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: After activation of the spliceosome, several proteins are required to promote the first catalytic reaction. Prp2 is first required to destabilize U2 components SF3a/b from binding to the branch site in an ATP-dependent manner (7,8), and its function requires Spp2 and Cwc22 (22,36). Yju2 and Cwc25 are then required to promote the first reaction in an ATP-independent manner. Since the N-terminal region of Ntc77 is not required for spliceosome activation, it may be required for the ATP-dependent step in supporting Prp2 function, or for the post-Prp2 step that requires Yju2 and Cwc25 to promote the catalytic reaction. We first examined whether deletion of the N-3 region of Ntc77 prevents Prp2 binding to the spliceosome (Figure 5A). To retain Prp2 on the spliceosome, we used a dominant-negative mutant of PRP2, S378L, which carries a mutation in the SAT motif (37). Splicing reactions were carried out in Prp19-depleted prp2-1 mutant extracts supplemented with N77 or ΔN3, with the addition of recombinant V5-tagged Prp2-S378L, which allows Prp2 binding but not its release from the spliceosome. The reaction mixtures were precipitated with anti-V5 antibody to assay for the association of Prp2 (lanes 4, 8 and 12), with Prp8 and Ntc20 as controls to reveal the amount of total spliceosome. The result shows that indeed Prp2-S378L was able to bind the spliceosome in the presence of ΔN3 (lane 12). We then examined whether deletion of the N-3 region impedes the function of Prp2 in mediating destabilization of SF3a/b from the spliceosome by examining the association of SF3a component Prp9 with the spliceosome (Figure 5B). Splicing was carried out using biotinylated ACAC pre-mRNA in NTC-depleted extracts supplemented with NTC, N77 or ΔN3, and the spliceosome was pulled down with streptavidin Sepharose to examine for its protein content by western blotting. The result showed that Prp9 accumulated in higher amounts in NTC-depleted extracts (lane 2), but was maintained at a low level in mock-treated extracts (lane 1) or upon addition of NTC (lane 3) or N77 (lane 4). Addition of ΔN3 also resulted in loss of Prp9 (lane 5), suggesting that the Prp2 function in destabilizing SF3a/b was not severely impaired by deletion of the N-3 region of Ntc77 (lane 5).
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.