{"gene":"PRPF38A","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":1992,"finding":"Yeast PRP38 encodes an essential pre-mRNA splicing factor required for the first cleavage-ligation step of intron excision; temperature-sensitive inactivation blocks the first step of splicing and causes a decrease in intracellular U6 snRNA levels.","method":"Genetic screen of temperature-sensitive mutants; in vitro splicing inactivation and complementation assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro splicing assay plus genetic complementation, foundational study replicated and extended by subsequent work","pmids":["1508195"],"is_preprint":false},{"year":1999,"finding":"Yeast Prp38p is required for U4 snRNA release from the spliceosome; dosage suppressor screen identified Spp381p (yeast ortholog of human MFAP1) as a direct binding partner that assists Prp38p function; the two-hybrid assay and immune precipitation showed Spp381p is present in the U4/U6.U5 tri-snRNP and directly interacts with the carboxyl half of Prp38p.","method":"Dosage suppressor screen; co-immunoprecipitation; yeast two-hybrid; in vitro splicing assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus two-hybrid plus in vitro splicing, replicated by later structural and evolutionary work","pmids":["9858581"],"is_preprint":false},{"year":2011,"finding":"The ubiquitin-like protein Hub1 binds non-covalently to a conserved HIND element present in spliceosomal proteins including Prp38 (in plants) and Snu66 (in yeast/mammals); this interaction modulates splice-site usage and alternative splicing without globally disrupting the spliceosome.","method":"Structural analysis (NMR/biochemical binding); genetic (splicing assays in Hub1-deficient yeast); domain mapping","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — structural and biochemical binding data combined with genetic functional validation, published in Nature","pmids":["21614000"],"is_preprint":false},{"year":2015,"finding":"Human PRPF38A contains an N-terminal domain organized around three pairs of antiparallel α-helices that acts as a multi-interface protein-protein interaction hub, binding at least 12 other spliceosomal proteins (most recruited at the same stage) via four distinct surfaces; addition of excess PRPF38A N-terminal domain to in vitro splicing assays stalled splicing at a pre-catalytic stage, while an interaction-deficient mutant did not.","method":"Crystal structure of human PRPF38A N-terminal domain; yeast two-hybrid (alanine surface-scanning); in vitro binding assays with recombinant proteins; in vitro splicing inhibition assay with domain mutants","journal":"RNA","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis and in vitro splicing assay with multiple orthogonal methods in one study","pmids":["26673105"],"is_preprint":false},{"year":2016,"finding":"Spliceosomal PRPF38A/Prp38 forms a complex with MFAP1 and Snu23; crystal structure analysis shows MFAP1 and Snu23 contact Prp38 via ER/K motif-stabilized single α-helices; helix-stabilizing mutations in the MFAP1 single α-helix reduce Prp38 binding affinity, demonstrating that the strength of these single-α-helix-based interactions can be tuned by helix stabilization in the unbound state.","method":"Crystal structure analysis; binding assays with mutant peptides; biophysical characterization (ITC/CD)","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional mutagenesis and quantitative binding measurements in one study","pmids":["27773687"],"is_preprint":false},{"year":2017,"finding":"In the cryo-EM structure of the yeast pre-catalytic B complex spliceosome, Prp38 (along with Snu23 and Spp381) binds the Prp8 N-terminal domain and stabilizes U6 ACAGAGA stem–pre-mRNA and Brr2-U4 snRNA interactions, providing a structural basis for Prp38's role in spliceosome stabilization prior to catalytic activation.","method":"Cryo-EM structure of yeast B complex spliceosome at near-atomic resolution","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-atomic cryo-EM structure providing direct structural evidence for protein location and interactions","pmids":["28530653"],"is_preprint":false},{"year":2017,"finding":"Human MFAP1 is a cryptic ortholog of yeast Spp381; in vitro binding studies showed that MFAP1 and Spp381 bind their respective Prp38 proteins via equivalent interfaces and cross-interact with Prp38 from the other species; MFAP1 and Spp381 both form higher-order complexes that additionally include Snu23, constituting an equivalent Snu23-Prp38-MFAP1/Spp381 sub-complex; MFAP1 partially rescued growth defects of temperature-sensitive prp38-1 yeast.","method":"In vitro binding assays; bioinformatics (InParanoid, BLAST); yeast complementation assay","journal":"BMC evolutionary biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro binding combined with yeast complementation, single lab study","pmids":["28335716"],"is_preprint":false},{"year":2017,"finding":"PRPF38A knockdown in breast cancer cells caused widespread intronic retention and altered splicing of transcripts involved in protein homeostasis, mitosis, and apoptosis, placing PRPF38A as