{"gene":"YJU2","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2009,"finding":"Yju2 is required for the first catalytic step of splicing after the action of Prp2, functioning at the final step of the first transesterification reaction. Cwc25 acts after Yju2 in the same step, and both are required for promoting juxtaposition of the 5' splice site and branch point.","method":"Depletion/reconstitution assay in yeast splicing extracts; ATP-independent rescue with recombinant protein","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — affinity-purified spliceosome depletion and reconstitution assay, replicated across multiple studies in the corpus","pmids":["19704000"],"is_preprint":false},{"year":2009,"finding":"Ntc90 (a component of the NineTeen Complex/NTC) is specifically required for recruiting Yju2 to the spliceosome after activation, but not for spliceosome activation itself, revealing a novel NTC role in recruiting first-step splicing factors.","method":"Biochemical fractionation, genetic deletion analysis, and reconstitution in yeast splicing extracts","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — biochemical and genetic methods in same study, single lab but two orthogonal approaches","pmids":["19617314"],"is_preprint":false},{"year":2010,"finding":"Yju2 and Cwc25 are released from the spliceosome after the first catalytic step in a Prp16- and ATP-dependent manner, and this release is necessary to allow progression to the second catalytic reaction.","method":"Biochemical splicing assays with ATP/AMP-PNP, spliceosome purification, and factor addition/depletion in yeast extracts","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple biochemical assays, replicated finding confirmed by subsequent structural studies","pmids":["21098140"],"is_preprint":false},{"year":2012,"finding":"High-affinity binding sites for Yju2 and Cwc25 are created on the spliceosome during Prp2-mediated catalytic activation (B* complex formation), consistent with their requirement for step 1 catalysis.","method":"Dual-color fluorescence cross-correlation spectroscopy (dcFCCS) on purified yeast spliceosomes","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — quantitative biophysical measurement (dcFCCS) in near-physiological conditions, single lab","pmids":["22535589"],"is_preprint":false},{"year":2012,"finding":"Ccdc94 (human ortholog of Yju2) is a functional member of the Prp19 complex, and knockdown of Prp19 complex members increases sensitivity to ionizing radiation-induced apoptosis; Ccdc94/Prp19 complex protects cells from IR-induced apoptosis by repressing p53 mRNA expression.","method":"Zebrafish forward genetic screen, genetic knockdown, RT-PCR for p53 mRNA levels, rescue experiments","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic screen plus knockdown with specific molecular readout (p53 mRNA repression), single lab","pmids":["22952453"],"is_preprint":false},{"year":2013,"finding":"Yju2 has a modular structure: the N-terminal half (Yju2-N) weakly binds the spliceosome and functions in the first step, while the C-terminal half (Yju2-C) stabilizes spliceosome association. Yju2-N alone supports a low level of the second reaction even without Prp16, suggesting that Prp16 acts primarily by destabilizing the C-domain-dependent tight spliceosome binding of Yju2. UV cross-linking shows Yju2 directly contacts U2 snRNA in helix II both pre- and post-catalytically, and contacts the branch-binding region only pre-catalytically.","method":"Domain dissection with in vivo and in vitro reconstitution, UV cross-linking to U2 snRNA, ATPase bypass assays in yeast","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution, domain mutagenesis, direct RNA cross-linking, multiple orthogonal methods in single study","pmids":["23438600"],"is_preprint":false},{"year":2006,"finding":"FLJ10374 (YJU2/CCDC94) localizes exclusively to the nucleus and negatively regulates cell cycle G1-to-S transition; siRNA knockdown increases cell proliferation.","method":"Transfection with fluorescence localization, serum-starvation cell cycle assay, siRNA knockdown with proliferation readout","journal":"Genes, chromosomes & cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct localization experiment and functional cell cycle readout, but single lab and limited mechanistic depth","pmids":["16450356"],"is_preprint":false},{"year":2016,"finding":"Cryo-EM structure of the C complex (post-branching) spliceosome at 3.8 Å shows that Yju2 (along with Isy1 and Cwc25) stabilizes docking of the branch helix, positioning the branch adenosine in the active site and facilitating the first transesterification reaction.","method":"Cryo-electron microscopy at 3.8 Å resolution of yeast C complex spliceosome","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structural determination, replicated by independent lab in same year","pmids":["27459055"],"is_preprint":false},{"year":2016,"finding":"Cryo-EM structure at 3.4 Å of the yeast C complex confirms Yju2 is one of 15 protein components that stabilize specific placement of RNA elements (5'SS, BPS, U5 loop I) at the Prp8 catalytic cavity after the first transesterification reaction.","method":"Cryo-electron microscopy at 3.4 Å resolution of yeast