{"gene":"PRPF40A","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2011,"finding":"Polyglutamine-expanded huntingtin sequesters HYPA/FBP11 (PRPF40A) to the cytosolic location, significantly reducing pre-mRNA splicing efficiency. NMR structural analysis showed that the proline-rich region (PRR) of huntingtin cooperatively interacts with the tandem WW domains of HYPA through a domain chaperoning effect of WW1 on WW2.","method":"NMR structural analysis, pre-mRNA splicing efficiency assay, subcellular localization experiments","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — NMR structure plus functional splicing assay and localization data, single lab with two orthogonal methods","pmids":["21566141"],"is_preprint":false},{"year":2004,"finding":"FBP11 (PRPF40A) inhibits N-WASP-dependent microspike formation by trapping N-WASP in the nucleus. Endogenous FBP11 localizes to nuclear speckles, and co-expression with N-WASP causes co-localization in the nucleus, preventing N-WASP from acting in the cytoplasm.","method":"Transient overexpression, subcellular localization by fluorescence microscopy, actin microspike formation assay in COS7 cells","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — localization and phenotypic rescue experiments, single lab, two methods but no direct binding assay","pmids":["14697212"],"is_preprint":false},{"year":2005,"finding":"The nuclear matrix protein NAKAP interacts with HYPA/FBP11 (PRPF40A) via a proline-rich domain in NAKAP binding to a WW domain of HYPA. NAKAP and HypA co-localize within the nucleus, co-purify with the nuclear matrix, and are components of nuclear aggregates in Huntington's disease neurons.","method":"Yeast two-hybrid screen, in vitro binding assay, deletion mapping, subcellular fractionation, co-immunoprecipitation from human brain tissue","journal":"Neuromolecular medicine","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid plus in vitro binding plus fractionation, single lab but multiple orthogonal methods","pmids":["16391387"],"is_preprint":false},{"year":2006,"finding":"The first WW domain of FBP11/HYPA (PRPF40A) adopts an antiparallel triple-stranded beta-sheet structure. It binds proline-rich sequences containing PL, PP, and PR motifs via both XP and XP2 grooves, with the XP2 groove being functionally important for ligand recognition. Dissociation constant for a PL motif peptide was 248 ± 27 µM.","method":"NMR solution structure determination, ligand titration by 1H-15N HSQC NMR, GST pulldown binding assay","journal":"Proteins","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — NMR structure with binding characterization, single lab, two orthogonal methods","pmids":["16463264","16472085"],"is_preprint":false},{"year":2024,"finding":"PRPF40A tandem WW domains adopt a defined solution structure characterized by NMR/SAXS. Both WW domains cooperate to bind a high-affinity SF1 peptide, enabling tryptophan sandwiching by two proline residues. An N-terminal proline-rich motif of PRPF40A mediates intramolecular autoinhibitory interactions with the WW tandem, acting as a selectivity filter for high-affinity proline-rich bona fide binding partners (SF1 and SF3A1). This was validated by NMR, ITC, mutagenesis, and immunoprecipitation in cells.","method":"NMR spectroscopy, SAXS, ITC, mutagenesis, co-immunoprecipitation in cells","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — structure by NMR+SAXS, binding by ITC, mutagenesis, and cellular IP, multiple orthogonal methods in a single rigorous study","pmids":["38719828"],"is_preprint":false},{"year":2024,"finding":"PRPF40A acts predominantly as an activator of cassette exon inclusion, particularly of exons flanked by short GC-rich introns that tend to localize to nuclear speckles. PRPF40A knockdown in HL-60 cells caused increased cell death, decreased proliferation, and slight myeloid differentiation phenotype. Cell death but not proliferation was rescued by overexpression of paralog PRPF40B. PRPF40A functions in a network with SRRM2, SON, PCBP1/2, TRA2B, and SRSF2.","method":"shRNA knockdown, transcriptomic analysis (RNA-seq), overexpression rescue experiments, cell viability and proliferation assays","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with defined cellular phenotype and transcriptome-wide splicing analysis, single lab with multiple orthogonal readouts","pmids":["38943321"],"is_preprint":false},{"year":2024,"finding":"PRPF40A is globally required for microexon splicing in mouse neuroblastoma cells, with a graded dependence on exon size (greater dependence for smaller exons). PRPF40A co-regulates microexons with SRRM4. PRPF40A knockdown also causes increased productive splicing of its spliceosomal binding partner Luc7l by skipping a 'poison exon', suggesting homeostatic cross-regulation between coupled spliceosomal components.","method":"siRNA knockdown, RNA-seq, splicing analysis in N2A cells","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with defined splicing phenotypes and transcriptome-wide analysis, single lab, multiple exon classes examined","pmids":["39389624"],"is_preprint":false},{"year":2025,"finding":"In yeast, Prp40 (ortholog of PRPF40A) interacts with RNA polymerase II through multiple domains, and this interaction does not require the CTD of pol II. While yeast Prp40 is stably associated with U1 snRNP and is the primary mediator of U1 snRNP-pol II interaction, its human homologs PRPF40A and PRPF40B are alternative splicing factors not integral to U1 snRNP.","method":"Co-immunoprecipitation, domain deletion analysis, yeast genetic experiments","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab, primarily yeast data with limited direct evidence for human PRPF40A mechanism","pmids":["bio_10.1101_2025.08.28.672894"],"is_preprint":true}],"current_model":"PRPF40A is a pre-mRNA splicing factor whose tandem WW domains bind proline-rich motifs in spliceosome components (SF1 and SF3A1) to bridge 5' and 3' splice site recognition during early spliceosome assembly, with binding selectivity regulated by an intramolecular autoinhibitory proline-rich motif; it is specifically required for inclusion of cassette exons—especially microexons—in GC-rich regions, localizes to nuclear speckles, can trap binding partners such as N-WASP in the nucleus to regulate their cytoplasmic activity, and its sequestration by polyglutamine-expanded huntingtin impairs pre-mRNA splicing efficiency."},"narrative":{"mechanistic_narrative":"PRPF40A is a pre-mRNA splicing factor that uses tandem WW domains to read proline-rich motifs in early spliceosome components and promote alternative exon inclusion [PMID:38719828, PMID:38943321]. Structural work established that the first WW domain folds into an antiparallel triple-stranded beta-sheet that engages PL, PP, and PR motifs through its XP and XP2 grooves [PMID:16463264, PMID:16472085], and that the two WW domains cooperate to bind a high-affinity SF1 peptide by sandwiching a tryptophan between two proline residues; an N-terminal autoinhibitory proline-rich motif folds back onto the WW tandem to act as a selectivity filter favoring bona fide partners SF1 and SF3A1 [PMID:38719828]. Functionally, PRPF40A acts predominantly as an activator of cassette exon inclusion, with strong preference for exons flanked by short GC-rich introns and for microexons, where its dependence is graded by exon size, and it operates within a splicing-factor network including SRRM2, SON, SRRM4, PCBP1/2, TRA2B, and SRSF2 [PMID:38943321, PMID:39389624]. It localizes to nuclear speckles and can sequester binding partners such as N-WASP in the nucleus, thereby restraining their cytoplasmic activity [PMID:14697212]. Polyglutamine-expanded huntingtin cooperatively engages the WW tandem via its proline-rich region and mislocalizes PRPF40A to the cytosol, reducing pre-mRNA splicing efficiency [PMID:21566141].","teleology":[{"year":2004,"claim":"Established that PRPF40A is a nuclear-speckle protein capable of controlling a partner's cytoplasmic activity by nuclear retention, first linking it to spatial regulation of interacting proteins.","evidence":"Overexpression, fluorescence localization, and actin microspike assays in COS7 cells","pmids":["14697212"],"confidence":"Medium","gaps":["No direct binding interface defined for N-WASP","Relationship between N-WASP trapping and splicing function not addressed"]},{"year":2005,"claim":"Showed PRPF40A engages additional nuclear-matrix proline-rich partners (NAKAP) through its WW domains and is recruited into Huntington's disease nuclear aggregates, extending its interaction repertoire beyond splicing.","evidence":"Yeast two-hybrid, in vitro binding, deletion mapping, fractionation, and co-IP from human brain","pmids":["16391387"],"confidence":"Medium","gaps":["Functional consequence of NAKAP binding unknown","Does not connect aggregate localization to splicing defects"]},{"year":2006,"claim":"Resolved how the first WW domain recognizes ligands, defining the structural basis (XP and XP2 grooves) for proline-rich motif binding by PRPF40A.","evidence":"NMR solution