{"gene":"PRPF19","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":1996,"finding":"PSO4 is allelic to PRP19, encoding a spliceosome-associated protein; gene disruption is lethal in haploid yeast, establishing it as an essential gene with dual roles in pre-mRNA splicing and DNA repair/recombination.","method":"Genetic complementation, gene disruption, sequence analysis in S. cerevisiae","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic complementation and gene disruption in yeast, replicated across multiple early studies establishing allelism and essentiality","pmids":["8918805"],"is_preprint":false},{"year":2003,"finding":"Human hPso4 (PRPF19) binds double-stranded DNA in a sequence-nonspecific manner but does not bind single-stranded DNA; it associates physically with terminal deoxynucleotidyl transferase (TdT) in lymphoid cells; siRNA-mediated loss of hPso4 causes accumulation of DNA double-strand breaks and apoptosis after DNA damage.","method":"Co-immunoprecipitation, DNA binding assays (purified protein), siRNA knockdown with γ-H2AX/comet assay readout","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and in vitro DNA binding assays, single lab with two orthogonal methods","pmids":["12960389"],"is_preprint":false},{"year":2005,"finding":"The Pso4/Prp19 complex (composed of Pso4/Prp19, Cdc5L, Plrg1, and Spf27) is required for processing of DNA interstrand cross-links (ICLs) in vitro; WRN helicase activity (but not exonuclease activity) is also required; WRN physically interacts with the Pso4 complex via direct binding to Cdc5L.","method":"In vitro ICL processing biochemical assay with site-specific psoralen substrate, co-immunoprecipitation, pulldown","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro activity assay with defined substrate, mutagenesis (helicase-dead vs exonuclease-dead WRN), and protein interaction mapping","pmids":["16223718"],"is_preprint":false},{"year":2005,"finding":"SNEV (PRPF19) has in vitro E3 ubiquitin ligase activity and interacts directly with the β7 subunit (PSMB4) of the 20S proteasome; upon proteasome inhibition, SNEV co-localizes with ubiquitin without itself being ubiquitinated, suggesting it escorts substrates to the proteasome; this interaction is evolutionarily conserved (yeast Prp19 also binds yeast β7).","method":"In vitro ubiquitination assay, co-immunoprecipitation, immunofluorescence microscopy, yeast two-hybrid","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro E3 ligase assay combined with Co-IP and localization, replicated in yeast ortholog","pmids":["15660529"],"is_preprint":false},{"year":2005,"finding":"SNEV (PRPF19) is the human ortholog of yeast Prp19; it functions in pre-mRNA splicing; its homo-oligomerization (self-interaction domain mapped to amino acids 56–74) is essential for spliceosome assembly and stability, as synthetic peptides from this region inhibit in vitro splicing by disrupting spliceosome formation.","method":"Yeast complementation, immunodepletion from HeLa nuclear extracts, in vitro splicing assay, peptide inhibition, yeast two-hybrid self-interaction mapping","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution (immunodepletion + rescue), mutational domain mapping, yeast complementation, multiple orthogonal methods in one study","pmids":["16332694"],"is_preprint":false},{"year":2007,"finding":"hPrp19 forms an ubiquitylated oligomeric species (likely via thiolester between ubiquitin and a cysteine in Prp19) that is enhanced upon DNA damage and associates more with chromatin; this ubiquitylated form fails to interact with Cdc5L or Plrg1, indicating DNA damage induces structural alterations to the Prp19 core complex.","method":"SDS-PAGE under non-reducing conditions, co-immunoprecipitation, chromatin fractionation, DNA damage treatment","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical fractionation and Co-IP with DNA damage perturbation, single lab, two orthogonal methods","pmids":["17276391"],"is_preprint":false},{"year":2007,"finding":"SNEV (PRPF19) is essential for early mouse development; SNEV-null embryos initiate blastocyst formation but cells of the inner cell mass fail to proliferate and die; heterozygous MEFs show decreased proliferative potential, demonstrating the protein is functionally non-redundant in vivo.","method":"Homologous recombination knockout in mice, blastocyst outgrowth assays, MEF proliferation assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout with defined developmental phenotype, replicated across embryo and cell culture models","pmids":["17283042"],"is_preprint":false},{"year":2008,"finding":"hPso4 (PRPF19) forms a stable complex with Metnase (SETMAR) on both TIR and non-TIR DNA; hPso4 is required to recruit Metnase to DNA double-strand break sites in vivo; siRNA depletion of hPso4 abolishes Metnase localization at DSBs and Metnase-mediated stimulation of DNA end joining.","method":"Co-immunoprecipitation, immunofluorescence co-localization after ionizing radiation, siRNA knockdown, DNA end-joining functional assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, localization with functional consequence (end-joining assay), single lab","pmids":["18263876"],"is_preprint":false},{"year":2010,"finding":"When hPso4 forms a complex with Metnase, hPso4 is solely responsible for DNA binding within the complex and negatively regulates Metnase's TIR-specific DNA binding activity, redirecting Metnase to non-TIR sites such as DSBs.","method":"Electrophoretic mobility shift assay, competitive inhibition assay, stoichiometric analysis of protein-DNA complexes","journal":"Archives of biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro binding assays with competitive inhibition and stoichiometric analysis, single lab","pmids":["20416268"],"is_preprint":false},{"year":2011,"finding":"Exo70, a subunit of the exocyst complex, directly interacts with SNEV (PRPF19) via its N-terminal 100 amino acids; Exo70 shuttles to the nucleus, associates with the spliceosome, and its N-terminal fragment inhibits pre-mRNA splicing in vitro; Exo70 influences splicing of a model substrate and its own pre-mRNA in vivo.","method":"Co-immunoprecipitation, yeast two-hybrid, in vitro splicing assay with peptide inhibition, minigene splicing assay in vivo","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct interaction mapped, in vitro functional splicing assay, in vivo minigene assay, single lab","pmids":["21639856"],"is_preprint":false},{"year":2012,"finding":"SNEVhPrp19/hPso4 is phosphorylated at serine 149 in an ATM-dependent manner in response to oxidative stress and DNA double-strand