Structural requirement of Ntc77 for spliceosome activation and first catalytic step.
Bottom Line: The Prp19-associated complex is required for spliceosome activation by stabilizing the binding of U5 and U6 on the spliceosome after the release of U4.Deletion of this region had no severe effect on the integrity of the NTC, binding of NTC to the spliceosome or spliceosome activation, but impaired splicing and exhibited a dominant-negative growth phenotype.Our data reveal functional roles of Ntc77 in both spliceosome activation and the first catalytic step, and distinct structural domains of Ntc77 required for these two steps.
Affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan 115, Republic of China Institute of Microbiology and Immunology, National Yang-Ming University, Shih-Pai, Taipei, Taiwan 112, Republic of China.Show MeSH
Mentions: To confirm that the ΔN3 complex is able to bind to the spliceosome, we analyzed the association of NTC with the spliceosome by immunoprecipitation with anti-Ntc20 antibody using a 3′-truncated actin pre-mRNA Ac/Cla, which retains only five bases downstream from the branchpoint (34). We have previously shown that the spliceosome can assemble on the Ac/Cla pre-mRNA up to the post-activation step, allowing binding of NTC and Prp2 but preventing the adenosine triphosphate (ATP)-dependent function of Prp2 in displacing SF3a/b (35). The result showed that the anti-Ntc20 antibody could precipitate the spliceosome formed in extracts containing N77 or ΔN3 complex (Figure 4A, lanes 6 and 9), indicating that deletion of the N-3 region of Ntc77 did not affect the binding of NTC to the spliceosome.
Affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan 115, Republic of China Institute of Microbiology and Immunology, National Yang-Ming University, Shih-Pai, Taipei, Taiwan 112, Republic of China.