{"gene":"CWC25","run_date":"2026-06-09T22:57:19","timeline":{"discoveries":[{"year":2009,"finding":"Cwc25 is required for the first catalytic step (step 1) of pre-mRNA splicing. In an in vitro reconstitution system, spliceosomes stalled before step 1 (purified from a Prp2 temperature-sensitive mutant) catalyzed efficient step 1 when supplemented with recombinant Prp2, Spp2, and Cwc25, demonstrating Cwc25's previously unknown role in promoting step 1.","method":"In vitro splicing reconstitution with purified spliceosomal components and recombinant proteins","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical reconstitution with purified components, replicated across multiple subsequent studies","pmids":["19935684"],"is_preprint":false},{"year":2009,"finding":"Cwc25 is not tightly associated with the NTC (Prp19-associated complex) and is not required for spliceosome activation, but functions in the final step of the first catalytic reaction after the action of Prp2, in an ATP-independent manner. Depleted spliceosomes could be chased into splicing intermediates by addition of recombinant Cwc25 without ATP.","method":"Affinity purification of spliceosomes from Cwc25-depleted extracts, complementation with recombinant Cwc25","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical reconstitution with defined factors, ATP-independence demonstrated directly, replicated by other labs","pmids":["19704000"],"is_preprint":false},{"year":2010,"finding":"Cwc25 and Yju2 are released from the spliceosome after the first catalytic reaction in a Prp16- and ATP-dependent manner. Binding of Cwc25 to the spliceosome is destabilized by mutations at the branchpoint sequence, suggesting Cwc25 contacts the branch site region.","method":"In vitro splicing assays with branchpoint mutant pre-mRNAs, spliceosome purification, ATP-dependence experiments","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal in vitro assays, replicated by subsequent structural and biochemical studies","pmids":["21098140"],"is_preprint":false},{"year":2012,"finding":"High-affinity binding sites for Cwc25 are created on the spliceosome during Prp2-mediated catalytic activation (transition from Bact to B* complex), consistent with Cwc25's requirement for step 1 catalysis. Cwc25 binding is enhanced after Prp2-mediated remodeling.","method":"Dual-color fluorescence cross-correlation spectroscopy (dcFCCS) on purified yeast spliceosomes","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — quantitative biophysical measurement (dcFCCS) on purified complexes, single lab with rigorous controls","pmids":["22535589"],"is_preprint":false},{"year":2013,"finding":"Cwc25 acts as a 'pawl' in a biased Brownian ratchet mechanism: after Prp2/Spp2/NTP rearranges the spliceosome to reversibly explore conformations with proximal 5' splice site and branch point, addition of Cwc25 strongly biases the equilibrium toward the proximal (reactive) conformation, promoting efficient first-step splicing.","method":"Single-molecule FRET on affinity-purified spliceosomes stalled by Prp2 mutation, with addition of recombinant factors","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — single-molecule FRET reconstitution with purified factors, directly monitors conformational dynamics","pmids":["24240612"],"is_preprint":false},{"year":2015,"finding":"Cwc25 contacts the branch-site region of the pre-mRNA after step 1 catalysis, as shown by UV-induced crosslinking. However, Cwc25's step 1-promoting activity was not dependent on its direct interaction with pre-mRNA, indicating it acts via protein-protein interactions rather than direct RNA contacts.","method":"UV-induced crosslinking of purified yeast spliceosomes formed on site-specifically labeled pre-mRNA, analyzed across B(act), B*, and C complexes","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct UV crosslinking with site-specifically labeled pre-mRNA in defined purified complexes, functional dissection of RNA vs. protein contacts","pmids":["26393790"],"is_preprint":false},{"year":2016,"finding":"Cryo-EM structure of the yeast C complex (step I spliceosome) at 3.4 Å shows Cwc25 as one of 15 protein components stabilizing the catalytic cavity of Prp8, with the branch adenosine covalently joined to the 5' splice site phosphate. Cwc25 participates in stabilizing specific placement of RNA elements at the catalytic cavity.","method":"Cryo-electron microscopy structure determination at 3.4 Å resolution","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-atomic resolution cryo-EM structure directly showing Cwc25 position in the catalytic complex","pmids":["27445308"],"is_preprint":false},{"year":2016,"finding":"Cryo-EM structure of the spliceosome immediately after branching (C complex) at 3.8 Å shows that Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix into the active site, with non-Watson-Crick interactions between the branch helix and the 5' splice site docking the branch adenosine into