Affinage

PRPF8

Pre-mRNA-processing-splicing factor 8 · UniProt Q6P2Q9

Length
2335 aa
Mass
273.6 kDa
Annotated
2026-04-28
100 papers in source corpus 46 papers cited in narrative 44 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

PRPF8 is the largest and most highly conserved protein in the spliceosome, functioning as the central scaffold of the catalytic core where it directly contacts the 5′ splice site, 3′ splice site, branch point, and U2/U5/U6 snRNAs to coordinate both transesterification steps of pre-mRNA splicing (PMID:7781612, PMID:10444595, PMID:23393194). Its multi-domain architecture—comprising reverse-transcriptase-like, RNase H-like, endonuclease-like, beta-finger, and Jab1/MPN domains—forms a large cavity that accommodates the catalytic RNA network; the RNase H domain undergoes a conformational toggle between catalytic steps that unmasks a metal-binding site promoting exon ligation, while two opposing classes of alleles bias the spliceosome between error-prone/efficient and hyperaccurate/inefficient states, establishing PRPF8 as a fidelity regulator (PMID:23354046, PMID:23686287, PMID:28416677). The Jab1/MPN C-terminal tail inserts into the Brr2 helicase RNA-binding tunnel to reversibly inhibit U4/U6 unwinding, providing a checkpoint for spliceosome activation; mutations in this domain that cause autosomal dominant retinitis pigmentosa (RP13) weaken Brr2 regulation, impair U5 snRNP biogenesis, and preferentially mis-splice introns with weak 5′ splice sites and retinal-specific transcripts (PMID:23704370, PMID:17934474, PMID:38605034). PRPF8 also serves as the platform for U5 snRNP assembly, carrying a nuclear localization signal and chaperoned by Ecdysoneless in the cytoplasm, with Aar2 and Brr2 binding mutually exclusively to direct sequential maturation of the particle (PMID:23727230, PMID:33444449).

Mechanistic history

Synthesis pass · year-by-year structured walk · 16 steps
  1. 1989 High

    Establishing that Prp8 is evolutionarily conserved and physically present in the U4/U6·U5 tri-snRNP and assembled spliceosomes resolved the question of whether this yeast-identified factor operates in mammalian splicing.

    Evidence Immunoprecipitation with anti-PRP8 antibodies and gradient fractionation of human nuclear extracts

    PMID:2138328 PMID:2139226 PMID:2479028

    Open questions at the time
    • Precise position within the spliceosome unknown
    • No direct RNA contacts yet mapped
  2. 1992 High

    Demonstrating that PRP8 is required for tri-snRNP assembly and spliceosome formation established it as an essential structural organizer rather than a peripheral accessory factor.

    Evidence Genetic depletion, temperature-sensitive inactivation, and antibody inhibition in yeast in vitro splicing

    PMID:1396567

    Open questions at the time
    • Mechanism of tri-snRNP assembly role undefined
    • No domain information available
  3. 1995 High

    Systematic site-specific crosslinking revealed that PRP8 directly contacts exon nucleotides flanking both splice sites and the polypyrimidine tract, placing it at the catalytic center rather than at a regulatory periphery.

    Evidence UV-crosslinking with 4-thiouridine-substituted pre-mRNAs at defined positions in yeast splicing extracts

    PMID:7781612 PMID:7885825

    Open questions at the time
    • Identity of crosslinked amino acids unknown
    • No structural context for RNA contacts
  4. 1996 High

    Identification of separable C-terminal functional regions governing 3′ splice site specificity versus fidelity demonstrated that Prp8 is not a monolithic scaffold but contains modular domains with distinct catalytic-core functions.

    Evidence Extensive PRP8 mutagenesis with in vivo splicing reporter assays in yeast

    PMID:8725222

    Open questions at the time
    • No structural data for these domains
    • Biochemical basis of fidelity control unclear
  5. 1998 High

    Identification of a stable RNA-free protein complex of Prp8 with Snu114 (GTPase), Brr2 (helicase), and U5-40kD defined the core protein machinery of U5 snRNP and revealed Prp8 as the central binding platform.