break-inducing agents; this S149 phosphorylation is necessary for mediating resistance to apoptosis upon oxidative stress and partially necessary for extending cellular life span.","method":"Mass spectrometry phosphorylation mapping, ATM inhibitor treatment, phosphorylation-deficient point-mutant (S149A) overexpression, apoptosis assays","journal":"Aging","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified PTM, site-directed mutagenesis with functional readout, single lab","pmids":["22529335"],"is_preprint":false},{"year":2014,"finding":"The PSO4 complex (PSO4/PRP19, CDC5L, PLRG1, BCAS2/SPF27) directly interacts and co-localizes with RPA; both BCAS2 and PSO4 interact with RPA1; depletion of BCAS2 or PSO4 impairs ATRIP recruitment to DNA damage sites and compromises CHK1 activation and RPA2 phosphorylation; both RPA1-binding ability of BCAS2 and E3 ligase activity of PSO4 are required for efficient ATR pathway activation.","method":"Co-immunoprecipitation, immunofluorescence, siRNA depletion, CHK1/RPA2 phosphorylation assays, E3 ligase activity mutant analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, localization, functional phosphorylation assays, E3 ligase mutant, multiple orthogonal methods in single study","pmids":["24443570"],"is_preprint":false},{"year":2014,"finding":"The hPso4 complex is required for timely S-phase progression and G2/M checkpoint transition; hPso4 depletion results in delayed replication fork restart after hydroxyurea-induced stalling, reduced DSB repair, increased sensitivity to PARP inhibitors, and impaired homologous recombination; hPso4 regulates BRCA1 protein levels and single-strand DNA generation at DSBs.","method":"siRNA knockdown, BrdU incorporation (replication assay), γ-H2AX foci, PARP inhibitor sensitivity, HR reporter assay, Western blot for BRCA1","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with multiple defined cellular phenotypes, single lab, multiple orthogonal methods","pmids":["24675077"],"is_preprint":false},{"year":2018,"finding":"Prp19 forms a homotetramer whose elongated coiled coils serve as an assembly axis for the NTC (nineteen complex); Prp19 is autoinhibited and inactive as an E3 ligase on its own; stepwise assembly of SPF27, CDC5L, and PLRG1 onto the Prp19 tetramer activates ubiquitin ligation; structural basis for autoinhibition was determined by crystallography; communication between PLRG1 and Prp19 enables E3 activity.","method":"X-ray crystallography, mutational analysis, in vitro ubiquitination assay, protein-protein crosslinking mass spectrometry, stepwise complex reconstitution","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis, in vitro reconstitution of E3 activity, and crosslinking MS; multiple orthogonal methods in one rigorous study","pmids":["29547724"],"is_preprint":false},{"year":2021,"finding":"PRPF19 knockdown causes a switch in MDM4 splicing from the stable full-length MDM4-FL isoform to the unstable MDM4-S isoform (lacking exon 6), thereby reducing MDM4-mediated p53 inactivation and inducing p53-p21-dependent cellular senescence; PRPF19 promotes the physical interaction between splicing factors PRPF3 and PRPF8 (components of U4/U6.U5 tri-snRNP).","method":"siRNA knockdown, RNA-seq, RT-PCR splicing isoform analysis, co-immunoprecipitation of PRPF3/PRPF8, p53-p21 pathway analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with RNA-seq and RT-PCR validation, Co-IP for protein interactions, single lab with multiple orthogonal methods","pmids":["34144037"],"is_preprint":false},{"year":2021,"finding":"Prpf19/prp19 promotes poly-ubiquitination and proteasomal degradation of mutant expanded ataxin-3 (ATXN3-polyQ); nuclear localization of Prpf19 is essential for its modulatory function; Exoc7/exo70 interacts with Prpf19 and opposes its E3 ligase function toward ATXN3-polyQ.","method":"Ubiquitination assay, proteasome inhibitor treatment, localization mutant analysis, co-immunoprecipitation, Drosophila and mammalian cell models","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ubiquitination assay with functional rescue, localization mutant, two model systems, single lab","pmids":["33542212"],"is_preprint":false},{"year":2022,"finding":"PRPF19 recruits the E3 ubiquitin ligase MARCH8 to the PEDV nucleocapsid (N) protein to mediate its ubiquitination; the ubiquitinated N protein is recognized by cargo receptor NDP52 and transported to autolysosomes for degradation via selective autophagy, thereby inhibiting PEDV replication.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, overexpression, selective autophagy assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP interaction mapping with ubiquitination assay and functional viral replication readout, single lab","pmids":["36541804"],"is_preprint":false},{"year":2023,"finding":"PRPF19 promotes K63-linked ubiquitination of myosin light chain 9 (MYL9), enhancing MYL9 stability, which activates the Src-YAP1 signaling cascade to promote migration and invasion in colorectal cancer cells.","method":"Co-immunoprecipitation, ubiquitination assay (K63-linkage specific), gain- and loss-of-function experiments, Src/YAP1 pathway analysis","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ubiquitination linkage-specific assay with substrate identification and downstream pathway analysis, single lab","pmids":["37031206"],"is_preprint":false},{"year":2024,"finding":"PRPF19 acts as a substrate recognition receptor that interacts with CUL4B, DDB1, and RBX1 to form a CRL4B-based E3 ubiquitin ligase complex; this complex catalyzes ubiquitination and proteasomal degradation of SARS-CoV-2 ORF6, relieving ORF6-mediated IFN suppression and inhibiting SARS-CoV-2 replication.","method":"Pulldown/Co-immunoprecipitation, ubiquitination assay, overexpression/knockdown with viral replication readout, mouse SARS-CoV-2 infection model","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP complex characterization, in vitro ubiquitination assay, functional viral assay in cell culture and mouse model, single lab","pmids":["38265236"],"is_preprint":false},{"year":2025,"finding":"PRPF19 mediates ubiquitin-dependent proteasomal degradation of the vitamin D receptor (VDR); loss of VDR reduces GPX4 transcription, promoting ferroptosis in renal tubular epithelial cells; berberine was identified as an inhibitor of PRPF19-VDR interaction via molecular docking and surface plasmon resonance.","method":"Proteomics, luciferase reporter assay, chromatin immunoprecipitation, co-immunoprecipitation, ubiquitination assay, SPR, VDR knockout mouse model","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with