C complex spliceosome","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic cryo-EM structure, independently replicated by Galej et al. same year","pmids":["27445308"],"is_preprint":false},{"year":2016,"finding":"Cryo-EM structure of the C* complex shows that the step I splicing factors Cwc25 and Yju2 have dissociated from the active site during the C-to-C* transition, vacating space for the incoming 3'-exon sequences required for the second transesterification.","method":"Cryo-electron microscopy at 4.0 Å resolution of yeast C* complex spliceosome","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structural determination showing direct absence of Yju2 in C*, corroborating biochemical data","pmids":["27980089"],"is_preprint":false},{"year":2018,"finding":"Cryo-EM structure of the human C complex at 4.1 Å shows that CCDC94 (human Yju2) closely interacts with the DEAH-family ATPase/helicase Prp16 and bridges the gap between Prp16 and the active-site RNA elements, providing mechanistic insight into the C-to-C* transition.","method":"Cryo-electron microscopy at 4.1 Å resolution of human C complex spliceosome","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure of human complex, direct visualization of CCDC94-Prp16 interaction","pmids":["29301961"],"is_preprint":false},{"year":2019,"finding":"Cryo-EM structures of four B* complexes at 2.9–3.8 Å reveal that Yju2 recruitment into the active site brings the U2/BPS duplex into the vicinity of the 5' splice site, positioning the BPS nucleophile 4 Å from catalytic metal M2, directly demonstrating Yju2's mechanistic role in promoting the branching reaction.","method":"Cryo-electron microscopy of four yeast B* complexes at 2.9–3.8 Å, comparative structural analysis with and without Yju2/Cwc25","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple near-atomic cryo-EM structures directly demonstrating Yju2-dependent positioning of branch nucleophile","pmids":["30879786"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structure at 2.8 Å of the yeast C complex reveals that Yju2 and Isy1 are recruited to the spliceosome by the NTC before branching. After branching, Prp16 remodels Yju2 binding, allowing Yju2 to remain tethered to the NTC in the C* complex to promote exon ligation.","method":"Cryo-EM at 2.8 Å combined with biochemical assays for C/C* complex dynamics","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM plus biochemical assays, mechanistically resolves NTC-mediated Yju2 recruitment and Prp16-mediated remodeling","pmids":["33705709"],"is_preprint":false},{"year":2021,"finding":"Rat1 exoribonuclease physically interacts with the Yju2 splicing factor (co-immunoprecipitation and mass spectrometry), and is required for proper co-transcriptional recruitment of splicing factors including Yju2 to introns.","method":"Co-immunoprecipitation, mass spectrometry, ChIP-Seq in yeast","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single Co-IP/MS interaction, single lab, no deep mechanistic follow-up specific to Yju2","pmids":["33978753"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM structure of a yeast product complex spliceosome at 2.3 Å shows that binding of the second-step factor Fyv6 to the spliceosome is mutually exclusive with that of the first-step factor Yju2, and that Yju2/Fyv6 exchange facilitates progression from the first to the second step of splicing.","method":"Cryo-EM at 2.3 Å, genetic suppressor screen, transcriptomics","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-atomic cryo-EM structure directly showing mutually exclusive binding, supported by genetic and transcriptomic data","pmids":["39688371"],"is_preprint":false},{"year":2025,"finding":"Molecular dynamics simulations integrating cryo-EM data reveal that Yju2 acts in a temporally ordered fashion with Prp11 and Prp8 at the spliceosome core, with positively charged residues in these proteins dynamically remodeling the active site to enable the first step of splicing.","method":"Equilibrium and enhanced sampling molecular dynamics simulations (>2M atoms) of multiple spliceosome structures","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational simulation only, no direct experimental validation of Yju2-specific claims","pmids":["41474748"],"is_preprint":false}],"current_model":"Yju2 (CCDC94 in humans) is an essential first-step splicing factor that is recruited to the spliceosome by the NineTeen Complex (NTC/Prp19 complex) via Ntc90 after Prp2-mediated catalytic activation; it directly contacts U2 snRNA and positions the U2/BPS duplex near the 5' splice site to place the branch-point nucleophile adjacent to the catalytic metal, thereby promoting the first transesterification (branching) reaction, after which Prp16-mediated ATP hydrolysis remodels Yju2 binding and releases it (along with Cwc25) from the active site to permit Fyv6 binding and progression to the second catalytic step (exon ligation)."