    Evidence Salt dissociation of U5 snRNP, sedimentation, and biochemical interaction assays in human extracts

    PMID:9774689

    Open questions at the time
    • Stoichiometry and order of assembly unknown
    • No structural data on complex architecture
  6. 1999 High

    Mapping the 5′ splice site crosslink to Prp8 residues 1894–1898 and demonstrating genetic suppression of U6 ACAGAG, 5′SS, and 3′SS mutations established that Prp8 participates in a tertiary RNA network at the catalytic center linking U6 snRNA to both intron termini.

    Evidence Peptide-level UV-crosslink mapping in human splicing complexes; genetic suppressor screens with splice-site and U6 mutants in yeast

    PMID:10024169 PMID:10444595 PMID:10444596

    Open questions at the time
    • No high-resolution structure of the catalytic center
    • Mechanism of Prp8-mediated RNA rearrangement unknown
  7. 1999 High

    Discovery that a prp8 suppressor mutation restores U4/U6 unwinding blocked by a cold-sensitive U4 mutant established Prp8 as a regulator of spliceosome activation that couples 5′SS recognition to U4/U6 duplex disruption.

    Evidence In vitro splicing and spliceosome complex analysis with U4-cs1 mutant and prp8-201 suppressor in yeast

    PMID:10024880

    Open questions at the time
    • Direct biochemical mechanism of Prp8-controlled unwinding undefined
    • Brr2 involvement not yet demonstrated
  8. 2002 High

    Systematic allele-specific genetic interactions between five Prp8 regions and Prp28, Brr2, and U6 RNA demonstrated that Prp8 coordinates multiple activation events—U1 displacement and U4/U6 unwinding—through spatially distinct domains.

    Evidence Large-scale PRP8 mutagenesis with epistasis analysis against helicase and snRNA mutations in yeast

    PMID:10924465 PMID:12087126

    Open questions at the time
    • Physical basis for intramolecular communication between Prp8 regions unknown
    • No structural data on domain organization
  9. 2007 High

    Crystal structure of the Jab1/MPN domain, discovery of a Prp8 nuclear localization signal, and demonstration that RP13 mutations disrupt Brr2 binding and nuclear U5 snRNP assembly established the molecular basis of retinitis pigmentosa pathogenesis through defective snRNP biogenesis.

    Evidence X-ray crystallography, yeast two-hybrid, subcellular fractionation, and co-immunoprecipitation in yeast

    PMID:17317632 PMID:17934474

    Open questions at the time
    • Full-length Prp8 structure unavailable
    • Why RP mutations cause retina-specific disease not explained
  10. 2007 High

    Two opposing classes of prp8 alleles that suppress first-step versus second-step splicing defects revealed that Prp8 functions as a conformational toggle—analogous to ribosomal accuracy mutants—that repositions the catalytic center between transesterification reactions.

    Evidence Genetic screen for prp8 alleles, in vivo and in vitro splicing, epistasis with prp16 and U6 mutants in yeast

    PMID:17486100

    Open questions at the time
    • Structural basis of the two conformational states unresolved
    • How Prp16 ATPase triggers the transition mechanistically unclear
  11. 2008 High

    Crystal structures of the beta-finger and RNase H (domain IV) domains, combined with EM localization of Prp8 centrally in the tri-snRNP, provided the first atomic views of Prp8's RNA-contacting surfaces and their spatial arrangement within the particle.

    Evidence X-ray crystallography at 1.85 Å resolution; EM of genetically tagged tri-snRNP in yeast

    PMID:18779563 PMID:18836455 PMID:18953335 PMID:19098916

    Open questions at the time
    • No structure of Prp8 in an assembled spliceosome
    • Conformational dynamics during catalysis unresolved at atomic level
  12. 2013 High

    The landmark crystal structure of the Prp8 large fragment (885–2413) revealed a bacterial group II intron RT-like architecture with an active-site cavity accommodating catalytic RNAs; structures of the Jab1/MPN tail in Brr2's RNA tunnel and the RNase H domain on U4/U6 defined dual inhibitory mechanisms controlling spliceosome activation, while RNase H conformational switching was shown to unmask a metal-binding site for exon ligation.