required for normal splicing of transcripts essential for basal-like TNBC cell survival.","method":"siRNA knockdown followed by RNA-seq splicing analysis in multiple human breast cancer cell lines","journal":"Molecular cancer therapeutics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KD with defined splicing phenotype, single lab with no additional mechanistic follow-up","pmids":["28878028"],"is_preprint":false},{"year":2020,"finding":"PRPF38A localizes to nuclear speckles; PRPF38A is a binding partner of MFAP1, and co-localization of MFAP1 and PRPF38A in droplet-like nuclear bodies precedes formation of nuclear speckles during telophase.","method":"Proximity labeling (TSA-MS ratio); live-cell imaging; co-localization analysis during telophase","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity labeling proteomics combined with imaging-based validation of localization and binding partner","pmids":["32609799"],"is_preprint":false},{"year":2022,"finding":"Partial re-localization of PRPF38A to the nuclear lamina in HEK293T cells using a reversible chemical dimerizer induced a moderate increase in intron retention, demonstrating that spatial positioning of PRPF38A within the nucleus is functionally important for its splicing activity.","method":"Chemical dimerizer-based reversible re-localization to nuclear lamina; intron retention quantification by RNA-seq in HEK293T cells","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct localization experiment with functional consequence (intron retention), single lab, single method","pmids":["35869234"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM structure of human spliceosomal B complex dimers revealed that PRPF38A (PRP38) makes molecular contacts with SNU23 and FBP21 around the U6/5' splice site helix, and that MFAP1 and UBL5 form a 5' exon binding channel in hB, refining the understanding of PRP38's interactions at this stage.","method":"Cryo-EM structure of human B complex dimers at enhanced resolution","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — near-atomic cryo-EM structure directly resolving protein contacts, single study but structural evidence","pmids":["38383864"],"is_preprint":false},{"year":2025,"finding":"Single-molecule imaging (CoSMoS) showed that yeast Prp38, Snu23, and Spp381 bind to and release from spliceosomes simultaneously after tri-snRNP binding, forming a B complex protein (BCP) subcomplex; BCP release predominantly occurs after U4 snRNP dissociation and NineTeen Complex (NTC) association; under low ATP, BCP pre-associates with the tri-snRNP; this recruitment pathway is conserved between S. cerevisiae and humans.","method":"Colocalization Single Molecule Spectroscopy (CoSMoS) real-time imaging of Prp38, Snu23, and Spp381 dynamics during splicing","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — single-molecule real-time imaging with direct observation of protein binding/release dynamics, peer-reviewed, mechanistically informative","pmids":["39995036"],"is_preprint":false},{"year":2022,"finding":"In C. elegans, the Hub1/UBL-5 ubiquitin-like protein binds to the HIND-containing splicing factor PRP-38 (C. elegans Prp38 ortholog) and Snu66/SART-1, and associates with other spliceosomal proteins; ubl-5 mutants show splicing defects for selected targets, demonstrating a conserved role of Hub1-Prp38 HIND interaction in pre-mRNA splicing in multicellular organisms.","method":"Biochemical binding assays; genetic analysis of C. elegans ubl-5 mutants; splicing assays; yeast complementation","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — binding assays plus genetic loss-of-function phenotype in C. elegans, cross-species complementation","pmids":["36480405"],"is_preprint":false}],"current_model":"PRPF38A/Prp38 is an essential pre-mRNA splicing factor that, together with Snu23 and MFAP1 (the metazoan ortholog of yeast Spp381), forms a B-complex protein (BCP) subcomplex that is recruited to the spliceosome after tri-snRNP binding; its N-terminal domain acts as a multi-interface protein-protein interaction hub that contacts Prp8, Snu23, MFAP1, and other B-complex proteins to stabilize the U6 ACAGAGA stem–pre-mRNA and Brr2-U4 snRNA interactions prior to catalytic activation, and its correct spatial positioning within nuclear speckles is required for normal splicing activity, as mislocalization to the nuclear lamina causes intron retention."