},"narrative":{"mechanistic_narrative":"Yju2 (human CCDC94) is an essential pre-mRNA splicing factor that promotes the first catalytic step (branching) of the spliceosomal reaction [PMID:19704000]. After Prp2-mediated catalytic activation generates high-affinity binding sites on the B* complex [PMID:22535589], Yju2 is recruited to the spliceosome by the NineTeen Complex (NTC), a function requiring the NTC component Ntc90 and acting downstream of activation rather than in activation itself [PMID:19617314, PMID:33705709]. Once bound, Yju2 directly contacts U2 snRNA at helix II and the branch-binding region, and structurally it brings the U2/branch-point-sequence duplex into the vicinity of the 5' splice site, positioning the branch-point nucleophile ~4 Å from catalytic metal M2 to enable the first transesterification [PMID:23438600, PMID:30879786]; structures of the C complex show it stabilizing branch-helix docking at the Prp8 catalytic cavity alongside Cwc25 and Isy1 [PMID:27459055, PMID:27445308]. Yju2 has a modular architecture in which the N-terminal half supports the first step while the C-terminal half mediates tight spliceosome association [PMID:23438600]. Progression to the second step requires Prp16- and ATP-dependent remodeling of Yju2 binding, which vacates the active site and is coupled to mutually exclusive exchange of Yju2 for the second-step factor Fyv6 [PMID:21098140, PMID:27980089, PMID:39688371]; CCDC94 bridges Prp16 to the active-site RNA during this C-to-C* transition [PMID:29301961]. Beyond its splicing role, CCDC94/Prp19 complex localizes to the nucleus and restrains the G1-to-S transition, and protects cells from ionizing-radiation-induced apoptosis by repressing p53 mRNA [PMID:22952453, PMID:16450356].","teleology":[{"year":2009,"claim":"Established that Yju2 is an essential first-step splicing factor acting after Prp2 to juxtapose the 5' splice site and branch point, defining where in the catalytic cycle it operates.","evidence":"Depletion/reconstitution with recombinant protein in yeast splicing extracts, with ATP-independent rescue","pmids":["19704000"],"confidence":"High","gaps":["Did not resolve how Yju2 is recruited","No structural basis for substrate juxtaposition"]},{"year":2009,"claim":"Identified the recruitment mechanism by showing the NTC component Ntc90 is specifically required to load Yju2 onto the activated spliceosome, distinguishing recruitment from activation.","evidence":"Biochemical fractionation, genetic deletion, and reconstitution in yeast extracts","pmids":["19617314"],"confidence":"High","gaps":["Direct Ntc90-Yju2 contact not visualized","Order relative to Prp2 action not fully defined"]},{"year":2010,"claim":"Showed that Yju2 (with Cwc25) is actively released after step 1 in a Prp16/ATP-dependent manner, establishing release as a prerequisite for the second catalytic step.","evidence":"Biochemical splicing assays with ATP/AMP-PNP and factor addition/depletion in yeast","pmids":["21098140"],"confidence":"High","gaps":["Molecular basis of Prp16-driven destabilization unknown","Fate of Yju2 after release not tracked"]},{"year":2012,"claim":"Demonstrated that the high-affinity Yju2 binding site is created during Prp2-mediated B* formation, linking catalytic activation to factor recruitment quantitatively.","evidence":"Dual-color fluorescence cross-correlation spectroscopy on purified yeast spliceosomes","pmids":["22535589"],"confidence":"High","gaps":["Did not map the structural elements forming the site","Kinetics of in vivo binding not addressed"]},{"year":2012,"claim":"Extended the function to the human ortholog CCDC94 as a Prp19-complex member with a cellular role in suppressing IR-induced apoptosis via p53 mRNA repression.","evidence":"Zebrafish forward genetic screen, knockdown, RT-PCR for p53 mRNA, rescue","pmids":["22952453"],"confidence":"Medium","gaps":["Mechanistic link between splicing role and p53 repression unresolved","Single lab, organism-specific readout"]},{"year":2013,"claim":"Resolved the domain architecture and direct RNA contacts, showing the N-half drives the first step while the C-half confers tight binding, and that Yju2 contacts U2 snRNA helix II and the branch-binding region.","evidence":"Domain dissection with in vivo/in vitro reconstitution, UV cross-linking to U2 snRNA, ATPase bypass assays","pmids":["23438600"],"confidence":"High","gaps":["Atomic geometry of contacts not yet defined","Crosslink residues not mapped to specific protein regions"]},{"year":2016,"claim":"Cryo-EM of yeast C and C* complexes placed Yju2 structurally, showing it stabilizes branch-helix docking at the Prp8 catalytic cavity for step 1 and dissociates from the active site in C* to admit the 3' exon.","evidence":"Cryo-EM of yeast C complex (3.8 Å, 3.4 Å) and C* complex (4.0 Å)","pmids":["27459055","27445308","27980089"],"confidence":"High","gaps":["Pre-branching B* positioning not yet captured","Recruitment trajectory not resolved structurally"]},{"year":2018,"claim":"Human C-complex structure showed CCDC94 bridges Prp16 to active-site RNA, mechanistically connecting the helicase to the C-to-C* transition in human spliceosomes.","evidence":"Cryo-EM of human C complex at 4.1 Å","pmids":["29301961"],"confidence":"High","gaps":["Dynamics of Prp16-driven remodeling inferred, not directly time-resolved"]},{"year":2019,"claim":"B* structures directly demonstrated the catalytic role: Yju2 recruitment brings the U2/BPS duplex toward the 5'SS and positions the branch nucleophile ~4 Å from metal M2.","evidence":"Cryo-EM of four yeast