP release and RNA movement","pmids":["31409651"],"is_preprint":false},{"year":2024,"finding":"C. elegans MOG-5 (PRP22/DHX8 ortholog) and DDX41/SACY-1 have overlapping roles in proofreading against proximal 3' splice site usage. Targeted mutations in MOG-5 in the region predicted to interact with SACY-1 (based on human C* spliceosome structure) cause increased usage of proximal alternative adjacent 3' splice sites, phenocopying SACY-1 perturbation.","method":"CRISPR-generated targeted alleles of mog-5, transcriptomic analysis (RNA-seq), genetic interaction analysis","journal":"RNA","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR alleles with transcriptomic readout, single lab, C. elegans model","pmids":["38282418"],"is_preprint":false},{"year":2025,"finding":"Prp22 promotes exon ligation by stabilizing Slu7's association with the spliceosome prior to the second transesterification step (ATP binding by Prp22, not hydrolysis, inhibits exon ligation of 3'SS mutant pre-mRNA). After exon ligation, Prp22-driven ATP hydrolysis induces a conformational change in Prp8 disrupting interdomain interactions to enable mRNA release; Prp22 and Cwc22 remain associated with the released mRNA while Slu7 and Fyv6 dissociate independently.","method":"In vitro splicing assay, ATP analog competition, spliceosome pull-down to track protein association, mutant analysis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical reconstitution with mechanistic dissection of ATP binding vs. hydrolysis steps and protein tracking","pmids":["40876859"],"is_preprint":false},{"year":2025,"finding":"DHX8 binds consecutive guanine sequences in RNA and interacts with lysosomal membrane protein SIDT2. DHX8 is partially localized to the cytoplasmic side of the lysosomal membrane and regulates intracellular RNA degradation via SIDT2-dependent RNautophagy. RNA binding (not ATPase activity) of DHX8 is important for this regulatory function. DHX8 also contributes to clearance of pathogenic CAG-repeat mRNA.","method":"Protein binding screen for G-rich RNA, co-immunoprecipitation with SIDT2, subcellular fractionation/localization, RNautophagy assay, ATPase-deficient mutant analysis","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with SIDT2, localization by fractionation, functional assay with RNA-binding vs. ATPase mutants, single lab","pmids":["40842239"],"is_preprint":false}],"current_model":"DHX8 (yeast Prp22) is a DEAH-box RNA helicase that acts at two distinct steps of pre-mRNA splicing: it promotes the second transesterification (exon ligation) step in an ATP-independent manner by stabilizing Slu7 association with the spliceosome, and it then uses ATP hydrolysis to remodel Prp8 and disrupt mRNA/U5 snRNP contacts, releasing mature mRNA from the spliceosome; its crystal structure reveals a hook-turn gatekeeper mechanism coupling RNA binding to ADP release, and it has an additional cytoplasmic role at the lysosomal membrane in regulating RNA degradation via SIDT2-dependent RNautophagy."},"narrative":{"mechanistic_narrative":"DHX8 (yeast Prp22) is a DEAH-box, RNA-dependent ATPase and ATP-dependent RNA helicase that acts at the final catalytic transition of pre-mRNA splicing [PMID:9524130, PMID:9582286]. It performs two mechanistically separable functions: an ATP-independent role promoting the second transesterification (exon ligation) step, and an ATP-hydrolysis-driven role releasing mature mRNA from the spliceosome [PMID:9524130, PMID:11856747]. Exon ligation is promoted by stabilizing Slu7 association with the spliceosome prior to the second step, with ATP binding (not hydrolysis) by DHX8 antagonizing this step [PMID:40876859]. Following exon ligation, the enzyme is deposited on the mRNA downstream of the exon-exon junction and, using 3'→5' helicase activity, disrupts mRNA/U5 snRNP contacts to liberate the mRNA — an interaction normally stabilized by Prp8, against which ATP-driven conformational remodeling acts [PMID:18570877, PMID:14688266, PMID:40876859]. Genetic and biochemical dissection establishes that ATP hydrolysis is necessary but not sufficient for release, with helicase activity directly coupled to the release function [PMID:11861548, PMID:11856747]. Crystal structures of the human helicase core show that RNA binding triggers ADP release through the DEAH-, P-loop, and hook-turn motifs, the hook-turn acting as a gatekeeper controlling directional movement of the 3' RNA end through the binding channel [PMID:31409651]. Its spliceosomal association is modulated by Prp45/SNW1 and by a cactin–DHX8–SRRM2 complex required for faithful splicing of substrates including sororin, linking DHX8 to sister chromatid cohesion and mitotic fidelity [PMID:19016306, PMID:28062851, PMID:22411201]. Beyond splicing, DHX8 binds G-rich RNA, localizes to the cytoplasmic side of the lysosomal membrane via interaction with SIDT2, and regulates RNA degradation through SIDT2-dependent RNautophagy in an RNA-binding-dependent, ATPase-independent manner [PMID:40842239].","teleology":[{"year":1991,"claim":"Established that the Prp22/DHX8 protein is required for a defined late step of splicing — release of spliced mRNA from the spliceosome — and predicted it to be an ATP-dependent RNA helicase.","evidence":"Genetic analysis and protein sequence homology in yeast","pmids":["1992352"],"confidence":"High","gaps":["Helicase and ATPase activities were predicted from sequence, not demonstrated biochemically","No direct RNA substrate or binding site defined"]},{"year":1994,"claim":"Demonstrated functional conservation of the human ortholog (HRH1/DHX8) by yeast rescue, and identified a human-specific RS domain mediating SR protein interaction not present in yeast Prp22.","evidence":"Yeast complementation, in vitro binding, yeast