the active site.","method":"Cryo-electron microscopy structure determination at 3.8 Å resolution","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-atomic resolution cryo-EM structure directly showing Cwc25 stabilizing the branch helix","pmids":["27459055"],"is_preprint":false},{"year":2016,"finding":"In the C* complex (step II catalytically activated spliceosome), step I splicing factors Cwc25 and Yju2 have been dissociated from the active site compared to the C complex, consistent with Prp16-mediated removal being required for the second transesterification step.","method":"Cryo-electron microscopy structure of C* complex at 4.0 Å resolution","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-atomic resolution cryo-EM structure directly showing Cwc25 absence in C* complex","pmids":["27980089"],"is_preprint":false},{"year":2017,"finding":"Structural comparison of C and C* complexes reveals that branching-specific factors such as Cwc25 lock the branch helix into position for nucleophilic attack of the branch adenosine at the 5' splice site in the C complex, and that Prp16-mediated remodeling destabilizes these factors and releases the branch helix from the active site.","method":"Cryo-EM structure of C* complex at 3.8 Å, compared with C complex structure","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-atomic resolution cryo-EM with direct structural comparison of successive complexes","pmids":["28076345"],"is_preprint":false},{"year":2017,"finding":"Cwc25 plays a central role in modulating the conformational state of the catalytic spliceosome: it binds tightly after the first reaction and stabilizes the first-step conformation. When Cwc25 is absent from the purified first-step spliceosome, both forward and reverse splicing reactions (debranching, spliced-exon-reopening) occur under normal splicing conditions without energy. Adding Cwc25 back inhibits both reactions by stabilizing the first-step conformation. Prp16 is dispensable for the second reaction under conditions that destabilize Cwc25.","method":"In vitro splicing assays with purified spliceosomes under varied ionic conditions, with and without recombinant Cwc25","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal biochemical assays (forward splicing, reverse splicing, debranching) with direct add-back experiments","pmids":["28057857"],"is_preprint":false},{"year":2018,"finding":"In the human C complex (step I spliceosome), the human ortholog of Cwc25 (CCDC49) closely interacts with the DEAH-family ATPase/helicase Prp16 and bridges the gap between Prp16 and the active-site RNA elements. Structural comparison of human C and C* complexes provides mechanistic insights into how CCDC49/Cwc25 is remodeled during the C-to-C* transition.","method":"Cryo-EM structure of human C complex at 4.1 Å resolution","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-atomic resolution cryo-EM of human spliceosome directly showing CCDC49 (CWC25) interactions with Prp16 and active-site RNA","pmids":["29301961"],"is_preprint":false},{"year":2019,"finding":"Cryo-EM structures of four distinct yeast B* complexes reveal that in B* complexes devoid of Yju2 and Cwc25, the U2/BPS duplex is discretely away from the 5' splice site. Recruitment of Yju2 brings the U2/BPS duplex into the vicinity of the 5' splice site, positioning the BPS nucleophile 4 Å from catalytic metal M2. This analysis reveals the functional mechanism: Cwc25 (together with Yju2) promotes branching by bringing the branch point nucleophile into proximity of the 5' splice site.","method":"Cryo-EM structures of four B* complexes at 2.9–3.8 Å resolution","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple near-atomic resolution cryo-EM structures of complexes with and without Cwc25/Yju2, directly revealing mechanism of branch point positioning","pmids":["30879786"],"is_preprint":false},{"year":2023,"finding":"Prp16's ATP-independent function in promoting first-step splicing of aberrant 5' splice sites and mutated branchpoints is mediated through the step-one factor Cwc25, establishing Cwc25 as an effector through which Prp16 exerts dual roles in splice site selection.","method":"In vitro splicing assays with mutant pre-mRNAs, genetic and biochemical epistasis between Prp16 and Cwc25","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and biochemical epistasis, single lab, multiple substrate contexts but no structural resolution of mechanism","pmids":["37858289"],"is_preprint":false}],"current_model":"CWC25 (human: CCDC49) is a step I-specific splicing factor that is recruited to the spliceosome after Prp2-mediated remodeling exposes high-affinity binding sites; it acts as a conformational 'pawl' that, together with Yju2, docks the branch helix into the active site and positions the branch-point adenosine nucleophile for the first transesterification reaction via protein-protein interactions rather than direct RNA contacts, after which it is removed by Prp16-mediated ATP hydrolysis to allow the conformational changes required for the second catalytic step."