},"narrative":{"mechanistic_narrative":"PRPF38A (yeast Prp38) is an essential pre-mRNA splicing factor required for the catalytic activation of the spliceosome [PMID:1508195]. It is recruited to the assembling spliceosome as part of a B-complex protein (BCP) subcomplex together with Snu23 and MFAP1 (metazoan ortholog of yeast Spp381), the three proteins binding and releasing as a unit after tri-snRNP association and dissociating predominantly after U4 snRNP release and NTC recruitment [PMID:39995036]. Its N-terminal domain is built from three pairs of antiparallel α-helices and functions as a multi-interface protein–protein interaction hub that engages at least twelve other spliceosomal proteins through four distinct surfaces; excess of this domain stalls splicing at a pre-catalytic stage, whereas an interaction-deficient mutant does not [PMID:26673105]. Within the assembled B complex, Prp38 contacts the Prp8 N-terminal domain and stabilizes the U6 ACAGAGA stem–pre-mRNA and Brr2–U4 snRNA interactions prior to catalytic activation, and in human B complex it contacts SNU23 and FBP21 around the U6/5' splice site helix [PMID:28530653, PMID:38383864]. The Snu23–Prp38–MFAP1/Spp381 architecture is built on ER/K motif–stabilized single α-helices whose binding strength is tunable by helix stabilization, and is conserved across species [PMID:27773687, PMID:28335716]. Functionally, loss of PRPF38A causes widespread intron retention and altered splicing of transcripts controlling protein homeostasis, mitosis, and apoptosis [PMID:28878028], and correct spatial positioning of PRPF38A within nuclear speckles is itself required for normal splicing, since mislocalization to the nuclear lamina induces intron retention [PMID:32609799, PMID:35869234]. Independently, the ubiquitin-like protein Hub1/UBL5 binds non-covalently to a conserved HIND element in Prp38 to modulate splice-site usage and alternative splicing [PMID:21614000, PMID:36480405].","teleology":[{"year":1992,"claim":"Established that Prp38 is an essential splicing factor acting specifically at the first catalytic step rather than during early assembly, defining its functional stage in the pathway.","evidence":"Temperature-sensitive genetic screen with in vitro splicing inactivation and complementation in yeast","pmids":["1508195"],"confidence":"High","gaps":["Molecular mechanism by which Prp38 promotes the first cleavage-ligation step not defined","Connection between Prp38 loss and reduced U6 snRNA levels unexplained at the time"]},{"year":1999,"claim":"Identified Spp381 (MFAP1 ortholog) as a direct binding partner of the Prp38 C-terminal half and placed both in the tri-snRNP, linking Prp38 to U4 snRNA release.","evidence":"Dosage suppressor screen, yeast two-hybrid, co-immunoprecipitation, and in vitro splicing in yeast","pmids":["9858581"],"confidence":"High","gaps":["Structural basis of the Prp38–Spp381 interface not resolved","Whether the interaction is direct in the assembled spliceosome not shown"]},{"year":2011,"claim":"Revealed a regulatory layer in which the ubiquitin-like Hub1 binds a conserved HIND element in Prp38, modulating alternative splice-site usage without disrupting the spliceosome.","evidence":"NMR/biochemical binding, domain mapping, and splicing assays in Hub1-deficient yeast and plants","pmids":["21614000"],"confidence":"High","gaps":["Mechanism by which HIND binding alters splice-site selection not defined","Conservation in animals addressed only later"]},{"year":2015,"claim":"Defined the human PRPF38A N-terminal domain as a multi-interface interaction hub whose contacts are required to progress past a pre-catalytic stage, explaining how it organizes B-complex protein assembly.","evidence":"Crystal structure, alanine surface-scanning two-hybrid, in vitro binding, and splicing inhibition with domain mutants","pmids":["26673105"],"confidence":"High","gaps":["Functional role of each of the four surfaces in vivo not dissected","Identities of all twelve binding partners not individually validated functionally"]},{"year":2016,"claim":"Showed the Snu23–Prp38–MFAP1 subcomplex is held together by ER/K-stabilized single α-helices whose affinity is tunable by helix stabilization, revealing a biophysical principle of its assembly.","evidence":"Crystal structure with mutant-peptide binding assays and biophysical characterization (ITC/CD)","pmids":["27773687"],"confidence":"High","gaps":["Whether helix tuning is exploited for regulation in vivo unknown","Dynamics of subcomplex assembly during spliceosome maturation not addressed"]},{"year":2017,"claim":"Placed Prp38 structurally in the pre-catalytic B complex, showing it binds the Prp8 N-terminal domain and stabilizes U6 ACAGAGA stem–pre-mRNA and Brr2–U4 interactions before catalytic activation.","evidence":"Near-atomic cryo-EM of the yeast B complex spliceosome","pmids":["28530653"],"confidence":"High","gaps":["Order of contact formation during assembly not resolved from a static structure","How these contacts are remodeled at activation not shown"]},{"year":2017,"claim":"Established evolutionary equivalence of human MFAP1 to yeast Spp381 and conservation of the Snu23-Prp38-MFAP1/Spp381 subcomplex across species.","evidence":"In vitro cross-species binding assays, bioinformatics, and yeast prp38-1 complementation","pmids":["28335716"],"confidence":"Medium","gaps":["Single-lab study","Only partial rescue of yeast growth defects achieved"]},{"year":2017,"claim":"Demonstrated a cellular requirement for PRPF38A in correct splicing of survival-relevant transcripts, linking its loss to intron retention and altered processing of homeostasis, mitosis, and apoptosis genes.","evidence":"siRNA knockdown with RNA-seq splicing analysis in human breast cancer cell lines","pmids":["28878028"],"confidence":"Medium","gaps":["Direct vs. indirect splicing targets