two-hybrid","pmids":["7935475"],"confidence":"Medium","gaps":["Functional role of the RS domain in human splicing not established","SR protein interaction shown in vitro/two-hybrid only"]},{"year":1998,"claim":"Resolved the dual nature of Prp22 by showing it is a bona fide RNA-dependent ATPase/helicase with an ATP-independent step-2 function and a separate ATP-requiring mRNA-release function, with ATP hydrolysis (not binding) driving unwinding.","evidence":"In vitro depletion/reconstitution splicing, purified recombinant ATPase/helicase assays, non-hydrolyzable analog competition, ATP-motif mutagenesis","pmids":["9524130","9582286"],"confidence":"High","gaps":["Molecular target of unwinding within the spliceosome not yet identified","Structural basis of activity unknown"]},{"year":2003,"claim":"Linked helicase catalytic motifs to release function and provided genetic evidence that Prp22 disrupts a specific RNA/protein contact stabilized by Prp8, identifying the spliceosomal target of its remodeling.","evidence":"Systematic motif mutagenesis, in vitro release/ATPase/helicase assays, intragenic and Prp8 extragenic suppressor genetics","pmids":["11861548","11856747","14688266"],"confidence":"High","gaps":["Direct physical contact between Prp22 and Prp8 not structurally resolved at this stage","Coupling between hydrolysis and disassembly defined genetically, not structurally"]},{"year":2008,"claim":"Defined where on the substrate Prp22 acts — binding the mRNA downstream of the exon-exon junction and requiring >13 nucleotides downstream of the 3' splice site to translocate and disrupt mRNA/U5 contacts.","evidence":"Site-specific crosslinking, RNase H protection, in vitro splicing","pmids":["18570877"],"confidence":"High","gaps":["How the rearrangement deposits Prp22 on mRNA mechanistically unresolved","Single-system biochemistry"]},{"year":2005,"claim":"Quantified substrate preference and mechanism, showing strong ssRNA binding, a ≥20 nt RNA requirement for ATPase activity, and 3'→5' unwinding directionality consistent with the release model.","evidence":"Filter binding, RNA-stimulated ATPase, directional unwinding assays","pmids":["16008364"],"confidence":"High","gaps":["In vitro substrate preference not validated on physiological spliceosomal substrates"]},{"year":2012,"claim":"Connected DHX8 to higher-order biology — its recruitment depends on Prp45/SNW1, its loss causes splicing-coupled hematopoietic and mitotic spindle defects in zebrafish and HeLa cells, and downstream NTR-complex disassembly is gated by prior Prp22 ATPase action.","evidence":"Affinity purification, in vivo splicing reporters and genetic rescue, ENU mutant cloning, siRNA with spindle imaging, in vitro disassembly of arrested intermediates","pmids":["19016306","22411201","23166295"],"confidence":"Medium","gaps":["Whether spindle defects are direct or secondary to global splicing failure not separated","Mechanism of Prp45-dependent recruitment unknown"]},{"year":2012,"claim":"Provided the first structural view by solving the human DHX8 C-terminal domain, revealing a fold related to other DEAH/helicase ratchet domains that contributes to ATPase activity.","evidence":"X-ray crystallography (MAD), ATPase assay of CTD deletions, homology modeling","pmids":["23096351"],"confidence":"High","gaps":["Full-length and RNA-bound conformations not captured","CTD role inferred from deletion, not mechanism"]},{"year":2017,"claim":"Placed DHX8 in a defined human splicing complex (cactin–DHX8–SRRM2) required for faithful splicing of cohesion regulator sororin, mechanistically linking DHX8-dependent splicing to chromosome segregation.","evidence":"Reciprocal co-IP, siRNA depletion, RNA-seq splicing analysis, sister chromatid cohesion assay","pmids":["28062851"],"confidence":"Medium","gaps":["Direct vs. complex-mediated DHX8 contribution to sororin splicing not isolated","Single-lab interaction data"]},{"year":2019,"claim":"Defined the structural mechanism coupling RNA binding to nucleotide cycling, showing RNA-triggered ADP release via DEAH/P-loop/hook-turn motifs and a hook-turn gatekeeper controlling 3' RNA-end movement.","evidence":"ADP- and RNA-bound crystal structures of the helicase core, ATPase/RNA-binding assays, R620 and hook-turn mutagenesis","pmids":["31409651"],"confidence":"High","gaps":["Structures of the full enzyme on an authentic spliceosome not determined","Adenine-rich RNA preference biological significance unclear"]},{"year":2024,"claim":"Revealed a proofreading function in 3' splice site fidelity, with the SACY-1/DDX41-interacting region of MOG-5/DHX8 required to suppress aberrant proximal 3' splice site usage.","evidence":"CRISPR-targeted mog-5 alleles, RNA-seq, genetic interaction analysis in C. elegans","pmids":["38282418"],"confidence":"Medium","gaps":["Interaction inferred from human C* structure, not directly demonstrated for MOG-5","Conservation of this proofreading role in humans not tested"]},{"year":2025,"claim":"Mechanistically separated DHX8's two splicing steps — ATP binding (not hydrolysis) gating Slu7-dependent exon ligation, and hydrolysis-driven Prp8 conformational remodeling driving release with defined protein co-tracking on released mRNA.","evidence":"In vitro splicing, ATP analog competition, spliceosome pull-down protein tracking, mutant analysis","pmids":["40876859"],"confidence":"High","gaps":["Structural intermediate of the Slu7-stabilized state not captured","How Cwc22 co-release is coordinated unresolved"]},{"year":2025,"claim":"Identified a non-splicing cytoplasmic role: G-rich-RNA-bound DHX8 localizes to the lysosomal membrane via SIDT2 and drives SIDT2-dependent RNautophagy, including clearance of pathogenic CAG-repeat mRNA, independent of its ATPase activity.","evidence":"G-rich RNA binding screen, co-IP with