},"narrative":{"mechanistic_narrative":"CWC25 (human CCDC49) is a step I-specific pre-mRNA splicing factor that promotes the first transesterification reaction by stabilizing the catalytic conformation of the spliceosome [PMID:19935684, PMID:19704000]. It is not a constitutive component of the NTC and is dispensable for spliceosome activation; instead, it acts after Prp2-mediated remodeling, which creates high-affinity Cwc25 binding sites during the Bact-to-B* transition, and it promotes step 1 in an ATP-independent manner [PMID:19704000, PMID:22535589]. Mechanistically, Cwc25 functions as a conformational 'pawl' in a biased Brownian ratchet: it shifts the reversibly fluctuating spliceosome toward the reactive conformation in which the 5' splice site and branch point are proximal, and together with Yju2 it docks the branch helix into the active site and positions the branch-point adenosine nucleophile within striking distance of the catalytic metal for nucleophilic attack [PMID:24240612, PMID:27459055, PMID:30879786]. This positioning is achieved through protein-protein interactions rather than direct pre-mRNA contacts, although Cwc25 does crosslink to the branch-site region after catalysis [PMID:26393790]. By locking the branch helix in place, Cwc25 stabilizes the first-step conformation and suppresses both forward and reverse reactions until it is destabilized; its removal is required to permit the conformational changes of the second catalytic step [PMID:28057857, PMID:27980089, PMID:28076345]. In the human spliceosome CCDC49 bridges the DEAH-box ATPase Prp16 and the active-site RNA, and Prp16-mediated ATP hydrolysis dissociates Cwc25 during the C-to-C* transition [PMID:29301961, PMID:21098140]. Cwc25 also serves as the effector through which Prp16 exerts its ATP-independent role in proofreading aberrant 5' splice sites and mutated branchpoints [PMID:37858289].","teleology":[{"year":2009,"claim":"Established that an uncharacterized factor is specifically required for the first catalytic step of splicing, defining Cwc25's core function.","evidence":"In vitro splicing reconstitution of Prp2-stalled spliceosomes complemented with recombinant Prp2, Spp2, and Cwc25; and Cwc25-depletion/add-back with ATP-independence demonstrated","pmids":["19935684","19704000"],"confidence":"High","gaps":["Did not resolve whether Cwc25 contacts RNA or acts purely through protein interactions","Structural position within the spliceosome unknown"]},{"year":2010,"claim":"Defined the temporal placement of Cwc25 action by showing it and Yju2 are released after step 1 and that its binding depends on the branchpoint sequence.","evidence":"In vitro splicing with branchpoint mutant pre-mRNAs and Prp16/ATP-dependent release assays","pmids":["21098140"],"confidence":"High","gaps":["Whether branchpoint dependence reflects direct RNA contact or conformational sensing unresolved"]},{"year":2012,"claim":"Showed that the requirement for prior Prp2 remodeling is explained by creation of high-affinity Cwc25 binding sites, coupling activation to step 1 recruitment.","evidence":"Dual-color fluorescence cross-correlation spectroscopy on purified yeast spliceosomes","pmids":["22535589"],"confidence":"High","gaps":["Molecular identity of the created binding surface not defined"]},{"year":2013,"claim":"Provided the mechanistic model: Cwc25 acts as a 'pawl' that biases a reversible conformational equilibrium toward the reactive proximal state.","evidence":"Single-molecule FRET on Prp2-stalled spliceosomes with recombinant factor add-back","pmids":["24240612"],"confidence":"High","gaps":["Did not show atomic basis of how Cwc25 biases the equilibrium"]},{"year":2015,"claim":"Dissected RNA versus protein contributions, establishing that Cwc25 promotes step 1 through protein-protein interactions despite contacting the branch site after catalysis.","evidence":"UV crosslinking of site-specifically labeled pre-mRNA across Bact, B*, and C complexes with functional dissection","pmids":["26393790"],"confidence":"High","gaps":["Identity of the protein partners mediating step 1 promotion not pinpointed"]},{"year":2016,"claim":"Cryo-EM structures placed Cwc25 in the catalytic complex, showing it and Yju2 stabilize docking of the branch helix into the active site after branching.","evidence":"Cryo-EM of yeast C complex at 3.4 and 3.8 Å resolution","pmids":["27445308","27459055"],"confidence":"High","gaps":["Captured post-catalytic C complex; did not directly visualize the pre-catalytic positioning step"]},{"year":2016,"claim":"Demonstrated that Cwc25 is dissociated in the step II-activated spliceosome, linking its removal to progression toward the second reaction.","evidence":"Cryo-EM of C* complex at 4.0 Å resolution","pmids":["27980089"],"confidence":"High","gaps":["Did not resolve the structural intermediate of Cwc25 displacement"]},{"year":2017,"claim":"Comparative