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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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Ntc77 is required for the integrity of NTC and for its binding to the spliceosome. (A) Western blotting of total proteins of extracts prepared from wild-type (lane 1) or Ntc77 metabolically depleted cells after incubation in the glucose-based media for 12 (lane 2) or 16 h (lane 3). (B) Extracts prepared from wild-type (lanes 1 and 2) or Ntc77-depleted cells (16 h in glucose) (lane 3) were immunoprecipitated with no antibody (lane 1) or anti-Ntc85 antibody (lanes 2 and 3), and probed with antibodies against Ntc90, Ntc85 or Prp19. (C) Splicing reactions were carried out in wild-type (lanes 1 and 2) or Ntc77-depleted (12 h in glucose) (lane 3) extracts using non-biotinylated (lane 1) or biotinylated (lanes 2 and 3) ACAC pre-mRNA, and the spliceosomes were pulled down with streptavidin Sepharose. WT, wild-type; d77, Ntc77-depleted extracts.
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Figure 1: Ntc77 is required for the integrity of NTC and for its binding to the spliceosome. (A) Western blotting of total proteins of extracts prepared from wild-type (lane 1) or Ntc77 metabolically depleted cells after incubation in the glucose-based media for 12 (lane 2) or 16 h (lane 3). (B) Extracts prepared from wild-type (lanes 1 and 2) or Ntc77-depleted cells (16 h in glucose) (lane 3) were immunoprecipitated with no antibody (lane 1) or anti-Ntc85 antibody (lanes 2 and 3), and probed with antibodies against Ntc90, Ntc85 or Prp19. (C) Splicing reactions were carried out in wild-type (lanes 1 and 2) or Ntc77-depleted (12 h in glucose) (lane 3) extracts using non-biotinylated (lane 1) or biotinylated (lanes 2 and 3) ACAC pre-mRNA, and the spliceosomes were pulled down with streptavidin Sepharose. WT, wild-type; d77, Ntc77-depleted extracts.

Mentions: Two components of NTC, Ntc90 and Ntc77, contain multiple copies of the TPR motif, which is implicated in protein–protein interactions (27). We have previously shown that Ntc90 is not required for spliceosome activation, and instead is essential for the recruitment of Yju2 for the first chemical reaction (28). To assess the function of Ntc77, we metabolically depleted Ntc77 using GAL1-promoter to test how formation of the NTC or the spliceosome was affected. Splicing extracts prepared from such cells after incubation in glucose-containing media for 12 or 16 h contained only residual amounts of Ntc77, while the amount of Ntc20 was not affected. The amounts of Prp19 and Ntc90 were slightly reduced, but that of Ntc85 and Ntc30 appeared to decrease more extensively after growth in glucose for 16 h (Figure 1A). The extract also contained residual amount of splicing activity, and could be rescued by the addition of purified NTC (Supplementary Figure S1).


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)

Ntc77 is required for the integrity of NTC and for its binding to the spliceosome. (A) Western blotting of total proteins of extracts prepared from wild-type (lane 1) or Ntc77 metabolically depleted cells after incubation in the glucose-based media for 12 (lane 2) or 16 h (lane 3). (B) Extracts prepared from wild-type (lanes 1 and 2) or Ntc77-depleted cells (16 h in glucose) (lane 3) were immunoprecipitated with no antibody (lane 1) or anti-Ntc85 antibody (lanes 2 and 3), and probed with antibodies against Ntc90, Ntc85 or Prp19. (C) Splicing reactions were carried out in wild-type (lanes 1 and 2) or Ntc77-depleted (12 h in glucose) (lane 3) extracts using non-biotinylated (lane 1) or biotinylated (lanes 2 and 3) ACAC pre-mRNA, and the spliceosomes were pulled down with streptavidin Sepharose. WT, wild-type; d77, Ntc77-depleted extracts.
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Related In: Results  -  Collection

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Figure 1: Ntc77 is required for the integrity of NTC and for its binding to the spliceosome. (A) Western blotting of total proteins of extracts prepared from wild-type (lane 1) or Ntc77 metabolically depleted cells after incubation in the glucose-based media for 12 (lane 2) or 16 h (lane 3). (B) Extracts prepared from wild-type (lanes 1 and 2) or Ntc77-depleted cells (16 h in glucose) (lane 3) were immunoprecipitated with no antibody (lane 1) or anti-Ntc85 antibody (lanes 2 and 3), and probed with antibodies against Ntc90, Ntc85 or Prp19. (C) Splicing reactions were carried out in wild-type (lanes 1 and 2) or Ntc77-depleted (12 h in glucose) (lane 3) extracts using non-biotinylated (lane 1) or biotinylated (lanes 2 and 3) ACAC pre-mRNA, and the spliceosomes were pulled down with streptavidin Sepharose. WT, wild-type; d77, Ntc77-depleted extracts.
Mentions: Two components of NTC, Ntc90 and Ntc77, contain multiple copies of the TPR motif, which is implicated in protein–protein interactions (27). We have previously shown that Ntc90 is not required for spliceosome activation, and instead is essential for the recruitment of Yju2 for the first chemical reaction (28). To assess the function of Ntc77, we metabolically depleted Ntc77 using GAL1-promoter to test how formation of the NTC or the spliceosome was affected. Splicing extracts prepared from such cells after incubation in glucose-containing media for 12 or 16 h contained only residual amounts of Ntc77, while the amount of Ntc20 was not affected. The amounts of Prp19 and Ntc90 were slightly reduced, but that of Ntc85 and Ntc30 appeared to decrease more extensively after growth in glucose for 16 h (Figure 1A). The extract also contained residual amount of splicing activity, and could be rescued by the addition of purified NTC (Supplementary Figure S1).

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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