SIDT2, subcellular fractionation, RNautophagy assay, ATPase-deficient mutant","pmids":["40842239"],"confidence":"Medium","gaps":["Mechanism of DHX8 targeting to the lysosomal membrane unknown","Single-lab interaction and localization data"]},{"year":null,"claim":"How DHX8's nuclear splicing role and its cytoplasmic lysosomal RNautophagy role are partitioned, regulated, and physiologically balanced within a cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of DHX8 engaged with an intact spliceosome","Determinants of nuclear vs. lysosomal localization unknown","Disease relevance of DHX8 dysfunction not directly established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[2,3,4,7]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[2,3,7,14]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[7,14,17]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[2,3,5]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[12,13]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[17]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,2,8,16]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[17]}],"complexes":["spliceosome","cactin-DHX8-SRRM2 complex"],"partners":["SLU7","PRP8","PRP45/SNW1","SRRM2","CACTIN","SIDT2","SACY-1/DDX41"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q14562","full_name":"ATP-dependent RNA helicase DHX8","aliases":["DEAH box protein 8","RNA helicase HRH1"],"length_aa":1220,"mass_kda":139.3,"function":"Involved in pre-mRNA splicing as component of the spliceosome (PubMed:11991638, PubMed:28076346, PubMed:28502770). Facilitates nuclear export of spliced mRNA by releasing the RNA from the spliceosome (PubMed:8608946)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q14562/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/DHX8","classification":"Common Essential","n_dependent_lines":1206,"n_total_lines":1208,"dependency_fraction":0.9983443708609272},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CPSF6","stoichiometry":0.2},{"gene":"SNRPA","stoichiometry":0.2},{"gene":"SNRPB","stoichiometry":0.2},{"gene":"SNRPC","stoichiometry":0.2},{"gene":"TNPO3","stoichiometry":0.2},{"gene":"TOP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/DHX8","total_profiled":1310},"omim":[{"mim_id":"618536","title":"CACTIN, SPLICEOSOME C COMPLEX SUBUNIT; CACTIN","url":"https://www.omim.org/entry/618536"},{"mim_id":"615475","title":"DExH-BOX HELICASE 34; DHX34","url":"https://www.omim.org/entry/615475"},{"mim_id":"607570","title":"DEAH-BOX HELICASE 40; DHX40","url":"https://www.omim.org/entry/607570"},{"mim_id":"600396","title":"DEAH-BOX HELICASE 8; DHX8","url":"https://www.omim.org/entry/600396"},{"mim_id":"301165","title":"RNA-BINDING MOTIF PROTEIN 41; RBM41","url":"https://www.omim.org/entry/301165"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nuclear bodies","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DHX8"},"hgnc":{"alias_symbol":["HRH1","Prp22","PRPF22","Dhr2"],"prev_symbol":["DDX8"]},"alphafold":{"accession":"Q14562","domains":[{"cath_id":"2.40.50.140","chopping":"265-354","consensus_level":"high","plddt":80.2248,"start":265,"end":354},{"cath_id":"3.40.50.300","chopping":"559-736","consensus_level":"medium","plddt":86.6023,"start":559,"end":736},{"cath_id":"3.40.50.300","chopping":"755-917","consensus_level":"medium","plddt":85.1236,"start":755,"end":917},{"cath_id":"1.20.120.1080","chopping":"983-1193","consensus_level":"high","plddt":84.9078,"start":983,"end":1193},{"cath_id":"1.10.10","chopping":"22-91","consensus_level":"high","plddt":80.6891,"start":22,"end":91}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14562","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q14562-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q14562-F1-predicted_aligned_error_v6.png","plddt_mean":73.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DHX8","jax_strain_url":"https://www.jax.org/strain/search?query=DHX8"},"sequence":{"accession":"Q14562","fasta_url":"https://rest.uniprot.org/uniprotkb/Q14562.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q14562/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14562"}},"corpus_meta":[{"pmid":"1992352","id":"PMC_1992352","title":"Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes.","date":"1991","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/1992352","citation_count":318,"is_preprint":false},{"pmid":"9524130","id":"PMC_9524130","title":"Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing.","date":"1998","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/9524130","citation_count":205,"is_preprint":false},{"pmid":"9582286","id":"PMC_9582286","title":"The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes.","date":"1998","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/9582286","citation_count":140,"is_preprint":false},{"pmid":"18570877","id":"PMC_18570877","title":"A conformational rearrangement in the spliceosome sets the stage for Prp22-dependent mRNA release.","date":"2008","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/18570877","citation_count":109,"is_preprint":false},{"pmid":"7935475","id":"PMC_7935475","title":"Identification of a putative RNA helicase (HRH1), a human homolog of yeast Prp22.","date":"1994","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/7935475","citation_count":66,"is_preprint":false},{"pmid":"16008364","id":"PMC_16008364","title":"Characterization of the NTPase, RNA-binding, and RNA helicase activities of the DEAH-box splicing factor Prp22.","date":"2005","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16008364","citation_count":63,"is_preprint":false},{"pmid":"10737793","id":"PMC_10737793","title":"The hermaphrodite sperm/oocyte switch requires the Caenorhabditis elegans homologs of PRP2 and PRP22.","date":"2000","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/10737793","citation_count":61,"is_preprint":false},{"pmid":"14688266","id":"PMC_14688266","title":"Motifs IV and V in the DEAH box splicing factor Prp22 are important for RNA unwinding, and helicase-defective Prp22 mutants are suppressed by Prp8.