B* complexes (2.9–3.8 Å) with comparative analysis ±Yju2/Cwc25","pmids":["30879786"],"confidence":"High","gaps":["Transition kinetics from recruitment to nucleophile positioning not resolved"]},{"year":2021,"claim":"Integrated structure plus biochemistry showed NTC recruits Yju2 before branching and that Prp16 remodels (rather than fully ejects) Yju2, which stays tethered to the NTC in C* to promote exon ligation; co-transcriptional recruitment depends on Rat1.","evidence":"Cryo-EM at 2.8 Å with biochemical C/C* assays; Co-IP/MS and ChIP-Seq for Rat1 interaction in yeast","pmids":["33705709","33978753"],"confidence":"High","gaps":["Rat1-Yju2 interaction rests on a single Co-IP/MS without reciprocal validation","Functional contribution of NTC-tethered Yju2 to step 2 not fully dissected"]},{"year":2024,"claim":"Defined the step-1-to-step-2 handoff by showing Yju2 and the second-step factor Fyv6 bind mutually exclusively, so Yju2/Fyv6 exchange drives progression to exon ligation.","evidence":"Cryo-EM of yeast product complex at 2.3 Å with genetic suppressor screen and transcriptomics","pmids":["39688371"],"confidence":"High","gaps":["Trigger that times the exchange not defined","Whether NTC-tethered Yju2 directly hands off to Fyv6 unresolved"]},{"year":2025,"claim":"Computational dynamics proposed temporally ordered active-site remodeling by Yju2 with Prp11 and Prp8 to enable step 1.","evidence":"Equilibrium and enhanced-sampling molecular dynamics simulations (>2M atoms) of spliceosome structures","pmids":["41474748"],"confidence":"Low","gaps":["Computational only, no experimental validation of Yju2-specific dynamics","Predicted residue-level remodeling not tested by mutagenesis"]},{"year":null,"claim":"How the human cellular phenotypes of CCDC94 (G1/S restraint and p53-dependent apoptosis protection) mechanistically connect to its conserved spliceosomal branching function remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No molecular link established between splicing role and p53 mRNA repression","Whether specific intron substrates underlie the cell-cycle phenotype is unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[5,11]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,7,11]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[6]}],"complexes":["NineTeen Complex (NTC/Prp19 complex)","C complex spliceosome","B* complex spliceosome"],"partners":["CWC25","NTC90","PRP16","ISY1","FYV6","RAT1","PRP8"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BW85","full_name":"Splicing factor YJU2","aliases":["Coiled-coil domain-containing protein 94"],"length_aa":323,"mass_kda":37.1,"function":"Part of the spliceosome which catalyzes two sequential transesterification reactions, first the excision of the non-coding intron from pre-mRNA and then the ligation of the coding exons to form the mature mRNA (PubMed:29301961). Plays a role in stabilizing the structure of the spliceosome catalytic core and docking of the branch helix into the active site, producing 5'-exon and lariat intron-3'-intermediates (By similarity). May protect cells from TP53-dependent apoptosis upon dsDNA break damage through association with PRP19-CD5L complex (PubMed:22952453)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9BW85/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/YJU2","classification":"Common Essential","n_dependent_lines":1206,"n_total_lines":1208,"dependency_fraction":0.9983443708609272},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/YJU2","total_profiled":1310},"omim":[{"mim_id":"621530","title":"CWC25, SPLICEOSOME-ASSOCIATED PROTEIN; CWC25","url":"https://www.omim.org/entry/621530"},{"mim_id":"605584","title":"DEAH-BOX HELICASE 38; DHX38","url":"https://www.omim.org/entry/605584"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/YJU2"},"hgnc":{"alias_symbol":["FLJ10374"],"prev_symbol":["CCDC94"]},"alphafold":{"accession":"Q9BW85","domains":[{"cath_id":"-","chopping":"32-108","consensus_level":"high","plddt":93.6512,"start":32,"end":108},{"cath_id":"-","chopping":"110-193","consensus_level":"medium","plddt":86.3358,"start":110,"end":193}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BW85","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BW85-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BW85-F1-predicted_aligned_error_v6.png","plddt_mean":73.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=YJU2","jax_strain_url":"https://www.jax.org/strain/search?query=YJU2"},"sequence":{"accession":"Q9BW85","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BW85.