not distinguished","No mechanistic follow-up beyond the descriptive phenotype"]},{"year":2020,"claim":"Localized PRPF38A to nuclear speckles and showed it co-localizes with MFAP1 in droplet-like bodies that precede speckle formation during telophase, connecting the subcomplex to speckle biogenesis.","evidence":"Proximity labeling (TSA-MS) with live-cell imaging and telophase co-localization","pmids":["32609799"],"confidence":"Medium","gaps":["Functional significance of pre-speckle droplet co-localization not tested","Whether speckle targeting is required for splicing addressed separately"]},{"year":2022,"claim":"Established that PRPF38A spatial positioning is itself functionally important, as forced re-localization to the nuclear lamina increases intron retention.","evidence":"Reversible chemical-dimerizer re-localization with RNA-seq intron-retention quantification in HEK293T cells","pmids":["35869234"],"confidence":"Medium","gaps":["Single method, single cell line","Whether the effect reflects loss of speckle context or sequestration not distinguished"]},{"year":2022,"claim":"Confirmed the Hub1/UBL5–HIND–Prp38 regulatory axis operates in a multicellular animal, generalizing the splice-site regulation mechanism beyond yeast and plants.","evidence":"Biochemical binding, C. elegans ubl-5 mutant genetics, splicing assays, and yeast complementation","pmids":["36480405"],"confidence":"Medium","gaps":["Only selected targets analyzed","Mechanism of splice-site modulation by the HIND interaction still undefined"]},{"year":2024,"claim":"Refined the human B-complex contacts of PRPF38A, resolving interactions with SNU23 and FBP21 around the U6/5' splice site helix and a MFAP1/UBL5 5' exon binding channel.","evidence":"Enhanced-resolution cryo-EM of human B complex dimers","pmids":["38383864"],"confidence":"High","gaps":["Functional consequence of each refined contact not individually tested","Significance of the dimeric arrangement unclear"]},{"year":2025,"claim":"Resolved the recruitment dynamics, showing Prp38, Snu23, and Spp381 bind and release as a single BCP subcomplex after tri-snRNP binding, with release after U4 dissociation/NTC association, conserved between yeast and humans.","evidence":"Single-molecule colocalization spectroscopy (CoSMoS) of real-time binding/release dynamics in yeast","pmids":["39995036"],"confidence":"High","gaps":["What triggers coordinated BCP release not defined","Human dynamics inferred from conservation rather than directly observed"]},{"year":null,"claim":"How the Hub1/UBL5-HIND regulatory input is integrated with PRPF38A's structural role in B-complex assembly to control specific splice-site choices in human cells remains open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No mechanistic link established between HIND-mediated regulation and the multi-interface hub function","Direct human substrate/target rules for PRPF38A-dependent splicing not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,4,5]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,7]}],"localization":[{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[8,9]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,3,5,11]}],"complexes":["spliceosome B complex","BCP (Snu23-Prp38-MFAP1/Spp381) subcomplex","U4/U6.U5 tri-snRNP"],"partners":["MFAP1","SNU23","PRPF8","FBP21","UBL5","SART1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8NAV1","full_name":"Pre-mRNA-splicing factor 38A","aliases":[],"length_aa":312,"mass_kda":37.5,"function":"Involved in pre-mRNA splicing as a component of the spliceosome","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q8NAV1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/PRPF38A","classification":"Common Essential","n_dependent_lines":1208,"n_total_lines":1208,"dependency_fraction":1.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SNRPC","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PRPF38A","total_profiled":1310},"omim":[{"mim_id":"617031","title":"PRE-mRNA-PROCESSING FACTOR 38A; PRPF38A","url":"https://www.omim.org/entry/617031"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PRPF38A"},"hgnc":{"alias_symbol":["FLJ14936","Prp38"],"prev_symbol":[]},"alphafold":{"accession":"Q8NAV1","domains":[{"cath_id":"-","chopping":"16-165","consensus_level":"high","plddt":90.5019,"start":16,"end":165},{"cath_id":"-","chopping":"166-197","consensus_level":"medium","plddt":68.2572,"start":166,"end":197}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NAV1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NAV1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NAV1-F1-predicted_aligned_error_v6.png","plddt_mean":71.