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/14688266","citation_count":52,"is_preprint":false},{"pmid":"11856747","id":"PMC_11856747","title":"Characterization of dominant-negative mutants of the DEAH-box splicing factors Prp22 and Prp16.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11856747","citation_count":50,"is_preprint":false},{"pmid":"23166295","id":"PMC_23166295","title":"Link of NTR-mediated spliceosome disassembly with DEAH-box ATPases Prp2, Prp16, and Prp22.","date":"2012","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/23166295","citation_count":35,"is_preprint":false},{"pmid":"19016306","id":"PMC_19016306","title":"Prp45 affects Prp22 partition in spliceosomal complexes and splicing efficiency of non-consensus substrates.","date":"2009","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19016306","citation_count":31,"is_preprint":false},{"pmid":"23096351","id":"PMC_23096351","title":"Structural analysis of the C-terminal domain of the spliceosomal helicase Prp22.","date":"2012","source":"Biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23096351","citation_count":25,"is_preprint":false},{"pmid":"22411201","id":"PMC_22411201","title":"Incomplete splicing, cell division defects, and hematopoietic blockage in dhx8 mutant zebrafish.","date":"2012","source":"Developmental dynamics : an official publication of the American Association of Anatomists","url":"https://pubmed.ncbi.nlm.nih.gov/22411201","citation_count":22,"is_preprint":false},{"pmid":"11861548","id":"PMC_11861548","title":"ATP-dependent remodeling of the spliceosome: intragenic suppressors of release-defective mutants of Saccharomyces cerevisiae Prp22.","date":"2002","source":"Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/11861548","citation_count":22,"is_preprint":false},{"pmid":"28062851","id":"PMC_28062851","title":"Human cactin interacts with DHX8 and SRRM2 to assure efficient pre-mRNA splicing and sister chromatid cohesion.","date":"2017","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/28062851","citation_count":21,"is_preprint":false},{"pmid":"31409651","id":"PMC_31409651","title":"Structural and functional characterisation of human RNA helicase DHX8 provides insights into the mechanism of RNA-stimulated ADP release.","date":"2019","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/31409651","citation_count":17,"is_preprint":false},{"pmid":"30191368","id":"PMC_30191368","title":"A tandem duplication of BRCA1 exons 1-19 through DHX8 exon 2 in four families with hereditary breast and ovarian cancer syndrome.","date":"2018","source":"Breast cancer research and treatment","url":"https://pubmed.ncbi.nlm.nih.gov/30191368","citation_count":10,"is_preprint":false},{"pmid":"24244416","id":"PMC_24244416","title":"Prp22 and spliceosome components regulate chromatin dynamics in germ-line polyploid cells.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24244416","citation_count":8,"is_preprint":false},{"pmid":"38282418","id":"PMC_38282418","title":"Spliceosomal helicases DDX41/SACY-1 and PRP22/MOG-5 both contribute to proofreading against proximal 3' splice site usage.","date":"2024","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/38282418","citation_count":7,"is_preprint":false},{"pmid":"40842239","id":"PMC_40842239","title":"DHX8 regulates degradation of RNA by RNautophagy.","date":"2025","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/40842239","citation_count":3,"is_preprint":false},{"pmid":"40876859","id":"PMC_40876859","title":"New mechanistic insights into Prp22-mediated exon ligation and mRNA release.","date":"2025","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/40876859","citation_count":2,"is_preprint":false},{"pmid":"41898898","id":"PMC_41898898","title":"DHX8 Plays a Critical Role in Larval Development in Lepidopteran Bombyx mori.","date":"2026","source":"Insects","url":"https://pubmed.ncbi.nlm.nih.gov/41898898","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.06.16.659908","title":"Control of 3’ Splice Site Selection in  <i>S. cerevisiae</i>  by a Highly Conserved Amino Acid within the Prp8 α-finger Domain","date":"2025-06-20","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.16.659908","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12640,"output_tokens":4475,"usd":0.052522,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12448,"output_tokens":4468,"usd":0.08697,"stage2_stop_reason":"end_turn"},"total_usd":0.139492,"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\": \"Yeast PRP22 (DHX8 ortholog) is required for release of spliced mRNA from the spliceosome after the second step of splicing. The protein shares extensive homology with PRP2 and PRP16, containing sequence elements characteristic of ATP-dependent RNA helicases and an RNA-binding motif.\",\n      \"method\": \"Genetic analysis, predicted protein sequence homology\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — foundational genetic study replicated and extended by multiple subsequent labs\",\n      \"pmids\": [\"1992352\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Human HRH1 (DHX8) is a functional homolog of yeast Prp22; it can partially rescue the temperature-sensitive phenotype of a yeast prp22 mutant. Unlike Prp22, HRH1 contains an RS domain that mediates interaction with SR protein family members in vitro and in the yeast two-hybrid system.