structural and biochemical analyses established that Cwc25 locks the first-step conformation and that Prp16 remodeling destabilizes it to release the branch helix.","evidence":"Cryo-EM comparison of C and C* complexes at 3.8 Å, plus in vitro splicing measuring forward/reverse/debranching reactions with Cwc25 add-back","pmids":["28076345","28057857"],"confidence":"High","gaps":["Mechanism of Prp16-driven Cwc25 destabilization not structurally captured"]},{"year":2018,"claim":"In the human spliceosome, placed CCDC49 (Cwc25) bridging Prp16 and the active-site RNA, providing the structural basis for ATPase-coupled remodeling.","evidence":"Cryo-EM of human C complex at 4.1 Å resolution","pmids":["29301961"],"confidence":"High","gaps":["Functional reconstitution of human CCDC49 not performed"]},{"year":2019,"claim":"Resolved how Cwc25 (with Yju2) actively positions the branch point nucleophile, showing the U2/BPS duplex moves toward the 5' splice site upon their recruitment.","evidence":"Cryo-EM of four distinct yeast B* complexes at 2.9–3.8 Å resolution with and without step-one factors","pmids":["30879786"],"confidence":"High","gaps":["Order of Yju2 versus Cwc25 contributions to nucleophile positioning not fully separated"]},{"year":2023,"claim":"Identified Cwc25 as the downstream effector of Prp16's ATP-independent splice-site proofreading function.","evidence":"In vitro splicing with mutant 5' splice sites and branchpoints, genetic/biochemical epistasis between Prp16 and Cwc25","pmids":["37858289"],"confidence":"Medium","gaps":["No structural resolution of how Prp16 signals through Cwc25","Single-lab epistasis without orthogonal validation"]},{"year":null,"claim":"How Cwc25 functions are regulated or specialized in human cells beyond the structural snapshot remains unresolved.","evidence":"No functional reconstitution or regulatory study of human CCDC49 in the available corpus","pmids":[],"confidence":"Medium","gaps":["No human in vitro reconstitution of CCDC49 step 1 activity","No disease or physiological phenotype linked in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,4,10]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[2,5]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6,11]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,6,11]}],"complexes":["spliceosome (C complex / step I spliceosome)"],"partners":["YJU2","PRP16","ISY1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NXE8","full_name":"Pre-mRNA-splicing factor CWC25 homolog","aliases":["Coiled-coil domain-containing protein 49","Spliceosome-associated protein homolog CWC25"],"length_aa":425,"mass_kda":49.6,"function":"Involved in pre-mRNA splicing as component of the spliceosome","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9NXE8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CWC25","classification":"Not Classified","n_dependent_lines":324,"n_total_lines":1208,"dependency_fraction":0.2682119205298013},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CWC25","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":"Enhanced","locations":[{"location":"Nuclear speckles","reliability":"Enhanced"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":58.7}],"url":"https://www.proteinatlas.org/search/CWC25"},"hgnc":{"alias_symbol":["FLJ20291"],"prev_symbol":["CCDC49"]},"alphafold":{"accession":"Q9NXE8","domains":[{"cath_id":"4.10.270","chopping":"8-76","consensus_level":"medium","plddt":84.1541,"start":8,"end":76}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NXE8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NXE8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NXE8-F1-predicted_aligned_error_v6.png","plddt_mean":65.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CWC25","jax_strain_url":"https://www.jax.org/strain/search?query=CWC25"},"sequence":{"accession":"Q9NXE8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NXE8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NXE8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NXE8"}},"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":"28076345","id":"PMC_28076345","title":"Structure of a spliceosome remodelled for exon ligation.","date":"2017","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/28076345","citation_count":146,"is_preprint":false},{"pmid":"19935684","id":"PMC_19935684","title":"Reconstitution of both steps of Saccharomyces cerevisiae splicing with purified spliceosomal components.","date":"2009","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/19935684","citation_count":144,"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 Branching.","date":"2019","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/30879786","citation_count":69,"is_preprint":false},{"pmid":"24240612","id":"PMC_24240612","title":"Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing.","date":"2013","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/24240612","citation_count":56,"is_preprint":false},{"pmid":"23260012","id":"PMC_23260012","title":"Structural