    Evidence X-ray crystallography of Prp8–Aar2, Prp8 Jab1/MPN–Brr2, and RNase H–U4/U6 complexes; in vitro helicase/ATPase assays; crosslinking-mass spectrometry; CLIP/CRAC RNA footprinting

    PMID:23124066 PMID:23354046 PMID:23393194 PMID:23686287 PMID:23704370 PMID:23727230

    Open questions at the time
    • No cryo-EM structure of Prp8 within a catalytically active spliceosome at this point
    • Mutual exclusivity of Aar2/Brr2 binding not visualized in a full U5 assembly intermediate
  13. 2015 High

    PRPF8 depletion in human cells preferentially impaired splicing of introns with weak 5′ splice sites, and site-specific crosslinking in purified spliceosomes showed dynamic Prp8–branch-site contacts during catalytic activation, establishing PRPF8 as a determinant of splice-site strength-dependent splicing kinetics.

    Evidence siRNA knockdown with iCLIP, RNA-seq, and minigene assays in human cells; UV-crosslinking in purified yeast spliceosomal complexes

    PMID:26385511 PMID:26392272 PMID:26393790

    Open questions at the time
    • Quantitative contribution of individual Prp8 domains to splice-site discrimination unclear
    • Human spliceosome structural snapshots lacking
  14. 2017 High

    Demonstration that prp8 RNase H-domain alleles partition into two structural classes—error-prone/efficient versus hyperaccurate/inefficient—unified the fidelity model by showing the spliceosome toggles between discrete RNase H conformations to balance accuracy against throughput.

    Evidence In vitro and in vivo splicing assays, genome-wide lariat sequencing, genetic interaction with prp2 in yeast

    PMID:28416677

    Open questions at the time
    • How Prp2 remodeling triggers the RNase H conformational switch structurally unresolved
    • Whether the same toggle operates in human spliceosomes untested
  15. 2021 High

    Identification of Ecdysoneless as a cytoplasmic chaperone that delivers Prp8 to the forming U5 snRNP defined a previously unknown step in U5 biogenesis upstream of the Aar2-to-Brr2 exchange.

    Evidence Drosophila genetics, proteomics, co-immunoprecipitation, and U5 snRNP assembly assays

    PMID:33444449

    Open questions at the time
    • Whether Ecd chaperone function is conserved in mammals needs direct demonstration
    • Structural basis of Ecd–Prp8 interaction unknown
  16. 2024 High

    Patient iPSC-derived retinal cells carrying the RP PRPF8-H2309P mutation showed impaired 5′SS selection in ciliary/retinal transcripts, altered nuclear speckle organization, and peripheral accumulation of active spliceosomes, providing the first mechanistic link between Prp8-Brr2 regulatory disruption and retina-specific pathology.

    Evidence iPSC-derived retinal organoids and RPE cells, transcriptomics, proteomics, co-IP with U6 snRNA, nuclear speckle imaging

    PMID:38605034

    Open questions at the time
    • Whether nuclear speckle disorganization is cause or consequence of splicing defects unclear
    • How retinal-specific transcript vulnerability arises from a ubiquitous splicing factor mutation remains incompletely explained
    • No therapeutic rescue demonstrated

Open questions

Synthesis pass · forward-looking unresolved questions
  • A complete structural trajectory of Prp8 conformational changes through all spliceosome states in human cells, the precise mechanism by which Prp8 fidelity toggling is coupled to Prp2/Prp16 ATPase action, and the basis for retina-specific vulnerability to PRPF8 mutations remain major open questions.
  • Full human spliceosome catalytic cycle with Prp8 conformational tracking not achieved
  • Quantitative kinetic framework for Prp8-dependent fidelity decisions lacking
  • Cell-type-specific splicing vulnerability mechanism for RP not resolved

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0003723 RNA binding 7 GO:0098772 molecular function regulator activity 6 GO:0005198 structural molecule activity 4
Localization
GO:0005634 nucleus 3 GO:0005829 cytosol 2
Pathway
R-HSA-8953854 Metabolism of RNA 14 R-HSA-1852241 Organelle biogenesis and maintenance 3
Partners
AAR2BRR2ECDPRPF28SNU114U5-40K
Complex memberships
U4/U6·U5 tri-snRNPU5 snRNPspliceosome (major and minor)