\",\n      \"method\": \"Yeast complementation assay, in vitro binding, yeast two-hybrid\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — complementation rescue plus two orthogonal interaction assays, single lab\",\n      \"pmids\": [\"7935475\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Purified recombinant yeast Prp22 is an RNA-dependent ATPase and an ATP-dependent RNA helicase. It has two distinct functions: an ATP-independent role in the second catalytic (transesterification) step of splicing, and an ATP-requiring function in mRNA release from the spliceosome. The ATP-independent role in step 2 depends on the distance between the branchpoint and the 3' splice site.\",\n      \"method\": \"In vitro depletion/reconstitution splicing assay, purified recombinant protein ATPase and helicase assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified protein, multiple orthogonal assays, replicated by companion paper\",\n      \"pmids\": [\"9524130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"PRP22 unwinds RNA duplexes in a concentration- and ATP-dependent manner; ATP hydrolysis (not just binding) is required for RNA unwinding. Non-hydrolyzable ATP analogs did not substitute. Mutation of the putative ATP phosphate-binding motif eliminated both ATPase and RNA-unwinding activities. mRNA release from the spliceosome is an ATP-dependent process.\",\n      \"method\": \"RNA duplex unwinding assay, ATPase assay, site-directed mutagenesis of ATP-binding motif, non-hydrolyzable analog competition\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical reconstitution with mutagenesis, confirms findings of companion paper 9524130\",\n      \"pmids\": [\"9582286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Prp22 motif III (SAT) mutations uncouple ATP hydrolysis from spliceosome disassembly, demonstrating that ATP hydrolysis is necessary but not sufficient for mRNA release. Intragenic suppressor mutations that restore RNA unwinding activity also restore mRNA release, linking helicase activity to the release function.\",\n      \"method\": \"Intragenic suppressor genetics, in vitro mRNA release assay, ATPase and helicase assays on purified mutant proteins\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — combined genetic suppressor analysis with in vitro biochemical characterization of multiple mutant proteins\",\n      \"pmids\": [\"11861548\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Lethal ATPase-defective mutations in Prp22 motifs II (D603A, E604A) and VI (Q804A, R808A) abolish mRNA release from the spliceosome but retain ATP-independent activity promoting step 2 transesterification. These mutant proteins bind spliceosomes and block wild-type Prp22 function in trans (dominant-negative effect).\",\n      \"method\": \"Site-directed mutagenesis, in vitro splicing assay, ATPase assay, spliceosome binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic mutagenesis with multiple orthogonal in vitro assays\",\n      \"pmids\": [\"11856747\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Residues in Prp22 motifs IV (F697) and V (T757, I764, T765) are critical for RNA unwinding and mRNA release. T757 and T765 couple ATP hydrolysis to RNA cofactor. A missense mutation in PRP8 (R1753K) suppresses helicase-deficient prp22 mutations, indicating that Prp22 disrupts an RNA/protein or RNA/RNA interaction normally stabilized by Prp8.\",\n      \"method\": \"Systematic mutagenesis, in vitro mRNA release assay, ATPase and helicase assays, extragenic suppressor screen\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutational analysis of 16 residues combined with genetic epistasis via suppressor screen\",\n      \"pmids\": [\"14688266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Prp22 preferentially binds single-stranded RNA (~20-fold higher affinity than ssDNA or duplexes); optimal ATPase activity requires RNA ≥20 nt. Inclusion of hydrolyzable ATP reduces RNA binding affinity 3-4-fold. Prp22 unwinds RNA duplexes with 3'→5' directionality and can hydrolyze all common NTPs and dNTPs with comparable efficiency.\",\n      \"method\": \"Nitrocellulose filter binding assay, RNA-stimulated ATPase assay, RNA duplex unwinding assay with directionality determination\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal quantitative biochemical assays with purified protein\",\n      \"pmids\": [\"16008364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Prp22 interacts with the mRNA downstream of the exon-exon junction prior to mRNA release (detected by site-specific crosslinking and RNase H protection). A rearrangement accompanying the second transesterification step deposits Prp22 on the mRNA, and the 3'→5' helicase then disrupts mRNA/U5 snRNP contacts to liberate the mRNA. Spliceosome disassembly requires >13 ribonucleotides downstream of the 3' splice site.\",\n      \"method\": \"Site-specific crosslinking, RNase H protection assay, in vitro splicing assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct RNA-protein crosslinking and protection assays defining binding site and mechanistic model\",\n      \"pmids\": [\"18570877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Prp45 (yeast ortholog of SNW1/SKIP) affects the stoichiometric association of Prp22 with the spliceosome. In prp45 mutant cells, Prp22 is underrepresented in Cwc2-associated spliceosomal complexes, and expression of Prp45(119-379) restores Prp22 partitioning and rescues splicing phenotypes, establishing a functional link between Prp45 and Prp22 recruitment.