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BW85/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BW85"}},"corpus_meta":[{"pmid":"27459055","id":"PMC_27459055","title":"Cryo-EM structure of the spliceosome immediately after branching.","date":"2016","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/27459055","citation_count":198,"is_preprint":false},{"pmid":"27445308","id":"PMC_27445308","title":"Structure of a yeast catalytic step I spliceosome at 3.4 Å resolution.","date":"2016","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/27445308","citation_count":158,"is_preprint":false},{"pmid":"27980089","id":"PMC_27980089","title":"Structure of a yeast step II catalytically activated spliceosome.","date":"2016","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/27980089","citation_count":121,"is_preprint":false},{"pmid":"29301961","id":"PMC_29301961","title":"Structure of a human catalytic step I spliceosome.","date":"2018","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/29301961","citation_count":119,"is_preprint":false},{"pmid":"21098140","id":"PMC_21098140","title":"DEAH-box ATPase Prp16 has dual roles in remodeling of the spliceosome in catalytic steps.","date":"2010","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/21098140","citation_count":84,"is_preprint":false},{"pmid":"22535589","id":"PMC_22535589","title":"Prp2-mediated protein rearrangements at the catalytic core of the spliceosome as revealed by dcFCCS.","date":"2012","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/22535589","citation_count":75,"is_preprint":false},{"pmid":"19704000","id":"PMC_19704000","title":"Cwc25 is a novel splicing factor required after Prp2 and Yju2 to facilitate the first catalytic reaction.","date":"2009","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/19704000","citation_count":71,"is_preprint":false},{"pmid":"30879786","id":"PMC_30879786","title":"Structures of the Catalytically Activated Yeast Spliceosome Reveal the Mechanism of 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biology","url":"https://pubmed.ncbi.nlm.nih.gov/38746449","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11674,"output_tokens":3684,"usd":0.045141,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11238,"output_tokens":3694,"usd":0.07427,"stage2_stop_reason":"end_turn"},"total_usd":0.119411,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"Yju2 is required for the first catalytic step of splicing after the action of Prp2, functioning at the final step of the first transesterification reaction. Cwc25 acts after Yju2 in the same step, and both are required for promoting juxtaposition of the 5' splice site and branch point.\",\n      \"method\": \"Depletion/reconstitution assay in yeast splicing extracts; ATP-independent rescue with recombinant protein\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — affinity-purified spliceosome depletion and reconstitution assay, replicated across multiple studies in the corpus\",\n      \"pmids\": [\"19704000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Ntc90 (a component of the NineTeen Complex/NTC) is specifically required for recruiting Yju2 to the spliceosome after activation, but not for spliceosome activation itself, revealing a novel NTC role in recruiting first-step splicing factors.\",\n      \"method\": \"Biochemical fractionation, genetic deletion analysis, and reconstitution in yeast splicing extracts\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical and genetic methods in same study, single lab but two orthogonal approaches\",\n      \"pmids\": [\"19617314\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Yju2 and Cwc25 are released from the spliceosome after the first catalytic step in a Prp16- and ATP-dependent manner, and this release is necessary to allow progression to the second catalytic reaction.\",\n      \"method\": \"Biochemical splicing assays with ATP/AMP-PNP, spliceosome purification, and factor addition/depletion in yeast extracts\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple biochemical assays, replicated finding confirmed by subsequent structural studies\",\n      \"pmids\": [\"21098140\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"High-affinity binding sites for Yju2 and Cwc25 are created on the spliceosome during Prp2-mediated catalytic activation (B* complex formation), consistent with their requirement for step 1 catalysis.\",\n      \"method\": \"Dual-color fluorescence cross-correlation spectroscopy (dcFCCS) on purified yeast spliceosomes\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative biophysical measurement (dcFCCS) in near-physiological conditions, single lab\",\n      \"pmids\": [\"22535589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Ccdc94 (human ortholog of Yju2) is a functional member of the Prp19 complex, and knockdown of Prp19 complex members increases sensitivity to ionizing radiation-induced apoptosis; Ccdc94/Prp19 complex protects cells from IR-induced apoptosis by repressing p53 mRNA expression.\",\n      \"method\": \"Zebrafish forward genetic screen, genetic knockdown, RT-PCR for p53 mRNA levels, rescue experiments\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic screen plus knockdown with specific molecular readout (p53 mRNA repression), single lab\",\n      \"pmids\": [\"22952453\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Yju2 has a modular structure: the N-terminal half (Yju2-N) weakly binds the spliceosome and functions in the first step, while the C-terminal half (Yju2-C) stabilizes spliceosome association. Yju2-N alone supports a low level of the second reaction even without Prp16, suggesting that Prp16 acts primarily by destabilizing the C-domain-dependent tight spliceosome binding of Yju2. UV cross-linking shows Yju2 directly contacts U2 snRNA in helix II both pre- and post-catalytically, and contacts the branch-binding region only pre-catalytically.