analysis of the genome of breast cancer cell line ZR-75-30 identifies twelve expressed fusion genes.","date":"2012","source":"BMC genomics","url":"https://pubmed.ncbi.nlm.nih.gov/23260012","citation_count":40,"is_preprint":false},{"pmid":"26393790","id":"PMC_26393790","title":"Dynamic Contacts of U2, RES, Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes.","date":"2015","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/26393790","citation_count":31,"is_preprint":false},{"pmid":"23438600","id":"PMC_23438600","title":"A weak spliceosome-binding domain of Yju2 functions in the first step and bypasses Prp16 in the second step of splicing.","date":"2013","source":"Molecular and cellular 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of Alternative Splicing Events and Differential Isoform Expression in Large Yellow Croaker (Larimichthys crocea) After Cryptocaryon irritans Infection.","date":"2022","source":"Marine biotechnology (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/35624193","citation_count":7,"is_preprint":false},{"pmid":"23865364","id":"PMC_23865364","title":"Serum proteome changes following human immunodeficiency virus infection.","date":"2013","source":"Clinical laboratory","url":"https://pubmed.ncbi.nlm.nih.gov/23865364","citation_count":4,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11074,"output_tokens":3439,"usd":0.042403,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10899,"output_tokens":3217,"usd":0.06746,"stage2_stop_reason":"end_turn"},"total_usd":0.109863,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"Cwc25 is required for the first catalytic step (step 1) of pre-mRNA splicing. In an in vitro reconstitution system, spliceosomes stalled before step 1 (purified from a Prp2 temperature-sensitive mutant) catalyzed efficient step 1 when supplemented with recombinant Prp2, Spp2, and Cwc25, demonstrating Cwc25's previously unknown role in promoting step 1.\",\n      \"method\": \"In vitro splicing reconstitution with purified spliceosomal components and recombinant proteins\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical reconstitution with purified components, replicated across multiple subsequent studies\",\n      \"pmids\": [\"19935684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Cwc25 is not tightly associated with the NTC (Prp19-associated complex) and is not required for spliceosome activation, but functions in the final step of the first catalytic reaction after the action of Prp2, in an ATP-independent manner. Depleted spliceosomes could be chased into splicing intermediates by addition of recombinant Cwc25 without ATP.\",\n      \"method\": \"Affinity purification of spliceosomes from Cwc25-depleted extracts, complementation with recombinant Cwc25\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical reconstitution with defined factors, ATP-independence demonstrated directly, replicated by other labs\",\n      \"pmids\": [\"19704000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Cwc25 and Yju2 are released from the spliceosome after the first catalytic reaction in a Prp16- and ATP-dependent manner. Binding of Cwc25 to the spliceosome is destabilized by mutations at the branchpoint sequence, suggesting Cwc25 contacts the branch site region.\",\n      \"method\": \"In vitro splicing assays with branchpoint mutant pre-mRNAs, spliceosome purification, ATP-dependence experiments\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal in vitro assays, replicated by subsequent structural and biochemical studies\",\n      \"pmids\": [\"21098140\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"High-affinity binding sites for Cwc25 are created on the spliceosome during Prp2-mediated catalytic activation (transition from Bact to B* complex), consistent with Cwc25's requirement for step 1 catalysis. Cwc25 binding is enhanced after Prp2-mediated remodeling.\",\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 2 / Moderate — quantitative biophysical measurement (dcFCCS) on purified complexes, single lab with rigorous controls\",\n      \"pmids\": [\"22535589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Cwc25 acts as a 'pawl' in a biased Brownian ratchet mechanism: after Prp2/Spp2/NTP rearranges the spliceosome to reversibly explore conformations with proximal 5' splice site and branch point, addition of Cwc25 strongly biases the equilibrium toward the proximal (reactive) conformation, promoting efficient first-step splicing.\",\n      \"method\": \"Single-molecule FRET on affinity-purified spliceosomes stalled by Prp2 mutation, with addition of recombinant factors\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — single-molecule FRET reconstitution with purified factors, directly monitors conformational dynamics\",\n      \"pmids\": [\"24240612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Cwc25 contacts the branch-site region of the pre-mRNA after step 1 catalysis, as shown by UV-induced crosslinking. However, Cwc25's step 1-promoting activity was not dependent on its direct interaction with pre-mRNA, indicating it acts via protein-protein interactions rather than direct RNA contacts.