ubiquitination assay and in vivo knockout validation, SPR for inhibitor binding, single lab with multiple methods","pmids":["40414879"],"is_preprint":false},{"year":2009,"finding":"Blom7α (a novel KH domain protein) directly interacts with SNEV(Prp19-Pso4), co-localizes and co-precipitates with splicing factors, and is present in affinity-purified spliceosomes; addition of Blom7α to HeLa nuclear extracts increases splicing activity; Blom7α overexpression alters both 5'- and 3'-splice site selection.","method":"Yeast two-hybrid, co-immunoprecipitation, affinity-purified spliceosome analysis, in vitro splicing assay, minigene splicing assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct interaction confirmed, spliceosome co-purification, in vitro and in vivo functional splicing assays, single lab","pmids":["19641227"],"is_preprint":false},{"year":2024,"finding":"The 5' UTR of PRPF19 mRNA contains an upstream open reading frame (uORF) that is translated in human cells; inactivation of this uORF reduces cell viability.","method":"Ribosome profiling, uORF mutagenesis, cell viability assays","journal":"Doklady. Biochemistry and biophysics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, limited mechanistic detail in abstract; functional consequence is cell viability without pathway placement","pmids":["38472668"],"is_preprint":false}],"current_model":"PRPF19 (PSO4/SNEV/hPrp19) is an essential, multifunctional nuclear E3 ubiquitin ligase that exists as a homotetramer in an autoinhibited state; stepwise assembly of SPF27, CDC5L, and PLRG1 onto this tetramer forms the NTC/Pso4 core complex and activates ubiquitin ligation. As a splicing factor, PRPF19 oligomerization is required for spliceosome assembly and stability, and it regulates alternative splicing (e.g., MDM4, TPT1). In the DNA damage response, the NTC complex interacts with RPA and its E3 ligase activity—together with BCAS2's RPA1-binding—is required for ATRIP recruitment, CHK1 activation, and RPA2 phosphorylation, positioning PRPF19 as an activator of ATR signaling; it also participates in ICL repair (via WRN helicase recruitment through Cdc5L), homologous recombination, and replication fork restart. ATM phosphorylates PRPF19 at S149 in response to DNA damage to modulate apoptosis resistance. Beyond these core functions, PRPF19 ubiquitinates diverse substrates (MYL9 via K63 linkage for stability; VDR, SARS-CoV-2 ORF6, and polyQ-expanded ATXN3 for degradation) and escorts substrates to the proteasome via direct binding to the PSMB4/β7 subunit of the 20S proteasome."},"narrative":{"mechanistic_narrative":"PRPF19 (PSO4/SNEV/hPrp19) is an essential, evolutionarily conserved nuclear protein that couples pre-mRNA splicing to genome maintenance, and its loss is lethal in yeast and arrests early mouse development at the inner cell mass stage [PMID:8918805, PMID:17283042]. Structurally, PRPF19 assembles into a homotetramer whose elongated coiled coils form an assembly axis for the NTC/Pso4 core complex; the tetramer is autoinhibited and catalytically silent as an E3 ubiquitin ligase until stepwise loading of SPF27, CDC5L, and PLRG1 activates ubiquitin ligation, with PLRG1–Prp19 communication relieving autoinhibition [PMID:29547724, PMID:15660529]. As a splicing factor, its self-interaction (mapped to residues 56–74) is required for spliceosome assembly and stability, and it promotes the PRPF3–PRPF8 interaction within the U4/U6.U5 tri-snRNP to control alternative splicing such as the MDM4 isoform switch that gates p53-p21–dependent senescence [PMID:16332694, PMID:34144037]. In the DNA damage response, the Pso4 complex binds RPA and, together with BCAS2's RPA1-binding and PRPF19's E3 activity, drives ATRIP recruitment, CHK1 activation, and RPA2 phosphorylation, while supporting interstrand crosslink repair through Cdc5L-mediated WRN recruitment, homologous recombination, replication fork restart, and recruitment of Metnase to double-strand breaks [PMID:24443570, PMID:16223718, PMID:24675077, PMID:18263876]. PRPF19 also functions broadly as a substrate-selective ubiquitin ligase—escorting substrates to the 20S proteasome via direct PSMB4/β7 binding and ubiquitinating diverse targets including expanded ataxin-3, viral proteins, MYL9, and VDR [PMID:15660529, PMID:33542212, PMID:37031206, PMID:40414879].","teleology":[{"year":1996,"claim":"Established that a single essential gene underlies both splicing and DNA repair, defining PRPF19's dual functional identity from the outset.","evidence":"Genetic complementation and gene disruption in S. cerevisiae showing PSO4/PRP19 allelism and haploid lethality","pmids":["8918805"],"confidence":"High","gaps":["Does not resolve whether splicing and repair roles are mechanistically coupled or separable","No molecular activity assigned to the protein"]},{"year":2003,"claim":"Showed the human protein directly engages DNA and protects against breaks, providing the first biochemical link to genome integrity.","evidence":"In vitro DNA-binding assays and Co-IP with TdT, plus siRNA knockdown with γ-H2AX/comet readout in lymphoid cells","pmids":["12960389"],"confidence":"Medium","gaps":["dsDNA binding is sequence-nonspecific, leaving target selectivity unexplained","Mechanism linking DNA binding to break suppression undefined"]},{"year":2005,"claim":"Resolved that PRPF19 is an E3 ubiquitin ligase that escorts substrates to the proteasome and that its oligomerization is structurally required for spliceosome assembly, unifying its catalytic and splicing roles.","evidence":"In vitro ubiquitination assay, PSMB4 Co-IP, yeast two-hybrid self-interaction mapping (aa 56–74), immunodepletion/rescue and peptide-inhibition splicing assays, plus reconstituted in vitro ICL processing with WRN","pmids":["15660529","16332694","16223718"],"confidence":"High","gaps":["Physiological ubiquitination substrates not yet identified in these studies","How proteasome escort relates to splicing function unclear"]},{"year":2007,"claim":"Defined PRPF19 as functionally non-redundant in vivo and showed DNA damage remodels its core complex, hinting at a damage-responsive conformational switch.","evidence":"Mouse knockout with blastocyst outgrowth/MEF proliferation assays; non-reducing SDS-PAGE and chromatin fractionation after DNA damage","pmids":["17283042","17276391"],"confidence":"High","gaps":["The ubiquitylated damage-induced species is biochemically inferred, not structurally proven","Developmental phenotype not assigned to splicing vs repair function"]},{"year":2008,"claim":"Demonstrated PRPF19 acts as a DNA-binding recruitment platform that targets repair