structure, HSQC ligand titration, and GST pulldown","pmids":["16463264","16472085"],"confidence":"Medium","gaps":["Single WW domain in isolation; tandem cooperativity not addressed","Low affinity (248 µM) leaves physiological partner specificity open"]},{"year":2011,"claim":"Provided a disease mechanism: polyQ-huntingtin cooperatively binds the WW tandem and relocalizes PRPF40A to the cytosol, mechanistically linking its sequestration to impaired splicing.","evidence":"NMR structural analysis, pre-mRNA splicing efficiency assay, and subcellular localization","pmids":["21566141"],"confidence":"Medium","gaps":["Which endogenous splicing targets are affected not identified","In vivo neuronal relevance not established"]},{"year":2024,"claim":"Defined the tandem-WW solution architecture and an intramolecular autoinhibitory selectivity filter, explaining how PRPF40A achieves high-affinity, selective binding to spliceosomal partners SF1 and SF3A1.","evidence":"NMR, SAXS, ITC, mutagenesis, and co-IP in cells","pmids":["38719828"],"confidence":"High","gaps":["How autoinhibition is relieved in vivo not defined","Structural state within assembled spliceosome unknown"]},{"year":2024,"claim":"Demonstrated PRPF40A is a transcriptome-wide activator of cassette exon and microexon inclusion biased toward short GC-rich introns and nuclear speckles, embedding it in a defined splicing-regulatory network with cellular phenotypes on viability and differentiation.","evidence":"shRNA/siRNA knockdown, RNA-seq splicing analysis, rescue and viability assays in HL-60 and N2A cells","pmids":["38943321","39389624"],"confidence":"Medium","gaps":["Direct RNA or factor contacts driving exon selection not mapped","Mechanism of homeostatic Luc7l poison-exon cross-regulation unresolved"]},{"year":null,"claim":"Whether human PRPF40A makes direct, regulated contacts with RNA polymerase II or the U1 snRNP during co-transcriptional splicing remains undefined.","evidence":"Yeast Prp40 ortholog data indicate U1/pol II coupling, but human PRPF40A is reported as a non-integral alternative splicing factor","pmids":[],"confidence":"Low","gaps":["No direct evidence for human PRPF40A–pol II interaction","Extent of co-transcriptional vs post-transcriptional action in human cells unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[5,6]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[5,6]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,2]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[4,5,6]}],"complexes":[],"partners":["SF1","SF3A1","N-WASP","HTT","SRRM4","SRRM2","SON","TRA2B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75400","full_name":"Pre-mRNA-processing factor 40 homolog A","aliases":["Fas ligand-associated factor 1","Formin-binding protein 11","Formin-binding protein 3","Huntingtin yeast partner A","Huntingtin-interacting protein 10","HIP-10","Huntingtin-interacting protein A","Renal carcinoma antigen NY-REN-6"],"length_aa":957,"mass_kda":108.8,"function":"Binds to WASL/N-WASP and suppresses its translocation from the nucleus to the cytoplasm, thereby inhibiting its cytoplasmic function (By similarity). Plays a role in the regulation of cell morphology and cytoskeletal organization. Required in the control of cell shape and migration. May play a role in cytokinesis. May be involved in pre-mRNA splicing","subcellular_location":"Nucleus speckle; Nucleus matrix","url":"https://www.uniprot.org/uniprotkb/O75400/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/PRPF40A","classification":"Common Essential","n_dependent_lines":1177,"n_total_lines":1208,"dependency_fraction":0.9743377483443708},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"RBM25","stoichiometry":4.0},{"gene":"AAMP","stoichiometry":0.2},{"gene":"CPSF6","stoichiometry":0.2},{"gene":"DDX21","stoichiometry":0.2},{"gene":"EMC10","stoichiometry":0.2},{"gene":"HUS1","stoichiometry":0.2},{"gene":"MARK2","stoichiometry":0.2},{"gene":"MARK4","stoichiometry":0.2},{"gene":"PBK","stoichiometry":0.2},{"gene":"PHKG2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PRPF40A","total_profiled":1310},"omim":[{"mim_id":"621019","title":"PRE-mRNA-PROCESSING FACTOR 40 HOMOLOG B; PRPF40B","url":"https://www.omim.org/entry/621019"},{"mim_id":"612941","title":"PRE-mRNA-PROCESSING FACTOR 40 HOMOLOG A; PRPF40A","url":"https://www.omim.org/entry/612941"},{"mim_id":"609475","title":"A-KINASE ANCHOR PROTEIN 8-LIKE PROTEIN; AKAP8L","url":"https://www.omim.org/entry/609475"},{"mim_id":"604981","title":"WW DOMAIN-CONTAINING BINDING PROTEIN 