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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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ΔN-3 destabilized the association of Yju2 with the spliceosome. (A) Splicing reactions were carried out in Cwc25- (lane 1) or Prp19- and Cwc25- doubly depleted extracts (lanes 2–5) without (lanes 1 and 2) or with the addition of NTC (lane 3), N77 (lane 4) or ΔN3 (lane 5) using biotinylated ACAC pre-mRNA, and the spliceosomes were pulled down with streptavidin Sepharose. CPX, complex; d25, Cwc25-depleted extracts; d25dNTC, Cwc25- and NTC-doubly depleted extracts. (B) Splicing reactions were carried out in Prp16- (lane 1) or Prp19- and Prp16-doubly depleted extracts (lanes 2–5) without (lanes 1 and 2) or with the addition of NTC (lane 3), N77 (lane 4) or ΔN3 (lane 5) using biotinylated ACAC pre-mRNA, and the spliceosomes were pulled down with streptavidin Sepharose. In both (A) and (B), components were probed with antibodies against Snu114, Ntc85, Yju2 and HA-epitope for Prp19 (lanes 1 and 3) and Ntc77 (lanes 4 and 5). CPX, complex; d16, Prp16-depleted extracts; d16dNTC, Prp16- and NTC-doubly depleted extracts.
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Figure 6: ΔN-3 destabilized the association of Yju2 with the spliceosome. (A) Splicing reactions were carried out in Cwc25- (lane 1) or Prp19- and Cwc25- doubly depleted extracts (lanes 2–5) without (lanes 1 and 2) or with the addition of NTC (lane 3), N77 (lane 4) or ΔN3 (lane 5) using biotinylated ACAC pre-mRNA, and the spliceosomes were pulled down with streptavidin Sepharose. CPX, complex; d25, Cwc25-depleted extracts; d25dNTC, Cwc25- and NTC-doubly depleted extracts. (B) Splicing reactions were carried out in Prp16- (lane 1) or Prp19- and Prp16-doubly depleted extracts (lanes 2–5) without (lanes 1 and 2) or with the addition of NTC (lane 3), N77 (lane 4) or ΔN3 (lane 5) using biotinylated ACAC pre-mRNA, and the spliceosomes were pulled down with streptavidin Sepharose. In both (A) and (B), components were probed with antibodies against Snu114, Ntc85, Yju2 and HA-epitope for Prp19 (lanes 1 and 3) and Ntc77 (lanes 4 and 5). CPX, complex; d16, Prp16-depleted extracts; d16dNTC, Prp16- and NTC-doubly depleted extracts.

Mentions: We then examined whether the N-terminal region of Ntc77 is required for the function of Yju2 and Cwc25. Yju2 is required for the association of Cwc25 with the spliceosome to promote the first reaction, and can be recruited to the spliceosome before or after Prp2 action (10). We have previously shown that Ntc90 is not required for NTC-mediated spliceosome activation, and is required only for the recruitment of Yju2 to the spliceosome (28). Yju2 interacts with both Ntc90 and Ntc77, with Ntc90 via its NTD (Yju2-N) and with Ntc77 via its C-terminal domain (Yju2-C) (29). Yju2-C alone can bind stably to the spliceosome, but does not function in the first reaction. Furthermore, Yju2-C stabilizes the association of Yju2-N with the spliceosome to restore its full activity. Together, these results suggest that Ntc77 also plays a role in stabilization of Yju2. We speculated that the N-terminal region of Ntc77 might be involved in its interaction with Yju2, and examined the association of Yju2 with the spliceosome in ΔN-3. Splicing was carried out with biotinylated ACAC pre-mRNA in extracts depleted of both NTC and Cwc25, and supplemented with NTC, N77 or ΔN3. Depletion of Cwc25 results in the arrest of the spliceosome immediately before the first reaction, and accumulation of Yju2 on the spliceosome. The spliceosome was pulled down with streptavidin Sepharose to analyze the protein components (Figure 6A). Consistent with previous observation, Yju2 was barely detected in the absence of NTC (lane 2). While Yju2 was accumulated in a large amount on the spliceosome with N77 (lanes 3 and 4), less than 40% of Yju2 was present with ΔN3 (lane 5). The amount of Snu114 or Ntc85 was not significantly affected. This result indicates that the deletion of the N-3 region of Ntc77 might inhibit the splicing activity by preventing stable association of Yju2, and consequently the recruitment of Cwc25 to the spliceosome. After the first reaction, Yju2 and Cwc25 become stably associated with the spliceosome, and need to be displaced from the spliceosome mediated by Prp16. We further examined whether stable association of Yju2 with the spliceosome in post-catalytic stage is also affected by the presence of the N-3 region of Ntc77. Splicing was carried out with biotinylated ACAC pre-mRNA in extracts depleted of both NTC and Prp16, and supplemented with NTC, N77 or ΔN3 (Figure 6B). Similarly, less Yju2 was seen to be associated with the pulled-down ΔN3 spliceosome (lane 5), suggesting that the N-3 region of Ntc77 is required for stable association of Yju2 with both the pre- and post-catalytic 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)

ΔN-3 destabilized the association of Yju2 with the spliceosome. (A) Splicing reactions were carried out in Cwc25- (lane 1) or Prp19- and Cwc25- doubly depleted extracts (lanes 2–5) without (lanes 1 and 2) or with the addition of NTC (lane 3), N77 (lane 4) or ΔN3 (lane 5) using biotinylated ACAC pre-mRNA, and the spliceosomes were pulled down with streptavidin Sepharose. CPX, complex; d25, Cwc25-depleted extracts; d25dNTC, Cwc25- and NTC-doubly depleted extracts. (B) Splicing reactions were carried out in Prp16- (lane 1) or Prp19- and Prp16-doubly depleted extracts (lanes 2–5) without (lanes 1 and 2) or with the addition of NTC (lane 3), N77 (lane 4) or ΔN3 (lane 5) using biotinylated ACAC pre-mRNA, and the spliceosomes were pulled down with streptavidin Sepharose. In both (A) and (B), components were probed with antibodies against Snu114, Ntc85, Yju2 and HA-epitope for Prp19 (lanes 1 and 3) and Ntc77 (lanes 4 and 5). CPX, complex; d16, Prp16-depleted extracts; d16dNTC, Prp16- and NTC-doubly depleted extracts.