effectors to break sites, extending its role beyond catalysis.","evidence":"Co-IP, IR-induced co-localization, siRNA depletion, and end-joining assays with Metnase; EMSA and competitive binding showing PRPF19 controls Metnase DNA selectivity","pmids":["18263876","20416268"],"confidence":"Medium","gaps":["Single lab characterization of the Metnase axis","Generality across repair pathways not established"]},{"year":2012,"claim":"Identified ATM-dependent S149 phosphorylation as a post-translational switch linking PRPF19 to stress-resistance and lifespan, placing it downstream of damage signaling.","evidence":"MS phosphosite mapping, ATM inhibitor treatment, and S149A mutant apoptosis assays under oxidative stress","pmids":["22529335"],"confidence":"Medium","gaps":["Downstream effectors of S149 phosphorylation unknown","Single-lab finding without structural context"]},{"year":2014,"claim":"Positioned PRPF19 as an upstream activator of ATR signaling and a regulator of replication-coupled repair, defining its mechanistic role in the DNA damage response.","evidence":"Co-IP and co-localization with RPA, siRNA depletion with ATRIP recruitment/CHK1/RPA2 readouts and E3 mutant analysis; BrdU, HR reporter, PARP-inhibitor sensitivity and BRCA1 Western assays","pmids":["24443570","24675077"],"confidence":"High","gaps":["Direct ubiquitination substrate driving ATR activation not identified","Mechanism of BRCA1 level regulation undefined"]},{"year":2018,"claim":"Provided the structural basis for activity by showing the Prp19 tetramer is autoinhibited and activated only by stepwise NTC assembly, explaining how E3 activity is gated.","evidence":"X-ray crystallography, mutagenesis, crosslinking MS, and stepwise in vitro reconstitution of E3 activity with SPF27/CDC5L/PLRG1","pmids":["29547724"],"confidence":"High","gaps":["How DNA damage signals release autoinhibition in vivo not addressed","Substrate engagement geometry of the active complex unresolved"]},{"year":2021,"claim":"Connected PRPF19's splicing activity to a defined biological output by showing it controls the MDM4 isoform switch governing p53-p21 senescence, and broadened its substrate repertoire to neurodegeneration.","evidence":"siRNA/RNA-seq/RT-PCR splicing analysis with PRPF3/PRPF8 Co-IP; ubiquitination and proteasome assays for ATXN3-polyQ in Drosophila and mammalian cells with Exo70 antagonism","pmids":["34144037","33542212"],"confidence":"Medium","gaps":["Whether MDM4 regulation requires E3 activity vs splicing alone unclear","Nuclear-localization requirement for ATXN3 clearance not mechanistically dissected"]},{"year":2024,"claim":"Established PRPF19 as a substrate-recognition receptor within a CRL4B complex and an antiviral/disease-relevant E3 ligase across multiple substrates.","evidence":"Co-IP/pulldown, ubiquitination, and viral replication assays for PEDV-N (via MARCH8/NDP52), SARS-CoV-2 ORF6 (via CUL4B/DDB1/RBX1), MYL9 (K63-linked), and VDR with knockout/SPR validation","pmids":["36541804","38265236","37031206","40414879"],"confidence":"Medium","gaps":["Each substrate characterized by a single lab","How PRPF19 selects among diverse substrates and ligase partners is unresolved"]},{"year":null,"claim":"How PRPF19's splicing, DNA-repair, and substrate-degradation activities are coordinately regulated and selectively deployed within a single autoinhibited scaffold remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking conformational state to substrate/pathway choice","Determinants of substrate selectivity for the E3 activity undefined","In vivo trigger releasing autoinhibition during DNA damage unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[3,13,11,15,17,18,19]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,13,17,18,19]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[1,8]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[11]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[5,15]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[5,11,7]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[4,14,20]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[2,11,12,7]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[3,13,15,18,19]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[11,10]}],"complexes":["NTC/Pso4 complex","CRL4B E3 ligase complex","spliceosome"],"partners":["CDC5L","PLRG1","BCAS2","RPA1","PSMB4","WRN","SETMAR","CUL4B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UMS4","full_name":"Pre-mRNA-processing factor 19","aliases":["Nuclear matrix protein 200","PRP19/PSO4 homolog","hPso4","RING-type E3 ubiquitin transferase PRP19","Senescence evasion factor"],"length_aa":504,"mass_kda":55.2,"function":"Ubiquitin-protein ligase which is a core component of several complexes mainly involved pre-mRNA splicing and DNA repair. Required for pre-mRNA splicing as component of the spliceosome (PubMed:28076346, PubMed:28502770, PubMed:29301961, PubMed:29360106, PubMed:30705154). Core component of the PRP19C/Prp19 complex/NTC/Nineteen complex which is part of the spliceosome and participates in its assembly, its remodeling and is required for its activity. During assembly of the spliceosome, mediates 'Lys-63'-linked polyubiquitination of the U4 spliceosomal protein PRPF3. Ubiquitination of PRPF3 allows its recognition by the U5 component PRPF8 and stabilizes the U4/U5/U6 tri-snRNP spliceosomal complex (PubMed:20595234). Recruited to RNA polymerase II C-terminal domain (CTD) and the pre-mRNA, it may also couple the transcriptional and spliceosomal machineries (PubMed:21536736). The XAB2 complex, which contains PRPF19, is also involved in pre-mRNA splicing, transcription and transcription-coupled repair (PubMed:17981804). Beside its role in pre-mRNA splicing PRPF19, as part of the PRP19-CDC5L complex, plays a role in the DNA damage response/DDR. It is recruited to the sites of DNA damage by the RPA complex where PRPF19 directly ubiquitinates RPA1 and RPA2. 