{"gene":"CDC40","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1998,"finding":"Human PRP17 (hPRP17/CDC40) is required specifically for the second catalytic step of pre-mRNA splicing. Immunodepletion of hPRP17 from splicing extracts blocks step II, which is rescued by recombinant hPRP17. Both hPRP16 and hPRP17 associate with the spliceosome late in the splicing pathway, at a stage prior to 3' splice site recognition.","method":"Immunodepletion from splicing extracts, rescue with recombinant protein, spliceosome association assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with immunodepletion and recombinant rescue, rigorous mechanistic controls","pmids":["9524131"],"is_preprint":false},{"year":1998,"finding":"hPRP17 (CDC40 human homolog) contains WD repeats and its C-terminal two-thirds (including WD repeats) are sufficient to complement both cell cycle and splicing defects of a yeast prp17 mutant. The yeast and chimeric proteins co-precipitate the intron-exon 2 lariat intermediate and the intron lariat product, demonstrating spliceosome association during catalysis.","method":"Complementation of yeast prp17 mutant, co-immunoprecipitation of splicing intermediates","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1–2 — functional complementation across species combined with co-IP of splicing intermediates","pmids":["9769104"],"is_preprint":false},{"year":1985,"finding":"CDC40 (yeast) has a role in DNA repair; cdc40 mutants are extremely sensitive to methyl methanesulfonate (MMS) at the restrictive temperature, suggesting the CDC40 gene product protects or holds together DNA during early stages of repair. The CDC40 gene was cloned and mapped to chromosome IV of S. cerevisiae.","method":"MMS sensitivity assay of cdc40 temperature-sensitive mutants, gene cloning and genetic mapping","journal":"Current genetics","confidence":"Medium","confidence_rationale":"Tier 3 — phenotypic characterization of mutants, no direct biochemical mechanism established","pmids":["3916722"],"is_preprint":false},{"year":1986,"finding":"Epistasis analysis shows that rad6-1 is epistatic to cdc40-1 for UV and MMS sensitivity, placing CDC40 in the RAD6 DNA repair pathway. rad50-1 is epistatic to cdc40-1 for MMS sensitivity in G1 but not in logarithmic cultures. cdc40-1 mutants are defective in UV-induced mutagenesis at the restrictive temperature.","method":"Double-mutant epistasis analysis, UV/MMS survival assays","journal":"Mutation research","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with multiple repair pathway mutants, pathway placement established","pmids":["3523226"],"is_preprint":false},{"year":1996,"finding":"Temperature-sensitive missense mutations in PRP17 map to the N-terminal nonconserved region of the protein, not to the WD (beta-transducin) repeat domain. The N-terminal domain mediates synthetic lethality with PRP16 and PRP18 mutations and shows allele-specific interactions with U5 snRNA (snr7 mutations), indicating this domain interacts functionally with other second-step factors and U5 snRNA.","method":"In vitro mutagenesis, genetic interaction analysis, synthetic lethality screens","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 — domain mapping by mutagenesis combined with genetic interaction screens","pmids":["8722761"],"is_preprint":false},{"year":2000,"finding":"PRP8 and PRP17/CDC40 have extensive genetic interactions: specific PRP8 mutations suppress the temperature-sensitive growth and splicing defects of prp17 null mutants, and suppress 3' splice site mutations. Conversely, other PRP8 alleles are synthetically lethal with prp17 absence. This supports a model where Prp8 and Prp17 interact functionally during the second catalytic step, particularly at the 3' splice site recognition stage.","method":"Genetic suppressor analysis, synthetic lethality screens, ACT1-CUP1 splicing reporter assay","journal":"Genetics","confidence":"High","confidence_rationale":"Tier 2 — bidirectional genetic interactions with multiple alleles and functional splicing reporter validation","pmids":["10628969"],"is_preprint":false},{"year":2001,"finding":"SKY1 (a SRPK-family kinase) genetically interacts with PRP17/SLU4 in 3' splice site recognition. Deletion of SKY1 is synthetically lethal with all prp17 mutants tested and suppresses 3'AG mutations in splicing reporters, suggesting that Sky1p-mediated phosphorylation regulates the 3' AG recognition step in which Prp17 participates.","method":"Synthetic lethality analysis, ACT1-CUP1 splicing reporter assay","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with functional splicing reporter readout","pmids":["11565750"],"is_preprint":false},{"year":2003,"finding":"PRP17/CDC40 is required for G1/S and G2/M cell cycle transitions. In prp17-null cells, splicing of TUB1 and TUB3 (alpha-tubulin) pre-mRNAs is specifically deficient; reduced alpha-tubulin protein underlies benomyl sensitivity and G2/M arrest. Genomic replacement with an intronless TUB1 gene relieves benomyl sensitivity, demonstrating that CDC40 controls mitosis through splicing of intron-containing tubulin genes.","method":"Cell cycle arrest/release experiments, in vitro splicing assays, intronless gene replacement, RT-PCR","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro splicing, in vivo genetic rescue with intronless gene, multiple orthogonal methods","pmids":["12711678"],"is_preprint":false},{"year":2004,"finding":"CDC40/PRP17 controls cell cycle progression through splicing of the ANC1 pre-mRNA. Deletion of the ANC1 intron relieves G2/M arrest and temperature sensitivity of cdc40 mutants. Point mutation analysis of the ANC1 intron identified specific residues required for Cdc40p-dependent splicing, revealing a mechanism where cell cycle regulation depends on differential splicing of a subset of introns by specific splicing factors.","method":"Intron deletion rescue genetics, point mutation analysis of intron sequences, temperature-sensitivity assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 — genetic rescue with intron deletion and point mutation analysis providing mechanistic dissection","pmids":["15133121"],"is_preprint":false},{"year":2004,"finding":"Genome-wide splicing microarray analysis shows that Prp17 is preferentially required for splicing of introns longer than 200 nt and is dispensable for introns with ≤13-nt spacing between branch point and 3' splice site. In vitro splicing with substrates of varying branch-to-3'SS distances confirmed these differential dependencies. In S. pombe, whose genome contains predominantly short introns, SpPrp17 is non-essential, linking the functional importance of this factor to intron architecture.","method":"Splicing-sensitive DNA microarrays, in vitro splicing assays with defined substrates, comparative genetics","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — genome-wide analysis validated by in vitro splicing with mechanistic substrate variation","pmids":["15452114"],"is_preprint":false},{"year":2006,"finding":"CDC40 (PRP17) is required for the G1/S transition (START) in addition to G2/M. Overexpression screening identified suppressors (chaperones, translation initiation factors, glycolytic enzymes) that relieve the G1/S transition delay of cdc40 cells. Enhanced temperature sensitivity of cdc40 combined with cln2Δ (G1 cyclin deletion) further supports a role for Cdc40p at START.","method":"cDNA overexpression suppressor screen, genetic interaction with cln2Δ, cell cycle timing assays","journal":"Current genetics","confidence":"Medium","confidence_rationale":"Tier 3 — genetic suppressor screen and double-mutant analysis, mechanism not fully resolved","pmids":["17171376"],"is_preprint":false},{"year":2008,"finding":"Prp17 interacts with U2, U5, and U6 snRNPs (but is not a core component of any single snRNP) as shown by co-immunoprecipitation of snRNAs. Prp17 joins the spliceosome at the pre-catalytic A1 complex (after U4 dissociation) and remains through catalytic and post-splicing complexes containing the lariat intron. In prp17Δ extracts, stalled spliceosomes are compromised for the Prp16 helicase-triggered conformational switch required for step II.","method":"Co-immunoprecipitation with epitope-tagged Prp17, snRNA analysis, in vitro spliceosome assembly on actin pre-mRNA","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal biochemical approaches, functional