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Structural requirement of Ntc77 for spliceosome activation and first catalytic step.

Chen HC, Chang KJ, Su YL, Huang YH, Cheng SC - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

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.

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Spliceosome activation was not affected by deletion of the N-3 region of Ntc77. (A) Splicing reactions were carried out in Prp19-depleted extracts supplemented without (lanes 1–3) or with N77 (lanes 4–6) or ΔN3 (lanes 7–9), and the reaction mixtures were precipitated with pre-immune serum (lanes 2, 5 and 8) or anti-Ntc20 antibody (lanes 3, 6 and 9). (B) Splicing reactions were carried out using Ac/Cla pre-mRNA in the presence of 0.05 mM (lanes 1, 3 and 5) or 2 mM (lanes 2, 4 and 6) ATP, in mock- (top panel) or Prp19-depleted Lsm-V5 extracts (lower panels) supplemented without (second panel) or with NTC (third panel), N77 (fourth panel) or ΔN3 (bottom panel). The reaction mixtures were immunoprecipitated with anti-Smd1 (lanes 3 and 4) or anti-V5 antibody (lanes 5 and 6). (C) Splicing reactions were carried out with biotinylated ACAC pre-mRNA in mock- (lanes 1–3) or Prp19-depleted extracts (lanes 4–15) supplemented without (lanes 1–6) or with NTC (lanes 7–9), N77 (lanes 10–12) or ΔN3 (lanes 13–15). The spliceosomes precipitated with streptavidin Sepharose were incubated in splicing buffer containing ATP at 25°C for 20 min, and pellet and supernatant fractions were separated and then analyzed by northern blotting. (D) Schematic of U6-pre-mRNA interactions and products of UV-crosslinking. (E) Splicing reactions were carried out as in (C) but using 32P-labeled Ac/Cla pre-mRNA, and the spliceosomes were immunoprecipitated with anti-Smd1 antibody. After irradiation with UV254 nm, RNA was extracted and fractionated on a 5% polyacrylamide gel. RXN, 1/10 of reaction mix; pre-imm, pre-immune serum; dNTC, Prp19-depleted extracts; L, low ATP concentration; H, high ATP concentration; CPX, complex; T, total precipitates; P, pellet; S, supernatant.
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Figure 4: Spliceosome activation was not affected by deletion of the N-3 region of Ntc77. (A) Splicing reactions were carried out in Prp19-depleted extracts supplemented without (lanes 1–3) or with N77 (lanes 4–6) or ΔN3 (lanes 7–9), and the reaction mixtures were precipitated with pre-immune serum (lanes 2, 5 and 8) or anti-Ntc20 antibody (lanes 3, 6 and 9). (B) Splicing reactions were carried out using Ac/Cla pre-mRNA in the presence of 0.05 mM (lanes 1, 3 and 5) or 2 mM (lanes 2, 4 and 6) ATP, in mock- (top panel) or Prp19-depleted Lsm-V5 extracts (lower panels) supplemented without (second panel) or with NTC (third panel), N77 (fourth panel) or ΔN3 (bottom panel). The reaction mixtures were immunoprecipitated with anti-Smd1 (lanes 3 and 4) or anti-V5 antibody (lanes 5 and 6). (C) Splicing reactions were carried out with biotinylated ACAC pre-mRNA in mock- (lanes 1–3) or Prp19-depleted extracts (lanes 4–15) supplemented without (lanes 1–6) or with NTC (lanes 7–9), N77 (lanes 10–12) or ΔN3 (lanes 13–15). The spliceosomes precipitated with streptavidin Sepharose were incubated in splicing buffer containing ATP at 25°C for 20 min, and pellet and supernatant fractions were separated and then analyzed by northern blotting. (D) Schematic of U6-pre-mRNA interactions and products of UV-crosslinking. (E) Splicing reactions were carried out as in (C) but using 32P-labeled Ac/Cla pre-mRNA, and the spliceosomes were immunoprecipitated with anti-Smd1 antibody. After irradiation with UV254 nm, RNA was extracted and fractionated on a 5% polyacrylamide gel. RXN, 1/10 of reaction mix; pre-imm, pre-immune serum; dNTC, Prp19-depleted extracts; L, low ATP concentration; H, high ATP concentration; CPX, complex; T, total precipitates; P, pellet; S, supernatant.

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.


Structural requirement of Ntc77 for spliceosome activation and first catalytic step.