\",\n      \"method\": \"Affinity purification of spliceosomal complexes, in vivo splicing assays with reporter constructs, genetic rescue experiments\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — affinity purification plus genetic rescue, single lab\",\n      \"pmids\": [\"19016306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Crystal structure of the C-terminal domain (CTD) of human Prp22 (DHX8) was determined by MAD. The CTD fold resembles yeast Prp43-CTD and shares structural similarity with winged-helix and ratchet domains of DNA helicase Hel308, suggesting analogous function in dsRNA binding/unwinding. The CTD has a significant impact on the ATPase activity of yPrp22 in vitro.\",\n      \"method\": \"X-ray crystallography (MAD phasing), in vitro ATPase assay of CTD deletion constructs, homology modeling\",\n      \"journal\": \"Biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional validation of CTD contribution to ATPase activity\",\n      \"pmids\": [\"23096351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"NTR complex (Prp43+Ntr1+Ntr2) can disassemble spliceosomes specifically arrested after the ATP-dependent action of Prp22 (or Prp2, Prp16), but not at steps before these ATPases act or upon their mere binding. This links spliceosome disassembly competence to prior Prp22 ATPase activity.\",\n      \"method\": \"Affinity purification of arrested spliceosome intermediates, in vitro disassembly assay with NTR complex\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro disassembly assay with defined arrested intermediates, single lab\",\n      \"pmids\": [\"23166295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Loss-of-function of dhx8 in zebrafish (mmy mutant, truncation) causes splicing defects in many genes including hematopoietic genes, hematopoietic blockage, and cell division defects (disorganized mitotic spindles, multipolar spindles). DHX8 knockdown in HeLa cells confirmed the cell division defects.\",\n      \"method\": \"Positional cloning of ENU mutant, splicing analysis, DHX8 siRNA knockdown in HeLa cells with mitotic spindle imaging\",\n      \"journal\": \"Developmental dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo vertebrate knockout combined with human cell knockdown confirming cell division phenotype\",\n      \"pmids\": [\"22411201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Human cactin physically interacts with DHX8 and SRRM2 (detected by co-immunoprecipitation). Cactin depletion impairs efficient pre-mRNA splicing; the cactin-DHX8-SRRM2 complex is required for faithful splicing of specific pre-mRNAs including sororin, whose mis-splicing causes premature sister chromatid separation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA depletion, RNA-seq splicing analysis, sister chromatid cohesion assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus functional depletion with splicing readout, single lab\",\n      \"pmids\": [\"28062851\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Crystal structures of human DHX8 helicase core (DHX8Δ547) bound to ADP and to poly(A)6 ssRNA reveal that RNA binding triggers ADP release through conformational changes in the DEAH-, P-loop, and hook-turn motifs. R620 and the hook-turn/hook-loop regions are required for helicase activity. The hook-turn acts as a gatekeeper regulating directional movement of the 3' RNA end through the RNA-binding channel. DHX8 has preferential in vitro binding for adenine-rich RNA.\",\n      \"method\": \"X-ray crystallography (ADP-bound and RNA-bound structures), ATPase assay, RNA-binding assay, site-directed mutagenesis of R620 and hook-turn\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — two crystal structures with mutagenesis and multiple biochemical assays characterizing mechanism of ADP release and RNA movement\",\n      \"pmids\": [\"31409651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"C. elegans MOG-5 (PRP22/DHX8 ortholog) and DDX41/SACY-1 have overlapping roles in proofreading against proximal 3' splice site usage. Targeted mutations in MOG-5 in the region predicted to interact with SACY-1 (based on human C* spliceosome structure) cause increased usage of proximal alternative adjacent 3' splice sites, phenocopying SACY-1 perturbation.\",\n      \"method\": \"CRISPR-generated targeted alleles of mog-5, transcriptomic analysis (RNA-seq), genetic interaction analysis\",\n      \"journal\": \"RNA\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR alleles with transcriptomic readout, single lab, C. elegans model\",\n      \"pmids\": [\"38282418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Prp22 promotes exon ligation by stabilizing Slu7's association with the spliceosome prior to the second transesterification step (ATP binding by Prp22, not hydrolysis, inhibits exon ligation of 3'SS mutant pre-mRNA). After exon ligation, Prp22-driven ATP hydrolysis induces a conformational change in Prp8 disrupting interdomain interactions to enable mRNA release; Prp22 and Cwc22 remain associated with the released mRNA while Slu7 and Fyv6 dissociate independently.\",\n      \"method\": \"In vitro splicing assay, ATP analog competition, spliceosome pull-down to track protein association, mutant analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical reconstitution with mechanistic dissection of ATP binding vs. hydrolysis steps and protein tracking\",\n      \"pmids\": [\"40876859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DHX8 binds consecutive guanine sequences in RNA and interacts with lysosomal membrane protein SIDT2. DHX8 is partially localized to the cytoplasmic side of the lysosomal membrane and regulates intracellular RNA degradation via SIDT2-dependent RNautophagy. RNA binding (not ATPase activity) of DHX8 is important for this regulatory function. DHX8 also contributes to clearance of pathogenic CAG-repeat mRNA.