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PRPF38A","jax_strain_url":"https://www.jax.org/strain/search?query=PRPF38A"},"sequence":{"accession":"Q8NAV1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8NAV1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8NAV1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NAV1"}},"corpus_meta":[{"pmid":"28530653","id":"PMC_28530653","title":"Structure of a pre-catalytic spliceosome.","date":"2017","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/28530653","citation_count":180,"is_preprint":false},{"pmid":"33397451","id":"PMC_33397451","title":"Genome-wide association study implicates novel loci and reveals candidate effector genes for longitudinal pediatric bone accrual.","date":"2021","source":"Genome biology","url":"https://pubmed.ncbi.nlm.nih.gov/33397451","citation_count":91,"is_preprint":false},{"pmid":"21614000","id":"PMC_21614000","title":"Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing.","date":"2011","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/21614000","citation_count":81,"is_preprint":false},{"pmid":"1508195","id":"PMC_1508195","title":"PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels.","date":"1992","source":"Molecular and cellular 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temperature-sensitive mutants; in vitro splicing inactivation and complementation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro splicing assay plus genetic complementation, foundational study replicated and extended by subsequent work\",\n      \"pmids\": [\"1508195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Yeast Prp38p is required for U4 snRNA release from the spliceosome; dosage suppressor screen identified Spp381p (yeast ortholog of human MFAP1) as a direct binding partner that assists Prp38p function; the two-hybrid assay and immune precipitation showed Spp381p is present in the U4/U6.U5 tri-snRNP and directly interacts with the carboxyl half of Prp38p.\",\n      \"method\": \"Dosage suppressor screen; co-immunoprecipitation; yeast two-hybrid; in vitro splicing assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus two-hybrid plus in vitro splicing, replicated by later structural and evolutionary work\",\n      \"pmids\": [\"9858581\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The ubiquitin-like protein Hub1 binds non-covalently to a conserved HIND element present in spliceosomal proteins including Prp38 (in plants) and Snu66 (in yeast/mammals); this interaction modulates splice-site usage and alternative splicing without globally disrupting the spliceosome.\",\n      \"method\": \"Structural analysis (NMR/biochemical binding); genetic (splicing assays in Hub1-deficient yeast); domain mapping\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structural and biochemical binding data combined with genetic functional validation, published in Nature\",\n      \"pmids\": [\"21614000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Human PRPF38A contains an N-terminal domain organized around three pairs of antiparallel α-helices that acts as a multi-interface protein-protein interaction hub, binding at least 12 other spliceosomal proteins (most recruited at the same stage) via four distinct surfaces; addition of excess PRPF38A N-terminal domain to in vitro splicing assays stalled splicing at a pre-catalytic stage, while an interaction-deficient mutant did not.\",\n      \"method\": \"Crystal structure of human PRPF38A N-terminal domain; yeast two-hybrid (alanine surface-scanning); in vitro binding assays with recombinant proteins; in vitro splicing inhibition assay with domain mutants\",\n      \"journal\": \"RNA\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis and in vitro splicing assay with multiple orthogonal methods in one study\",\n      \"pmids\": [\"26673105\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Spliceosomal PRPF38A/Prp38 forms a complex with MFAP1 and Snu23; crystal structure analysis shows MFAP1 and Snu23 contact Prp38 via ER/K motif-stabilized single α-helices; helix-stabilizing mutations in the MFAP1 single α-helix reduce Prp38 binding affinity, demonstrating that the strength of these single-α-helix-based interactions can be tuned by helix stabilization in the unbound state.\",\n      \"method\": \"Crystal structure analysis; binding assays with mutant peptides; biophysical characterization (ITC/CD)\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional mutagenesis and quantitative binding measurements in one study\",\n      \"pmids\": [\"27773687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In the cryo-EM structure of the yeast pre-catalytic B complex spliceosome, Prp38 (along with Snu23 and Spp381) binds the Prp8 N-terminal domain and stabilizes U6 ACAGAGA stem–pre-mRNA and Brr2-U4 snRNA interactions, providing a structural basis for Prp38's role in spliceosome stabilization prior to catalytic activation.\",\n      \"method\": \"Cryo-EM structure of yeast B complex spliceosome at near-atomic resolution\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-atomic cryo-EM structure providing direct structural evidence for protein location and interactions\",\n      \"pmids\": [\"28530653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Human MFAP1 is a cryptic ortholog of yeast Spp381; in vitro binding studies showed that MFAP1 and Spp381 bind their respective Prp38 proteins via equivalent interfaces and cross-interact with Prp38 from the other species; MFAP1 and Spp381 both form higher-order complexes that additionally include Snu23, constituting an equivalent Snu23-Prp38-MFAP1/Spp381 sub-complex; MFAP1 partially rescued growth defects of temperature-sensitive prp38-1 yeast.