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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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Overexpression of ΔN-3 of Ntc77 caused dominant-negative growth phenotype with a splicing defect. (A) Growth analysis for the overexpression of NTC77 deletion mutants. Predicted TPR motifs of Ntc77 are shown in gray boxes. Black lines represent retained regions of the protein in deletion mutants. Cells harboring pRS426 plasmid containing NTC77 deletion mutants under the control of GAL1-promoter were analyzed for growth in glucose- and galactose-based media by spot assays with 10-fold serial dilutions. (B) Western blotting of total proteins from lysates extracted from Ntc77- (lanes 1 and 2) or ΔN-3-overexpressed cells (lanes 3 and 4) after incubation in raffinose- (lanes 1 and 3) or galactose-based media (lanes 2 and 4). (C) Primer extension analysis. Total RNA extracted from Ntc77- or ΔN-3-overexpressed cells was analyzed by primer extension using a U3 primer R13. The prp2-1 mutant was grown at 37°C for 2 h before harvest. WT, wild-type; R, raffinose; G, galactose.
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Figure 2: Overexpression of ΔN-3 of Ntc77 caused dominant-negative growth phenotype with a splicing defect. (A) Growth analysis for the overexpression of NTC77 deletion mutants. Predicted TPR motifs of Ntc77 are shown in gray boxes. Black lines represent retained regions of the protein in deletion mutants. Cells harboring pRS426 plasmid containing NTC77 deletion mutants under the control of GAL1-promoter were analyzed for growth in glucose- and galactose-based media by spot assays with 10-fold serial dilutions. (B) Western blotting of total proteins from lysates extracted from Ntc77- (lanes 1 and 2) or ΔN-3-overexpressed cells (lanes 3 and 4) after incubation in raffinose- (lanes 1 and 3) or galactose-based media (lanes 2 and 4). (C) Primer extension analysis. Total RNA extracted from Ntc77- or ΔN-3-overexpressed cells was analyzed by primer extension using a U3 primer R13. The prp2-1 mutant was grown at 37°C for 2 h before harvest. WT, wild-type; R, raffinose; G, galactose.

Mentions: Ntc77 contains 15 tandem TPR motifs flanked by short N-terminal and C-terminal domains (24). We deleted several TPR motifs in Ntc77 to seek for dominant-negative mutants of NTC77 (Figure 2A). The deletion mutants of NTC77 were placed under the control of GAL1-promoter on 2μ-based plasmid vector pRS426, and overexpression of the mutant proteins were induced by incubation in the presence of galactose. The result showed that overexpression of the mutant with deletion of the first 129 amino acid residues of Ntc77, encompassing NTD and the first three TPR elements (ΔN-3), caused a dominant-negative phenotype (Figure 2B). Western blotting confirmed expression of the ΔN-3 protein when grown in galactose-containing media (Figure 2B, lane 4). The ΔN-3 mutant also conferred splicing defect as revealed by primer extension analysis using U3 as a probe (Figure 2C, lane 4).


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)

Overexpression of ΔN-3 of Ntc77 caused dominant-negative growth phenotype with a splicing defect. (A) Growth analysis for the overexpression of NTC77 deletion mutants. Predicted TPR motifs of Ntc77 are shown in gray boxes. Black lines represent retained regions of the protein in deletion mutants. Cells harboring pRS426 plasmid containing NTC77 deletion mutants under the control of GAL1-promoter were analyzed for growth in glucose- and galactose-based media by spot assays with 10-fold serial dilutions. (B) Western blotting of total proteins from lysates extracted from Ntc77- (lanes 1 and 2) or ΔN-3-overexpressed cells (lanes 3 and 4) after incubation in raffinose- (lanes 1 and 3) or galactose-based media (lanes 2 and 4). (C) Primer extension analysis. Total RNA extracted from Ntc77- or ΔN-3-overexpressed cells was analyzed by primer extension using a U3 primer R13. The prp2-1 mutant was grown at 37°C for 2 h before harvest. WT, wild-type; R, raffinose; G, galactose.
© Copyright Policy - creative-commons
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Figure 2: Overexpression of ΔN-3 of Ntc77 caused dominant-negative growth phenotype with a splicing defect. (A) Growth analysis for the overexpression of NTC77 deletion mutants. Predicted TPR motifs of Ntc77 are shown in gray boxes. Black lines represent retained regions of the protein in deletion mutants. Cells harboring pRS426 plasmid containing NTC77 deletion mutants under the control of GAL1-promoter were analyzed for growth in glucose- and galactose-based media by spot assays with 10-fold serial dilutions. (B) Western blotting of total proteins from lysates extracted from Ntc77- (lanes 1 and 2) or ΔN-3-overexpressed cells (lanes 3 and 4) after incubation in raffinose- (lanes 1 and 3) or galactose-based media (lanes 2 and 4). (C) Primer extension analysis. Total RNA extracted from Ntc77- or ΔN-3-overexpressed cells was analyzed by primer extension using a U3 primer R13. The prp2-1 mutant was grown at 37°C for 2 h before harvest. WT, wild-type; R, raffinose; G, galactose.
Mentions: Ntc77 contains 15 tandem TPR motifs flanked by short N-terminal and C-terminal domains (24). We deleted several TPR motifs in Ntc77 to seek for dominant-negative mutants of NTC77 (Figure 2A). The deletion mutants of NTC77 were placed under the control of GAL1-promoter on 2μ-based plasmid vector pRS426, and overexpression of the mutant proteins were induced by incubation in the presence of galactose. The result showed that overexpression of the mutant with deletion of the first 129 amino acid residues of Ntc77, encompassing NTD and the first three TPR elements (ΔN-3), caused a dominant-negative phenotype (Figure 2B). Western blotting confirmed expression of the ΔN-3 protein when grown in galactose-containing media (Figure 2B, lane 4). The ΔN-3 mutant also conferred splicing defect as revealed by primer extension analysis using U3 as a probe (Figure 2C, lane 4).

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.

Show MeSH
Related in: MedlinePlus