Evidence

Reading pass · 44 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1989 The mammalian Prp8 homolog (p220/200 kDa protein) is a component of the U4/U6.U5 tri-snRNP complex and is present in affinity-purified spliceosomes, establishing its conservation from yeast to mammals and its location in the spliceosome. Immunoprecipitation with anti-PRP8 antibodies, gradient fractionation, UV-crosslinking Proceedings of the National Academy of Sciences of the United States of America High 2138328 2139226 2479028
1991 PRP8 protein directly contacts pre-mRNA in the spliceosome via UV-crosslinking; this interaction is ATP-dependent, splicing-specific, and requires a functional 5' splice site or branchpoint, establishing PRP8 as a direct RNA-binding component of the spliceosome. UV-crosslinking combined with immunoprecipitation using anti-PRP8 antibodies in yeast in vitro splicing reactions Nucleic acids research High 1945827
1992 PRP8 is required for stable formation of U4/U6.U5 triple snRNP complexes; depletion or inactivation of PRP8 prevents U5 and U4/U6 snRNPs from assembling into spliceosomes, and also causes decline in U4, U5, and U6 snRNA levels. Genetic depletion in vivo, heat inactivation of temperature-sensitive alleles, antibody inhibition in vitro splicing assays The EMBO journal High 1396567
1995 PRP8 interacts extensively with at least 8 exon nucleotides at the 5' splice site prior to step 1 of splicing and with at least 13 exon nucleotides plus polypyrimidine tract at the 3' splice site region after step 1, suggesting PRP8 stabilizes U5 snRNA interactions with exons at the catalytic core. UV-crosslinking with 4-thiouridine-substituted pre-mRNAs at specific positions, site-directed mutagenesis of splice sites The EMBO journal High 7781612
1995 PRP8 protein maintains stable association with the spliceosome during both steps of splicing and continues with the excised intron, contacting the 5' splice site region and the branchpoint-3' splice site region as mapped by RNase T1 protection and immunoprecipitation. UV-crosslinking combined with PRP8-specific immunoprecipitation, RNase T1 protection, analysis of coprecipitated RNA species Nucleic acids research High 7885825
1996 Distinct domains of yeast PRP8 govern specificity of 3' splice site selection (uridine tract recognition) and fidelity of 3' splice site utilization (PyAG recognition); mutagenesis identifies two separable functional regions in the C-terminal portion of Prp8. Extensive mutagenesis of PRP8, genetic selection for phenotypic alleles, in vivo splicing reporter assays Genetics High 8725222
1998 Human Prp8 (hPrp8/U5-220kD) forms a stable, RNA-free protein complex with U5-116kD (EF-2 homologue/Snu114), U5-200kD (RNA unwindase/Brr2), and U5-40kD (a novel WD-40 protein); Prp8 binds simultaneously to the 40kD and 116kD proteins and likely also to the 200kD protein. Sodium thiocyanate dissociation of U5 snRNP, sedimentation analysis, cDNA cloning, biochemical protein-protein interaction assays Molecular and cellular biology High 9774689
1999 The C-terminal region of hPrp8 (positions 1894–1898) directly crosslinks to the GU dinucleotide at the 5' splice site, mapping the functional RNA-contact domain within the protein. UV-induced crosslinking in splicing complex B, immunoprecipitation with truncation constructs, peptide mapping by proteolysis and size comparison RNA (New York, N.Y.) High 10024169
1999 Yeast prp8 alleles suppress mutations at position 2 of the 5' GU, all positions of the 3' YAG, and position A51 of the U6 ACAGAG motif (which crosslinks position 2 of 5' GU), establishing through genetic epistasis that Prp8 participates in a tertiary interaction between U6 snRNA and both 5' and 3' ends of the intron at the catalytic core. Genetic suppressor screen, in vivo splicing assays with splice-site mutants and U6 mutants, allele specificity analysis Genes & development High 10444595 10444596
1999 Mutagenesis of the yeast Prp8 C-terminal region yields alleles that suppress both 5' splice site and 3' splice site mutations, indicating Prp8 functionally interacts with both splice sites at the catalytic center and is required for the second transesterification step. In vivo mutagenesis, genetic suppression screen, ACT1-CUP1 splicing reporter, U1 snRNA suppressor epistasis Genes & development High 10444596
1999 Prp8 governs U4/U6 RNA unwinding during spliceosome activation; a prp8 suppressor mutation (prp8-201) restores U4/U6 unwinding blocked by U4-cs1, indicating wild-type Prp8 triggers unwinding only after correct 5' splice site recognition by the U6 ACAGAG box. In vitro splicing assay with cold-sensitive U4 mutant, spliceosome complex analysis, genetic suppression Molecular cell High 10024880
1999 Human Prp8 is a core component of both the major U2-dependent and minor U12-dependent spliceosomes, as antibodies against hPrp8 immunoprecipitate both spliceosome types; it is encoded by a single gene and is the first splicing factor (beyond Sm proteins) common to both spliceosomes. Cloning, Northern/Southern analysis, immunoprecipitation of U2 and U12 spliceosomes with anti-hPrp8 antibodies RNA (New York, N.Y.) High 10411133