\",\n      \"method\": \"UV-induced crosslinking of purified yeast spliceosomes formed on site-specifically labeled pre-mRNA, analyzed across B(act), B*, and C complexes\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct UV crosslinking with site-specifically labeled pre-mRNA in defined purified complexes, functional dissection of RNA vs. protein contacts\",\n      \"pmids\": [\"26393790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cryo-EM structure of the yeast C complex (step I spliceosome) at 3.4 Å shows Cwc25 as one of 15 protein components stabilizing the catalytic cavity of Prp8, with the branch adenosine covalently joined to the 5' splice site phosphate. Cwc25 participates in stabilizing specific placement of RNA elements at the catalytic cavity.\",\n      \"method\": \"Cryo-electron microscopy structure determination at 3.4 Å resolution\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-atomic resolution cryo-EM structure directly showing Cwc25 position in the catalytic complex\",\n      \"pmids\": [\"27445308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cryo-EM structure of the spliceosome immediately after branching (C complex) at 3.8 Å shows that Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix into the active site, with non-Watson-Crick interactions between the branch helix and the 5' splice site docking the branch adenosine into the active site.\",\n      \"method\": \"Cryo-electron microscopy structure determination at 3.8 Å resolution\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-atomic resolution cryo-EM structure directly showing Cwc25 stabilizing the branch helix\",\n      \"pmids\": [\"27459055\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In the C* complex (step II catalytically activated spliceosome), step I splicing factors Cwc25 and Yju2 have been dissociated from the active site compared to the C complex, consistent with Prp16-mediated removal being required for the second transesterification step.\",\n      \"method\": \"Cryo-electron microscopy structure of C* complex at 4.0 Å resolution\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-atomic resolution cryo-EM structure directly showing Cwc25 absence in C* complex\",\n      \"pmids\": [\"27980089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Structural comparison of C and C* complexes reveals that branching-specific factors such as Cwc25 lock the branch helix into position for nucleophilic attack of the branch adenosine at the 5' splice site in the C complex, and that Prp16-mediated remodeling destabilizes these factors and releases the branch helix from the active site.\",\n      \"method\": \"Cryo-EM structure of C* complex at 3.8 Å, compared with C complex structure\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-atomic resolution cryo-EM with direct structural comparison of successive complexes\",\n      \"pmids\": [\"28076345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cwc25 plays a central role in modulating the conformational state of the catalytic spliceosome: it binds tightly after the first reaction and stabilizes the first-step conformation. When Cwc25 is absent from the purified first-step spliceosome, both forward and reverse splicing reactions (debranching, spliced-exon-reopening) occur under normal splicing conditions without energy. Adding Cwc25 back inhibits both reactions by stabilizing the first-step conformation. Prp16 is dispensable for the second reaction under conditions that destabilize Cwc25.\",\n      \"method\": \"In vitro splicing assays with purified spliceosomes under varied ionic conditions, with and without recombinant Cwc25\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal biochemical assays (forward splicing, reverse splicing, debranching) with direct add-back experiments\",\n      \"pmids\": [\"28057857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In the human C complex (step I spliceosome), the human ortholog of Cwc25 (CCDC49) closely interacts with the DEAH-family ATPase/helicase Prp16 and bridges the gap between Prp16 and the active-site RNA elements. Structural comparison of human C and C* complexes provides mechanistic insights into how CCDC49/Cwc25 is remodeled during the C-to-C* transition.\",\n      \"method\": \"Cryo-EM structure of human C complex at 4.1 Å resolution\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-atomic resolution cryo-EM of human spliceosome directly showing CCDC49 (CWC25) interactions with Prp16 and active-site RNA\",\n      \"pmids\": [\"29301961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cryo-EM structures of four distinct yeast B* complexes reveal that in B* complexes devoid of Yju2 and Cwc25, the U2/BPS duplex is discretely away from the 5' splice site. Recruitment of Yju2 brings the U2/BPS duplex into the vicinity of the 5' splice site, positioning the BPS nucleophile 4 Å from catalytic metal M2. This analysis reveals the functional mechanism: Cwc25 (together with Yju2) promotes branching by bringing the branch point nucleophile into proximity of the 5' splice site.