\",\n      \"method\": \"Protein binding screen for G-rich RNA, co-immunoprecipitation with SIDT2, subcellular fractionation/localization, RNautophagy assay, ATPase-deficient mutant analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with SIDT2, localization by fractionation, functional assay with RNA-binding vs. ATPase mutants, single lab\",\n      \"pmids\": [\"40842239\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DHX8 (yeast Prp22) is a DEAH-box RNA helicase that acts at two distinct steps of pre-mRNA splicing: it promotes the second transesterification (exon ligation) step in an ATP-independent manner by stabilizing Slu7 association with the spliceosome, and it then uses ATP hydrolysis to remodel Prp8 and disrupt mRNA/U5 snRNP contacts, releasing mature mRNA from the spliceosome; its crystal structure reveals a hook-turn gatekeeper mechanism coupling RNA binding to ADP release, and it has an additional cytoplasmic role at the lysosomal membrane in regulating RNA degradation via SIDT2-dependent RNautophagy.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DHX8 (yeast Prp22) is a DEAH-box, RNA-dependent ATPase and ATP-dependent RNA helicase that acts at the final catalytic transition of pre-mRNA splicing [#2, #3]. It performs two mechanistically separable functions: an ATP-independent role promoting the second transesterification (exon ligation) step, and an ATP-hydrolysis-driven role releasing mature mRNA from the spliceosome [#2, #5]. Exon ligation is promoted by stabilizing Slu7 association with the spliceosome prior to the second step, with ATP binding (not hydrolysis) by DHX8 antagonizing this step [#16]. Following exon ligation, the enzyme is deposited on the mRNA downstream of the exon-exon junction and, using 3'→5' helicase activity, disrupts mRNA/U5 snRNP contacts to liberate the mRNA — an interaction normally stabilized by Prp8, against which ATP-driven conformational remodeling acts [#8, #6, #16]. Genetic and biochemical dissection establishes that ATP hydrolysis is necessary but not sufficient for release, with helicase activity directly coupled to the release function [#4, #5]. Crystal structures of the human helicase core show that RNA binding triggers ADP release through the DEAH-, P-loop, and hook-turn motifs, the hook-turn acting as a gatekeeper controlling directional movement of the 3' RNA end through the binding channel [#14]. Its spliceosomal association is modulated by Prp45/SNW1 and by a cactin–DHX8–SRRM2 complex required for faithful splicing of substrates including sororin, linking DHX8 to sister chromatid cohesion and mitotic fidelity [#9, #13, #12]. Beyond splicing, DHX8 binds G-rich RNA, localizes to the cytoplasmic side of the lysosomal membrane via interaction with SIDT2, and regulates RNA degradation through SIDT2-dependent RNautophagy in an RNA-binding-dependent, ATPase-independent manner [#17].\",\n  \"teleology\": [\n    {\n      \"year\": 1991,\n      \"claim\": \"Established that the Prp22/DHX8 protein is required for a defined late step of splicing — release of spliced mRNA from the spliceosome — and predicted it to be an ATP-dependent RNA helicase.\",\n      \"evidence\": \"Genetic analysis and protein sequence homology in yeast\",\n      \"pmids\": [\"1992352\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Helicase and ATPase activities were predicted from sequence, not demonstrated biochemically\", \"No direct RNA substrate or binding site defined\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Demonstrated functional conservation of the human ortholog (HRH1/DHX8) by yeast rescue, and identified a human-specific RS domain mediating SR protein interaction not present in yeast Prp22.\",\n      \"evidence\": \"Yeast complementation, in vitro binding, yeast two-hybrid\",\n      \"pmids\": [\"7935475\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional role of the RS domain in human splicing not established\", \"SR protein interaction shown in vitro/two-hybrid only\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Resolved the dual nature of Prp22 by showing it is a bona fide RNA-dependent ATPase/helicase with an ATP-independent step-2 function and a separate ATP-requiring mRNA-release function, with ATP hydrolysis (not binding) driving unwinding.\",\n      \"evidence\": \"In vitro depletion/reconstitution splicing, purified recombinant ATPase/helicase assays, non-hydrolyzable analog competition, ATP-motif mutagenesis\",\n      \"pmids\": [\"9524130\", \"9582286\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular target of unwinding within the spliceosome not yet identified\", \"Structural basis of activity unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Linked helicase catalytic motifs to release function and provided genetic evidence that Prp22 disrupts a specific RNA/protein contact stabilized by Prp8, identifying the spliceosomal target of its remodeling.\",\n      \"evidence\": \"Systematic motif mutagenesis, in vitro release/ATPase/helicase assays, intragenic and Prp8 extragenic suppressor genetics\",\n      \"pmids\": [\"11861548\", \"11856747\", \"14688266\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct physical contact between Prp22 and Prp8 not structurally resolved at this stage\", \"Coupling between hydrolysis and disassembly defined genetically, not structurally\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined where on the substrate Prp22 acts — binding the mRNA downstream of the exon-exon junction and requiring >13 nucleotides downstream of the 3' splice site to translocate and disrupt mRNA/U5 contacts.\",\n      \"evidence\": \"Site-specific crosslinking, RNase H protection, in vitro splicing\",\n      \"pmids\": [\"18570877\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the rearrangement deposits Prp22 on mRNA mechanistically unresolved\", \"Single-system biochemistry\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Quantified substrate preference and mechanism, showing strong ssRNA binding, a ≥20 nt RNA requirement for ATPase activity, and 3'→5' unwinding directionality consistent with the release model.