'Lys-63'-linked polyubiquitination of the RPA complex allows the recruitment of the ATR-ATRIP complex and the activation of ATR, a master regulator of the DNA damage response (PubMed:24332808). May also play a role in DNA double-strand break (DSB) repair by recruiting the repair factor SETMAR to altered DNA (PubMed:18263876). As part of the PSO4 complex may also be involved in the DNA interstrand cross-links/ICLs repair process (PubMed:16223718). In addition, may also mediate 'Lys-48'-linked polyubiquitination of substrates and play a role in proteasomal degradation (PubMed:11435423). May play a role in the biogenesis of lipid droplets (By similarity). May play a role in neural differentiation possibly through its function as part of the spliceosome (By similarity)","subcellular_location":"Nucleus; Nucleus, nucleoplasm; Cytoplasm, cytoskeleton, spindle; Cytoplasm; Lipid droplet","url":"https://www.uniprot.org/uniprotkb/Q9UMS4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/PRPF19","classification":"Common Essential","n_dependent_lines":1208,"n_total_lines":1208,"dependency_fraction":1.0},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000110107","cell_line_id":"CID001764","localizations":[{"compartment":"chromatin","grade":3},{"compartment":"nucleoplasm","grade":2}],"interactors":[{"gene":"CTNNBL1","stoichiometry":10.0},{"gene":"PRPF4B","stoichiometry":10.0},{"gene":"PSMB4","stoichiometry":10.0},{"gene":"CDC5L","stoichiometry":4.0},{"gene":"PLRG1","stoichiometry":4.0},{"gene":"BCAS2","stoichiometry":4.0},{"gene":"RBM39","stoichiometry":4.0},{"gene":"SF3A1","stoichiometry":4.0},{"gene":"SF3B1","stoichiometry":4.0},{"gene":"SNRPA","stoichiometry":4.0}],"url":"https://opencell.sf.czbiohub.org/target/CID001764","total_profiled":1310},"omim":[{"mim_id":"621530","title":"CWC25, SPLICEOSOME-ASSOCIATED PROTEIN; CWC25","url":"https://www.omim.org/entry/621530"},{"mim_id":"621501","title":"CWC15, SPLICEOSOME-ASSOCIATED PROTEIN; CWC15","url":"https://www.omim.org/entry/621501"},{"mim_id":"619370","title":"KH DOMAIN-CONTAINING PROTEIN 4, PRE-mRNA SPLICING FACTOR; KHDC4","url":"https://www.omim.org/entry/619370"},{"mim_id":"617079","title":"LONG INTERGENIC NONCODING RNA 673; LINC00673","url":"https://www.omim.org/entry/617079"},{"mim_id":"617037","title":"NONCODING RNA ACTIVATED BY DNA DAMAGE; NORAD","url":"https://www.omim.org/entry/617037"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nuclear speckles","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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it associates physically with terminal deoxynucleotidyl transferase (TdT) in lymphoid cells; siRNA-mediated loss of hPso4 causes accumulation of DNA double-strand breaks and apoptosis after DNA damage.\",\n      \"method\": \"Co-immunoprecipitation, DNA binding assays (purified protein), siRNA knockdown with γ-H2AX/comet assay readout\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and in vitro DNA binding assays, single lab with two orthogonal methods\",\n      \"pmids\": [\"12960389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The Pso4/Prp19 complex (composed of Pso4/Prp19, Cdc5L, Plrg1, and Spf27) is required for processing of DNA interstrand cross-links (ICLs) in vitro; WRN helicase activity (but not exonuclease activity) is also required; WRN physically interacts with the Pso4 complex via direct binding to Cdc5L.\",\n      \"method\": \"In vitro ICL processing biochemical assay with site-specific psoralen substrate, co-immunoprecipitation, pulldown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro activity assay with defined substrate, mutagenesis (helicase-dead vs exonuclease-dead WRN), and protein interaction mapping\",\n      \"pmids\": [\"16223718\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"SNEV (PRPF19) has in vitro E3 ubiquitin ligase activity and interacts directly with the β7 subunit (PSMB4) of the 20S proteasome; upon proteasome inhibition, SNEV co-localizes with ubiquitin without itself being ubiquitinated, suggesting it escorts substrates to the proteasome; this interaction is evolutionarily conserved (yeast Prp19 also binds yeast β7).\",\n      \"method\": \"In vitro ubiquitination assay, co-immunoprecipitation, immunofluorescence microscopy, yeast two-hybrid\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro E3 ligase assay combined with Co-IP and localization, replicated in yeast ortholog\",\n      \"pmids\": [\"15660529\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"SNEV (PRPF19) is the human ortholog of yeast Prp19; it functions in pre-mRNA splicing; its homo-oligomerization (self-interaction domain mapped to amino acids 56–74) is essential for spliceosome assembly and stability, as synthetic peptides from this region inhibit in vitro splicing by disrupting spliceosome formation.\",\n      \"method\": \"Yeast complementation, immunodepletion from HeLa nuclear extracts, in vitro splicing assay, peptide inhibition, yeast two-hybrid self-interaction mapping\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution (immunodepletion + rescue), mutational domain mapping, yeast complementation, multiple orthogonal methods in one study\",\n      \"pmids\": [\"16332694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"hPrp19 forms an ubiquitylated oligomeric species (likely via thiolester between ubiquitin and a cysteine in Prp19) that is enhanced upon DNA damage and associates more with chromatin; this ubiquitylated form fails to interact with Cdc5L or Plrg1, indicating DNA damage induces structural alterations to the Prp19 core complex.\",\n      \"method\": \"SDS-PAGE under non-reducing conditions, co-immunoprecipitation, chromatin fractionation, DNA damage treatment\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical fractionation and Co-IP with DNA damage perturbation, single lab, two orthogonal methods\",\n      \"pmids\": [\"17276391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SNEV (PRPF19) is essential for early mouse development; SNEV-null embryos initiate blastocyst formation but cells of the inner cell mass fail to proliferate and die; heterozygous MEFs show decreased proliferative potential, demonstrating the protein is functionally non-redundant in vivo.\",\n      \"method\": \"Homologous recombination knockout in mice, blastocyst outgrowth assays, MEF proliferation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout with defined developmental phenotype, replicated across embryo and cell culture models\",\n      \"pmids\": [\"17283042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"hPso4 (PRPF19) forms a stable complex with Metnase (SETMAR) on both TIR and non-TIR DNA; hPso4 is required to recruit Metnase to DNA double-strand break sites in vivo; siRNA depletion of hPso4 abolishes Metnase localization at DSBs and Metnase-mediated stimulation of DNA end joining.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization after ionizing radiation, siRNA knockdown, DNA end-joining functional assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, localization with functional consequence (end-joining assay), single lab\",\n      \"pmids\": [\"18263876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"When hPso4 forms a complex with Metnase, hPso4 is solely responsible for DNA binding within the complex and negatively regulates Metnase's TIR-specific DNA binding activity, redirecting Metnase to non-TIR sites such as DSBs.