{"gene":"PRPF40A","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":1998,"finding":"PRPF40A (HYPA/FBP11) was identified as a WW domain-containing protein that binds huntingtin's proline-rich N-terminal region via its WW domains. The interaction is enhanced by lengthening the adjacent polyglutamine tract and is mediated by huntingtin's proline-rich region. HYPA was recognized as a protein implicated in spliceosome function, establishing its role as an RNA processing factor.","method":"Yeast two-hybrid screen, confirmed by interaction with huntingtin in extracts of HD lymphoblastoid cells","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal binding confirmed in cell extracts, single lab","pmids":["9700202"],"is_preprint":false},{"year":2004,"finding":"FBP11 (PRPF40A) sequesters N-WASP in the nucleus, thereby inhibiting N-WASP-dependent actin microspike formation in the cytoplasm. Endogenous FBP11 localizes to nuclear speckles, and co-expression with N-WASP leads to nuclear co-localization. EGF-induced microspike formation was suppressed by FBP11 overexpression, demonstrating that FBP11 negatively regulates cytoplasmic N-WASP function by controlling its subcellular localization.","method":"Transient transfection, confocal immunofluorescence, EGF stimulation assay in COS7 cells, loss-of-function/gain-of-function with defined phenotypic readout","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization experiment with functional consequence, single lab","pmids":["14697212"],"is_preprint":false},{"year":2005,"finding":"PRPF40A (HypA/FBP11) interacts with the nuclear scaffold protein NAKAP via a proline-rich domain in NAKAP engaging a WW domain of PRPF40A. Both NAKAP and PRPF40A co-purify with the nuclear matrix in cultured cells and in human brain, and NAKAP associates with PRPF40A from HD patient brain tissue where they co-localize with mutant huntingtin in nuclear aggregates, suggesting PRPF40A-NAKAP forms a nuclear docking scaffold for huntingtin.","method":"Yeast two-hybrid, in vitro binding assay, deletion mapping, co-purification with nuclear matrix fraction, immunofluorescence in HD neurons","journal":"Neuromolecular medicine","confidence":"Medium","confidence_rationale":"Tier 2-3 — multiple methods (Y2H, in vitro binding, fractionation, IF), single lab","pmids":["16391387"],"is_preprint":false},{"year":2006,"finding":"The first WW domain of FBP11/HYPA (PRPF40A) adopts an antiparallel triple-stranded beta-sheet with XP and XP2 grooves on its surface. NMR titration showed the WW domain binds proline-rich peptides containing PL, PP, and PR motifs, with the XP2 groove being functionally important for ligand recognition, establishing PRPF40A as a Group-II/III WW domain protein with broad proline-rich motif binding specificity.","method":"Solution NMR structure determination, 1H-15N HSQC chemical shift perturbation titration","journal":"Proteins","confidence":"High","confidence_rationale":"Tier 1 — NMR structure with functional binding validation","pmids":["16463264"],"is_preprint":false},{"year":2011,"finding":"Polyglutamine-expanded huntingtin sequesters PRPF40A (HYPA/FBP11) to cytosolic locations and significantly reduces pre-mRNA splicing efficiency. NMR structural analysis showed that the proline-rich region (PRR) of huntingtin cooperatively interacts with PRPF40A's tandem WW domains through a domain-chaperoning effect of WW1 on WW2, establishing a mechanism whereby polyQ-expanded Htt causes dysfunction of cellular RNA processing by mislocating PRPF40A.","method":"NMR structural analysis of WW domain-PRR interaction, cellular fractionation, pre-mRNA splicing efficiency assays, immunofluorescence co-localization","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — NMR structure plus functional splicing assay and localization, single lab with multiple orthogonal methods","pmids":["21566141"],"is_preprint":false},{"year":2024,"finding":"PRPF40A acts predominantly as an activator of cassette exon inclusion, particularly for exons flanked by short, GC-rich introns that tend to localize to nuclear speckles in the nuclear center. PRPF40A knockdown in HL-60 cells caused increased cell death, decreased proliferation, and a slight differentiation phenotype with upregulation of immune activation genes. Cell death but not proliferation defects were rescued by overexpression of its paralog PRPF40B. PRPF40A shares regulatory features with SRRM2, SON, PCBP1/2, TRA2B, and SRSF2, forming a functional network regulating splicing partly via co-localization in the nucleus.","method":"siRNA knockdown in HL-60 cells, transcriptomic (RNA-seq) analysis, rescue experiments with PRPF40B overexpression, nuclear speckle localization analysis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 — clean KD with defined cellular phenotype, transcriptome-wide mechanistic characterization, rescue experiment, multiple orthogonal methods","pmids":["38943321"],"is_preprint":false},{"year":2024,"finding":"The PRPF40A tandem WW domains adopt a specific solution structure (determined by NMR and SAXS) in which both WW domains cooperate to bind high-affinity SF1 proline-rich peptide, enabling tryptophan sandwiching by two proline residues. Unexpectedly, a proline-rich motif in the N-terminal region of PRPF40A itself mediates intramolecular interactions with the WW tandem, acting as an autoinhibitory filter that selectively gates binding to high-affinity proline-rich motifs in bona fide partners such as SF1 and SF3A1. This autoinhibitory mechanism was validated by NMR, ITC, mutational analysis in vitro, and immunoprecipitation in cells, establishing that PRPF40A promotes spliceosome assembly at early E and A complexes by bridging 5' and 3' splice site recognition.","method":"NMR, SAXS, ITC, mutational analysis, immunoprecipitation in cells","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — structural determination plus ITC binding thermodynamics plus mutagenesis plus cellular IP, multiple orthogonal methods in one study","pmids":["38719828"],"is_preprint":false},{"year":2024,"finding":"PRPF40A