Chen HC, Chang KJ, Su YL, Huang YH, Cheng SC - Nucleic Acids Res. (2014)

Spliceosome activation was not affected by deletion of the N-3 region of Ntc77. (A) Splicing reactions were carried out in Prp19-depleted extracts supplemented without (lanes 1–3) or with N77 (lanes 4–6) or ΔN3 (lanes 7–9), and the reaction mixtures were precipitated with pre-immune serum (lanes 2, 5 and 8) or anti-Ntc20 antibody (lanes 3, 6 and 9). (B) Splicing reactions were carried out using Ac/Cla pre-mRNA in the presence of 0.05 mM (lanes 1, 3 and 5) or 2 mM (lanes 2, 4 and 6) ATP, in mock- (top panel) or Prp19-depleted Lsm-V5 extracts (lower panels) supplemented without (second panel) or with NTC (third panel), N77 (fourth panel) or ΔN3 (bottom panel). The reaction mixtures were immunoprecipitated with anti-Smd1 (lanes 3 and 4) or anti-V5 antibody (lanes 5 and 6). (C) Splicing reactions were carried out with biotinylated ACAC pre-mRNA in mock- (lanes 1–3) or Prp19-depleted extracts (lanes 4–15) supplemented without (lanes 1–6) or with NTC (lanes 7–9), N77 (lanes 10–12) or ΔN3 (lanes 13–15). The spliceosomes precipitated with streptavidin Sepharose were incubated in splicing buffer containing ATP at 25°C for 20 min, and pellet and supernatant fractions were separated and then analyzed by northern blotting. (D) Schematic of U6-pre-mRNA interactions and products of UV-crosslinking. (E) Splicing reactions were carried out as in (C) but using 32P-labeled Ac/Cla pre-mRNA, and the spliceosomes were immunoprecipitated with anti-Smd1 antibody. After irradiation with UV254 nm, RNA was extracted and fractionated on a 5% polyacrylamide gel. RXN, 1/10 of reaction mix; pre-imm, pre-immune serum; dNTC, Prp19-depleted extracts; L, low ATP concentration; H, high ATP concentration; CPX, complex; T, total precipitates; P, pellet; S, supernatant.
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Figure 4: Spliceosome activation was not affected by deletion of the N-3 region of Ntc77. (A) Splicing reactions were carried out in Prp19-depleted extracts supplemented without (lanes 1–3) or with N77 (lanes 4–6) or ΔN3 (lanes 7–9), and the reaction mixtures were precipitated with pre-immune serum (lanes 2, 5 and 8) or anti-Ntc20 antibody (lanes 3, 6 and 9). (B) Splicing reactions were carried out using Ac/Cla pre-mRNA in the presence of 0.05 mM (lanes 1, 3 and 5) or 2 mM (lanes 2, 4 and 6) ATP, in mock- (top panel) or Prp19-depleted Lsm-V5 extracts (lower panels) supplemented without (second panel) or with NTC (third panel), N77 (fourth panel) or ΔN3 (bottom panel). The reaction mixtures were immunoprecipitated with anti-Smd1 (lanes 3 and 4) or anti-V5 antibody (lanes 5 and 6). (C) Splicing reactions were carried out with biotinylated ACAC pre-mRNA in mock- (lanes 1–3) or Prp19-depleted extracts (lanes 4–15) supplemented without (lanes 1–6) or with NTC (lanes 7–9), N77 (lanes 10–12) or ΔN3 (lanes 13–15). The spliceosomes precipitated with streptavidin Sepharose were incubated in splicing buffer containing ATP at 25°C for 20 min, and pellet and supernatant fractions were separated and then analyzed by northern blotting. (D) Schematic of U6-pre-mRNA interactions and products of UV-crosslinking. (E) Splicing reactions were carried out as in (C) but using 32P-labeled Ac/Cla pre-mRNA, and the spliceosomes were immunoprecipitated with anti-Smd1 antibody. After irradiation with UV254 nm, RNA was extracted and fractionated on a 5% polyacrylamide gel. RXN, 1/10 of reaction mix; pre-imm, pre-immune serum; dNTC, Prp19-depleted extracts; L, low ATP concentration; H, high ATP concentration; CPX, complex; T, total precipitates; P, pellet; S, supernatant.
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.

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.

View Article: PubMed Central - PubMed

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.

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