2000 Mutagenesis screen identifies five distinct regions (a–e) of yeast Prp8 involved in control of spliceosome activation (U4/U6 unwinding); genetic interactions between regions suggest a long-range intramolecular fold, and two regions implicated in direct and indirect contacts to U1 snRNP. Large-scale PRP8 mutagenesis suppressor screen (suppressing U4-cs1 cold sensitivity), two-hybrid analysis, genetic interaction mapping Genetics High 10924465
2002 Distinct domains of Prp8 mediate different aspects of spliceosome activation: regions a, d, and e show allele-specific genetic interactions with Prp28, Brr2/Prp44, and U6 RNA respectively, demonstrating Prp8 coordinates multiple processes including U1 displacement and U4/U6 unwinding. Allele-specific genetic interaction analysis between PRP8 regions and Prp28, Brr2, U6 RNA mutations Proceedings of the National Academy of Sciences of the United States of America High 12087126
2003 A missense mutation in Prp8 (R1753K) suppresses multiple helicase-deficient prp22 alleles, suggesting Prp8 stabilizes an RNA/protein or RNA/RNA interaction in the spliceosome that Prp22 must disrupt for mRNA release. Extragenic suppressor screen, in vitro splicing assays, mRNA release assay with helicase-dead Prp22 mutants The Journal of biological chemistry High 14688266
2006 Prp8 interacts directly with multiple catalytic core RNAs (U5, U6 snRNAs, and pre-mRNA splice sites/branch region) through contacts in its central region, as mapped by transposon-based domain insertion analysis; a discrete highly conserved central region is implicated as a splicing cofactor. Transposon-based domain dissection strategy mapping RNA interactions, RNA binding assays with Prp8 fragments RNA (New York, N.Y.) High 16431982
2006 Snu114 assembly into U5 snRNP requires Prp8 and a functional GTPase domain; GTPase domain mutations in Snu114 prevent its interaction with Prp8 and with U5 snRNA, while C-terminal truncation of Snu114 allows snRNP assembly but blocks U4 snRNP release during splicing. Genetic analysis, snRNP and spliceosome assembly assays in SNU114 mutant extracts, co-immunoprecipitation RNA (New York, N.Y.) High 16540695
2007 The C-terminal domain of yeast Prp8 has a Jab1/MPN-like fold (crystallographically determined); the RP13-linked region in its C-terminal appendix is essential for binding of human Brr2 and Snu114, and RP13 point mutations weaken these interactions, establishing the Jab1/MPN domain as a protein-protein interaction platform. X-ray crystallography of Prp8 C-terminal domain, targeted yeast two-hybrid analysis, RP13 point mutation functional testing Molecular cell High 17317632
2007 RP13 mutations in Prp8 in yeast cause nuclear accumulation of a precursor U5 snRNP that lacks Brr2p; Prp8 contains a functional nuclear localization signal, and RP mutations disrupt the Prp8-Brr2 interaction required for nuclear assembly of mature U5 snRNP, defining a U5 snRNP assembly pathway. Yeast genetics, subcellular fractionation, co-immunoprecipitation of U5 snRNP components, NLS mutagenesis Nature structural & molecular biology High 17934474
2007 Two opposing classes of prp8 alleles modulate the transition between the two catalytic steps of splicing: one class suppresses first-step defects, the opposing class suppresses second-step defects, analogous to ribosomal ram/restrictive mutants; Prp8-mediated repositioning occurs between catalytic-center opening (U6 snRNA) and closure (Prp16 ATPase). Genetic screen for new prp8 alleles, in vivo and in vitro splicing assays, genetic interaction analysis with prp16 and U6 mutants Nature structural & molecular biology High 17486100
2008 Crystal structure of the beta-finger domain of Prp8 (residues 1822–2095) reveals a beta-hairpin protruding from the protein; mutations throughout the beta-finger change the conformational equilibrium between the two catalytic steps, and mutations at the base affect U4/U6 unwinding-mediated activation. X-ray crystallography (1.85 Å), mutant yeast phenotype analysis, splicing assays Proceedings of the National Academy of Sciences of the United States of America High 18779563
2008 Brr2-dependent U4/U6 snRNA unwinding in vitro is activated by a fragment from the C-terminus of Prp8; paradoxically, this fragment inhibits Brr2 U4/U6-dependent ATPase activity; RP-linked Prp8 alleles in the C-terminus fail to stimulate U4/U6 unwinding. In vitro U4/U6 unwinding assay, in vitro ATPase assay with Prp8 C-terminal fragments, RP mutation functional testing Nature structural & molecular biology High 19098916
2008 Crystal structure of PRP8 domain IV core (RNase H fold linked to a five-helix assembly at 1.85 Å) reveals a surface that directly contacts RNA structures at the catalytic core of the spliceosome, consistent with mutant yeast allele phenotypes and RNA binding studies. X-ray crystallography, RNA binding studies with Prp8 domain IV, mutant yeast allele analysis Nature structural & molecular biology High 18836455