is globally required for microexon splicing in mouse neuroblastoma cells, with dependence on PRPF40A exhibiting a graded decrease as exon size increases (rather than a sharp threshold). PRPF40A co-regulates microexons together with SRRM4, a neuron-specific microexon regulator. Additionally, PRPF40A knockdown causes increased productive splicing of its spliceosomal binding partner Luc7l by skipping a small poison exon, revealing homeostatic cross-regulation between functionally coupled but evolutionarily unrelated spliceosomal components.","method":"siRNA knockdown in mouse neuroblastoma cells, RNA-seq, comparison with SRRM4 knockdown datasets","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 — clean KD with transcriptome-wide mechanistic readout, conserved function validated across species","pmids":["39389624"],"is_preprint":false},{"year":2025,"finding":"In yeast, Prp40 (the PRPF40A ortholog) is stably associated with U1 snRNP and multiple domains of Prp40 directly interact with RNA polymerase II independently of the pol II CTD. In contrast, human PRPF40A and PRPF40B are alternative splicing factors that are not integral components of U1 snRNP, indicating a functional divergence of Prp40 homologs during evolution: the transcription-splicing coupling function shifted from Prp40/PRPF40A to U1-70K in humans.","method":"Co-immunoprecipitation of yeast snRNPs with pol II, domain-deletion analysis of Prp40, comparison with human PRPF40A behavior","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2-3 — yeast ortholog with direct IP evidence, preprint, functional inference about human PRPF40A divergence","pmids":[],"is_preprint":true}],"current_model":"PRPF40A is a nuclear splicing factor that promotes cassette exon inclusion (especially in GC-rich, short-intron contexts) and is globally required for microexon splicing; it bridges early spliceosome assembly (E and A complexes) by engaging SF1 and SF3A1 proline-rich motifs through its tandem WW domains, whose selectivity is regulated by an intramolecular autoinhibitory interaction with its own N-terminal proline-rich region, while it also controls subcellular localization of N-WASP and is sequestered from the nucleus by polyglutamine-expanded huntingtin, impairing pre-mRNA splicing."},"narrative":{"teleology":[{"year":1998,"claim":"Identification of PRPF40A as a WW-domain protein that binds huntingtin's proline-rich region established its first molecular interaction and implicated it in spliceosome-related biology, though its precise splicing function was unknown.","evidence":"Yeast two-hybrid screen and binding confirmed in HD lymphoblastoid cell extracts","pmids":["9700202"],"confidence":"Medium","gaps":["No direct evidence of splicing function at this stage","Interaction with huntingtin not validated by reciprocal IP from endogenous sources","Polyglutamine-length dependence mechanism unclear"]},{"year":2004,"claim":"Demonstration that PRPF40A sequesters N-WASP in the nucleus and suppresses EGF-induced actin microspike formation revealed a non-canonical function in controlling cytoplasmic actin dynamics via subcellular partitioning of a binding partner.","evidence":"Transient transfection, confocal immunofluorescence, and EGF stimulation assay in COS7 cells","pmids":["14697212"],"confidence":"Medium","gaps":["Endogenous N-WASP nuclear retention by PRPF40A not shown under physiological conditions","Relevance to splicing function unclear","Single cell type tested"]},{"year":2005,"claim":"Co-purification of PRPF40A with the nuclear scaffold protein NAKAP and their co-localization in Huntington's disease nuclear aggregates suggested PRPF40A forms part of a nuclear docking platform that is disrupted by mutant huntingtin.","evidence":"Yeast two-hybrid, in vitro binding, nuclear matrix fractionation, and immunofluorescence in HD patient neurons","pmids":["16391387"],"confidence":"Medium","gaps":["Functional consequence of NAKAP-PRPF40A interaction on splicing not tested","Co-localization in aggregates does not prove functional sequestration","Single lab, no independent replication"]},{"year":2006,"claim":"Determination of the NMR structure of PRPF40A's first WW domain and mapping of its proline-rich peptide binding grooves established the molecular basis for its Group-II/III WW domain specificity.","evidence":"Solution NMR structure and 1H-15N HSQC chemical shift perturbation titration","pmids":["16463264"],"confidence":"High","gaps":["Only WW1 solved; tandem WW domain architecture unknown at this point","Binding to physiological spliceosomal partners not yet demonstrated structurally","No cellular functional validation of identified binding grooves"]},{"year":2011,"claim":"Structural and functional evidence that polyglutamine-expanded huntingtin sequesters PRPF40A to the cytoplasm via cooperative binding of the huntingtin proline-rich region to the WW tandem, directly reducing pre-mRNA splicing efficiency, provided the first mechanistic link between PRPF40A mislocalization and Huntington's disease-associated splicing defects.","evidence":"NMR of WW-PRR interaction, cellular fractionation, splicing efficiency assays, immunofluorescence","pmids":["21566141"],"confidence":"High","gaps":["Splicing defects not mapped to specific target transcripts","In vivo relevance in HD model organisms not tested","Whether PRPF40B can compensate for PRPF40A mislocalization unknown"]},{"year":2024,"claim":"Transcriptome-wide characterization revealed PRPF40A as an activator of cassette exon inclusion for exons with short, GC-rich flanking introns and as a factor essential for cell viability in hematopoietic cells, with partial functional overlap with PRPF40B.","evidence":"siRNA knockdown in HL-60 cells, RNA-seq, rescue with PRPF40B overexpression, nuclear speckle localization analysis","pmids":["38943321"],"confidence":"High","gaps":["Mechanism by which GC-richness or intron length determines PRPF40A dependence not elucidated","Tissue-specificity of PRPF40A requirement beyond HL-60 cells unclear","Extent of functional