consequence of absence characterized","pmids":["18691155"],"is_preprint":false},{"year":2020,"finding":"Biallelic loss-of-function mutations in PRP17 (CDC40) cause autosomal-recessive pontocerebellar hypoplasia with microcephaly (PCHM) in humans. PPIL1 and PRP17 form an active isomerase-substrate interaction, but isomerase activity of PPIL1 is not critical for function. Loss of PRP17 affects splicing integrity, predominantly affecting short, high-GC-content introns and genes involved in brain disorders.","method":"Human genetics (biallelic mutations in patients), mouse knockouts (embryonic lethal), knockin patient mutation mice (neuron-specific apoptosis), splicing analysis","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 — human genetics combined with mouse knockout/knockin models and splicing integrity analysis","pmids":["33220177"],"is_preprint":false},{"year":2016,"finding":"In hepatocellular carcinoma cells, CDC40 is upregulated by HBx-induced miR-1269b (miR-1269b paradoxically increases CDC40 protein despite being a miRNA). CDC40 overexpression increases cell cycle progression, cell proliferation, and migration in HCC cells.","method":"Western blot, colony formation, flow cytometry, cell migration assays, rescue experiments","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 3 — cell-based phenotypic assays with rescue experiment, but no direct molecular mechanism for CDC40 action characterized","pmids":["27349221"],"is_preprint":false},{"year":2025,"finding":"CDC40 knockdown in lung cancer cell lines induces cell cycle defects, growth inhibition, and apoptosis. Mechanistically, CDC40 loss causes retention of the first intron of CDCA5 pre-mRNA, leading to increased unspliced CDCA5 transcript and decreased CDCA5 protein expression. Protein-protein interaction analysis identifies spliceosome components as the main CDC40 binding partners in human cells.","method":"siRNA knockdown, global transcriptomics and splicing analysis, RT-PCR validation of CDCA5 intron retention, co-immunoprecipitation/proteomics for interaction partners","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2–3 — functional knockdown with specific splicing phenotype and interactome characterization, single study","pmids":["39747150"],"is_preprint":false},{"year":2024,"finding":"Among 18 spliceosome components analyzed, CDC40 depletion (along with AQR, SF3B1, SF3B4) may have a more direct role in nonsense-mediated mRNA decay (NMD) beyond indirect effects of general splicing perturbation, as suggested by transcriptome-wide analysis of NMD-targeted mRNA isoform upregulation.","method":"Bioinformatic analysis of publicly available RNA-seq datasets from cells depleted of spliceosome components","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 4 — computational/bioinformatic analysis of existing datasets, no direct experimental validation of CDC40-NMD mechanism","pmids":[],"is_preprint":true}],"current_model":"CDC40/PRP17 is a WD-repeat spliceosomal protein that joins the spliceosome at the pre-catalytic A1 complex (after U4 dissociation), interacts with U2, U5, and U6 snRNPs, facilitates the Prp16 helicase-driven conformational switch required for the second catalytic step of pre-mRNA splicing (particularly for introns >200 nt or with long branch-point-to-3'SS distances), and controls cell cycle progression at both G1/S and G2/M transitions by enabling efficient splicing of a subset of intron-containing cell cycle regulators including ANC1 and the alpha-tubulin genes TUB1/TUB3; in humans, loss-of-function mutations cause pontocerebellar hypoplasia with microcephaly, and CDC40 loss in cancer cells specifically induces CDCA5 intron retention and growth arrest."},"narrative":{"teleology":[{"year":1985,"claim":"Initial characterization of CDC40 in yeast revealed it as a cell-cycle gene with an unexpected role in DNA damage tolerance, linking it to the RAD6 repair pathway, though the molecular function was unknown.","evidence":"MMS/UV sensitivity assays of cdc40 temperature-sensitive mutants and epistasis analysis with rad6 and rad50 in S. cerevisiae","pmids":["3916722","3523226"],"confidence":"Medium","gaps":["DNA repair phenotype likely indirect via splicing of repair gene transcripts — not resolved at this stage","No biochemical activity identified"]},{"year":1996,"claim":"Domain mapping established that the N-terminal region of PRP17, rather than its WD repeats, mediates functional interactions with other second-step splicing factors (PRP16, PRP18) and U5 snRNA, positioning PRP17 within the catalytic core of the spliceosome.","evidence":"Temperature-sensitive missense mutagenesis, synthetic lethality screens, and allele-specific interactions with snr7 in yeast","pmids":["8722761"],"confidence":"Medium","gaps":["Physical basis of N-terminal domain interactions unresolved","No structural information for PRP17 in spliceosome context"]},{"year":1998,"claim":"Biochemical reconstitution demonstrated that CDC40/PRP17 is specifically required for the second catalytic step of pre-mRNA splicing, resolving the molecular basis of its spliceosome function and showing conservation from yeast to human.","evidence":"Immunodepletion of hPRP17 from HeLa splicing extracts blocks step II, rescued by recombinant protein; cross-species complementation of yeast prp17 mutant by human C-terminal WD-repeat domain","pmids":["9524131","9769104"],"confidence":"High","gaps":["Mechanism of step II promotion not yet distinguished from structural scaffolding versus catalytic roles","Spliceosome entry point unknown"]},{"year":2000,"claim":"Extensive bidirectional genetic interactions with PRP8 placed PRP17 at the 3' splice site recognition stage of step II, establishing that these two factors collaborate during exon ligation.","evidence":"Genetic suppressor analysis showing PRP8 mutations suppress prp17Δ splicing defects and 3'SS mutations; reciprocal synthetic lethality using ACT1-CUP1 splicing reporter","pmids":["10628969"],"confidence":"High","gaps":["Direct physical interaction between PRP8 and PRP17 not demonstrated","Structural basis of functional cooperation unknown"]},{"year":2003,"claim":"The long-standing cell cycle phenotype of cdc40 mutants was mechanistically explained: PRP17 controls G2/M progression by splicing alpha-tubulin pre-mRNAs, and cell cycle control of ANC1 splicing explains additional arrest phenotypes, demonstrating that a specific spliceosome factor governs cell division through differential intron processing.","evidence":"Intronless TUB1 gene replacement rescues benomyl sensitivity; ANC1 intron deletion rescues G2/M arrest and temperature sensitivity of cdc40 mutants; point mutation analysis of intron sequences","pmids":["12711678","15133121"],"confidence":"High","gaps":["Full inventory of CDC40-dependent introns in yeast not established","Why these particular introns require PRP17 while others do not — cis-element rules incomplete"]},{"year":2004,"claim":"Genome-wide splicing microarray analysis revealed that PRP17 is preferentially required for introns >200 nt or with long branch-point-to-3'SS distances, establishing the intron-architecture determinants of PRP17 dependency.","evidence":"Splicing-sensitive microarrays in yeast prp17Δ, validated by in vitro splicing with defined substrates; comparative genetics in S. pombe where short introns dominate and Prp17 is non-essential","pmids":["15452114"],"confidence":"High","gaps":["Molecular basis of distance-dependent PRP17 requirement unknown","Whether these rules fully apply to mammalian CDC40 not tested"]},{"year":2008,"claim":"Biochemical dissection defined the spliceosome entry point and mechanistic role: PRP17 joins at the A1 complex (post-U4 release), associates with U2/U5/U6 snRNPs, and is required for the Prp16-triggered conformational switch that enables step II catalysis.","evidence":"Co-immunoprecipitation of snRNAs with epitope-tagged Prp17, in vitro spliceosome assembly and stalling in prp17Δ extracts","pmids":["18691155"],"confidence":"High","gaps":["Whether PRP17 directly contacts RNA or acts solely through protein-protein interactions unresolved","Structural position within C*/P complex not determined"]},{"year":2020,"claim":"Human genetic studies established that biallelic CDC40 loss-of-function causes pontocerebellar hypoplasia with microcephaly, revealing an essential neurodevelopmental