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DHX8

ATP-dependent RNA helicase DHX8 · UniProt Q14562

Length
1220 aa
Mass
139.3 kDa
Annotated
2026-06-09
22 papers in source corpus 18 papers cited in narrative 18 extracted findings
Cross-family judge vs UniProt: tie faithfulness: 8/8 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 13 steps
  1. 1991 High

    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

    PMID:1992352

    Open questions at the time
    • Helicase and ATPase activities were predicted from sequence, not demonstrated biochemically
    • No direct RNA substrate or binding site defined
  2. 1994 Medium

    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

    PMID:7935475

    Open questions at the time
    • Functional role of the RS domain in human splicing not established
    • SR protein interaction shown in vitro/two-hybrid only
  3. 1998 High

    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

    PMID:9524130 PMID:9582286

    Open questions at the time
    • Molecular target of unwinding within the spliceosome not yet identified
    • Structural basis of activity unknown
  4. 2003 High

    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

    PMID:11856747 PMID:11861548 PMID:14688266

    Open questions at the time
    • Direct physical contact between Prp22 and Prp8 not structurally resolved at this stage
    • Coupling between hydrolysis and disassembly defined genetically, not structurally
  5. 2008 High

    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

    PMID:18570877

    Open questions at the time
    • How the rearrangement deposits Prp22 on mRNA mechanistically unresolved
    • Single-system biochemistry
  6. 2005 High

    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

    PMID:16008364

    Open questions at the time
    • In vitro substrate preference not validated on physiological spliceosomal substrates
  7. 2012 Medium

    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

    PMID:19016306 PMID:22411201 PMID:23166295

    Open questions at the time
    • Whether spindle defects are direct or secondary to global splicing failure not separated
    • Mechanism of Prp45-dependent recruitment unknown
  8. 2012 High

    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

    PMID:23096351

    Open questions at the time
    • Full-length and RNA-bound conformations not captured
    • CTD role inferred from deletion, not mechanism
  9. 2017 Medium

    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

    PMID:28062851

    Open questions at the time
    • Direct vs. complex-mediated DHX8 contribution to sororin splicing not isolated
    • Single-lab interaction data
  10. 2019 High

    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

    PMID:31409651

    Open questions at the time
    • Structures of the full enzyme on an authentic spliceosome not determined
    • Adenine-rich RNA preference biological significance unclear
  11. 2024 Medium

    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

    PMID:38282418

    Open questions at the time
    • Interaction inferred from human C* structure, not directly demonstrated for MOG-5
    • Conservation of this proofreading role in humans not tested
  12. 2025 High

    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

    PMID:40876859

    Open questions at the time
    • Structural intermediate of the Slu7-stabilized state not captured
    • How Cwc22 co-release is coordinated unresolved
  13. 2025 Medium

    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

    PMID:40842239

    Open questions at the time
    • Mechanism of DHX8 targeting to the lysosomal membrane unknown
    • Single-lab interaction and localization data

Open questions

Synthesis pass · forward-looking unresolved questions
  • How DHX8's nuclear splicing role and its cytoplasmic lysosomal RNautophagy role are partitioned, regulated, and physiologically balanced within a cell remains unresolved.
  • 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

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140098 catalytic activity, acting on RNA 4 GO:0140657 ATP-dependent activity 4 GO:0003723 RNA binding 3 GO:0016787 hydrolase activity 3
Localization
GO:0005634 nucleus 2 GO:0005764 lysosome 1
Pathway
R-HSA-8953854 Metabolism of RNA 4 R-HSA-9612973 Autophagy 1
Partners
CACTINPRP45/SNW1PRP8SACY-1/DDX41SIDT2SLU7SRRM2
Complex memberships
cactin-DHX8-SRRM2 complexspliceosome