\",\n      \"method\": \"In vitro binding assays; bioinformatics (InParanoid, BLAST); yeast complementation assay\",\n      \"journal\": \"BMC evolutionary biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro binding combined with yeast complementation, single lab study\",\n      \"pmids\": [\"28335716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PRPF38A knockdown in breast cancer cells caused widespread intronic retention and altered splicing of transcripts involved in protein homeostasis, mitosis, and apoptosis, placing PRPF38A as required for normal splicing of transcripts essential for basal-like TNBC cell survival.\",\n      \"method\": \"siRNA knockdown followed by RNA-seq splicing analysis in multiple human breast cancer cell lines\",\n      \"journal\": \"Molecular cancer therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KD with defined splicing phenotype, single lab with no additional mechanistic follow-up\",\n      \"pmids\": [\"28878028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PRPF38A localizes to nuclear speckles; PRPF38A is a binding partner of MFAP1, and co-localization of MFAP1 and PRPF38A in droplet-like nuclear bodies precedes formation of nuclear speckles during telophase.\",\n      \"method\": \"Proximity labeling (TSA-MS ratio); live-cell imaging; co-localization analysis during telophase\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity labeling proteomics combined with imaging-based validation of localization and binding partner\",\n      \"pmids\": [\"32609799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Partial re-localization of PRPF38A to the nuclear lamina in HEK293T cells using a reversible chemical dimerizer induced a moderate increase in intron retention, demonstrating that spatial positioning of PRPF38A within the nucleus is functionally important for its splicing activity.\",\n      \"method\": \"Chemical dimerizer-based reversible re-localization to nuclear lamina; intron retention quantification by RNA-seq in HEK293T cells\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct localization experiment with functional consequence (intron retention), single lab, single method\",\n      \"pmids\": [\"35869234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structure of human spliceosomal B complex dimers revealed that PRPF38A (PRP38) makes molecular contacts with SNU23 and FBP21 around the U6/5' splice site helix, and that MFAP1 and UBL5 form a 5' exon binding channel in hB, refining the understanding of PRP38's interactions at this stage.\",\n      \"method\": \"Cryo-EM structure of human B complex dimers at enhanced resolution\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — near-atomic cryo-EM structure directly resolving protein contacts, single study but structural evidence\",\n      \"pmids\": [\"38383864\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Single-molecule imaging (CoSMoS) showed that yeast Prp38, Snu23, and Spp381 bind to and release from spliceosomes simultaneously after tri-snRNP binding, forming a B complex protein (BCP) subcomplex; BCP release predominantly occurs after U4 snRNP dissociation and NineTeen Complex (NTC) association; under low ATP, BCP pre-associates with the tri-snRNP; this recruitment pathway is conserved between S. cerevisiae and humans.\",\n      \"method\": \"Colocalization Single Molecule Spectroscopy (CoSMoS) real-time imaging of Prp38, Snu23, and Spp381 dynamics during splicing\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — single-molecule real-time imaging with direct observation of protein binding/release dynamics, peer-reviewed, mechanistically informative\",\n      \"pmids\": [\"39995036\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In C. elegans, the Hub1/UBL-5 ubiquitin-like protein binds to the HIND-containing splicing factor PRP-38 (C. elegans Prp38 ortholog) and Snu66/SART-1, and associates with other spliceosomal proteins; ubl-5 mutants show splicing defects for selected targets, demonstrating a conserved role of Hub1-Prp38 HIND interaction in pre-mRNA splicing in multicellular organisms.\",\n      \"method\": \"Biochemical binding assays; genetic analysis of C. elegans ubl-5 mutants; splicing assays; yeast complementation\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — binding assays plus genetic loss-of-function phenotype in C. elegans, cross-species complementation\",\n      \"pmids\": [\"36480405\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PRPF38A/Prp38 is an essential pre-mRNA splicing factor that, together with Snu23 and MFAP1 (the metazoan ortholog of yeast Spp381), forms a B-complex protein (BCP) subcomplex that is recruited to the spliceosome after tri-snRNP binding; its N-terminal domain acts as a multi-interface protein-protein interaction hub that contacts Prp8, Snu23, MFAP1, and other B-complex proteins to stabilize the U6 ACAGAGA stem–pre-mRNA and Brr2-U4 snRNA interactions prior to catalytic activation, and its correct spatial positioning within nuclear speckles is required for normal splicing activity, as mislocalization to the nuclear lamina causes intron retention.