redundancy with PRPF40B for splicing targets unknown"]},{"year":2024,"claim":"Structural determination of the PRPF40A tandem WW domains and discovery of an intramolecular autoinhibitory mechanism by its own proline-rich region explained how PRPF40A selectively engages high-affinity spliceosomal partners SF1 and SF3A1 to bridge 5′ and 3′ splice-site recognition during early spliceosome assembly.","evidence":"NMR, SAXS, ITC, mutational analysis, and immunoprecipitation in cells","pmids":["38719828"],"confidence":"High","gaps":["Structural basis of autoinhibition release upon partner engagement not fully resolved","No cryo-EM or crystal structure of PRPF40A within the spliceosome","Whether autoinhibition is regulated by post-translational modification unknown"]},{"year":2024,"claim":"PRPF40A was shown to be globally required for microexon splicing with a graded size dependence, and to engage in homeostatic cross-regulation with spliceosomal partner Luc7l, revealing an unexpected feedback circuit between functionally coupled spliceosomal factors.","evidence":"siRNA knockdown in mouse neuroblastoma cells, RNA-seq, comparison with SRRM4 knockdown datasets","pmids":["39389624"],"confidence":"High","gaps":["Molecular mechanism by which PRPF40A preferentially supports microexon recognition unknown","Whether PRPF40A-Luc7l cross-regulation occurs in human neurons not tested","Contribution of PRPF40A to neurodevelopmental microexon programs in vivo uncharacterized"]},{"year":null,"claim":"Key unresolved questions include the structural basis of PRPF40A within assembled spliceosomal complexes, the molecular determinants of its microexon selectivity, whether post-translational modifications regulate autoinhibition, and the in vivo contribution of PRPF40A mislocalization to Huntington's disease pathology.","evidence":"","pmids":[],"confidence":"High","gaps":["No cryo-EM structure of PRPF40A in spliceosomal context","In vivo HD model studies with PRPF40A manipulation absent","Post-translational regulation of autoinhibitory mechanism untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[5,6,7]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[6]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,2,4,5]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[5,6]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[4,5,6,7]}],"complexes":[],"partners":["SF1","SF3A1","HTT","WASL","SRRM4","LUC7L","NAKAP"],"other_free_text":[]},"mechanistic_narrative":"PRPF40A is a nuclear splicing factor that promotes early spliceosome assembly by bridging 5′ and 3′ splice-site recognition components through its tandem WW domains, which engage proline-rich motifs in SF1 and SF3A1 at the E and A complex stages; selectivity for these partners is enforced by an intramolecular autoinhibitory interaction between the WW tandem and PRPF40A's own N-terminal proline-rich region [PMID:38719828]. Functionally, PRPF40A acts predominantly as an activator of cassette exon inclusion—especially for exons flanked by short, GC-rich introns—and is globally required for microexon splicing, co-regulating this program with SRRM4 [PMID:38943321, PMID:39389624]. PRPF40A localizes to nuclear speckles and its cytoplasmic sequestration by polyglutamine-expanded huntingtin impairs pre-mRNA splicing, linking PRPF40A mislocalization to Huntington's disease-associated RNA processing defects [PMID:21566141, PMID:9700202]. Loss of PRPF40A in hematopoietic cells causes increased cell death and reduced proliferation, with cell death partially rescued by its paralog PRPF40B [PMID:38943321]."},"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":270,"is_preprint":false,"source_track":"pubmed_title"},{"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":155,"is_preprint":false,"source_track":"pubmed_title"},{"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,"source_track":"pubmed_title"},{"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,"source_track":"pubmed_title"},{"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":64,"is_preprint":false,"source_track":"pubmed_title"},{"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,"source_track":"pubmed_title"},{"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":54,"is_preprint":false,"source_track":"pubmed_title"},{"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":46,"is_preprint":false,"source_track":"pubmed_title"},{"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":45,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"16972939","id":"PMC_16972939","title":"Mutagenesis of hydrogenase accessory genes of Synechocystis sp. 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reducing pre-mRNA splicing efficiency, linking polyQ-mediated mislocalization to splicing dysfunction.\",\n      \"method\": \"NMR structural analysis of WW domain–PRR interaction, cellular localization experiments (co-expression), pre-mRNA splicing assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — NMR structure plus splicing functional assay in cells, single lab\",\n      \"pmids\": [\"21566141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"FBP11 (PRPF40A) localizes to nuclear speckles and, upon co-expression with N-WASP, sequesters N-WASP in the nucleus, thereby inhibiting N-WASP-dependent actin microspike formation in the cytoplasm.\",\n      \"method\": \"Fluorescence localization/co-localization, transient overexpression, actin microspike formation assay (EGF-stimulated COS7 cells)\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization experiment with defined functional consequence, single lab\",\n      \"pmids\": [\"14697212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PRPF40A (HypA/FBP11) interacts via its WW domain with the proline-rich domain of the nuclear matrix protein NAKAP; both proteins co-localize within the nucleus and co-purify with the nuclear matrix, and NAKAP–HYPA is a nuclear docking site for huntingtin.