Affinage

CWC25

Pre-mRNA-splicing factor CWC25 homolog · UniProt Q9NXE8

Length
425 aa
Mass
49.6 kDa
Annotated
2026-06-09
19 papers in source corpus 14 papers cited in narrative 14 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 7/7 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 11 steps
  1. 2009 High

    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

    PMID:19704000 PMID:19935684

    Open questions at the time
    • Did not resolve whether Cwc25 contacts RNA or acts purely through protein interactions
    • Structural position within the spliceosome unknown
  2. 2010 High

    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

    PMID:21098140

    Open questions at the time
    • Whether branchpoint dependence reflects direct RNA contact or conformational sensing unresolved
  3. 2012 High

    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

    PMID:22535589

    Open questions at the time
    • Molecular identity of the created binding surface not defined
  4. 2013 High

    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

    PMID:24240612

    Open questions at the time
    • Did not show atomic basis of how Cwc25 biases the equilibrium
  5. 2015 High

    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

    PMID:26393790

    Open questions at the time
    • Identity of the protein partners mediating step 1 promotion not pinpointed
  6. 2016 High

    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

    PMID:27445308 PMID:27459055

    Open questions at the time
    • Captured post-catalytic C complex; did not directly visualize the pre-catalytic positioning step
  7. 2016 High

    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

    PMID:27980089

    Open questions at the time
    • Did not resolve the structural intermediate of Cwc25 displacement
  8. 2017 High

    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

    PMID:28057857 PMID:28076345

    Open questions at the time
    • Mechanism of Prp16-driven Cwc25 destabilization not structurally captured
  9. 2018 High

    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

    PMID:29301961

    Open questions at the time
    • Functional reconstitution of human CCDC49 not performed
  10. 2019 High

    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

    PMID:30879786

    Open questions at the time
    • Order of Yju2 versus Cwc25 contributions to nucleophile positioning not fully separated
  11. 2023 Medium

    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

    PMID:37858289

    Open questions at the time
    • No structural resolution of how Prp16 signals through Cwc25
    • Single-lab epistasis without orthogonal validation

Open questions

Synthesis pass · forward-looking unresolved questions
  • How Cwc25 functions are regulated or specialized in human cells beyond the structural snapshot remains unresolved.
  • No human in vitro reconstitution of CCDC49 step 1 activity
  • No disease or physiological phenotype linked in the corpus

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0098772 molecular function regulator activity 3 GO:0003723 RNA binding 2
Localization
GO:0005634 nucleus 2
Pathway
R-HSA-8953854 Metabolism of RNA 3
Partners
ISY1PRP16YJU2
Complex memberships
spliceosome (C complex / step I spliceosome)

Evidence

Reading pass · 14 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2009 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. In vitro splicing reconstitution with purified spliceosomal components and recombinant proteins Nature structural & molecular biology High 19935684
2009 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. Affinity purification of spliceosomes from Cwc25-depleted extracts, complementation with recombinant Cwc25 Molecular and cellular biology High 19704000
2010 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. In vitro splicing assays with branchpoint mutant pre-mRNAs, spliceosome purification, ATP-dependence experiments RNA (New York, N.Y.) High 21098140
2012 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. Dual-color fluorescence cross-correlation spectroscopy (dcFCCS) on purified yeast spliceosomes RNA (New York, N.Y.) High 22535589
2013 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. Single-molecule FRET on affinity-purified spliceosomes stalled by Prp2 mutation, with addition of recombinant factors Nature structural & molecular biology High 24240612
2015 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. UV-induced crosslinking of purified yeast spliceosomes formed on site-specifically labeled pre-mRNA, analyzed across B(act), B*, and C complexes PLoS genetics High 26393790
2016 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. Cryo-electron microscopy structure determination at 3.4 Å resolution Science (New York, N.Y.) High 27445308
2016 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. Cryo-electron microscopy structure determination at 3.8 Å resolution Nature High 27459055
2016 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. Cryo-electron microscopy structure of C* complex at 4.0 Å resolution Science (New York, N.Y.) High 27980089
2017 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. Cryo-EM structure of C* complex at 3.8 Å, compared with C complex structure Nature High 28076345
2017 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. In vitro splicing assays with purified spliceosomes under varied ionic conditions, with and without recombinant Cwc25 RNA (New York, N.Y.) High 28057857
2018 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. Cryo-EM structure of human C complex at 4.1 Å resolution Science (New York, N.Y.) High 29301961
2019 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. Cryo-EM structures of four B* complexes at 2.9–3.8 Å resolution Cell High 30879786
2023 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. In vitro splicing assays with mutant pre-mRNAs, genetic and biochemical epistasis between Prp16 and Cwc25 Nucleic acids research Medium 37858289

Source papers

Stage 0 corpus · 19 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2016 Cryo-EM structure of the spliceosome immediately after branching. Nature 198 27459055
2016 Structure of a yeast catalytic step I spliceosome at 3.4 Å resolution. Science (New York, N.Y.) 158 27445308
2017 Structure of a spliceosome remodelled for exon ligation. Nature 146 28076345
2009 Reconstitution of both steps of Saccharomyces cerevisiae splicing with purified spliceosomal components. Nature structural & molecular biology 144 19935684
2016 Structure of a yeast step II catalytically activated spliceosome. Science (New York, N.Y.) 121 27980089
2018 Structure of a human catalytic step I spliceosome. Science (New York, N.Y.) 119 29301961
2010 DEAH-box ATPase Prp16 has dual roles in remodeling of the spliceosome in catalytic steps. RNA (New York, N.Y.) 84 21098140
2012 Prp2-mediated protein rearrangements at the catalytic core of the spliceosome as revealed by dcFCCS. RNA (New York, N.Y.) 75 22535589
2009 Cwc25 is a novel splicing factor required after Prp2 and Yju2 to facilitate the first catalytic reaction. Molecular and cellular biology 71 19704000
2019 Structures of the Catalytically Activated Yeast Spliceosome Reveal the Mechanism of Branching. Cell 69 30879786
2013 Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing. Nature structural & molecular biology 56 24240612
2012 Structural analysis of the genome of breast cancer cell line ZR-75-30 identifies twelve expressed fusion genes. BMC genomics 40 23260012
2015 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. PLoS genetics 31 26393790
2013 A weak spliceosome-binding domain of Yju2 functions in the first step and bypasses Prp16 in the second step of splicing. Molecular and cellular biology 18 23438600
2017 A central role of Cwc25 in spliceosome dynamics during the catalytic phase of pre-mRNA splicing. RNA (New York, N.Y.) 14 28057857
2014 Remodeling of U2-U6 snRNA helix I during pre-mRNA splicing by Prp16 and the NineTeen Complex protein Cwc2. Nucleic acids research 13 24848011
2023 An ATP-independent role for Prp16 in promoting aberrant splicing. Nucleic acids research 12 37858289
2022 Tissue-Specific Analysis of Alternative Splicing Events and Differential Isoform Expression in Large Yellow Croaker (Larimichthys crocea) After Cryptocaryon irritans Infection. Marine biotechnology (New York, N.Y.) 7 35624193
2013 Serum proteome changes following human immunodeficiency virus infection. Clinical laboratory 4 23865364

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