\",\n      \"evidence\": \"Filter binding, RNA-stimulated ATPase, directional unwinding assays\",\n      \"pmids\": [\"16008364\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro substrate preference not validated on physiological spliceosomal substrates\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Connected DHX8 to higher-order biology — its recruitment depends on Prp45/SNW1, its loss causes splicing-coupled hematopoietic and mitotic spindle defects in zebrafish and HeLa cells, and downstream NTR-complex disassembly is gated by prior Prp22 ATPase action.\",\n      \"evidence\": \"Affinity purification, in vivo splicing reporters and genetic rescue, ENU mutant cloning, siRNA with spindle imaging, in vitro disassembly of arrested intermediates\",\n      \"pmids\": [\"19016306\", \"22411201\", \"23166295\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether spindle defects are direct or secondary to global splicing failure not separated\", \"Mechanism of Prp45-dependent recruitment unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Provided the first structural view by solving the human DHX8 C-terminal domain, revealing a fold related to other DEAH/helicase ratchet domains that contributes to ATPase activity.\",\n      \"evidence\": \"X-ray crystallography (MAD), ATPase assay of CTD deletions, homology modeling\",\n      \"pmids\": [\"23096351\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length and RNA-bound conformations not captured\", \"CTD role inferred from deletion, not mechanism\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Placed DHX8 in a defined human splicing complex (cactin–DHX8–SRRM2) required for faithful splicing of cohesion regulator sororin, mechanistically linking DHX8-dependent splicing to chromosome segregation.\",\n      \"evidence\": \"Reciprocal co-IP, siRNA depletion, RNA-seq splicing analysis, sister chromatid cohesion assay\",\n      \"pmids\": [\"28062851\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. complex-mediated DHX8 contribution to sororin splicing not isolated\", \"Single-lab interaction data\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined the structural mechanism coupling RNA binding to nucleotide cycling, showing RNA-triggered ADP release via DEAH/P-loop/hook-turn motifs and a hook-turn gatekeeper controlling 3' RNA-end movement.\",\n      \"evidence\": \"ADP- and RNA-bound crystal structures of the helicase core, ATPase/RNA-binding assays, R620 and hook-turn mutagenesis\",\n      \"pmids\": [\"31409651\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structures of the full enzyme on an authentic spliceosome not determined\", \"Adenine-rich RNA preference biological significance unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed a proofreading function in 3' splice site fidelity, with the SACY-1/DDX41-interacting region of MOG-5/DHX8 required to suppress aberrant proximal 3' splice site usage.\",\n      \"evidence\": \"CRISPR-targeted mog-5 alleles, RNA-seq, genetic interaction analysis in C. elegans\",\n      \"pmids\": [\"38282418\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interaction inferred from human C* structure, not directly demonstrated for MOG-5\", \"Conservation of this proofreading role in humans not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mechanistically separated DHX8's two splicing steps — ATP binding (not hydrolysis) gating Slu7-dependent exon ligation, and hydrolysis-driven Prp8 conformational remodeling driving release with defined protein co-tracking on released mRNA.\",\n      \"evidence\": \"In vitro splicing, ATP analog competition, spliceosome pull-down protein tracking, mutant analysis\",\n      \"pmids\": [\"40876859\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural intermediate of the Slu7-stabilized state not captured\", \"How Cwc22 co-release is coordinated unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified a non-splicing cytoplasmic role: G-rich-RNA-bound DHX8 localizes to the lysosomal membrane via SIDT2 and drives SIDT2-dependent RNautophagy, including clearance of pathogenic CAG-repeat mRNA, independent of its ATPase activity.\",\n      \"evidence\": \"G-rich RNA binding screen, co-IP with SIDT2, subcellular fractionation, RNautophagy assay, ATPase-deficient mutant\",\n      \"pmids\": [\"40842239\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of DHX8 targeting to the lysosomal membrane unknown\", \"Single-lab interaction and localization data\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How DHX8's nuclear splicing role and its cytoplasmic lysosomal RNautophagy role are partitioned, regulated, and physiologically balanced within a cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of DHX8 engaged with an intact spliceosome\", \"Determinants of nuclear vs. lysosomal localization unknown\", \"Disease relevance of DHX8 dysfunction not directly established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [2, 3, 4, 7]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [2, 3, 7, 14]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [7, 14, 17]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [2, 3, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [12, 13]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 2, 8, 16]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"complexes\": [\n      \"spliceosome\",\n      \"cactin-DHX8-SRRM2 complex\"\n    ],\n    \"partners\": [\n      \"SLU7\",\n      \"PRP8\",\n      \"PRP45/SNW1\",\n      \"SRRM2\",\n      \"cactin\",\n      \"SIDT2\",\n      \"SACY-1/DDX41\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":8,"faith_total":8,"faith_pct":100.0}}