\",\n      \"method\": \"Electrophoretic mobility shift assay, competitive inhibition assay, stoichiometric analysis of protein-DNA complexes\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro binding assays with competitive inhibition and stoichiometric analysis, single lab\",\n      \"pmids\": [\"20416268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Exo70, a subunit of the exocyst complex, directly interacts with SNEV (PRPF19) via its N-terminal 100 amino acids; Exo70 shuttles to the nucleus, associates with the spliceosome, and its N-terminal fragment inhibits pre-mRNA splicing in vitro; Exo70 influences splicing of a model substrate and its own pre-mRNA in vivo.\",\n      \"method\": \"Co-immunoprecipitation, yeast two-hybrid, in vitro splicing assay with peptide inhibition, minigene splicing assay in vivo\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct interaction mapped, in vitro functional splicing assay, in vivo minigene assay, single lab\",\n      \"pmids\": [\"21639856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SNEVhPrp19/hPso4 is phosphorylated at serine 149 in an ATM-dependent manner in response to oxidative stress and DNA double-strand break-inducing agents; this S149 phosphorylation is necessary for mediating resistance to apoptosis upon oxidative stress and partially necessary for extending cellular life span.\",\n      \"method\": \"Mass spectrometry phosphorylation mapping, ATM inhibitor treatment, phosphorylation-deficient point-mutant (S149A) overexpression, apoptosis assays\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified PTM, site-directed mutagenesis with functional readout, single lab\",\n      \"pmids\": [\"22529335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The PSO4 complex (PSO4/PRP19, CDC5L, PLRG1, BCAS2/SPF27) directly interacts and co-localizes with RPA; both BCAS2 and PSO4 interact with RPA1; depletion of BCAS2 or PSO4 impairs ATRIP recruitment to DNA damage sites and compromises CHK1 activation and RPA2 phosphorylation; both RPA1-binding ability of BCAS2 and E3 ligase activity of PSO4 are required for efficient ATR pathway activation.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, siRNA depletion, CHK1/RPA2 phosphorylation assays, E3 ligase activity mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, localization, functional phosphorylation assays, E3 ligase mutant, multiple orthogonal methods in single study\",\n      \"pmids\": [\"24443570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The hPso4 complex is required for timely S-phase progression and G2/M checkpoint transition; hPso4 depletion results in delayed replication fork restart after hydroxyurea-induced stalling, reduced DSB repair, increased sensitivity to PARP inhibitors, and impaired homologous recombination; hPso4 regulates BRCA1 protein levels and single-strand DNA generation at DSBs.\",\n      \"method\": \"siRNA knockdown, BrdU incorporation (replication assay), γ-H2AX foci, PARP inhibitor sensitivity, HR reporter assay, Western blot for BRCA1\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with multiple defined cellular phenotypes, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"24675077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Prp19 forms a homotetramer whose elongated coiled coils serve as an assembly axis for the NTC (nineteen complex); Prp19 is autoinhibited and inactive as an E3 ligase on its own; stepwise assembly of SPF27, CDC5L, and PLRG1 onto the Prp19 tetramer activates ubiquitin ligation; structural basis for autoinhibition was determined by crystallography; communication between PLRG1 and Prp19 enables E3 activity.\",\n      \"method\": \"X-ray crystallography, mutational analysis, in vitro ubiquitination assay, protein-protein crosslinking mass spectrometry, stepwise complex reconstitution\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis, in vitro reconstitution of E3 activity, and crosslinking MS; multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"29547724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PRPF19 knockdown causes a switch in MDM4 splicing from the stable full-length MDM4-FL isoform to the unstable MDM4-S isoform (lacking exon 6), thereby reducing MDM4-mediated p53 inactivation and inducing p53-p21-dependent cellular senescence; PRPF19 promotes the physical interaction between splicing factors PRPF3 and PRPF8 (components of U4/U6.U5 tri-snRNP).\",\n      \"method\": \"siRNA knockdown, RNA-seq, RT-PCR splicing isoform analysis, co-immunoprecipitation of PRPF3/PRPF8, p53-p21 pathway analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with RNA-seq and RT-PCR validation, Co-IP for protein interactions, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"34144037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Prpf19/prp19 promotes poly-ubiquitination and proteasomal degradation of mutant expanded ataxin-3 (ATXN3-polyQ); nuclear localization of Prpf19 is essential for its modulatory function; Exoc7/exo70 interacts with Prpf19 and opposes its E3 ligase function toward ATXN3-polyQ.\",\n      \"method\": \"Ubiquitination assay, proteasome inhibitor treatment, localization mutant analysis, co-immunoprecipitation, Drosophila and mammalian cell models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ubiquitination assay with functional rescue, localization mutant, two model systems, single lab\",\n      \"pmids\": [\"33542212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PRPF19 recruits the E3 ubiquitin ligase MARCH8 to the PEDV nucleocapsid (N) protein to mediate its ubiquitination; the ubiquitinated N protein is recognized by cargo receptor NDP52 and transported to autolysosomes for degradation via selective autophagy, thereby inhibiting PEDV replication.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, overexpression, selective autophagy assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP interaction mapping with ubiquitination assay and functional viral replication readout, single lab\",\n      \"pmids\": [\"36541804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PRPF19 promotes K63-linked ubiquitination of myosin light chain 9 (MYL9), enhancing MYL9 stability, which activates the Src-YAP1 signaling cascade to promote migration and invasion in colorectal cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (K63-linkage specific), gain- and loss-of-function experiments, Src/YAP1 pathway analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ubiquitination linkage-specific assay with substrate identification and downstream pathway analysis, single lab\",\n      \"pmids\": [\"37031206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PRPF19 acts as a substrate recognition receptor that interacts with CUL4B, DDB1, and RBX1 to form a CRL4B-based E3 ubiquitin ligase complex; this complex catalyzes ubiquitination and proteasomal degradation of SARS-CoV-2 ORF6, relieving ORF6-mediated IFN suppression and inhibiting SARS-CoV-2 replication.