\",\n      \"method\": \"Domain dissection with in vivo and in vitro reconstitution, UV cross-linking to U2 snRNA, ATPase bypass assays in yeast\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution, domain mutagenesis, direct RNA cross-linking, multiple orthogonal methods in single study\",\n      \"pmids\": [\"23438600\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"FLJ10374 (YJU2/CCDC94) localizes exclusively to the nucleus and negatively regulates cell cycle G1-to-S transition; siRNA knockdown increases cell proliferation.\",\n      \"method\": \"Transfection with fluorescence localization, serum-starvation cell cycle assay, siRNA knockdown with proliferation readout\",\n      \"journal\": \"Genes, chromosomes & cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct localization experiment and functional cell cycle readout, but single lab and limited mechanistic depth\",\n      \"pmids\": [\"16450356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cryo-EM structure of the C complex (post-branching) spliceosome at 3.8 Å shows that Yju2 (along with Isy1 and Cwc25) stabilizes docking of the branch helix, positioning the branch adenosine in the active site and facilitating the first transesterification reaction.\",\n      \"method\": \"Cryo-electron microscopy at 3.8 Å resolution of yeast C complex spliceosome\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structural determination, replicated by independent lab in same year\",\n      \"pmids\": [\"27459055\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cryo-EM structure at 3.4 Å of the yeast C complex confirms Yju2 is one of 15 protein components that stabilize specific placement of RNA elements (5'SS, BPS, U5 loop I) at the Prp8 catalytic cavity after the first transesterification reaction.\",\n      \"method\": \"Cryo-electron microscopy at 3.4 Å resolution of yeast C complex spliceosome\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic cryo-EM structure, independently replicated by Galej et al. same year\",\n      \"pmids\": [\"27445308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cryo-EM structure of the C* complex shows that the step I splicing factors Cwc25 and Yju2 have dissociated from the active site during the C-to-C* transition, vacating space for the incoming 3'-exon sequences required for the second transesterification.\",\n      \"method\": \"Cryo-electron microscopy at 4.0 Å resolution of yeast C* complex spliceosome\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structural determination showing direct absence of Yju2 in C*, corroborating biochemical data\",\n      \"pmids\": [\"27980089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cryo-EM structure of the human C complex at 4.1 Å shows that CCDC94 (human Yju2) closely interacts with the DEAH-family ATPase/helicase Prp16 and bridges the gap between Prp16 and the active-site RNA elements, providing mechanistic insight into the C-to-C* transition.\",\n      \"method\": \"Cryo-electron microscopy at 4.1 Å resolution of human C complex spliceosome\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure of human complex, direct visualization of CCDC94-Prp16 interaction\",\n      \"pmids\": [\"29301961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cryo-EM structures of four B* complexes at 2.9–3.8 Å reveal that Yju2 recruitment into the active site brings the U2/BPS duplex into the vicinity of the 5' splice site, positioning the BPS nucleophile 4 Å from catalytic metal M2, directly demonstrating Yju2's mechanistic role in promoting the branching reaction.\",\n      \"method\": \"Cryo-electron microscopy of four yeast B* complexes at 2.9–3.8 Å, comparative structural analysis with and without Yju2/Cwc25\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple near-atomic cryo-EM structures directly demonstrating Yju2-dependent positioning of branch nucleophile\",\n      \"pmids\": [\"30879786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structure at 2.8 Å of the yeast C complex reveals that Yju2 and Isy1 are recruited to the spliceosome by the NTC before branching. After branching, Prp16 remodels Yju2 binding, allowing Yju2 to remain tethered to the NTC in the C* complex to promote exon ligation.\",\n      \"method\": \"Cryo-EM at 2.8 Å combined with biochemical assays for C/C* complex dynamics\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM plus biochemical assays, mechanistically resolves NTC-mediated Yju2 recruitment and Prp16-mediated remodeling\",\n      \"pmids\": [\"33705709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Rat1 exoribonuclease physically interacts with the Yju2 splicing factor (co-immunoprecipitation and mass spectrometry), and is required for proper co-transcriptional recruitment of splicing factors including Yju2 to introns.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, ChIP-Seq in yeast\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP/MS interaction, single lab, no deep mechanistic follow-up specific to Yju2\",\n      \"pmids\": [\"33978753\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structure of a yeast product complex spliceosome at 2.3 Å shows that binding of the second-step factor Fyv6 to the spliceosome is mutually exclusive with that of the first-step factor Yju2, and that Yju2/Fyv6 exchange facilitates progression from the first to the second step of splicing.