2008 By electron microscopy, Prp8 and GTPase Snu114 are located centrally in the yeast tri-snRNP, while Brr2 occupies a separate head domain, and U4/U6 snRNP forms the arm domain; the head and arm show variable relative positions suggesting structural dynamics during activation. Electron microscopy of genetically tagged tri-snRNP proteins, EM projection structure analysis Nature structural & molecular biology High 18953335
2013 Crystal structure of yeast Prp8 (residues 885–2413) in complex with Aar2 reveals tightly associated domains resembling a bacterial group II intron reverse transcriptase and a type II restriction endonuclease; a large cavity formed by reverse transcriptase thumb, endonuclease-like and RNaseH-like domains accommodates the catalytic core RNA, with splice site suppressor mutations mapping to this cavity. X-ray crystallography of Prp8–Aar2 complex, mapping of suppressor mutations onto structure Nature High 23354046
2013 The C-terminal tail of Prp8 inserts into Brr2's RNA-binding tunnel, blocking Brr2's RNA-binding, ATPase, and U4/U6 unwinding activities; RP-linked Prp8 mutations that map to the C-terminal tail cause inefficient Brr2 repression, establishing a direct inhibitory mechanism. Crystal structure of Prp8 C-terminal tail bound in Brr2 RNA tunnel, in vitro ATPase and helicase assays, RP mutant functional testing Science (New York, N.Y.) High 23704370
2013 The RNase H domain of Prp8 binds U4/U6 snRNA (single-stranded regions preceding U4/U6 stem I) and blocks Brr2 loading onto U4 snRNA by competing for the same U4 single-stranded region that Brr2 uses to initiate translocation; identified by crosslinking-mass spectrometry. RNA binding assays, crosslinking coupled with mass spectrometry (CLMS) of RNase H domain–U4/U6 contacts, Brr2 competition unwinding assays Genes & development High 23124066
2013 The RNase H domain of Prp8 undergoes a conformational switch between the two catalytic steps of splicing; this switch unmasks a metal-binding site involved in exon ligation (step 2), establishing Prp8 as a metalloprotein that promotes exon ligation within the spliceosome. Structural analysis of RNase H domain conformations, metal coordination assays, prp8 allele phenotyping rationalizing step 1/step 2 effects Nature structural & molecular biology High 23686287
2013 Yeast Brr2 in complex with the Jab1/MPN domain of Prp8 (crystal structure) shows the Jab1/MPN domain binds exclusively to the N-terminal helicase cassette of Brr2 and stimulates Brr2 activity; RP-linked residues in Jab1/MPN are at the Brr2 interface; Aar2 and Brr2 binding to Prp8 are mutually exclusive explaining cytoplasmic precursor U5 snRNP assembly. X-ray crystallography of Brr2–Prp8 Jab1/MPN complex, mutagenesis, biochemical Brr2 activity assays Structure (London, England : 1993) High 23727230
2013 In vivo CLIP/CRAC footprinting of Prp8 in yeast reveals direct contacts with U2, U5, and U6 snRNAs and pre-mRNA in purified activated spliceosomes; novel contacts with U1 snRNA are discovered, and disruption of Prp8–U1 snRNA interaction reduces tri-snRNP levels in the spliceosome. CLIP/CRAC (crosslinking and analysis of cDNAs) with next-generation sequencing, immunoprecipitation, affinity-purified activated spliceosomes Nucleic acids research High 23393194
2014 PRPF8 knockdown in K562 and CD34+ bone marrow cells increases proliferative capacity and causes missplicing defects; yeast homologous mutations in Prp8 abrogate a second-step splicing block, indicating PRPF8 has proofreading/fidelity functions in the second step of splicing. Knockdown in human cell lines, whole-RNA deep sequencing, yeast complementation with homologous mutations, splicing reporter assays Leukemia High 24781015
2014 Enterovirus 3D polymerase (RdRp) enters the nucleus via its NLS and associates with the C-terminal Jab1/MPN domain of Prp8, interfering with the second catalytic step of splicing and causing accumulation of lariat splicing intermediates. Co-immunoprecipitation of 3Dpol and Prp8, in vitro splicing assays showing lariat accumulation, domain mapping of interaction PLoS pathogens High 24968230
2015 PRPF8 depletion in human cells preferentially impairs splicing of introns with weak 5' splice sites; iCLIP shows PRPF8 depletion decreases RNP complex formation at most splice sites, and enhancing splice-site strength overcomes PRPF8 depletion effects on splicing kinetics and fidelity. siRNA knockdown, iCLIP of spliceosome components, RNA-seq, minigene splicing assays with altered splice-site strength Genome biology High 26392272
2015 Enhanced Prp8 crosslinking to nucleotides surrounding the branch-site is observed upon step 1 catalysis in yeast spliceosomes (Bact to C complex transition), and Prp8 interactions with the intron are dynamic during catalytic activation. UV-induced crosslinking of purified yeast spliceosomes assembled on site-specifically labeled pre-mRNA, mass spectrometry PLoS genetics High 26393790