\",\n      \"method\": \"Yeast two-hybrid screen, in vitro pulldown, deletion mapping, co-purification with nuclear matrix, co-immunoprecipitation from human brain tissue\",\n      \"journal\": \"Neuromolecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — multiple methods (Y2H, pulldown, nuclear fractionation) in single lab\",\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 β-sheet and binds proline-rich peptides containing PL, PP, and PR motifs through both XP and XP2 grooves, as shown by solution NMR structure determination and ligand-titration NMR.\",\n      \"method\": \"Solution NMR structure determination; 1H-15N HSQC NMR ligand titration\",\n      \"journal\": \"Proteins\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with functional binding characterization\",\n      \"pmids\": [\"16463264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The WW domains of FBP11/HYPA (PRPF40A) bind PL-motif peptides with measurable affinity (KD ~248 µM for WW domain of FBP11), confirmed by GST-fusion pulldown and binding assay.\",\n      \"method\": \"GST-fusion protein production, peptide binding assay (dissociation constant determination)\",\n      \"journal\": \"Protein and peptide letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single method, single lab, modest characterization\",\n      \"pmids\": [\"16472085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PRPF40A acts as an activator of cassette exon inclusion, preferentially regulating exons flanked by short, GC-rich introns that tend to localize to nuclear speckles; its function is partly redundant with PRPF40B for cell death but not proliferation, and it operates in a functional network with SRRM2, SON, PCBP1/2, TRA2B, and SRSF2.\",\n      \"method\": \"siRNA knockdown in HL-60 cells, RNA-seq transcriptomic analysis, overexpression rescue experiment, nuclear speckle co-localization analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO with transcriptome-wide phenotype plus rescue and network analysis, multiple orthogonal methods\",\n      \"pmids\": [\"38943321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The PRPF40A tandem WW domains bind SF1 and SF3A1 proline-rich motifs to bridge 5′ and 3′ splice site recognition during early spliceosome assembly; an intramolecular autoinhibitory interaction between a proline-rich motif in PRPF40A's own N-terminal region and its WW tandem acts as a selectivity filter for high-affinity binding partners.\",\n      \"method\": \"NMR, SAXS, ITC, mutational analysis in vitro, immunoprecipitation in cells\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — structure (NMR+SAXS), binding thermodynamics (ITC), mutagenesis, and cell-based IP, all in one study\",\n      \"pmids\": [\"38719828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PRPF40A (ortholog PRP-40 in C. elegans) is globally required for microexon splicing in mouse neuroblastoma cells, co-regulating microexons with SRRM4; PRPF40A dependence decreases gradually with increasing exon size, and PRPF40A knockdown increases productive splicing of its binding partner Luc7l by skipping a poison exon.\",\n      \"method\": \"siRNA knockdown in mouse neuroblastoma cells, RNA-seq analysis, comparison with SRRM4 knockdown\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with transcriptome-wide readout, mechanistic epistasis with SRRM4, multiple orthogonal analyses\",\n      \"pmids\": [\"39389624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In yeast, Prp40 (ortholog of PRPF40A) mediates interaction between U1 snRNP and RNA polymerase II through multiple Prp40 domains independently of the pol II CTD; in contrast, human PRPF40A and PRPF40B are not integral U1 snRNP components but act as alternative splicing factors, indicating an evolutionary shift in function.\",\n      \"method\": \"Co-immunoprecipitation, deletion analysis of Prp40 domains, comparison of yeast and human systems\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — reciprocal Co-IP and domain deletion in yeast with functional comparison to human; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.08.28.672894\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PRPF40A is a splicing factor that localizes to nuclear speckles and promotes cassette exon inclusion—particularly of microexons and exons in GC-rich contexts—by bridging early spliceosome assembly via its tandem WW domains, which bind proline-rich motifs in SF1 and SF3A1 at the 5′ and 3′ splice sites; binding selectivity is tuned by an intramolecular autoinhibitory interaction, and PRPF40A also sequesters N-WASP and huntingtin-interacting partners in the nucleus with consequences for actin regulation and RNA processing.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries only from KEEP papers.\n\n**Classification:**\n- Papers about bacterial/archaeal HypA (nickel metallochaperone): EXCLUDE (symbol collision - different protein entirely)\n- Papers about fungal HypA (hydrophobin, morphogenesis): EXCLUDE (symbol collision)\n- Papers about plant FBP11 (MADS box): EXCLUDE (symbol collision)\n- Papers about MBL2 HYPA haplotype: EXCLUDE (alias collision - MBL2 haplotype name)\n- Papers about PRPF40A/FBP11/HYPA the human splicing factor: KEEP\n- Large proteomics/interactome screens that detected PRPF40A: KEEP if mechanistic\n- NMR methods papers using FF domain of HYPA as a test protein: borderline - only KEEP if mechanistic findings about PRPF40A itself\n\n**KEEP papers:**\n- PMID:38943321 (PRPF40A in myeloid cell splicing)\n- PMID:38719828 (PRPF40A tandem WW domains, autoinhibition)\n- PMID:39389624 (PRPF40A microexon splicing, RNA published)\n- PMID:39386728 (preprint version of same)\n- PMID:21566141 (HYPA/FBP11 interaction with huntingtin, RNA processing)\n- PMID:16391387 (NAKAP-HypA interaction, nuclear matrix)\n- PMID:9700202 (Huntingtin interacts with WW domain proteins including HYPA)\n- PMID:16463264 (FBP11/HYPA WW domain structure and binding)\n- PMID:14697212 (FBP11 regulates N-WASP nuclear localization)\n- PMID:16472085 (WW domains of FBP11 binding)\n- bio_10.1101_2025.08.28.672894 (Prp40/PRPF40A interaction with pol II)\n\nLarge interactome screens (BioPlex, HuRI etc.): These detect PRPF40A interactions but provide no mechanistic insight beyond co-association - EXCLUDE from discoveries.