\",\n      \"method\": \"Pulldown/Co-immunoprecipitation, ubiquitination assay, overexpression/knockdown with viral replication readout, mouse SARS-CoV-2 infection model\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP complex characterization, in vitro ubiquitination assay, functional viral assay in cell culture and mouse model, single lab\",\n      \"pmids\": [\"38265236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PRPF19 mediates ubiquitin-dependent proteasomal degradation of the vitamin D receptor (VDR); loss of VDR reduces GPX4 transcription, promoting ferroptosis in renal tubular epithelial cells; berberine was identified as an inhibitor of PRPF19-VDR interaction via molecular docking and surface plasmon resonance.\",\n      \"method\": \"Proteomics, luciferase reporter assay, chromatin immunoprecipitation, co-immunoprecipitation, ubiquitination assay, SPR, VDR knockout mouse model\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with ubiquitination assay and in vivo knockout validation, SPR for inhibitor binding, single lab with multiple methods\",\n      \"pmids\": [\"40414879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Blom7α (a novel KH domain protein) directly interacts with SNEV(Prp19-Pso4), co-localizes and co-precipitates with splicing factors, and is present in affinity-purified spliceosomes; addition of Blom7α to HeLa nuclear extracts increases splicing activity; Blom7α overexpression alters both 5'- and 3'-splice site selection.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, affinity-purified spliceosome analysis, in vitro splicing assay, minigene splicing assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct interaction confirmed, spliceosome co-purification, in vitro and in vivo functional splicing assays, single lab\",\n      \"pmids\": [\"19641227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The 5' UTR of PRPF19 mRNA contains an upstream open reading frame (uORF) that is translated in human cells; inactivation of this uORF reduces cell viability.\",\n      \"method\": \"Ribosome profiling, uORF mutagenesis, cell viability assays\",\n      \"journal\": \"Doklady. Biochemistry and biophysics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, limited mechanistic detail in abstract; functional consequence is cell viability without pathway placement\",\n      \"pmids\": [\"38472668\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PRPF19 (PSO4/SNEV/hPrp19) is an essential, multifunctional nuclear E3 ubiquitin ligase that exists as a homotetramer in an autoinhibited state; stepwise assembly of SPF27, CDC5L, and PLRG1 onto this tetramer forms the NTC/Pso4 core complex and activates ubiquitin ligation. As a splicing factor, PRPF19 oligomerization is required for spliceosome assembly and stability, and it regulates alternative splicing (e.g., MDM4, TPT1). In the DNA damage response, the NTC complex interacts with RPA and its E3 ligase activity—together with BCAS2's RPA1-binding—is required for ATRIP recruitment, CHK1 activation, and RPA2 phosphorylation, positioning PRPF19 as an activator of ATR signaling; it also participates in ICL repair (via WRN helicase recruitment through Cdc5L), homologous recombination, and replication fork restart. ATM phosphorylates PRPF19 at S149 in response to DNA damage to modulate apoptosis resistance. Beyond these core functions, PRPF19 ubiquitinates diverse substrates (MYL9 via K63 linkage for stability; VDR, SARS-CoV-2 ORF6, and polyQ-expanded ATXN3 for degradation) and escorts substrates to the proteasome via direct binding to the PSMB4/β7 subunit of the 20S proteasome.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PRPF19 (PSO4/SNEV/hPrp19) is an essential, evolutionarily conserved nuclear protein that couples pre-mRNA splicing to genome maintenance, and its loss is lethal in yeast and arrests early mouse development at the inner cell mass stage [#0, #6]. Structurally, PRPF19 assembles into a homotetramer whose elongated coiled coils form an assembly axis for the NTC/Pso4 core complex; the tetramer is autoinhibited and catalytically silent as an E3 ubiquitin ligase until stepwise loading of SPF27, CDC5L, and PLRG1 activates ubiquitin ligation, with PLRG1–Prp19 communication relieving autoinhibition [#13, #3]. As a splicing factor, its self-interaction (mapped to residues 56–74) is required for spliceosome assembly and stability, and it promotes the PRPF3–PRPF8 interaction within the U4/U6.U5 tri-snRNP to control alternative splicing such as the MDM4 isoform switch that gates p53-p21–dependent senescence [#4, #14]. In the DNA damage response, the Pso4 complex binds RPA and, together with BCAS2's RPA1-binding and PRPF19's E3 activity, drives ATRIP recruitment, CHK1 activation, and RPA2 phosphorylation, while supporting interstrand crosslink repair through Cdc5L-mediated WRN recruitment, homologous recombination, replication fork restart, and recruitment of Metnase to double-strand breaks [#11, #2, #12, #7]. PRPF19 also functions broadly as a substrate-selective ubiquitin ligase—escorting substrates to the 20S proteasome via direct PSMB4/β7 binding and ubiquitinating diverse targets including expanded ataxin-3, viral proteins, MYL9, and VDR [#3, #15, #17, #19].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established that a single essential gene underlies both splicing and DNA repair, defining PRPF19's dual functional identity from the outset.\",\n      \"evidence\": \"Genetic complementation and gene disruption in S. cerevisiae showing PSO4/PRP19 allelism and haploid lethality\",\n      \"pmids\": [\"8918805\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not resolve whether splicing and repair roles are mechanistically coupled or separable\", \"No molecular activity assigned to the protein\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showed the human protein directly engages DNA and protects against breaks, providing the first biochemical link to genome integrity.