\",\n      \"method\": \"Cryo-EM structures of four B* complexes at 2.9–3.8 Å resolution\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple near-atomic resolution cryo-EM structures of complexes with and without Cwc25/Yju2, directly revealing mechanism of branch point positioning\",\n      \"pmids\": [\"30879786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Prp16's ATP-independent function in promoting first-step splicing of aberrant 5' splice sites and mutated branchpoints is mediated through the step-one factor Cwc25, establishing Cwc25 as an effector through which Prp16 exerts dual roles in splice site selection.\",\n      \"method\": \"In vitro splicing assays with mutant pre-mRNAs, genetic and biochemical epistasis between Prp16 and Cwc25\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and biochemical epistasis, single lab, multiple substrate contexts but no structural resolution of mechanism\",\n      \"pmids\": [\"37858289\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CWC25 (human: CCDC49) is a step I-specific splicing factor that is recruited to the spliceosome after Prp2-mediated remodeling exposes high-affinity binding sites; it acts as a conformational 'pawl' that, together with Yju2, docks the branch helix into the active site and positions the branch-point adenosine nucleophile for the first transesterification reaction via protein-protein interactions rather than direct RNA contacts, after which it is removed by Prp16-mediated ATP hydrolysis to allow the conformational changes required for the second catalytic step.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CWC25 (human CCDC49) is a step I-specific pre-mRNA splicing factor that promotes the first transesterification reaction by stabilizing the catalytic conformation of the spliceosome [#0, #1]. It is not a constitutive component of the NTC and is dispensable for spliceosome activation; instead, it acts after Prp2-mediated remodeling, which creates high-affinity Cwc25 binding sites during the Bact-to-B* transition, and it promotes step 1 in an ATP-independent manner [#1, #3]. Mechanistically, Cwc25 functions as a conformational 'pawl' in a biased Brownian ratchet: it shifts the reversibly fluctuating spliceosome toward the reactive conformation in which the 5' splice site and branch point are proximal, and together with Yju2 it docks the branch helix into the active site and positions the branch-point adenosine nucleophile within striking distance of the catalytic metal for nucleophilic attack [#4, #7, #12]. This positioning is achieved through protein-protein interactions rather than direct pre-mRNA contacts, although Cwc25 does crosslink to the branch-site region after catalysis [#5]. By locking the branch helix in place, Cwc25 stabilizes the first-step conformation and suppresses both forward and reverse reactions until it is destabilized; its removal is required to permit the conformational changes of the second catalytic step [#10, #8, #9]. In the human spliceosome CCDC49 bridges the DEAH-box ATPase Prp16 and the active-site RNA, and Prp16-mediated ATP hydrolysis dissociates Cwc25 during the C-to-C* transition [#11, #2]. Cwc25 also serves as the effector through which Prp16 exerts its ATP-independent role in proofreading aberrant 5' splice sites and mutated branchpoints [#13].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established that an uncharacterized factor is specifically required for the first catalytic step of splicing, defining Cwc25's core function.\",\n      \"evidence\": \"In vitro splicing reconstitution of Prp2-stalled spliceosomes complemented with recombinant Prp2, Spp2, and Cwc25; and Cwc25-depletion/add-back with ATP-independence demonstrated\",\n      \"pmids\": [\"19935684\", \"19704000\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve whether Cwc25 contacts RNA or acts purely through protein interactions\", \"Structural position within the spliceosome unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined the temporal placement of Cwc25 action by showing it and Yju2 are released after step 1 and that its binding depends on the branchpoint sequence.\",\n      \"evidence\": \"In vitro splicing with branchpoint mutant pre-mRNAs and Prp16/ATP-dependent release assays\",\n      \"pmids\": [\"21098140\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether branchpoint dependence reflects direct RNA contact or conformational sensing unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed that the requirement for prior Prp2 remodeling is explained by creation of high-affinity Cwc25 binding sites, coupling activation to step 1 recruitment.