Evidence

Reading pass · 18 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1991 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. Genetic analysis, predicted protein sequence homology Nature High 1992352
1994 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. Yeast complementation assay, in vitro binding, yeast two-hybrid Molecular and cellular biology Medium 7935475
1998 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. In vitro depletion/reconstitution splicing assay, purified recombinant protein ATPase and helicase assays The EMBO journal High 9524130
1998 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. RNA duplex unwinding assay, ATPase assay, site-directed mutagenesis of ATP-binding motif, non-hydrolyzable analog competition The EMBO journal High 9582286
2002 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. Intragenic suppressor genetics, in vitro mRNA release assay, ATPase and helicase assays on purified mutant proteins Genetics High 11861548
2002 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). Site-directed mutagenesis, in vitro splicing assay, ATPase assay, spliceosome binding assay The Journal of biological chemistry High 11856747
2003 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. Systematic mutagenesis, in vitro mRNA release assay, ATPase and helicase assays, extragenic suppressor screen The Journal of biological chemistry High 14688266
2005 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. Nitrocellulose filter binding assay, RNA-stimulated ATPase assay, RNA duplex unwinding assay with directionality determination Biochemistry High 16008364
2008 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. Site-specific crosslinking, RNase H protection assay, in vitro splicing assay Molecular cell High 18570877
2009 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. Affinity purification of spliceosomal complexes, in vivo splicing assays with reporter constructs, genetic rescue experiments Journal of cellular biochemistry Medium 19016306
2012 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. X-ray crystallography (MAD phasing), in vitro ATPase assay of CTD deletion constructs, homology modeling Biological chemistry High 23096351
2012 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. Affinity purification of arrested spliceosome intermediates, in vitro disassembly assay with NTR complex Molecular and cellular biology Medium 23166295
2012 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. Positional cloning of ENU mutant, splicing analysis, DHX8 siRNA knockdown in HeLa cells with mitotic spindle imaging Developmental dynamics Medium 22411201
2017 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. Co-immunoprecipitation, siRNA depletion, RNA-seq splicing analysis, sister chromatid cohesion assay Journal of cell science Medium 28062851
2019 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. X-ray crystallography (ADP-bound and RNA-bound structures), ATPase assay, RNA-binding assay, site-directed mutagenesis of R620 and hook-turn The Biochemical journal High 31409651
2024 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. CRISPR-generated targeted alleles of mog-5, transcriptomic analysis (RNA-seq), genetic interaction analysis RNA Medium 38282418
2025 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. In vitro splicing assay, ATP analog competition, spliceosome pull-down to track protein association, mutant analysis Nucleic acids research High 40876859
2025 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. Protein binding screen for G-rich RNA, co-immunoprecipitation with SIDT2, subcellular fractionation/localization, RNautophagy assay, ATPase-deficient mutant analysis Nucleic acids research Medium 40842239

Source papers

Stage 0 corpus · 22 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1991 Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes. Nature 318 1992352
1998 Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing. The EMBO journal 205 9524130
1998 The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes. The EMBO journal 140 9582286
2008 A conformational rearrangement in the spliceosome sets the stage for Prp22-dependent mRNA release. Molecular cell 109 18570877
1994 Identification of a putative RNA helicase (HRH1), a human homolog of yeast Prp22. Molecular and cellular biology 66 7935475
2005 Characterization of the NTPase, RNA-binding, and RNA helicase activities of the DEAH-box splicing factor Prp22. Biochemistry 63 16008364
2000 The hermaphrodite sperm/oocyte switch requires the Caenorhabditis elegans homologs of PRP2 and PRP22. Proceedings of the National Academy of Sciences of the United States of America 61 10737793
2003 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. The Journal of biological chemistry 52 14688266
2002 Characterization of dominant-negative mutants of the DEAH-box splicing factors Prp22 and Prp16. The Journal of biological chemistry 50 11856747
2012 Link of NTR-mediated spliceosome disassembly with DEAH-box ATPases Prp2, Prp16, and Prp22. Molecular and cellular biology 35 23166295
2009 Prp45 affects Prp22 partition in spliceosomal complexes and splicing efficiency of non-consensus substrates. Journal of cellular biochemistry 31 19016306
2012 Structural analysis of the C-terminal domain of the spliceosomal helicase Prp22. Biological chemistry 25 23096351
2012 Incomplete splicing, cell division defects, and hematopoietic blockage in dhx8 mutant zebrafish. Developmental dynamics : an official publication of the American Association of Anatomists 22 22411201
2002 ATP-dependent remodeling of the spliceosome: intragenic suppressors of release-defective mutants of Saccharomyces cerevisiae Prp22. Genetics 22 11861548
2017 Human cactin interacts with DHX8 and SRRM2 to assure efficient pre-mRNA splicing and sister chromatid cohesion. Journal of cell science 21 28062851
2019 Structural and functional characterisation of human RNA helicase DHX8 provides insights into the mechanism of RNA-stimulated ADP release. The Biochemical journal 17 31409651
2018 A tandem duplication of BRCA1 exons 1-19 through DHX8 exon 2 in four families with hereditary breast and ovarian cancer syndrome. Breast cancer research and treatment 10 30191368
2013 Prp22 and spliceosome components regulate chromatin dynamics in germ-line polyploid cells. PloS one 8 24244416
2024 Spliceosomal helicases DDX41/SACY-1 and PRP22/MOG-5 both contribute to proofreading against proximal 3' splice site usage. RNA (New York, N.Y.) 7 38282418
2025 DHX8 regulates degradation of RNA by RNautophagy. Nucleic acids research 3 40842239
2025 New mechanistic insights into Prp22-mediated exon ligation and mRNA release. Nucleic acids research 2 40876859
2026 DHX8 Plays a Critical Role in Larval Development in Lepidopteran Bombyx mori. Insects 0 41898898

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