role, with splicing defects predominantly affecting short, high-GC-content introns in brain-disorder genes — a substrate preference distinct from the yeast long-intron rule.","evidence":"Patient mutation identification, embryonic-lethal mouse knockout, knockin patient-mutation mice showing neuron-specific apoptosis, transcriptome-wide splicing analysis","pmids":["33220177"],"confidence":"High","gaps":["Discrepancy between yeast (long introns affected) and human (short high-GC introns affected) substrate rules not reconciled","Neuron-specific vulnerability mechanism unclear","PPIL1–PRP17 interaction demonstrated but isomerase activity dispensable — functional role of interaction unknown"]},{"year":2025,"claim":"In human cancer cells, CDC40 depletion was shown to cause intron retention in CDCA5, linking spliceosome function to a specific cell-cycle regulator and identifying the CDC40 interactome as predominantly spliceosomal in human cells.","evidence":"siRNA knockdown in lung cancer lines, RNA-seq with RT-PCR validation of CDCA5 intron 1 retention, co-IP/mass spectrometry","pmids":["39747150"],"confidence":"Medium","gaps":["CDCA5 intron retention as the primary cause of growth arrest versus broader splicing disruption not fully distinguished","Single study, not independently replicated"]},{"year":null,"claim":"Key unresolved questions include the structural basis of CDC40/PRP17 action within the human spliceosome, the molecular rules governing which introns require CDC40 in mammalian cells, and the mechanistic basis of neuron-specific vulnerability to CDC40 loss.","evidence":"","pmids":[],"confidence":"High","gaps":["No high-resolution structure of PRP17 within human C*/P spliceosome complex","Cis-element determinants of CDC40 dependency in mammalian introns undefined","No mechanistic explanation for tissue-specific (neuronal) sensitivity"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1,11]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,11,12]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,1,9,11,12,14]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[7,8,10]}],"complexes":["Spliceosome (catalytic step II complex)"],"partners":["PRP8","PRP16","PRP18","PPIL1","SKY1"],"other_free_text":[]},"mechanistic_narrative":"CDC40 (PRP17) is a WD-repeat spliceosomal protein that functions specifically in the second catalytic step of pre-mRNA splicing, joining the spliceosome at the pre-catalytic A1 complex after U4 dissociation and facilitating the Prp16 helicase-driven conformational switch required for 3' splice site recognition [PMID:9524131, PMID:18691155]. It associates with U2, U5, and U6 snRNPs and cooperates genetically with PRP8, PRP16, PRP18, and U5 snRNA during exon ligation, with preferential requirement for introns longer than 200 nt or with extended branch-point-to-3' splice site distances [PMID:10628969, PMID:15452114]. CDC40 controls cell cycle progression at G1/S and G2/M by enabling efficient splicing of a subset of intron-containing cell cycle regulators including alpha-tubulin genes (TUB1/TUB3) and ANC1, and in human cells its loss causes CDCA5 intron retention and growth arrest [PMID:12711678, PMID:15133121, PMID:39747150]. Biallelic loss-of-function mutations in CDC40 cause autosomal-recessive pontocerebellar hypoplasia with microcephaly in humans [PMID:33220177]."},"prefetch_data":{"uniprot":{"accession":"O60508","full_name":"Pre-mRNA-processing factor 17","aliases":["Cell division cycle 40 homolog","EH-binding protein 3","Ehb3","PRP17 homolog","hPRP17"],"length_aa":579,"mass_kda":65.5,"function":"Required for pre-mRNA splicing as component of the activated spliceosome (PubMed:33220177). Plays an important role in embryonic brain development; this function does not require proline isomerization (PubMed:33220177)","subcellular_location":"Nucleus; Nucleus speckle","url":"https://www.uniprot.org/uniprotkb/O60508/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CDC40","classification":"Common Essential","n_dependent_lines":1067,"n_total_lines":1208,"dependency_fraction":0.8832781456953642},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CAPZB","stoichiometry":0.2},{"gene":"CPSF6","stoichiometry":0.2},{"gene":"DDX39B","stoichiometry":0.2},{"gene":"RBM39","stoichiometry":0.2},{"gene":"RTCB","stoichiometry":0.2},{"gene":"SF3A1","stoichiometry":0.2},{"gene":"SF3A2","stoichiometry":0.2},{"gene":"SF3B1","stoichiometry":0.2},{"gene":"SNRPA","stoichiometry":0.2},{"gene":"SNRPB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CDC40","total_profiled":1310},"omim":[{"mim_id":"619302","title":"PONTOCEREBELLAR HYPOPLASIA, TYPE 15; PCH15","url":"https://www.omim.org/entry/619302"},{"mim_id":"618059","title":"WD REPEAT-CONTAINING PROTEIN 25; WDR25","url":"https://www.omim.org/entry/618059"},{"mim_id":"607596","title":"PONTOCEREBELLAR HYPOPLASIA, TYPE 1A; PCH1A","url":"https://www.omim.org/entry/607596"},{"mim_id":"605585","title":"CELL DIVISION CYCLE 40; CDC40","url":"https://www.omim.org/entry/605585"},{"mim_id":"601301","title":"PEPTIDYL-PROLYL ISOMERASE-LIKE 1; PPIL1","url":"https://www.omim.org/entry/601301"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CDC40"},"hgnc":{"alias_symbol":["PRP17","EHB3","PRPF17","FLJ10564"],"prev_symbol":[]},"alphafold":{"accession":"O60508","domains":[{"cath_id":"-","chopping":"121-161","consensus_level":"high","plddt":89.3034,"start":121,"end":161},{"cath_id":"2.130.10.10","chopping":"239-577","consensus_level":"medium","plddt":93.9001,"start":239,"end":577}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O60508","model_url":"https://alphafold.ebi.ac.uk/files/AF-O60508-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O60508-F1-predicted_aligned_error_v6.png","plddt_mean":85.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CDC40","jax_strain_url":"https://www.jax.org/strain/search?query=CDC40"},"sequence":{"accession":"O60508","fasta_url":"https://rest.uniprot.org/uniprotkb/O60508.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O60508/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O60508"}},"corpus_meta":[{"pmid":"9524131","id":"PMC_9524131","title":"Human homologs of yeast prp16 and prp17 reveal conservation of the mechanism for catalytic step II of pre-mRNA splicing.","date":"1998","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/9524131","citation_count":64,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"20419786","id":"PMC_20419786","title":"PRP-17 and the pre-mRNA splicing pathway are preferentially required for the proliferation versus meiotic development decision and germline sex determination in Caenorhabditis elegans.","date":"2010","source":"Developmental dynamics : an official publication of the American Association of Anatomists","url":"https://pubmed.ncbi.nlm.nih.gov/20419786","citation_count":63,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"33220177","id":"PMC_33220177","title":"Mutations in Spliceosomal Genes PPIL1 and PRP17 Cause Neurodegenerative Pontocerebellar Hypoplasia with 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splicing of the ANC1 gene.","date":"2004","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/15133121","citation_count":37,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"27349221","id":"PMC_27349221","title":"HBx-induced MiR-1269b in NF-κB dependent manner upregulates cell division cycle 40 homolog (CDC40) to promote proliferation and migration in hepatoma cells.","date":"2016","source":"Journal of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/27349221","citation_count":33,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"3916722","id":"PMC_3916722","title":"Cloning and mapping of CDC40, a Saccharomyces cerevisiae gene with a role in DNA repair.","date":"1985","source":"Current genetics","url":"https://pubmed.ncbi.nlm.nih.gov/3916722","citation_count":33,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"11565750","id":"PMC_11565750","title":"Evidence for a role of Sky1p-mediated phosphorylation in 3' splice site 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[\"9524131\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"A yeast-human chimera carrying the C-terminal two-thirds of hPRP17 (containing the WD repeats) complements yeast prp17 cell cycle and splicing defects; yeast Prp17p and the chimeric protein co-precipitate the intron-exon2 lariat intermediate and intron lariat product, demonstrating spliceosome association.