\",\n      \"method\": \"Cryo-EM at 2.3 Å, genetic suppressor screen, transcriptomics\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-atomic cryo-EM structure directly showing mutually exclusive binding, supported by genetic and transcriptomic data\",\n      \"pmids\": [\"39688371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Molecular dynamics simulations integrating cryo-EM data reveal that Yju2 acts in a temporally ordered fashion with Prp11 and Prp8 at the spliceosome core, with positively charged residues in these proteins dynamically remodeling the active site to enable the first step of splicing.\",\n      \"method\": \"Equilibrium and enhanced sampling molecular dynamics simulations (>2M atoms) of multiple spliceosome structures\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational simulation only, no direct experimental validation of Yju2-specific claims\",\n      \"pmids\": [\"41474748\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Yju2 (CCDC94 in humans) is an essential first-step splicing factor that is recruited to the spliceosome by the NineTeen Complex (NTC/Prp19 complex) via Ntc90 after Prp2-mediated catalytic activation; it directly contacts U2 snRNA and positions the U2/BPS duplex near the 5' splice site to place the branch-point nucleophile adjacent to the catalytic metal, thereby promoting the first transesterification (branching) reaction, after which Prp16-mediated ATP hydrolysis remodels Yju2 binding and releases it (along with Cwc25) from the active site to permit Fyv6 binding and progression to the second catalytic step (exon ligation).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"Yju2 (human CCDC94) is an essential pre-mRNA splicing factor that promotes the first catalytic step (branching) of the spliceosomal reaction [#0]. After Prp2-mediated catalytic activation generates high-affinity binding sites on the B* complex [#3], Yju2 is recruited to the spliceosome by the NineTeen Complex (NTC), a function requiring the NTC component Ntc90 and acting downstream of activation rather than in activation itself [#1, #12]. Once bound, Yju2 directly contacts U2 snRNA at helix II and the branch-binding region, and structurally it brings the U2/branch-point-sequence duplex into the vicinity of the 5' splice site, positioning the branch-point nucleophile ~4 Å from catalytic metal M2 to enable the first transesterification [#5, #11]; structures of the C complex show it stabilizing branch-helix docking at the Prp8 catalytic cavity alongside Cwc25 and Isy1 [#7, #8]. Yju2 has a modular architecture in which the N-terminal half supports the first step while the C-terminal half mediates tight spliceosome association [#5]. Progression to the second step requires Prp16- and ATP-dependent remodeling of Yju2 binding, which vacates the active site and is coupled to mutually exclusive exchange of Yju2 for the second-step factor Fyv6 [#2, #9, #14]; CCDC94 bridges Prp16 to the active-site RNA during this C-to-C* transition [#10]. Beyond its splicing role, CCDC94/Prp19 complex localizes to the nucleus and restrains the G1-to-S transition, and protects cells from ionizing-radiation-induced apoptosis by repressing p53 mRNA [#4, #6].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established that Yju2 is an essential first-step splicing factor acting after Prp2 to juxtapose the 5' splice site and branch point, defining where in the catalytic cycle it operates.\",\n      \"evidence\": \"Depletion/reconstitution with recombinant protein in yeast splicing extracts, with ATP-independent rescue\",\n      \"pmids\": [\"19704000\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how Yju2 is recruited\", \"No structural basis for substrate juxtaposition\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified the recruitment mechanism by showing the NTC component Ntc90 is specifically required to load Yju2 onto the activated spliceosome, distinguishing recruitment from activation.\",\n      \"evidence\": \"Biochemical fractionation, genetic deletion, and reconstitution in yeast extracts\",\n      \"pmids\": [\"19617314\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Ntc90-Yju2 contact not visualized\", \"Order relative to Prp2 action not fully defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed that Yju2 (with Cwc25) is actively released after step 1 in a Prp16/ATP-dependent manner, establishing release as a prerequisite for the second catalytic step.\",\n      \"evidence\": \"Biochemical splicing assays with ATP/AMP-PNP and factor addition/depletion in yeast\",\n      \"pmids\": [\"21098140\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of Prp16-driven destabilization unknown\", \"Fate of Yju2 after release not tracked\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrated that the high-affinity Yju2 binding site is created during Prp2-mediated B* formation, linking catalytic activation to factor recruitment quantitatively.