\nPMID:17081983, 16169070, etc. - large phosphoproteomics/interactome: no specific mechanistic finding about PRPF40A mechanism - EXCLUDE.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"PRPF40A (HYPA/FBP11) was identified as a WW domain-containing protein that binds huntingtin's proline-rich N-terminal region via its WW domains. The interaction is enhanced by lengthening the adjacent polyglutamine tract and is mediated by huntingtin's proline-rich region. HYPA was recognized as a protein implicated in spliceosome function, establishing its role as an RNA processing factor.\",\n      \"method\": \"Yeast two-hybrid screen, confirmed by interaction with huntingtin in extracts of HD lymphoblastoid cells\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal binding confirmed in cell extracts, single lab\",\n      \"pmids\": [\"9700202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"FBP11 (PRPF40A) sequesters N-WASP in the nucleus, thereby inhibiting N-WASP-dependent actin microspike formation in the cytoplasm. Endogenous FBP11 localizes to nuclear speckles, and co-expression with N-WASP leads to nuclear co-localization. EGF-induced microspike formation was suppressed by FBP11 overexpression, demonstrating that FBP11 negatively regulates cytoplasmic N-WASP function by controlling its subcellular localization.\",\n      \"method\": \"Transient transfection, confocal immunofluorescence, EGF stimulation assay in COS7 cells, loss-of-function/gain-of-function with defined phenotypic readout\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization experiment with functional consequence, single lab\",\n      \"pmids\": [\"14697212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PRPF40A (HypA/FBP11) interacts with the nuclear scaffold protein NAKAP via a proline-rich domain in NAKAP engaging a WW domain of PRPF40A. Both NAKAP and PRPF40A co-purify with the nuclear matrix in cultured cells and in human brain, and NAKAP associates with PRPF40A from HD patient brain tissue where they co-localize with mutant huntingtin in nuclear aggregates, suggesting PRPF40A-NAKAP forms a nuclear docking scaffold for huntingtin.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding assay, deletion mapping, co-purification with nuclear matrix fraction, immunofluorescence in HD neurons\",\n      \"journal\": \"Neuromolecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — multiple methods (Y2H, in vitro binding, fractionation, IF), single lab\",\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 with XP and XP2 grooves on its surface. NMR titration showed the WW domain binds proline-rich peptides containing PL, PP, and PR motifs, with the XP2 groove being functionally important for ligand recognition, establishing PRPF40A as a Group-II/III WW domain protein with broad proline-rich motif binding specificity.\",\n      \"method\": \"Solution NMR structure determination, 1H-15N HSQC chemical shift perturbation titration\",\n      \"journal\": \"Proteins\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with functional binding validation\",\n      \"pmids\": [\"16463264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Polyglutamine-expanded huntingtin sequesters PRPF40A (HYPA/FBP11) to cytosolic locations and significantly reduces pre-mRNA splicing efficiency. NMR structural analysis showed that the proline-rich region (PRR) of huntingtin cooperatively interacts with PRPF40A's tandem WW domains through a domain-chaperoning effect of WW1 on WW2, establishing a mechanism whereby polyQ-expanded Htt causes dysfunction of cellular RNA processing by mislocating PRPF40A.\",\n      \"method\": \"NMR structural analysis of WW domain-PRR interaction, cellular fractionation, pre-mRNA splicing efficiency assays, immunofluorescence co-localization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — NMR structure plus functional splicing assay and localization, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"21566141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PRPF40A acts predominantly as an activator of cassette exon inclusion, particularly for exons flanked by short, GC-rich introns that tend to localize to nuclear speckles in the nuclear center. PRPF40A knockdown in HL-60 cells caused increased cell death, decreased proliferation, and a slight differentiation phenotype with upregulation of immune activation genes. Cell death but not proliferation defects were rescued by overexpression of its paralog PRPF40B. PRPF40A shares regulatory features with SRRM2, SON, PCBP1/2, TRA2B, and SRSF2, forming a functional network regulating splicing partly via co-localization in the nucleus.\",\n      \"method\": \"siRNA knockdown in HL-60 cells, transcriptomic (RNA-seq) analysis, rescue experiments with PRPF40B overexpression, nuclear speckle localization analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with defined cellular phenotype, transcriptome-wide mechanistic characterization, rescue experiment, multiple orthogonal methods\",\n      \"pmids\": [\"38943321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The PRPF40A tandem WW domains adopt a specific solution structure (determined by NMR and SAXS) in which both WW domains cooperate to bind high-affinity SF1 proline-rich peptide, enabling tryptophan sandwiching by two proline residues. Unexpectedly, a proline-rich motif in the N-terminal region of PRPF40A itself mediates intramolecular interactions with the WW tandem, acting as an autoinhibitory filter that selectively gates binding to high-affinity proline-rich motifs in bona fide partners such as SF1 and SF3A1. This autoinhibitory mechanism was validated by NMR, ITC, mutational analysis in vitro, and immunoprecipitation in cells, establishing that PRPF40A promotes spliceosome assembly at early E and A complexes by bridging 5' and 3' splice site recognition.