\",\n      \"evidence\": \"In vitro DNA-binding assays and Co-IP with TdT, plus siRNA knockdown with γ-H2AX/comet readout in lymphoid cells\",\n      \"pmids\": [\"12960389\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"dsDNA binding is sequence-nonspecific, leaving target selectivity unexplained\", \"Mechanism linking DNA binding to break suppression undefined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Resolved that PRPF19 is an E3 ubiquitin ligase that escorts substrates to the proteasome and that its oligomerization is structurally required for spliceosome assembly, unifying its catalytic and splicing roles.\",\n      \"evidence\": \"In vitro ubiquitination assay, PSMB4 Co-IP, yeast two-hybrid self-interaction mapping (aa 56–74), immunodepletion/rescue and peptide-inhibition splicing assays, plus reconstituted in vitro ICL processing with WRN\",\n      \"pmids\": [\"15660529\", \"16332694\", \"16223718\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological ubiquitination substrates not yet identified in these studies\", \"How proteasome escort relates to splicing function unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined PRPF19 as functionally non-redundant in vivo and showed DNA damage remodels its core complex, hinting at a damage-responsive conformational switch.\",\n      \"evidence\": \"Mouse knockout with blastocyst outgrowth/MEF proliferation assays; non-reducing SDS-PAGE and chromatin fractionation after DNA damage\",\n      \"pmids\": [\"17283042\", \"17276391\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The ubiquitylated damage-induced species is biochemically inferred, not structurally proven\", \"Developmental phenotype not assigned to splicing vs repair function\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated PRPF19 acts as a DNA-binding recruitment platform that targets repair effectors to break sites, extending its role beyond catalysis.\",\n      \"evidence\": \"Co-IP, IR-induced co-localization, siRNA depletion, and end-joining assays with Metnase; EMSA and competitive binding showing PRPF19 controls Metnase DNA selectivity\",\n      \"pmids\": [\"18263876\", \"20416268\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab characterization of the Metnase axis\", \"Generality across repair pathways not established\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified ATM-dependent S149 phosphorylation as a post-translational switch linking PRPF19 to stress-resistance and lifespan, placing it downstream of damage signaling.\",\n      \"evidence\": \"MS phosphosite mapping, ATM inhibitor treatment, and S149A mutant apoptosis assays under oxidative stress\",\n      \"pmids\": [\"22529335\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Downstream effectors of S149 phosphorylation unknown\", \"Single-lab finding without structural context\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Positioned PRPF19 as an upstream activator of ATR signaling and a regulator of replication-coupled repair, defining its mechanistic role in the DNA damage response.\",\n      \"evidence\": \"Co-IP and co-localization with RPA, siRNA depletion with ATRIP recruitment/CHK1/RPA2 readouts and E3 mutant analysis; BrdU, HR reporter, PARP-inhibitor sensitivity and BRCA1 Western assays\",\n      \"pmids\": [\"24443570\", \"24675077\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ubiquitination substrate driving ATR activation not identified\", \"Mechanism of BRCA1 level regulation undefined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Provided the structural basis for activity by showing the Prp19 tetramer is autoinhibited and activated only by stepwise NTC assembly, explaining how E3 activity is gated.\",\n      \"evidence\": \"X-ray crystallography, mutagenesis, crosslinking MS, and stepwise in vitro reconstitution of E3 activity with SPF27/CDC5L/PLRG1\",\n      \"pmids\": [\"29547724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How DNA damage signals release autoinhibition in vivo not addressed\", \"Substrate engagement geometry of the active complex unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected PRPF19's splicing activity to a defined biological output by showing it controls the MDM4 isoform switch governing p53-p21 senescence, and broadened its substrate repertoire to neurodegeneration.\",\n      \"evidence\": \"siRNA/RNA-seq/RT-PCR splicing analysis with PRPF3/PRPF8 Co-IP; ubiquitination and proteasome assays for ATXN3-polyQ in Drosophila and mammalian cells with Exo70 antagonism\",\n      \"pmids\": [\"34144037\", \"33542212\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether MDM4 regulation requires E3 activity vs splicing alone unclear\", \"Nuclear-localization requirement for ATXN3 clearance not mechanistically dissected\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established PRPF19 as a substrate-recognition receptor within a CRL4B complex and an antiviral/disease-relevant E3 ligase across multiple substrates.\",\n      \"evidence\": \"Co-IP/pulldown, ubiquitination, and viral replication assays for PEDV-N (via MARCH8/NDP52), SARS-CoV-2 ORF6 (via CUL4B/DDB1/RBX1), MYL9 (K63-linked), and VDR with knockout/SPR validation\",\n      \"pmids\": [\"36541804\", \"38265236\", \"37031206\", \"40414879\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Each substrate characterized by a single lab\", \"How PRPF19 selects among diverse substrates and ligase partners is unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PRPF19's splicing, DNA-repair, and substrate-degradation activities are coordinately regulated and selectively deployed within a single autoinhibited scaffold remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking conformational state to substrate/pathway choice\", \"Determinants of substrate selectivity for the E3 activity undefined\", \"In vivo trigger releasing autoinhibition during DNA damage unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [3, 13, 11, 15, 17, 18, 19]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 13, 17, 18, 19]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [1, 8]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5, 15]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [5, 11, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [4, 14, 20]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [2, 11, 12, 7]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [3, 13, 15, 18, 19]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [11, 10]}\n    ],\n    \"complexes\": [\"NTC/Pso4 complex\", \"CRL4B E3 ligase complex\", \"spliceosome\"],\n    \"partners\": [\"CDC5L\", \"PLRG1\", \"BCAS2\", \"RPA1\", \"PSMB4\", \"WRN\", \"SETMAR\", \"CUL4B\"],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}