\",\n      \"method\": \"Complementation of yeast prp17 mutant, co-immunoprecipitation of splicing intermediates\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal functional rescue plus co-IP of splicing intermediates, two independent labs\",\n      \"pmids\": [\"9769104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1985,\n      \"finding\": \"The CDC40 gene product has a role in DNA repair: cdc40 mutants are hypersensitive to MMS at restrictive temperature, and the CDC40 gene was cloned and mapped to chromosome IV.\",\n      \"method\": \"MMS sensitivity assay, gene cloning and chromosomal mapping\",\n      \"journal\": \"Current genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean loss-of-function with specific phenotypic readout, but single lab\",\n      \"pmids\": [\"3916722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1986,\n      \"finding\": \"Double-mutant epistasis shows rad6-1 is epistatic to cdc40-1 for UV and MMS sensitivity, and rad50-1 is epistatic to cdc40-1 for MMS sensitivity in G1, placing CDC40 in a DNA repair pathway involving RAD6 and RAD50.\",\n      \"method\": \"Double-mutant epistasis analysis, survival assays after UV/MMS treatment\",\n      \"journal\": \"Mutation research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with defined pathway placement, single lab\",\n      \"pmids\": [\"3523226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Missense mutations in three temperature-sensitive prp17 alleles map to the N-terminal non-conserved region; the N-terminal region interacts functionally with Prp18p, Prp16p, and U5 snRNA (allele-specific synthetic lethality), while WD repeats are required but missense mutations in most conserved WD residues lack phenotypic effects.\",\n      \"method\": \"In vitro mutagenesis, temperature-sensitive allele mapping, synthetic lethality analysis, allele-specific interactions\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic mutagenesis with genetic interaction mapping, single lab\",\n      \"pmids\": [\"8722761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Specific PRP8 mutations suppress both the temperature-sensitive growth and splicing defects of prp17/cdc40 null mutants and can suppress 3' splice site mutations; other PRP8 alleles are synthetically lethal with prp17 deletion, establishing genetic epistasis between Prp8 and Prp17 during the second catalytic step of splicing and 3' splice site recognition.\",\n      \"method\": \"Genetic suppressor analysis, ACT1-CUP1 splicing reporter system, synthetic lethality\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — extensive genetic epistasis with splicing reporter, replicated interactions\",\n      \"pmids\": [\"10628969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SKY1 (SRPK kinase) genetically interacts with PRP17/SLU4 in 3' splice site recognition: deletion of SKY1 is synthetically lethal with all prp17 mutants tested, and sky1 deletion suppresses 3'AG mutations in splicing reporters, suggesting phosphorylation regulates 3'AG recognition at the same step as Prp17.\",\n      \"method\": \"Synthetic lethality, ACT1-CUP1 splicing reporter, genetic epistasis\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic interaction with splicing reporter, single lab\",\n      \"pmids\": [\"11565750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Prp17/Cdc40p is required for efficient G1/S and G2/M cell cycle transitions in yeast; prp17 null cells have reduced alpha-tubulin protein due to inefficient splicing of TUB1 and TUB3 pre-mRNAs; intronless TUB1 replacement relieves benomyl sensitivity of prp17 mutants, demonstrating that Prp17 controls mitosis through splicing of tubulin transcripts.\",\n      \"method\": \"Arrest/release cell cycle experiments, in vitro splicing, intronless TUB1 genomic replacement, RT-PCR\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro splicing plus genetic rescue with intronless gene, direct mechanistic link\",\n      \"pmids\": [\"12711678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CDC40/PRP17 controls cell cycle progression through splicing of the ANC1 intron-containing pre-mRNA; deletion of the ANC1 intron relieves cdc40 temperature sensitivity and cell cycle arrest, and point mutations in the ANC1 intron identify residues critical for its Cdc40p-dependent splicing.\",\n      \"method\": \"Intron deletion rescue, point mutation analysis of intron, cell cycle arrest assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic rescue with defined intron deletion plus mutational dissection, single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"15133121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Genome-wide splicing microarray analysis reveals that Prp17 is preferentially required for splicing introns longer than 200 nt and is dispensable for introns with ≤13 nt spacing between branch point and 3' splice site; in vitro splicing with substrates of varying branch-point to 3'SS distances confirms this differential Prp17 dependency.\",\n      \"method\": \"Splicing-sensitive DNA microarrays, in vitro splicing with defined substrates\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — genome-wide analysis confirmed by in vitro splicing with defined substrates\",\n      \"pmids\": [\"15452114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"cdc40 mutants show a delayed G1/S transition; overexpression of cDNAs encoding chaperones, translation initiation factors, and glycolytic enzymes suppresses the HU/MMS sensitivity and G1/S delay, and enhanced temperature sensitivity with cln2Δ supports a role for Cdc40p at START (G1/S).\",\n      \"method\": \"cDNA overexpression suppressor screen, cell cycle synchronization, genetic interaction with cln2Δ\",\n      \"journal\": \"Current genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — genetic suppressor screen with cell cycle readout, single lab\",\n      \"pmids\": [\"17171376\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Prp17 co-immunoprecipitates with U2, U5, and U6 snRNPs but is not a core component of any single snRNP; it joins the spliceosome at the pre-catalytic A1 complex (after U4 dissociation), remains associated through catalytic steps and post-splicing complexes; prp17Δ extracts fail to undergo the Prp16 helicase-triggered conformational switch required for step II.\",\n      \"method\": \"Co-immunoprecipitation of snRNAs with epitope-tagged Prp17, in vitro spliceosome assembly on actin pre-mRNA, in vitro splicing kinetics\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal biochemical methods (co-IP, spliceosome assembly, conformational switch assay) in single rigorous study\",\n      \"pmids\": [\"18691155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"C. elegans PRP-17 (ortholog of PRP17/CDC40) functions downstream of GLP-1 Notch signaling to promote meiotic entry via the GLD-1 pathway and in female germline sex determination; PRP-17 can rescue temperature-sensitive lethality of yeast PRP17 mutants, confirming functional conservation.\",\n      \"method\": \"RNAi knockdown, genetic epistasis in C. elegans, yeast complementation\",\n      \"journal\": \"Developmental dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with pathway placement and cross-species complementation, single lab\",\n      \"pmids\": [\"20419786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In hepatoma cells, miR-1269b directly targets and upregulates CDC40; elevated CDC40 increases cell cycle progression, proliferation, and migration; rescue experiments demonstrate CDC40 mediates the malignant phenotype induced by miR-1269b.\",\n      \"method\": \"Luciferase reporter assay, Western blot, colony formation, flow cytometry, cell migration assay, rescue experiment\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — luciferase reporter confirms direct targeting, functional rescue confirms CDC40 as mediator, single lab\",\n      \"pmids\": [\"27349221\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PPIL1 and PRP17 form an active isomerase-substrate interaction in the spliceosome; loss of either protein affects splicing integrity predominantly at short, high-GC-content introns; isomerase activity of PPIL1 is not critical for its function (non-enzymatic role established by patient mutations), and mouse knockouts of either gene are embryonic lethal.