\",\n      \"method\": \"NMR, SAXS, ITC, mutational analysis, immunoprecipitation in cells\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural determination plus ITC binding thermodynamics plus mutagenesis plus cellular IP, multiple orthogonal methods in one study\",\n      \"pmids\": [\"38719828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PRPF40A is globally required for microexon splicing in mouse neuroblastoma cells, with dependence on PRPF40A exhibiting a graded decrease as exon size increases (rather than a sharp threshold). PRPF40A co-regulates microexons together with SRRM4, a neuron-specific microexon regulator. Additionally, PRPF40A knockdown causes increased productive splicing of its spliceosomal binding partner Luc7l by skipping a small poison exon, revealing homeostatic cross-regulation between functionally coupled but evolutionarily unrelated spliceosomal components.\",\n      \"method\": \"siRNA knockdown in mouse neuroblastoma cells, RNA-seq, comparison with SRRM4 knockdown datasets\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with transcriptome-wide mechanistic readout, conserved function validated across species\",\n      \"pmids\": [\"39389624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In yeast, Prp40 (the PRPF40A ortholog) is stably associated with U1 snRNP and multiple domains of Prp40 directly interact with RNA polymerase II independently of the pol II CTD. In contrast, human PRPF40A and PRPF40B are alternative splicing factors that are not integral components of U1 snRNP, indicating a functional divergence of Prp40 homologs during evolution: the transcription-splicing coupling function shifted from Prp40/PRPF40A to U1-70K in humans.\",\n      \"method\": \"Co-immunoprecipitation of yeast snRNPs with pol II, domain-deletion analysis of Prp40, comparison with human PRPF40A behavior\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — yeast ortholog with direct IP evidence, preprint, functional inference about human PRPF40A divergence\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PRPF40A is a nuclear splicing factor that promotes cassette exon inclusion (especially in GC-rich, short-intron contexts) and is globally required for microexon splicing; it bridges early spliceosome assembly (E and A complexes) by engaging SF1 and SF3A1 proline-rich motifs through its tandem WW domains, whose selectivity is regulated by an intramolecular autoinhibitory interaction with its own N-terminal proline-rich region, while it also controls subcellular localization of N-WASP and is sequestered from the nucleus by polyglutamine-expanded huntingtin, impairing pre-mRNA splicing.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PRPF40A is a nuclear speckle-localized splicing factor that promotes cassette exon inclusion, with a particularly strong requirement for microexon splicing and exons flanked by short, GC-rich introns [PMID:38943321, PMID:39389624]. Its tandem WW domains bridge early spliceosome assembly by simultaneously binding proline-rich motifs in SF1 (at the branchpoint/5′ splice site) and SF3A1 (at the 3′ splice site), with binding selectivity governed by an intramolecular autoinhibitory interaction between its own N-terminal proline-rich region and the WW tandem [PMID:38719828]. PRPF40A also sequesters cytoplasmic interaction partners such as N-WASP and huntingtin into the nucleus via WW–proline-rich interactions, linking it to actin regulation and polyglutamine disease pathology in which expanded huntingtin mislocalizes PRPF40A and impairs pre-mRNA splicing [PMID:14697212, PMID:21566141]. PRPF40A functions in a regulatory network with SRRM2, SON, SRSF2, TRA2B, and PCBP1/2, and is partly redundant with its paralog PRPF40B for cell viability but not for proliferation [PMID:38943321].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing that PRPF40A resides in nuclear speckles and can sequester cytoplasmic WW-binding partners—demonstrated by its ability to relocalize N-WASP to the nucleus and thereby inhibit actin microspike formation—revealed a mechanism by which a splicing factor could modulate cytoskeletal signaling.\",\n      \"evidence\": \"Fluorescence co-localization and actin microspike assay in EGF-stimulated COS7 cells\",\n      \"pmids\": [\"14697212\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Overexpression-based relocalization; physiological relevance of N-WASP sequestration at endogenous levels unknown\",\n        \"No demonstration that endogenous PRPF40A regulates actin dynamics\",\n        \"Mechanism of nuclear retention of N-WASP not defined\"\n      ]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identification of NAKAP as a nuclear matrix-associated WW-domain partner of PRPF40A established that PRPF40A is anchored within the nuclear matrix and can serve as a docking site for huntingtin, connecting it to Huntington's disease protein networks.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro pulldown, nuclear matrix co-purification, co-IP from human brain tissue\",\n      \"pmids\": [\"16391387\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional consequence of NAKAP–PRPF40A interaction on splicing not tested\",\n        \"Single-lab study without independent replication\",\n        \"Relevance of huntingtin docking to PRPF40A splicing activity unexplored\"\n      ]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Solving the NMR structure of the first WW domain revealed the canonical triple-stranded β-sheet fold and mapped the XP/XP2 grooves used for proline-rich peptide recognition, providing the structural basis for PRPF40A's diverse WW-mediated interactions.\",\n      \"evidence\": \"Solution NMR structure determination and ¹H-¹⁵N HSQC ligand titration\",\n      \"pmids\": [\"16463264\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Only the first WW domain was structurally characterized; tandem domain architecture not resolved\",\n        \"No structure of WW domain in complex with full-length partner proteins\",\n        \"Binding affinities for physiological partners not determined in this study\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrating that polyglutamine-expanded huntingtin sequesters PRPF40A to the cytosol and reduces pre-mRNA splicing efficiency linked PRPF40A mislocalization to a concrete disease-relevant splicing defect, answering how polyQ expansions could cause transcriptomic dysfunction.