4; WBP4","url":"https://www.omim.org/entry/604981"},{"mim_id":"301158","title":"TRANSCRIPTION ELONGATION FACTOR A-LIKE 9; TCEAL9","url":"https://www.omim.org/entry/301158"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nuclear speckles","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PRPF40A"},"hgnc":{"alias_symbol":["FLJ20585","FBP11","HYPA","NY-REN-6","HIP10","FBP-11","FLAF1","Prp40"],"prev_symbol":["FNBP3"]},"alphafold":{"accession":"O75400","domains":[{"cath_id":"1.10.10.440","chopping":"394-522","consensus_level":"medium","plddt":90.6591,"start":394,"end":522},{"cath_id":"-","chopping":"599-736","consensus_level":"medium","plddt":93.8472,"start":599,"end":736},{"cath_id":"1.10.10.440","chopping":"758-804","consensus_level":"high","plddt":86.9381,"start":758,"end":804}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75400","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75400-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75400-F1-predicted_aligned_error_v6.png","plddt_mean":66.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PRPF40A","jax_strain_url":"https://www.jax.org/strain/search?query=PRPF40A"},"sequence":{"accession":"O75400","fasta_url":"https://rest.uniprot.org/uniprotkb/O75400.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75400/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75400"}},"corpus_meta":[{"pmid":"8535139","id":"PMC_8535139","title":"The petunia MADS box gene FBP11 determines ovule identity.","date":"1995","source":"The Plant cell","url":"https://pubmed.ncbi.nlm.nih.gov/8535139","citation_count":271,"is_preprint":false},{"pmid":"11123699","id":"PMC_11123699","title":"Requirement of nickel metabolism proteins HypA and HypB for full activity of both hydrogenase and urease in Helicobacter pylori.","date":"2001","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/11123699","citation_count":156,"is_preprint":false},{"pmid":"12081959","id":"PMC_12081959","title":"Network of hydrogenase maturation in Escherichia coli: role of accessory proteins HypA and HybF.","date":"2002","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/12081959","citation_count":79,"is_preprint":false},{"pmid":"8632464","id":"PMC_8632464","title":"The Agaricus bisporus hypA gene encodes a hydrophobin and specifically accumulates in peel tissue of mushroom caps during fruit body development.","date":"1996","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/8632464","citation_count":73,"is_preprint":false},{"pmid":"19621959","id":"PMC_19621959","title":"Structure of a nickel chaperone, HypA, from Helicobacter pylori reveals two distinct metal binding sites.","date":"2009","source":"Journal of the American Chemical Society","url":"https://pubmed.ncbi.nlm.nih.gov/19621959","citation_count":65,"is_preprint":false},{"pmid":"28258342","id":"PMC_28258342","title":"COL1A1, PRPF40A, and UCP2 correlate with hypoxia markers in non-small cell lung cancer.","date":"2017","source":"Journal of cancer research and clinical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/28258342","citation_count":57,"is_preprint":false},{"pmid":"15995183","id":"PMC_15995183","title":"Escherichia coli HypA is a zinc metalloprotein with a weak affinity for nickel.","date":"2005","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/15995183","citation_count":55,"is_preprint":false},{"pmid":"22179820","id":"PMC_22179820","title":"Metallo-GTPase HypB from Helicobacter pylori and its interaction with nickel chaperone protein HypA.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22179820","citation_count":47,"is_preprint":false},{"pmid":"20662514","id":"PMC_20662514","title":"Communication between the zinc and nickel sites in dimeric HypA: metal recognition and pH sensing.","date":"2010","source":"Journal of the American Chemical Society","url":"https://pubmed.ncbi.nlm.nih.gov/20662514","citation_count":46,"is_preprint":false},{"pmid":"16972939","id":"PMC_16972939","title":"Mutagenesis of hydrogenase accessory genes of Synechocystis sp. 