2015 Stable tri-snRNP integration into the spliceosome requires interaction of Prp8 with nucleotides at the exon-intron junction (5' splice site); addition of 5'ss RNA oligonucleotide alone to affinity-purified cross-exon complexes is sufficient to trigger a major structural rearrangement and stable tri-snRNP incorporation. Affinity purification of spliceosomal complexes, addition of 5'ss RNA oligonucleotides, electron microscopy structural analysis RNA (New York, N.Y.) High 26385511
2016 RP-linked mutations in the Prp8 Jab1/MPN hinge region cause defects in the transition between first and second catalytic steps of splicing in yeast, reduce overall splicing efficiency without affecting fidelity, and genetic analysis links Snu114 GTP/GDP occupancy to Prp8-dependent Brr2 regulation. In vivo and in vitro splicing assays in yeast with RP alleles, genetic interaction analysis with Snu114 GTPase mutants and Brr2 RNA (New York, N.Y.) High 26968627
2017 prp8 alleles mapping to a 17-aa extension in the RNase H domain fall into two functional classes associated with two mutually exclusive RNaseH domain structures: error-prone/efficient splicing and hyperaccurate/inefficient splicing; the spliceosome toggles between these conformations to regulate splicing fidelity. Characterization of prp8 alleles by in vitro and in vivo reporter splicing assays, lariat sequencing for global splice-site activation, genetic interaction with prp2 mutant Proceedings of the National Academy of Sciences of the United States of America High 28416677
2017 Prp8 interacts physically with the androgen receptor (AR) nuclear export signal (NESAR) via co-immunoprecipitation in prostate cancer cells; Prp8 knockdown induces nuclear accumulation of GFP-tagged AR and increases AR polyubiquitination, indicating Prp8 regulates AR nucleocytoplasmic trafficking and stability. Co-immunoprecipitation and deletion mutagenesis in mammalian cells, shRNA knockdown with rapamycin export assay, luciferase reporter assay Molecular endocrinology (Baltimore, Md.) Medium 26371515
2018 A dynamic region in human Prp8 stabilizes the pre-mRNA in the spliceosome active site through interactions with U5 snRNA; mutagenesis of the identified Prp8 residues in yeast impairs 5' splice site recognition, and genetic interactions with Isy1 and Snu114 corroborate a role in substrate positioning. Amino acid probing strategy (novel), yeast mutagenesis, genetic interaction analysis with Isy1 and Snu114 RNA (New York, N.Y.) High 29487104
2018 PRPF8 is required for hypoxia-induced mitophagy; PRPF8 knockdown impairs mitophagosome formation and mitochondrial clearance through aberrant mRNA splicing of ULK1 (autophagy initiator); RP-associated mutant PRPF8 R2310K is defective in regulating this mitophagy. RNAi screen using mt-Keima fluorescent reporter, knockdown validation, ULK1 splicing analysis, mitophagy flux assays Autophagy Medium 30103670
2019 prp8 alleles in C. elegans alter cryptic splice site usage frequency without broadly changing alternative splicing patterns; yeast allele analysis implicates these residues in stability of the spliceosome's catalytic core, suggesting intrinsic spliceosome mechanisms suppress cryptic splicing independently of alternative splicing regulation. C. elegans genetic screen, high-throughput mRNA sequencing of prp-8 mutants, complementary yeast allele analysis Proceedings of the National Academy of Sciences of the United States of America High 30674666
2021 The evolutionarily conserved protein Ecdysoneless (Ecd) chaperones Prp8 to the forming U5 snRNP in the cytoplasm; Ecd deficiency reduces Prp8 protein levels and compromises U5 snRNP biogenesis, causing splicing fidelity loss; SmD3 is identified as a novel Ecd interaction partner. Drosophila genetics, proteomic approaches, co-immunoprecipitation, U5 snRNP assembly assays, splicing fidelity analysis Nucleic acids research High 33444449
2023 PRPF8 silencing in hepatocellular carcinoma cells modulates fibronectin (FN1) splicing by promoting exclusion of exon 40.2, which reduces integrin binding, leading to decreased FAK/AKT phosphorylation and reduced stress fiber formation and invasion; CLIP-seq shows PRPF8 binds preferentially to exons of protein-coding genes. siRNA knockdown, CLIPseq, RNAseq, in vitro invasion assays, xenograft in vivo experiments, FAK/AKT phosphorylation analysis Experimental & molecular medicine Medium 36609600
2024 PRPF8/Brr2 regulatory disruption (H2309P RP mutation) impairs 5' splice site selection by spliceosomes, particularly affecting weak/suboptimal 5'SS and cryptic splicing in ciliary and retinal-specific transcripts; mutant PRPF8 alters nuclear speckle organization, PRPF8 interaction with U6 snRNA, and causes accumulation of active spliceosomes and poly(A)+ mRNA in unique splicing clusters at the nuclear periphery of photoreceptors. Patient iPSC-derived retinal and RPE cells, transcriptomic, proteomic, and molecular analyses, co-immunoprecipitation with U6 snRNA, nuclear speckle imaging Nature communications High 38605034