\",\n      \"method\": \"Patient mutation analysis, knockin mouse model, mouse knockout, splicing assays, protein interaction studies\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods including knockin/KO mouse models and direct protein interaction with mutagenesis of enzymatic activity\",\n      \"pmids\": [\"33220177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CDC40 knockdown in lung cancer cells causes intron retention in CDCA5 pre-mRNA (specifically the first intron), leading to decreased CDCA5 protein expression; proteomic analysis identifies spliceosome components as CDC40's main binding partners; CDC40 KD induces cell cycle defects, growth inhibition, and apoptosis.\",\n      \"method\": \"siRNA knockdown, RNA-seq/splicing analysis, mass spectrometry protein-protein interaction, Western blot, flow cytometry\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNA-seq with specific substrate identification plus MS interactome, single lab\",\n      \"pmids\": [\"39747150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Depletion of CDC40 in human K562 and HepG2 cells upregulates natural NMD-targeted mRNA isoforms, suggesting CDC40 may have a direct role in nonsense-mediated mRNA decay beyond general splicing effects.\",\n      \"method\": \"RNA-seq analysis of publicly available CDC40-depletion datasets, bioinformatic NMD substrate analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — computational/bioinformatic analysis of existing datasets, no direct mechanistic experiment\",\n      \"pmids\": [\"bio_10.1101_2024.12.27.630533\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CDC40/PRP17 is a WD-repeat spliceosomal protein that joins the pre-catalytic spliceosome (after U4 snRNP dissociation) in association with U2, U5, and U6 snRNPs, facilitates the Prp16 helicase-triggered conformational switch required for the second catalytic step of pre-mRNA splicing, preferentially acts on introns longer than 200 nt with extended branch-point to 3'-splice-site distances, and controls cell cycle progression (G1/S via ANC1 and CLN1/CLB5, G2/M via TUB1/TUB3 alpha-tubulin splicing) by ensuring efficient splicing of a specific subset of intron-containing transcripts; it also forms a direct protein interaction with the prolyl isomerase PPIL1 within the spliceosome, and its loss in human cells causes splicing defects—including CDCA5 intron retention—that result in apoptosis and growth arrest.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"Human PRP17 (hPRP17/CDC40) is required specifically for the second catalytic step of pre-mRNA splicing. Immunodepletion of hPRP17 from splicing extracts blocks step II, which is rescued by recombinant hPRP17. Both hPRP16 and hPRP17 associate with the spliceosome late in the splicing pathway, at a stage prior to 3' splice site recognition.\",\n      \"method\": \"Immunodepletion from splicing extracts, rescue with recombinant protein, spliceosome association assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with immunodepletion and recombinant rescue, rigorous mechanistic controls\",\n      \"pmids\": [\"9524131\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"hPRP17 (CDC40 human homolog) contains WD repeats and its C-terminal two-thirds (including WD repeats) are sufficient to complement both cell cycle and splicing defects of a yeast prp17 mutant. The yeast and chimeric proteins co-precipitate the intron-exon 2 lariat intermediate and the intron lariat product, demonstrating spliceosome association during catalysis.\",\n      \"method\": \"Complementation of yeast prp17 mutant, co-immunoprecipitation of splicing intermediates\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — functional complementation across species combined with co-IP of splicing intermediates\",\n      \"pmids\": [\"9769104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1985,\n      \"finding\": \"CDC40 (yeast) has a role in DNA repair; cdc40 mutants are extremely sensitive to methyl methanesulfonate (MMS) at the restrictive temperature, suggesting the CDC40 gene product protects or holds together DNA during early stages of repair. The CDC40 gene was cloned and mapped to chromosome IV of S. cerevisiae.\",\n      \"method\": \"MMS sensitivity assay of cdc40 temperature-sensitive mutants, gene cloning and genetic mapping\",\n      \"journal\": \"Current genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — phenotypic characterization of mutants, no direct biochemical mechanism established\",\n      \"pmids\": [\"3916722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1986,\n      \"finding\": \"Epistasis analysis shows that rad6-1 is epistatic to cdc40-1 for UV and MMS sensitivity, placing CDC40 in the RAD6 DNA repair pathway. rad50-1 is epistatic to cdc40-1 for MMS sensitivity in G1 but not in logarithmic cultures. cdc40-1 mutants are defective in UV-induced mutagenesis at the restrictive temperature.\",\n      \"method\": \"Double-mutant epistasis analysis, UV/MMS survival assays\",\n      \"journal\": \"Mutation research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with multiple repair pathway mutants, pathway placement established\",\n      \"pmids\": [\"3523226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Temperature-sensitive missense mutations in PRP17 map to the N-terminal nonconserved region of the protein, not to the WD (beta-transducin) repeat domain. The N-terminal domain mediates synthetic lethality with PRP16 and PRP18 mutations and shows allele-specific interactions with U5 snRNA (snr7 mutations), indicating this domain interacts functionally with other second-step factors and U5 snRNA.\",\n      \"method\": \"In vitro mutagenesis, genetic interaction analysis, synthetic lethality screens\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain mapping by mutagenesis combined with genetic interaction screens\",\n      \"pmids\": [\"8722761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"PRP8 and PRP17/CDC40 have extensive genetic interactions: specific PRP8 mutations suppress the temperature-sensitive growth and splicing defects of prp17 null mutants, and suppress 3' splice site mutations. Conversely, other PRP8 alleles are synthetically lethal with prp17 absence. This supports a model where Prp8 and Prp17 interact functionally during the second catalytic step, particularly at the 3' splice site recognition stage.\",\n      \"method\": \"Genetic suppressor analysis, synthetic lethality screens, ACT1-CUP1 splicing reporter assay\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — bidirectional genetic interactions with multiple alleles and functional splicing reporter validation\",\n      \"pmids\": [\"10628969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SKY1 (a SRPK-family kinase) genetically interacts with PRP17/SLU4 in 3' splice site recognition. Deletion of SKY1 is synthetically lethal with all prp17 mutants tested and suppresses 3'AG mutations in splicing reporters, suggesting that Sky1p-mediated phosphorylation regulates the 3' AG recognition step in which Prp17 participates.\",\n      \"method\": \"Synthetic lethality analysis, ACT1-CUP1 splicing reporter assay\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with functional splicing reporter readout\",\n      \"pmids\": [\"11565750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PRP17/CDC40 is required for G1/S and G2/M cell cycle transitions. In prp17-null cells, splicing of TUB1 and TUB3 (alpha-tubulin) pre-mRNAs is specifically deficient; reduced alpha-tubulin protein underlies benomyl sensitivity and G2/M arrest. Genomic replacement with an intronless TUB1 gene relieves benomyl sensitivity, demonstrating that CDC40 controls mitosis through splicing of intron-containing tubulin genes.\",\n      \"method\": \"Cell cycle arrest/release experiments, in vitro splicing assays, intronless gene replacement, RT-PCR\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro splicing, in vivo genetic rescue with intronless gene, multiple orthogonal methods\",\n      \"pmids\": [\"12711678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CDC40/PRP17 controls cell cycle progression through splicing of the ANC1 pre-mRNA. Deletion of the ANC1 intron relieves G2/M arrest and temperature sensitivity of cdc40 mutants. Point mutation analysis of the ANC1 intron identified specific residues required for Cdc40p-dependent splicing, revealing a mechanism where cell cycle regulation depends on differential splicing of a subset of introns by specific splicing factors.