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PRPF38A (yeast Prp38) is an essential pre-mRNA splicing factor required for the catalytic activation of the spliceosome [#0]. It is recruited to the assembling spliceosome as part of a B-complex protein (BCP) subcomplex together with Snu23 and MFAP1 (metazoan ortholog of yeast Spp381), the three proteins binding and releasing as a unit after tri-snRNP association and dissociating predominantly after U4 snRNP release and NTC recruitment [#11]. Its N-terminal domain is built from three pairs of antiparallel \\u03b1-helices and functions as a multi-interface protein\\u2013protein interaction hub that engages at least twelve other spliceosomal proteins through four distinct surfaces; excess of this domain stalls splicing at a pre-catalytic stage, whereas an interaction-deficient mutant does not [#3]. Within the assembled B complex, Prp38 contacts the Prp8 N-terminal domain and stabilizes the U6 ACAGAGA stem\\u2013pre-mRNA and Brr2\\u2013U4 snRNA interactions prior to catalytic activation, and in human B complex it contacts SNU23 and FBP21 around the U6/5' splice site helix [#5, #10]. The Snu23\\u2013Prp38\\u2013MFAP1/Spp381 architecture is built on ER/K motif\\u2013stabilized single \\u03b1-helices whose binding strength is tunable by helix stabilization, and is conserved across species [#4, #6]. Functionally, loss of PRPF38A causes widespread intron retention and altered splicing of transcripts controlling protein homeostasis, mitosis, and apoptosis [#7], and correct spatial positioning of PRPF38A within nuclear speckles is itself required for normal splicing, since mislocalization to the nuclear lamina induces intron retention [#8, #9]. Independently, the ubiquitin-like protein Hub1/UBL5 binds non-covalently to a conserved HIND element in Prp38 to modulate splice-site usage and alternative splicing [#2, #12].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Established that Prp38 is an essential splicing factor acting specifically at the first catalytic step rather than during early assembly, defining its functional stage in the pathway.\",\n      \"evidence\": \"Temperature-sensitive genetic screen with in vitro splicing inactivation and complementation in yeast\",\n      \"pmids\": [\"1508195\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism by which Prp38 promotes the first cleavage-ligation step not defined\", \"Connection between Prp38 loss and reduced U6 snRNA levels unexplained at the time\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identified Spp381 (MFAP1 ortholog) as a direct binding partner of the Prp38 C-terminal half and placed both in the tri-snRNP, linking Prp38 to U4 snRNA release.\",\n      \"evidence\": \"Dosage suppressor screen, yeast two-hybrid, co-immunoprecipitation, and in vitro splicing in yeast\",\n      \"pmids\": [\"9858581\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the Prp38\\u2013Spp381 interface not resolved\", \"Whether the interaction is direct in the assembled spliceosome not shown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Revealed a regulatory layer in which the ubiquitin-like Hub1 binds a conserved HIND element in Prp38, modulating alternative splice-site usage without disrupting the spliceosome.\",\n      \"evidence\": \"NMR/biochemical binding, domain mapping, and splicing assays in Hub1-deficient yeast and plants\",\n      \"pmids\": [\"21614000\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which HIND binding alters splice-site selection not defined\", \"Conservation in animals addressed only later\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined the human PRPF38A N-terminal domain as a multi-interface interaction hub whose contacts are required to progress past a pre-catalytic stage, explaining how it organizes B-complex protein assembly.\",\n      \"evidence\": \"Crystal structure, alanine surface-scanning two-hybrid, in vitro binding, and splicing inhibition with domain mutants\",\n      \"pmids\": [\"26673105\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of each of the four surfaces in vivo not dissected\", \"Identities of all twelve binding partners not individually validated functionally\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed the Snu23\\u2013Prp38\\u2013MFAP1 subcomplex is held together by ER/K-stabilized single \\u03b1-helices whose affinity is tunable by helix stabilization, revealing a biophysical principle of its assembly.\",\n      \"evidence\": \"Crystal structure with mutant-peptide binding assays and biophysical characterization (ITC/CD)\",\n      \"pmids\": [\"27773687\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether helix tuning is exploited for regulation in vivo unknown\", \"Dynamics of subcomplex assembly during spliceosome maturation not addressed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Placed Prp38 structurally in the pre-catalytic B complex, showing it binds the Prp8 N-terminal domain and stabilizes U6 ACAGAGA stem\\u2013pre-mRNA and Brr2\\u2013U4 interactions before catalytic activation.