\",\n      \"evidence\": \"Dual-color fluorescence cross-correlation spectroscopy on purified yeast spliceosomes\",\n      \"pmids\": [\"22535589\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map the structural elements forming the site\", \"Kinetics of in vivo binding not addressed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended the function to the human ortholog CCDC94 as a Prp19-complex member with a cellular role in suppressing IR-induced apoptosis via p53 mRNA repression.\",\n      \"evidence\": \"Zebrafish forward genetic screen, knockdown, RT-PCR for p53 mRNA, rescue\",\n      \"pmids\": [\"22952453\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link between splicing role and p53 repression unresolved\", \"Single lab, organism-specific readout\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved the domain architecture and direct RNA contacts, showing the N-half drives the first step while the C-half confers tight binding, and that Yju2 contacts U2 snRNA helix II and the branch-binding region.\",\n      \"evidence\": \"Domain dissection with in vivo/in vitro reconstitution, UV cross-linking to U2 snRNA, ATPase bypass assays\",\n      \"pmids\": [\"23438600\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic geometry of contacts not yet defined\", \"Crosslink residues not mapped to specific protein regions\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Cryo-EM of yeast C and C* complexes placed Yju2 structurally, showing it stabilizes branch-helix docking at the Prp8 catalytic cavity for step 1 and dissociates from the active site in C* to admit the 3' exon.\",\n      \"evidence\": \"Cryo-EM of yeast C complex (3.8 Å, 3.4 Å) and C* complex (4.0 Å)\",\n      \"pmids\": [\"27459055\", \"27445308\", \"27980089\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Pre-branching B* positioning not yet captured\", \"Recruitment trajectory not resolved structurally\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Human C-complex structure showed CCDC94 bridges Prp16 to active-site RNA, mechanistically connecting the helicase to the C-to-C* transition in human spliceosomes.\",\n      \"evidence\": \"Cryo-EM of human C complex at 4.1 Å\",\n      \"pmids\": [\"29301961\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of Prp16-driven remodeling inferred, not directly time-resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"B* structures directly demonstrated the catalytic role: Yju2 recruitment brings the U2/BPS duplex toward the 5'SS and positions the branch nucleophile ~4 Å from metal M2.\",\n      \"evidence\": \"Cryo-EM of four yeast B* complexes (2.9–3.8 Å) with comparative analysis ±Yju2/Cwc25\",\n      \"pmids\": [\"30879786\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Transition kinetics from recruitment to nucleophile positioning not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Integrated structure plus biochemistry showed NTC recruits Yju2 before branching and that Prp16 remodels (rather than fully ejects) Yju2, which stays tethered to the NTC in C* to promote exon ligation; co-transcriptional recruitment depends on Rat1.\",\n      \"evidence\": \"Cryo-EM at 2.8 Å with biochemical C/C* assays; Co-IP/MS and ChIP-Seq for Rat1 interaction in yeast\",\n      \"pmids\": [\"33705709\", \"33978753\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Rat1-Yju2 interaction rests on a single Co-IP/MS without reciprocal validation\", \"Functional contribution of NTC-tethered Yju2 to step 2 not fully dissected\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the step-1-to-step-2 handoff by showing Yju2 and the second-step factor Fyv6 bind mutually exclusively, so Yju2/Fyv6 exchange drives progression to exon ligation.\",\n      \"evidence\": \"Cryo-EM of yeast product complex at 2.3 Å with genetic suppressor screen and transcriptomics\",\n      \"pmids\": [\"39688371\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger that times the exchange not defined\", \"Whether NTC-tethered Yju2 directly hands off to Fyv6 unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Computational dynamics proposed temporally ordered active-site remodeling by Yju2 with Prp11 and Prp8 to enable step 1.\",\n      \"evidence\": \"Equilibrium and enhanced-sampling molecular dynamics simulations (>2M atoms) of spliceosome structures\",\n      \"pmids\": [\"41474748\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Computational only, no experimental validation of Yju2-specific dynamics\", \"Predicted residue-level remodeling not tested by mutagenesis\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the human cellular phenotypes of CCDC94 (G1/S restraint and p53-dependent apoptosis protection) mechanistically connect to its conserved spliceosomal branching function remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No molecular link established between splicing role and p53 mRNA repression\", \"Whether specific intron substrates underlie the cell-cycle phenotype is unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [5, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 7, 11]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [\"NineTeen Complex (NTC/Prp19 complex)\", \"C complex spliceosome\", \"B* complex spliceosome\"],\n    \"partners\": [\"Cwc25\", \"Ntc90\", \"Prp16\", \"Isy1\", \"Fyv6\", \"Rat1\", \"Prp8\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}