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mechanism.","date":"2026","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/42001940","citation_count":0,"is_preprint":false},{"pmid":"41950867","id":"PMC_41950867","title":"Dose-dependent biphasic effect of environmental UVA on stem cell function through PRPF40A, TGF-β1, NFATc1 signaling.","date":"2026","source":"Journal of photochemistry and photobiology. B, Biology","url":"https://pubmed.ncbi.nlm.nih.gov/41950867","citation_count":0,"is_preprint":false},{"pmid":"42217269","id":"PMC_42217269","title":"Nickel binding shifts Helicobacter pylori HypA toward compact conformations.","date":"2026","source":"Journal of inorganic biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/42217269","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.30.685498","title":"Two-dimensional NMR from a Single Pulse: Reconstructing Heteronuclear 2D spectra via off-resonance decoupling and Deep Neural Networks","date":"2025-10-31","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.30.685498","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.10.07.616944","title":"Autonomous adaptive optimization of NMR experimental conditions for precise inference of minor conformational states of proteins based on chemical exchange saturation transfer","date":"2024-10-07","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.07.616944","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.08.28.672894","title":"U1 snRNP and RNA polymerase II interaction is predominantly mediated by Prp40 rather than U1-70K in yeast","date":"2025-08-30","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.28.672894","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":24520,"output_tokens":2226,"usd":0.053475,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9194,"output_tokens":2424,"usd":0.053285,"stage2_stop_reason":"end_turn"},"total_usd":0.10676,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"Polyglutamine-expanded huntingtin sequesters HYPA/FBP11 (PRPF40A) to the cytosolic location, significantly reducing pre-mRNA splicing efficiency. NMR structural analysis showed that the proline-rich region (PRR) of huntingtin cooperatively interacts with the tandem WW domains of HYPA through a domain chaperoning effect of WW1 on WW2.\",\n      \"method\": \"NMR structural analysis, pre-mRNA splicing efficiency assay, subcellular localization experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — NMR structure plus functional splicing assay and localization data, single lab with two orthogonal methods\",\n      \"pmids\": [\"21566141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"FBP11 (PRPF40A) inhibits N-WASP-dependent microspike formation by trapping N-WASP in the nucleus. Endogenous FBP11 localizes to nuclear speckles, and co-expression with N-WASP causes co-localization in the nucleus, preventing N-WASP from acting in the cytoplasm.\",\n      \"method\": \"Transient overexpression, subcellular localization by fluorescence microscopy, actin microspike formation assay in COS7 cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — localization and phenotypic rescue experiments, single lab, two methods but no direct binding assay\",\n      \"pmids\": [\"14697212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The nuclear matrix protein NAKAP interacts with HYPA/FBP11 (PRPF40A) via a proline-rich domain in NAKAP binding to a WW domain of HYPA. NAKAP and HypA co-localize within the nucleus, co-purify with the nuclear matrix, and are components of nuclear aggregates in Huntington's disease neurons.\",\n      \"method\": \"Yeast two-hybrid screen, in vitro binding assay, deletion mapping, subcellular fractionation, co-immunoprecipitation from human brain tissue\",\n      \"journal\": \"Neuromolecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid plus in vitro binding plus fractionation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"16391387\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The first WW domain of FBP11/HYPA (PRPF40A) adopts an antiparallel triple-stranded beta-sheet structure. It binds proline-rich sequences containing PL, PP, and PR motifs via both XP and XP2 grooves, with the XP2 groove being functionally important for ligand recognition. Dissociation constant for a PL motif peptide was 248 ± 27 µM.\",\n      \"method\": \"NMR solution structure determination, ligand titration by 1H-15N HSQC NMR, GST pulldown binding assay\",\n      \"journal\": \"Proteins\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with binding characterization, single lab, two orthogonal methods\",\n      \"pmids\": [\"16463264\", \"16472085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PRPF40A tandem WW domains adopt a defined solution structure characterized by NMR/SAXS. Both WW domains cooperate to bind a high-affinity SF1 peptide, enabling tryptophan sandwiching by two proline residues. An N-terminal proline-rich motif of PRPF40A mediates intramolecular autoinhibitory interactions with the WW tandem, acting as a selectivity filter for high-affinity proline-rich bona fide binding partners (SF1 and SF3A1). This was validated by NMR, ITC, mutagenesis, and immunoprecipitation in cells.