\",\n      \"evidence\": \"NMR structural analysis of WW–PRR interaction, cellular co-expression localization, pre-mRNA splicing assay\",\n      \"pmids\": [\"21566141\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Splicing defect shown with reporter; genome-wide splicing impact of huntingtin-mediated mislocalization not assessed\",\n        \"Not tested in neuronal disease models or patient-derived cells\",\n        \"Relative contribution of PRPF40A mislocalization versus other splicing factors to HD pathology unclear\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Transcriptome-wide analysis established PRPF40A as a general activator of cassette exon inclusion—preferentially for exons flanked by short, GC-rich introns—and placed it in a functional network with SRRM2, SON, and SR proteins, resolving its splicing-regulatory specificity and partial redundancy with PRPF40B.\",\n      \"evidence\": \"siRNA knockdown in HL-60 cells, RNA-seq, overexpression rescue, nuclear speckle co-localization analysis\",\n      \"pmids\": [\"38943321\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism by which GC-richness or intron length dictates PRPF40A dependence is unknown\",\n        \"Functional distinction from PRPF40B at the molecular level not delineated\",\n        \"Analysis limited to a single cell line (HL-60)\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Structural and biophysical dissection of the tandem WW domains showed that PRPF40A bridges SF1 and SF3A1 to couple 5′ and 3′ splice-site recognition during early spliceosome assembly, with an autoinhibitory proline-rich motif in its own N-terminus tuning partner selectivity—answering how PRPF40A mechanistically contributes to cross-intron bridging.\",\n      \"evidence\": \"NMR, SAXS, ITC, mutational analysis in vitro, immunoprecipitation in cells\",\n      \"pmids\": [\"38719828\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No cryo-EM or crystal structure of PRPF40A within an assembled spliceosome complex\",\n        \"In vivo contribution of autoinhibition to splicing regulation not tested by mutagenesis in cells\",\n        \"Stoichiometry and dynamics of SF1–PRPF40A–SF3A1 ternary complex in the spliceosome remain undefined\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identifying PRPF40A as globally required for microexon splicing—with dependence decreasing as exon size increases—and showing epistasis with SRRM4 revealed a size-dependent mechanism and a co-regulatory logic for the shortest regulated exons in the transcriptome.\",\n      \"evidence\": \"siRNA knockdown in mouse neuroblastoma cells, RNA-seq, comparison with SRRM4 knockdown\",\n      \"pmids\": [\"39389624\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether PRPF40A directly contacts microexon-proximal sequences or acts indirectly through spliceosome kinetics is unknown\",\n        \"PRPF40A–SRRM4 physical interaction not demonstrated\",\n        \"In vivo neuronal phenotype of PRPF40A loss not assessed in animal models\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PRPF40A is recruited to specific pre-mRNAs, whether it functions within the assembled spliceosome or solely during early complex formation, and what determines its differential requirement across tissues and exon classes remain open mechanistic questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structural visualization of PRPF40A in an assembled spliceosomal complex\",\n        \"Tissue-specific splicing regulation by PRPF40A versus PRPF40B not systematically compared\",\n        \"No animal knockout phenotype reported\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [5, 6, 7]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 2, 5]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [5, 6, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"SF1\",\n      \"SF3A1\",\n      \"HTT\",\n      \"WASL\",\n      \"SRRM2\",\n      \"NAKAP95\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"PRPF40A is a nuclear splicing factor that promotes early spliceosome assembly by bridging 5′ and 3′ splice-site recognition components through its tandem WW domains, which engage proline-rich motifs in SF1 and SF3A1 at the E and A complex stages; selectivity for these partners is enforced by an intramolecular autoinhibitory interaction between the WW tandem and PRPF40A's own N-terminal proline-rich region [PMID:38719828]. Functionally, PRPF40A acts predominantly as an activator of cassette exon inclusion—especially for exons flanked by short, GC-rich introns—and is globally required for microexon splicing, co-regulating this program with SRRM4 [PMID:38943321, PMID:39389624]. PRPF40A localizes to nuclear speckles and its cytoplasmic sequestration by polyglutamine-expanded huntingtin impairs pre-mRNA splicing, linking PRPF40A mislocalization to Huntington's disease-associated RNA processing defects [PMID:21566141, PMID:9700202]. Loss of PRPF40A in hematopoietic cells causes increased cell death and reduced proliferation, with cell death partially rescued by its paralog PRPF40B [PMID:38943321].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Identification of PRPF40A as a WW-domain protein that binds huntingtin's proline-rich region established its first molecular interaction and implicated it in spliceosome-related biology, though its precise splicing function was unknown.\",\n      \"evidence\": \"Yeast two-hybrid screen and binding confirmed in HD lymphoblastoid cell extracts\",\n      \"pmids\": [\"9700202\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct evidence of splicing function at this stage\", \"Interaction with huntingtin not validated by reciprocal IP from endogenous sources\", \"Polyglutamine-length dependence mechanism unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstration that PRPF40A sequesters N-WASP in the nucleus and suppresses EGF-induced actin microspike formation revealed a non-canonical function in controlling cytoplasmic actin dynamics via subcellular partitioning of a binding partner.