\",\n      \"method\": \"Intron deletion rescue genetics, point mutation analysis of intron sequences, temperature-sensitivity assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic rescue with intron deletion and point mutation analysis providing mechanistic dissection\",\n      \"pmids\": [\"15133121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Genome-wide splicing microarray analysis shows that Prp17 is preferentially required for splicing of introns longer than 200 nt and is dispensable for introns with ≤13-nt spacing between branch point and 3' splice site. In vitro splicing with substrates of varying branch-to-3'SS distances confirmed these differential dependencies. In S. pombe, whose genome contains predominantly short introns, SpPrp17 is non-essential, linking the functional importance of this factor to intron architecture.\",\n      \"method\": \"Splicing-sensitive DNA microarrays, in vitro splicing assays with defined substrates, comparative genetics\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — genome-wide analysis validated by in vitro splicing with mechanistic substrate variation\",\n      \"pmids\": [\"15452114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CDC40 (PRP17) is required for the G1/S transition (START) in addition to G2/M. Overexpression screening identified suppressors (chaperones, translation initiation factors, glycolytic enzymes) that relieve the G1/S transition delay of cdc40 cells. Enhanced temperature sensitivity of cdc40 combined with cln2Δ (G1 cyclin deletion) further supports a role for Cdc40p at START.\",\n      \"method\": \"cDNA overexpression suppressor screen, genetic interaction with cln2Δ, cell cycle timing assays\",\n      \"journal\": \"Current genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — genetic suppressor screen and double-mutant analysis, mechanism not fully resolved\",\n      \"pmids\": [\"17171376\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Prp17 interacts with U2, U5, and U6 snRNPs (but is not a core component of any single snRNP) as shown by co-immunoprecipitation of snRNAs. Prp17 joins the spliceosome at the pre-catalytic A1 complex (after U4 dissociation) and remains through catalytic and post-splicing complexes containing the lariat intron. In prp17Δ extracts, stalled spliceosomes are compromised for the Prp16 helicase-triggered conformational switch required for step II.\",\n      \"method\": \"Co-immunoprecipitation with epitope-tagged Prp17, snRNA analysis, in vitro spliceosome assembly on actin pre-mRNA\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal biochemical approaches, functional consequence of absence characterized\",\n      \"pmids\": [\"18691155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Biallelic loss-of-function mutations in PRP17 (CDC40) cause autosomal-recessive pontocerebellar hypoplasia with microcephaly (PCHM) in humans. PPIL1 and PRP17 form an active isomerase-substrate interaction, but isomerase activity of PPIL1 is not critical for function. Loss of PRP17 affects splicing integrity, predominantly affecting short, high-GC-content introns and genes involved in brain disorders.\",\n      \"method\": \"Human genetics (biallelic mutations in patients), mouse knockouts (embryonic lethal), knockin patient mutation mice (neuron-specific apoptosis), splicing analysis\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — human genetics combined with mouse knockout/knockin models and splicing integrity analysis\",\n      \"pmids\": [\"33220177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In hepatocellular carcinoma cells, CDC40 is upregulated by HBx-induced miR-1269b (miR-1269b paradoxically increases CDC40 protein despite being a miRNA). CDC40 overexpression increases cell cycle progression, cell proliferation, and migration in HCC cells.\",\n      \"method\": \"Western blot, colony formation, flow cytometry, cell migration assays, rescue experiments\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — cell-based phenotypic assays with rescue experiment, but no direct molecular mechanism for CDC40 action characterized\",\n      \"pmids\": [\"27349221\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CDC40 knockdown in lung cancer cell lines induces cell cycle defects, growth inhibition, and apoptosis. Mechanistically, CDC40 loss causes retention of the first intron of CDCA5 pre-mRNA, leading to increased unspliced CDCA5 transcript and decreased CDCA5 protein expression. Protein-protein interaction analysis identifies spliceosome components as the main CDC40 binding partners in human cells.\",\n      \"method\": \"siRNA knockdown, global transcriptomics and splicing analysis, RT-PCR validation of CDCA5 intron retention, co-immunoprecipitation/proteomics for interaction partners\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — functional knockdown with specific splicing phenotype and interactome characterization, single study\",\n      \"pmids\": [\"39747150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Among 18 spliceosome components analyzed, CDC40 depletion (along with AQR, SF3B1, SF3B4) may have a more direct role in nonsense-mediated mRNA decay (NMD) beyond indirect effects of general splicing perturbation, as suggested by transcriptome-wide analysis of NMD-targeted mRNA isoform upregulation.\",\n      \"method\": \"Bioinformatic analysis of publicly available RNA-seq datasets from cells depleted of spliceosome components\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — computational/bioinformatic analysis of existing datasets, no direct experimental validation of CDC40-NMD mechanism\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CDC40/PRP17 is a WD-repeat spliceosomal protein that joins the spliceosome at the pre-catalytic A1 complex (after U4 dissociation), interacts with U2, U5, and U6 snRNPs, facilitates the Prp16 helicase-driven conformational switch required for the second catalytic step of pre-mRNA splicing (particularly for introns >200 nt or with long branch-point-to-3'SS distances), and controls cell cycle progression at both G1/S and G2/M transitions by enabling efficient splicing of a subset of intron-containing cell cycle regulators including ANC1 and the alpha-tubulin genes TUB1/TUB3; in humans, loss-of-function mutations cause pontocerebellar hypoplasia with microcephaly, and CDC40 loss in cancer cells specifically induces CDCA5 intron retention and growth arrest.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CDC40 (PRP17) is a WD-repeat spliceosomal protein that functions specifically in the second catalytic step of pre-mRNA splicing, facilitating 3' splice site recognition and the Prp16 helicase-triggered conformational switch required for exon ligation [PMID:9524131, PMID:18691155]. It joins the spliceosome after U4 snRNP dissociation and remains associated through catalytic and post-splicing complexes in partnership with U2, U5, and U6 snRNPs; it preferentially acts on introns longer than 200 nt with extended branch-point to 3' splice-site distances and forms a direct interaction with the prolyl isomerase PPIL1 within the spliceosome [PMID:18691155, PMID:15452114, PMID:33220177]. CDC40 controls cell cycle progression by ensuring efficient splicing of specific intron-containing transcripts: in yeast, loss of CDC40 impairs G2/M transition through defective TUB1/TUB3 alpha-tubulin pre-mRNA splicing and delays G1/S through ANC1 intron retention, while in human cells its depletion causes CDCA5 intron retention, growth arrest, and apoptosis [PMID:12711678, PMID:15133121, PMID:39747150]. Mouse knockout of CDC40 is embryonic lethal, and its functional conservation extends from yeast to C. elegans and humans [PMID:33220177, PMID:9769104, PMID:20419786].\",\n  \"teleology\": [\n    {\n      \"year\": 1985,\n      \"claim\": \"The initial identification of CDC40 as a cell-division-cycle gene revealed an unexpected connection to DNA damage sensitivity, establishing that its loss confers MMS hypersensitivity and linking it to genome integrity pathways.