\",\n      \"method\": \"NMR spectroscopy, SAXS, ITC, mutagenesis, co-immunoprecipitation in cells\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structure by NMR+SAXS, binding by ITC, mutagenesis, and cellular IP, multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"38719828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PRPF40A acts predominantly as an activator of cassette exon inclusion, particularly of exons flanked by short GC-rich introns that tend to localize to nuclear speckles. PRPF40A knockdown in HL-60 cells caused increased cell death, decreased proliferation, and slight myeloid differentiation phenotype. Cell death but not proliferation was rescued by overexpression of paralog PRPF40B. PRPF40A functions in a network with SRRM2, SON, PCBP1/2, TRA2B, and SRSF2.\",\n      \"method\": \"shRNA knockdown, transcriptomic analysis (RNA-seq), overexpression rescue experiments, cell viability and proliferation assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with defined cellular phenotype and transcriptome-wide splicing analysis, single lab with multiple orthogonal readouts\",\n      \"pmids\": [\"38943321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PRPF40A is globally required for microexon splicing in mouse neuroblastoma cells, with a graded dependence on exon size (greater dependence for smaller exons). PRPF40A co-regulates microexons with SRRM4. PRPF40A knockdown also causes increased productive splicing of its spliceosomal binding partner Luc7l by skipping a 'poison exon', suggesting homeostatic cross-regulation between coupled spliceosomal components.\",\n      \"method\": \"siRNA knockdown, RNA-seq, splicing analysis in N2A cells\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with defined splicing phenotypes and transcriptome-wide analysis, single lab, multiple exon classes examined\",\n      \"pmids\": [\"39389624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In yeast, Prp40 (ortholog of PRPF40A) interacts with RNA polymerase II through multiple domains, and this interaction does not require the CTD of pol II. While yeast Prp40 is stably associated with U1 snRNP and is the primary mediator of U1 snRNP-pol II interaction, its human homologs PRPF40A and PRPF40B are alternative splicing factors not integral to U1 snRNP.\",\n      \"method\": \"Co-immunoprecipitation, domain deletion analysis, yeast genetic experiments\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab, primarily yeast data with limited direct evidence for human PRPF40A mechanism\",\n      \"pmids\": [\"bio_10.1101_2025.08.28.672894\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PRPF40A is a pre-mRNA splicing factor whose tandem WW domains bind proline-rich motifs in spliceosome components (SF1 and SF3A1) to bridge 5' and 3' splice site recognition during early spliceosome assembly, with binding selectivity regulated by an intramolecular autoinhibitory proline-rich motif; it is specifically required for inclusion of cassette exons—especially microexons—in GC-rich regions, localizes to nuclear speckles, can trap binding partners such as N-WASP in the nucleus to regulate their cytoplasmic activity, and its sequestration by polyglutamine-expanded huntingtin impairs pre-mRNA splicing efficiency.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PRPF40A is a pre-mRNA splicing factor that uses tandem WW domains to read proline-rich motifs in early spliceosome components and promote alternative exon inclusion [#4, #5]. Structural work established that the first WW domain folds into an antiparallel triple-stranded beta-sheet that engages PL, PP, and PR motifs through its XP and XP2 grooves [#3], and that the two WW domains cooperate to bind a high-affinity SF1 peptide by sandwiching a tryptophan between two proline residues; an N-terminal autoinhibitory proline-rich motif folds back onto the WW tandem to act as a selectivity filter favoring bona fide partners SF1 and SF3A1 [#4]. Functionally, PRPF40A acts predominantly as an activator of cassette exon inclusion, with strong preference for exons flanked by short GC-rich introns and for microexons, where its dependence is graded by exon size, and it operates within a splicing-factor network including SRRM2, SON, SRRM4, PCBP1/2, TRA2B, and SRSF2 [#5, #6]. It localizes to nuclear speckles and can sequester binding partners such as N-WASP in the nucleus, thereby restraining their cytoplasmic activity [#1]. Polyglutamine-expanded huntingtin cooperatively engages the WW tandem via its proline-rich region and mislocalizes PRPF40A to the cytosol, reducing pre-mRNA splicing efficiency [#0].