\",\n      \"evidence\": \"Transient transfection, confocal immunofluorescence, and EGF stimulation assay in COS7 cells\",\n      \"pmids\": [\"14697212\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous N-WASP nuclear retention by PRPF40A not shown under physiological conditions\", \"Relevance to splicing function unclear\", \"Single cell type tested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Co-purification of PRPF40A with the nuclear scaffold protein NAKAP and their co-localization in Huntington's disease nuclear aggregates suggested PRPF40A forms part of a nuclear docking platform that is disrupted by mutant huntingtin.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro binding, nuclear matrix fractionation, and immunofluorescence in HD patient neurons\",\n      \"pmids\": [\"16391387\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of NAKAP-PRPF40A interaction on splicing not tested\", \"Co-localization in aggregates does not prove functional sequestration\", \"Single lab, no independent replication\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Determination of the NMR structure of PRPF40A's first WW domain and mapping of its proline-rich peptide binding grooves established the molecular basis for its Group-II/III WW domain specificity.\",\n      \"evidence\": \"Solution NMR structure and 1H-15N HSQC chemical shift perturbation titration\",\n      \"pmids\": [\"16463264\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Only WW1 solved; tandem WW domain architecture unknown at this point\", \"Binding to physiological spliceosomal partners not yet demonstrated structurally\", \"No cellular functional validation of identified binding grooves\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Structural and functional evidence that polyglutamine-expanded huntingtin sequesters PRPF40A to the cytoplasm via cooperative binding of the huntingtin proline-rich region to the WW tandem, directly reducing pre-mRNA splicing efficiency, provided the first mechanistic link between PRPF40A mislocalization and Huntington's disease-associated splicing defects.\",\n      \"evidence\": \"NMR of WW-PRR interaction, cellular fractionation, splicing efficiency assays, immunofluorescence\",\n      \"pmids\": [\"21566141\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Splicing defects not mapped to specific target transcripts\", \"In vivo relevance in HD model organisms not tested\", \"Whether PRPF40B can compensate for PRPF40A mislocalization unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Transcriptome-wide characterization revealed PRPF40A as an activator of cassette exon inclusion for exons with short, GC-rich flanking introns and as a factor essential for cell viability in hematopoietic cells, with partial functional overlap with PRPF40B.\",\n      \"evidence\": \"siRNA knockdown in HL-60 cells, RNA-seq, rescue with PRPF40B overexpression, nuclear speckle localization analysis\",\n      \"pmids\": [\"38943321\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which GC-richness or intron length determines PRPF40A dependence not elucidated\", \"Tissue-specificity of PRPF40A requirement beyond HL-60 cells unclear\", \"Extent of functional redundancy with PRPF40B for splicing targets unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Structural determination of the PRPF40A tandem WW domains and discovery of an intramolecular autoinhibitory mechanism by its own proline-rich region explained how PRPF40A selectively engages high-affinity spliceosomal partners SF1 and SF3A1 to bridge 5′ and 3′ splice-site recognition during early spliceosome assembly.\",\n      \"evidence\": \"NMR, SAXS, ITC, mutational analysis, and immunoprecipitation in cells\",\n      \"pmids\": [\"38719828\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of autoinhibition release upon partner engagement not fully resolved\", \"No cryo-EM or crystal structure of PRPF40A within the spliceosome\", \"Whether autoinhibition is regulated by post-translational modification unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"PRPF40A was shown to be globally required for microexon splicing with a graded size dependence, and to engage in homeostatic cross-regulation with spliceosomal partner Luc7l, revealing an unexpected feedback circuit between functionally coupled spliceosomal factors.\",\n      \"evidence\": \"siRNA knockdown in mouse neuroblastoma cells, RNA-seq, comparison with SRRM4 knockdown datasets\",\n      \"pmids\": [\"39389624\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism by which PRPF40A preferentially supports microexon recognition unknown\", \"Whether PRPF40A-Luc7l cross-regulation occurs in human neurons not tested\", \"Contribution of PRPF40A to neurodevelopmental microexon programs in vivo uncharacterized\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of PRPF40A within assembled spliceosomal complexes, the molecular determinants of its microexon selectivity, whether post-translational modifications regulate autoinhibition, and the in vivo contribution of PRPF40A mislocalization to Huntington's disease pathology.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No cryo-EM structure of PRPF40A in spliceosomal context\", \"In vivo HD model studies with PRPF40A manipulation absent\", \"Post-translational regulation of autoinhibitory mechanism untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [5, 6, 7]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 2, 4, 5]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [5, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [4, 5, 6, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SF1\", \"SF3A1\", \"HTT\", \"WASL\", \"SRRM4\", \"LUC7L\", \"NAKAP\"],\n    \"other_free_text\": []\n  }\n}\n```"}