\",\n      \"evidence\": \"MMS sensitivity assays and gene cloning in S. cerevisiae\",\n      \"pmids\": [\"3916722\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; DNA repair role not independently confirmed\", \"Whether MMS sensitivity is a direct or indirect (splicing-dependent) effect was unknown\", \"No molecular function assigned\"]\n    },\n    {\n      \"year\": 1986,\n      \"claim\": \"Epistasis analysis placed CDC40 in DNA repair pathways involving RAD6 and RAD50, suggesting it operates at the intersection of cell cycle control and damage response.\",\n      \"evidence\": \"Double-mutant epistasis with rad6 and rad50 for UV/MMS survival in yeast\",\n      \"pmids\": [\"3523226\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Mechanism of CDC40 contribution to repair unknown\", \"Splicing function not yet discovered\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Systematic mutagenesis revealed that the N-terminal non-conserved region of Prp17 mediates functional interactions with second-step splicing factors Prp18, Prp16, and U5 snRNA, while the C-terminal WD repeats are structurally required but tolerant of individual missense mutations.\",\n      \"evidence\": \"Temperature-sensitive allele mapping and allele-specific synthetic lethality in yeast\",\n      \"pmids\": [\"8722761\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct physical interaction data\", \"Biochemical role in step II not yet demonstrated\", \"Single lab\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Reconstitution experiments established that human PRP17/CDC40 is specifically required for the second catalytic step of pre-mRNA splicing, and cross-species complementation demonstrated functional conservation of the WD-repeat domain from human to yeast.\",\n      \"evidence\": \"Immunodepletion/add-back in HeLa splicing extracts; yeast-human chimera complementation and co-IP of splicing intermediates\",\n      \"pmids\": [\"9524131\", \"9769104\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise timing of spliceosome association unresolved\", \"Whether PRP17 has catalytic or structural role unclear\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Genetic suppressor analysis revealed that PRP8 mutations can bypass the requirement for Prp17 in the second step, establishing that Prp17 functionally cooperates with the spliceosome's catalytic core component Prp8 during 3' splice site recognition.\",\n      \"evidence\": \"PRP8 suppressor/synthetic lethal analysis with prp17Δ using ACT1-CUP1 splicing reporter in yeast\",\n      \"pmids\": [\"10628969\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physical interaction between Prp8 and Prp17 not shown\", \"Mechanism of suppression (conformational vs. catalytic) unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The long-standing question of how a splicing factor controls cell division was resolved by showing that Prp17 is required for efficient splicing of TUB1/TUB3 alpha-tubulin pre-mRNAs, and that replacing TUB1 with an intronless version rescues the mitotic defect—demonstrating that CDC40's cell cycle role is an indirect consequence of substrate-specific splicing.\",\n      \"evidence\": \"Cell cycle arrest/release, in vitro splicing, intronless TUB1 genomic replacement, RT-PCR in yeast\",\n      \"pmids\": [\"12711678\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full spectrum of Prp17-dependent transcripts unknown\", \"G1/S mechanism not yet explained\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Two complementary studies extended the substrate-specific model: ANC1 intron deletion rescued cdc40 temperature sensitivity and cell cycle arrest (explaining the G1/S defect), and genome-wide microarray analysis showed Prp17 preferentially acts on introns >200 nt with extended branch-point to 3' splice-site distances.\",\n      \"evidence\": \"Intron deletion rescue and mutagenesis for ANC1; splicing-sensitive microarrays plus in vitro splicing with defined substrates for intron-length dependency\",\n      \"pmids\": [\"15133121\", \"15452114\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for intron-length selectivity unknown\", \"Whether selectivity is conserved in metazoans untested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Biochemical dissection of spliceosome assembly kinetics showed Prp17 is not a core snRNP subunit but joins after U4 dissociation and is essential for the Prp16-triggered conformational rearrangement required for step II, defining its precise mechanistic point of action.\",\n      \"evidence\": \"Co-IP of snRNAs with tagged Prp17, spliceosome assembly on actin pre-mRNA, conformational switch assay in yeast extracts\",\n      \"pmids\": [\"18691155\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structural data for Prp17 in the spliceosome\", \"Molecular contacts mediating the conformational switch unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Functional conservation was extended to metazoans by showing C. elegans PRP-17 acts downstream of Notch signaling to promote meiotic entry and can rescue yeast prp17 mutants, indicating the splicing-to-cell-fate link is evolutionarily ancient.\",\n      \"evidence\": \"RNAi knockdown and genetic epistasis in C. elegans germline, yeast complementation\",\n      \"pmids\": [\"20419786\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific splicing targets in C. elegans germline unknown\", \"Single lab\", \"Whether meiotic entry phenotype is direct or indirect through specific transcript splicing unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identification of the PPIL1–PRP17 interaction as an isomerase-substrate pair within the spliceosome established a direct physical partnership; loss of either protein causes splicing defects at short high-GC introns, and both are essential for embryonic development in mice.\",\n      \"evidence\": \"Patient mutation analysis, knockin/KO mouse models, splicing assays, protein interaction studies\",\n      \"pmids\": [\"33220177\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the PPIL1-PRP17 interaction not resolved at atomic level\", \"How prolyl isomerization of PRP17 affects spliceosome conformation unknown\", \"Specific neural transcripts affected not comprehensively catalogued\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"In human lung cancer cells, CDC40 depletion was shown to cause intron retention in CDCA5 (first intron), leading to reduced CDCA5 protein, cell cycle defects, and apoptosis—identifying a specific human splicing substrate through which CDC40 controls proliferation.\",\n      \"evidence\": \"siRNA knockdown, RNA-seq splicing analysis, mass spectrometry interactome, Western blot, flow cytometry in lung cancer cell lines\",\n      \"pmids\": [\"39747150\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; whether CDCA5 is the primary mediator or one of many affected transcripts remains unclear\", \"Genome-wide intron retention analysis in human cells incomplete\", \"No rescue with intronless CDCA5\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the high-resolution structural basis for PRP17's intron-length selectivity, the full catalogue of PRP17-dependent transcripts in mammalian cells, and whether the early DNA-repair phenotype in yeast reflects splicing of a specific repair-pathway transcript or an additional non-splicing function.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No cryo-EM or crystal structure of PRP17 in the human C* spliceosome complex\", \"Systematic identification of PRP17-dependent introns in human cells not performed\", \"The RAD6/RAD50 epistasis has never been revisited in light of the splicing function\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1, 11]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 11, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 9, 11, 14, 15]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [7, 8, 15]}\n    ],\n    \"complexes\": [\n      \"spliceosome (C complex / step II)\"\n    ],\n    \"partners\": [\n      \"PPIL1\",\n      \"PRP8\",\n      \"PRP16\",\n      \"PRP18\",\n      \"SKY1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"CDC40 (PRP17) is a WD-repeat spliceosomal protein that functions specifically in the second catalytic step of pre-mRNA splicing, joining the spliceosome at the pre-catalytic A1 complex after U4 dissociation and facilitating the Prp16 helicase-driven conformational switch required for 3' splice site recognition [PMID:9524131, PMID:18691155]. It associates with U2, U5, and U6 snRNPs and cooperates genetically with PRP8, PRP16, PRP18, and U5 snRNA during exon ligation, with preferential requirement for introns longer than 200 nt or with extended branch-point-to-3' splice site distances [PMID:10628969, PMID:15452114]. CDC40 controls cell cycle progression at G1/S and G2/M by enabling efficient splicing of a subset of intron-containing cell cycle regulators including alpha-tubulin genes (TUB1/TUB3) and ANC1, and in human cells its loss causes CDCA5 intron retention and growth arrest [PMID:12711678, PMID:15133121, PMID:39747150]. Biallelic loss-of-function mutations in CDC40 cause autosomal-recessive pontocerebellar hypoplasia with microcephaly in humans [PMID:33220177].