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established that PRPF40A is a nuclear-speckle protein capable of controlling a partner's cytoplasmic activity by nuclear retention, first linking it to spatial regulation of interacting proteins.\",\n      \"evidence\": \"Overexpression, fluorescence localization, and actin microspike assays in COS7 cells\",\n      \"pmids\": [\"14697212\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct binding interface defined for N-WASP\", \"Relationship between N-WASP trapping and splicing function not addressed\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed PRPF40A engages additional nuclear-matrix proline-rich partners (NAKAP) through its WW domains and is recruited into Huntington's disease nuclear aggregates, extending its interaction repertoire beyond splicing.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro binding, deletion mapping, fractionation, and co-IP from human brain\",\n      \"pmids\": [\"16391387\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of NAKAP binding unknown\", \"Does not connect aggregate localization to splicing defects\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Resolved how the first WW domain recognizes ligands, defining the structural basis (XP and XP2 grooves) for proline-rich motif binding by PRPF40A.\",\n      \"evidence\": \"NMR solution structure, HSQC ligand titration, and GST pulldown\",\n      \"pmids\": [\"16463264\", \"16472085\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single WW domain in isolation; tandem cooperativity not addressed\", \"Low affinity (248 \\u00b5M) leaves physiological partner specificity open\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Provided a disease mechanism: polyQ-huntingtin cooperatively binds the WW tandem and relocalizes PRPF40A to the cytosol, mechanistically linking its sequestration to impaired splicing.\",\n      \"evidence\": \"NMR structural analysis, pre-mRNA splicing efficiency assay, and subcellular localization\",\n      \"pmids\": [\"21566141\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which endogenous splicing targets are affected not identified\", \"In vivo neuronal relevance not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the tandem-WW solution architecture and an intramolecular autoinhibitory selectivity filter, explaining how PRPF40A achieves high-affinity, selective binding to spliceosomal partners SF1 and SF3A1.\",\n      \"evidence\": \"NMR, SAXS, ITC, mutagenesis, and co-IP in cells\",\n      \"pmids\": [\"38719828\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How autoinhibition is relieved in vivo not defined\", \"Structural state within assembled spliceosome unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated PRPF40A is a transcriptome-wide activator of cassette exon and microexon inclusion biased toward short GC-rich introns and nuclear speckles, embedding it in a defined splicing-regulatory network with cellular phenotypes on viability and differentiation.\",\n      \"evidence\": \"shRNA/siRNA knockdown, RNA-seq splicing analysis, rescue and viability assays in HL-60 and N2A cells\",\n      \"pmids\": [\"38943321\", \"39389624\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct RNA or factor contacts driving exon selection not mapped\", \"Mechanism of homeostatic Luc7l poison-exon cross-regulation unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether human PRPF40A makes direct, regulated contacts with RNA polymerase II or the U1 snRNP during co-transcriptional splicing remains undefined.\",\n      \"evidence\": \"Yeast Prp40 ortholog data indicate U1/pol II coupling, but human PRPF40A is reported as a non-integral alternative splicing factor\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct evidence for human PRPF40A\\u2013pol II interaction\", \"Extent of co-transcriptional vs post-transcriptional action in human cells unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [5, 6]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [5, 6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0016607\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [4, 5, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SF1\", \"SF3A1\", \"N-WASP\", \"HTT\", \"SRRM4\", \"SRRM2\", \"SON\", \"TRA2B\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}