\",\n      \"evidence\": \"Dual-color fluorescence cross-correlation spectroscopy on purified yeast spliceosomes\",\n      \"pmids\": [\"22535589\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular identity of the created binding surface not defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Provided the mechanistic model: Cwc25 acts as a 'pawl' that biases a reversible conformational equilibrium toward the reactive proximal state.\",\n      \"evidence\": \"Single-molecule FRET on Prp2-stalled spliceosomes with recombinant factor add-back\",\n      \"pmids\": [\"24240612\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not show atomic basis of how Cwc25 biases the equilibrium\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Dissected RNA versus protein contributions, establishing that Cwc25 promotes step 1 through protein-protein interactions despite contacting the branch site after catalysis.\",\n      \"evidence\": \"UV crosslinking of site-specifically labeled pre-mRNA across Bact, B*, and C complexes with functional dissection\",\n      \"pmids\": [\"26393790\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the protein partners mediating step 1 promotion not pinpointed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Cryo-EM structures placed Cwc25 in the catalytic complex, showing it and Yju2 stabilize docking of the branch helix into the active site after branching.\",\n      \"evidence\": \"Cryo-EM of yeast C complex at 3.4 and 3.8 Å resolution\",\n      \"pmids\": [\"27445308\", \"27459055\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Captured post-catalytic C complex; did not directly visualize the pre-catalytic positioning step\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated that Cwc25 is dissociated in the step II-activated spliceosome, linking its removal to progression toward the second reaction.\",\n      \"evidence\": \"Cryo-EM of C* complex at 4.0 Å resolution\",\n      \"pmids\": [\"27980089\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural intermediate of Cwc25 displacement\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Comparative structural and biochemical analyses established that Cwc25 locks the first-step conformation and that Prp16 remodeling destabilizes it to release the branch helix.\",\n      \"evidence\": \"Cryo-EM comparison of C and C* complexes at 3.8 Å, plus in vitro splicing measuring forward/reverse/debranching reactions with Cwc25 add-back\",\n      \"pmids\": [\"28076345\", \"28057857\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of Prp16-driven Cwc25 destabilization not structurally captured\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"In the human spliceosome, placed CCDC49 (Cwc25) bridging Prp16 and the active-site RNA, providing the structural basis for ATPase-coupled remodeling.\",\n      \"evidence\": \"Cryo-EM of human C complex at 4.1 Å resolution\",\n      \"pmids\": [\"29301961\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional reconstitution of human CCDC49 not performed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Resolved how Cwc25 (with Yju2) actively positions the branch point nucleophile, showing the U2/BPS duplex moves toward the 5' splice site upon their recruitment.\",\n      \"evidence\": \"Cryo-EM of four distinct yeast B* complexes at 2.9–3.8 Å resolution with and without step-one factors\",\n      \"pmids\": [\"30879786\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Order of Yju2 versus Cwc25 contributions to nucleophile positioning not fully separated\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified Cwc25 as the downstream effector of Prp16's ATP-independent splice-site proofreading function.\",\n      \"evidence\": \"In vitro splicing with mutant 5' splice sites and branchpoints, genetic/biochemical epistasis between Prp16 and Cwc25\",\n      \"pmids\": [\"37858289\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural resolution of how Prp16 signals through Cwc25\", \"Single-lab epistasis without orthogonal validation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How Cwc25 functions are regulated or specialized in human cells beyond the structural snapshot remains unresolved.\",\n      \"evidence\": \"No functional reconstitution or regulatory study of human CCDC49 in the available corpus\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No human in vitro reconstitution of CCDC49 step 1 activity\", \"No disease or physiological phenotype linked in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 4, 10]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [2, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6, 11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 6, 11]}\n    ],\n    \"complexes\": [\"spliceosome (C complex / step I spliceosome)\"],\n    \"partners\": [\"YJU2\", \"PRP16\", \"ISY1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}