Source papers

Stage 0 corpus · 100 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2005 Prp8 protein: at the heart of the spliceosome. RNA (New York, N.Y.) 300 15840809
2001 Mutations in the pre-mRNA splicing factor gene PRPC8 in autosomal dominant retinitis pigmentosa (RP13). Human molecular genetics 235 11468273
2013 Crystal structure of Prp8 reveals active site cavity of the spliceosome. Nature 188 23354046
1995 Extensive interactions of PRP8 protein with the 5' and 3' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA. The EMBO journal 149 7781612
1998 The human U5-220kD protein (hPrp8) forms a stable RNA-free complex with several U5-specific proteins, including an RNA unwindase, a homologue of ribosomal elongation factor EF-2, and a novel WD-40 protein. Molecular and cellular biology 124 9774689
2013 Inhibition of RNA helicase Brr2 by the C-terminal tail of the spliceosomal protein Prp8. Science (New York, N.Y.) 118 23704370
2007 Structure of a multipartite protein-protein interaction domain in splicing factor prp8 and its link to retinitis pigmentosa. Molecular cell 118 17317632
2008 ATP-dependent unwinding of U4/U6 snRNAs by the Brr2 helicase requires the C terminus of Prp8. Nature structural & molecular biology 105 19098916
2014 PRPF8 defects cause missplicing in myeloid malignancies. Leukemia 104 24781015
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