\",\n      \"evidence\": \"Near-atomic cryo-EM of the yeast B complex spliceosome\",\n      \"pmids\": [\"28530653\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Order of contact formation during assembly not resolved from a static structure\", \"How these contacts are remodeled at activation not shown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established evolutionary equivalence of human MFAP1 to yeast Spp381 and conservation of the Snu23-Prp38-MFAP1/Spp381 subcomplex across species.\",\n      \"evidence\": \"In vitro cross-species binding assays, bioinformatics, and yeast prp38-1 complementation\",\n      \"pmids\": [\"28335716\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study\", \"Only partial rescue of yeast growth defects achieved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated a cellular requirement for PRPF38A in correct splicing of survival-relevant transcripts, linking its loss to intron retention and altered processing of homeostasis, mitosis, and apoptosis genes.\",\n      \"evidence\": \"siRNA knockdown with RNA-seq splicing analysis in human breast cancer cell lines\",\n      \"pmids\": [\"28878028\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. indirect splicing targets not distinguished\", \"No mechanistic follow-up beyond the descriptive phenotype\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Localized PRPF38A to nuclear speckles and showed it co-localizes with MFAP1 in droplet-like bodies that precede speckle formation during telophase, connecting the subcomplex to speckle biogenesis.\",\n      \"evidence\": \"Proximity labeling (TSA-MS) with live-cell imaging and telophase co-localization\",\n      \"pmids\": [\"32609799\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional significance of pre-speckle droplet co-localization not tested\", \"Whether speckle targeting is required for splicing addressed separately\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established that PRPF38A spatial positioning is itself functionally important, as forced re-localization to the nuclear lamina increases intron retention.\",\n      \"evidence\": \"Reversible chemical-dimerizer re-localization with RNA-seq intron-retention quantification in HEK293T cells\",\n      \"pmids\": [\"35869234\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single method, single cell line\", \"Whether the effect reflects loss of speckle context or sequestration not distinguished\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Confirmed the Hub1/UBL5\\u2013HIND\\u2013Prp38 regulatory axis operates in a multicellular animal, generalizing the splice-site regulation mechanism beyond yeast and plants.\",\n      \"evidence\": \"Biochemical binding, C. elegans ubl-5 mutant genetics, splicing assays, and yeast complementation\",\n      \"pmids\": [\"36480405\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Only selected targets analyzed\", \"Mechanism of splice-site modulation by the HIND interaction still undefined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Refined the human B-complex contacts of PRPF38A, resolving interactions with SNU23 and FBP21 around the U6/5' splice site helix and a MFAP1/UBL5 5' exon binding channel.\",\n      \"evidence\": \"Enhanced-resolution cryo-EM of human B complex dimers\",\n      \"pmids\": [\"38383864\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of each refined contact not individually tested\", \"Significance of the dimeric arrangement unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved the recruitment dynamics, showing Prp38, Snu23, and Spp381 bind and release as a single BCP subcomplex after tri-snRNP binding, with release after U4 dissociation/NTC association, conserved between yeast and humans.\",\n      \"evidence\": \"Single-molecule colocalization spectroscopy (CoSMoS) of real-time binding/release dynamics in yeast\",\n      \"pmids\": [\"39995036\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"What triggers coordinated BCP release not defined\", \"Human dynamics inferred from conservation rather than directly observed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the Hub1/UBL5-HIND regulatory input is integrated with PRPF38A's structural role in B-complex assembly to control specific splice-site choices in human cells remains open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No mechanistic link established between HIND-mediated regulation and the multi-interface hub function\", \"Direct human substrate/target rules for PRPF38A-dependent splicing not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 4, 5]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [8, 9]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 3, 5, 11]}\n    ],\n    \"complexes\": [\"spliceosome B complex\", \"BCP (Snu23-Prp38-MFAP1/Spp381) subcomplex\", \"U4/U6.U5 tri-snRNP\"],\n    \"partners\": [\"MFAP1\", \"SNU23\", \"PRPF8\", \"FBP21\", \"UBL5\", \"SART1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":7,"faith_total":7,"faith_pct":100.0}}