\",\n  \"teleology\": [\n    {\n      \"year\": 1985,\n      \"claim\": \"Initial characterization of CDC40 in yeast revealed it as a cell-cycle gene with an unexpected role in DNA damage tolerance, linking it to the RAD6 repair pathway, though the molecular function was unknown.\",\n      \"evidence\": \"MMS/UV sensitivity assays of cdc40 temperature-sensitive mutants and epistasis analysis with rad6 and rad50 in S. cerevisiae\",\n      \"pmids\": [\"3916722\", \"3523226\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"DNA repair phenotype likely indirect via splicing of repair gene transcripts — not resolved at this stage\", \"No biochemical activity identified\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Domain mapping established that the N-terminal region of PRP17, rather than its WD repeats, mediates functional interactions with other second-step splicing factors (PRP16, PRP18) and U5 snRNA, positioning PRP17 within the catalytic core of the spliceosome.\",\n      \"evidence\": \"Temperature-sensitive missense mutagenesis, synthetic lethality screens, and allele-specific interactions with snr7 in yeast\",\n      \"pmids\": [\"8722761\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physical basis of N-terminal domain interactions unresolved\", \"No structural information for PRP17 in spliceosome context\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Biochemical reconstitution demonstrated that CDC40/PRP17 is specifically required for the second catalytic step of pre-mRNA splicing, resolving the molecular basis of its spliceosome function and showing conservation from yeast to human.\",\n      \"evidence\": \"Immunodepletion of hPRP17 from HeLa splicing extracts blocks step II, rescued by recombinant protein; cross-species complementation of yeast prp17 mutant by human C-terminal WD-repeat domain\",\n      \"pmids\": [\"9524131\", \"9769104\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of step II promotion not yet distinguished from structural scaffolding versus catalytic roles\", \"Spliceosome entry point unknown\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Extensive bidirectional genetic interactions with PRP8 placed PRP17 at the 3' splice site recognition stage of step II, establishing that these two factors collaborate during exon ligation.\",\n      \"evidence\": \"Genetic suppressor analysis showing PRP8 mutations suppress prp17Δ splicing defects and 3'SS mutations; reciprocal synthetic lethality using ACT1-CUP1 splicing reporter\",\n      \"pmids\": [\"10628969\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct physical interaction between PRP8 and PRP17 not demonstrated\", \"Structural basis of functional cooperation unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The long-standing cell cycle phenotype of cdc40 mutants was mechanistically explained: PRP17 controls G2/M progression by splicing alpha-tubulin pre-mRNAs, and cell cycle control of ANC1 splicing explains additional arrest phenotypes, demonstrating that a specific spliceosome factor governs cell division through differential intron processing.\",\n      \"evidence\": \"Intronless TUB1 gene replacement rescues benomyl sensitivity; ANC1 intron deletion rescues G2/M arrest and temperature sensitivity of cdc40 mutants; point mutation analysis of intron sequences\",\n      \"pmids\": [\"12711678\", \"15133121\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full inventory of CDC40-dependent introns in yeast not established\", \"Why these particular introns require PRP17 while others do not — cis-element rules incomplete\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Genome-wide splicing microarray analysis revealed that PRP17 is preferentially required for introns >200 nt or with long branch-point-to-3'SS distances, establishing the intron-architecture determinants of PRP17 dependency.\",\n      \"evidence\": \"Splicing-sensitive microarrays in yeast prp17Δ, validated by in vitro splicing with defined substrates; comparative genetics in S. pombe where short introns dominate and Prp17 is non-essential\",\n      \"pmids\": [\"15452114\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of distance-dependent PRP17 requirement unknown\", \"Whether these rules fully apply to mammalian CDC40 not tested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Biochemical dissection defined the spliceosome entry point and mechanistic role: PRP17 joins at the A1 complex (post-U4 release), associates with U2/U5/U6 snRNPs, and is required for the Prp16-triggered conformational switch that enables step II catalysis.\",\n      \"evidence\": \"Co-immunoprecipitation of snRNAs with epitope-tagged Prp17, in vitro spliceosome assembly and stalling in prp17Δ extracts\",\n      \"pmids\": [\"18691155\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PRP17 directly contacts RNA or acts solely through protein-protein interactions unresolved\", \"Structural position within C*/P complex not determined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Human genetic studies established that biallelic CDC40 loss-of-function causes pontocerebellar hypoplasia with microcephaly, revealing an essential neurodevelopmental role, with splicing defects predominantly affecting short, high-GC-content introns in brain-disorder genes — a substrate preference distinct from the yeast long-intron rule.\",\n      \"evidence\": \"Patient mutation identification, embryonic-lethal mouse knockout, knockin patient-mutation mice showing neuron-specific apoptosis, transcriptome-wide splicing analysis\",\n      \"pmids\": [\"33220177\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Discrepancy between yeast (long introns affected) and human (short high-GC introns affected) substrate rules not reconciled\", \"Neuron-specific vulnerability mechanism unclear\", \"PPIL1–PRP17 interaction demonstrated but isomerase activity dispensable — functional role of interaction unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"In human cancer cells, CDC40 depletion was shown to cause intron retention in CDCA5, linking spliceosome function to a specific cell-cycle regulator and identifying the CDC40 interactome as predominantly spliceosomal in human cells.\",\n      \"evidence\": \"siRNA knockdown in lung cancer lines, RNA-seq with RT-PCR validation of CDCA5 intron 1 retention, co-IP/mass spectrometry\",\n      \"pmids\": [\"39747150\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CDCA5 intron retention as the primary cause of growth arrest versus broader splicing disruption not fully distinguished\", \"Single study, not independently replicated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of CDC40/PRP17 action within the human spliceosome, the molecular rules governing which introns require CDC40 in mammalian cells, and the mechanistic basis of neuron-specific vulnerability to CDC40 loss.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure of PRP17 within human C*/P spliceosome complex\", \"Cis-element determinants of CDC40 dependency in mammalian introns undefined\", \"No mechanistic explanation for tissue-specific (neuronal) sensitivity\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 11, 12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 1, 9, 11, 12, 14]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [7, 8, 10]}\n    ],\n    \"complexes\": [\n      \"Spliceosome (catalytic step II complex)\"\n    ],\n    \"partners\": [\n      \"PRP8\",\n      \"PRP16\",\n      \"PRP18\",\n      \"PPIL1\",\n      \"SKY1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}