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Figure 6: ΔN-3 destabilized the association of Yju2 with the spliceosome. (A) Splicing reactions were carried out in Cwc25- (lane 1) or Prp19- and Cwc25- doubly depleted extracts (lanes 2–5) without (lanes 1 and 2) or with the addition of NTC (lane 3), N77 (lane 4) or ΔN3 (lane 5) using biotinylated ACAC pre-mRNA, and the spliceosomes were pulled down with streptavidin Sepharose. CPX, complex; d25, Cwc25-depleted extracts; d25dNTC, Cwc25- and NTC-doubly depleted extracts. (B) Splicing reactions were carried out in Prp16- (lane 1) or Prp19- and Prp16-doubly depleted extracts (lanes 2–5) without (lanes 1 and 2) or with the addition of NTC (lane 3), N77 (lane 4) or ΔN3 (lane 5) using biotinylated ACAC pre-mRNA, and the spliceosomes were pulled down with streptavidin Sepharose. In both (A) and (B), components were probed with antibodies against Snu114, Ntc85, Yju2 and HA-epitope for Prp19 (lanes 1 and 3) and Ntc77 (lanes 4 and 5). CPX, complex; d16, Prp16-depleted extracts; d16dNTC, Prp16- and NTC-doubly depleted extracts.
Mentions: We then examined whether the N-terminal region of Ntc77 is required for the function of Yju2 and Cwc25. Yju2 is required for the association of Cwc25 with the spliceosome to promote the first reaction, and can be recruited to the spliceosome before or after Prp2 action (10). We have previously shown that Ntc90 is not required for NTC-mediated spliceosome activation, and is required only for the recruitment of Yju2 to the spliceosome (28). Yju2 interacts with both Ntc90 and Ntc77, with Ntc90 via its NTD (Yju2-N) and with Ntc77 via its C-terminal domain (Yju2-C) (29). Yju2-C alone can bind stably to the spliceosome, but does not function in the first reaction. Furthermore, Yju2-C stabilizes the association of Yju2-N with the spliceosome to restore its full activity. Together, these results suggest that Ntc77 also plays a role in stabilization of Yju2. We speculated that the N-terminal region of Ntc77 might be involved in its interaction with Yju2, and examined the association of Yju2 with the spliceosome in ΔN-3. Splicing was carried out with biotinylated ACAC pre-mRNA in extracts depleted of both NTC and Cwc25, and supplemented with NTC, N77 or ΔN3. Depletion of Cwc25 results in the arrest of the spliceosome immediately before the first reaction, and accumulation of Yju2 on the spliceosome. The spliceosome was pulled down with streptavidin Sepharose to analyze the protein components (Figure 6A). Consistent with previous observation, Yju2 was barely detected in the absence of NTC (lane 2). While Yju2 was accumulated in a large amount on the spliceosome with N77 (lanes 3 and 4), less than 40% of Yju2 was present with ΔN3 (lane 5). The amount of Snu114 or Ntc85 was not significantly affected. This result indicates that the deletion of the N-3 region of Ntc77 might inhibit the splicing activity by preventing stable association of Yju2, and consequently the recruitment of Cwc25 to the spliceosome. After the first reaction, Yju2 and Cwc25 become stably associated with the spliceosome, and need to be displaced from the spliceosome mediated by Prp16. We further examined whether stable association of Yju2 with the spliceosome in post-catalytic stage is also affected by the presence of the N-3 region of Ntc77. Splicing was carried out with biotinylated ACAC pre-mRNA in extracts depleted of both NTC and Prp16, and supplemented with NTC, N77 or ΔN3 (Figure 6B). Similarly, less Yju2 was seen to be associated with the pulled-down ΔN3 spliceosome (lane 5), suggesting that the N-3 region of Ntc77 is required for stable association of Yju2 with both the pre- and post-catalytic 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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