FIG. 5.
Cwc22 and Prp2 bind to the spliceosome independently of each other. Splicing was carried with ACAC pre-mRNA in these experiments. (A) Splicing was carried out with mock-treated (lane 1) or Cwc22-depleted (lanes 2 to 7) extracts without (lanes 2 to 6) or with (lane 7) the addition of recombinant Cwc22. Glucose (lanes 2 and 3) or ATP (lanes 4 and 5) was added to the reaction mixtures and incubated for 5 min. Cwc22 was then added, and the reaction mixture was incubated for 20 min. (B) Splicing was carried out with wild-type (lane 1), mock-treated (lanes 2 to 5), NTC-depleted (lanes 6 to 9), or Cwc22-depleted (lanes 10 to 13) extracts. After the addition of recombinant V5-tagged prp2S378L, the reaction mixtures were precipitated with anti-Ntc20 or anti-V5 antibody. RXN, 1/5 of the reaction mixture used for immunoprecipitation; PAS, protein A-Sepharose. (C) Splicing was carried out with mock-treated (lanes 1 to 8) or Cwc22-depleted (lanes 9 to 16) Prp2-V5 extracts. Following the addition of glucose (lanes 1 to 4 and 9 to 12) or ATP (lanes 5 to 8 and 13 to 16) and incubation for 5 min, the reaction mixtures were precipitated with anti-Ntc20 or anti-V5 antibody. RXN, 1/10 of the reaction mixture used for immunoprecipitation; PAS, protein A-Sepharose. (D) Splicing was carried out with mock-treated (lanes 1 to 7) or Cwc22-depleted (lanes 8 to 14) Prp2-V5 extracts or with Cwc22-depleted extracts with the addition of the prp2S378L-V5 protein (lanes 15 to 20). Following the addition of glucose and incubation for 5 min, the reaction mixtures were precipitated with anti-V5 antibody. The precipitates were reincubated in splicing buffer containing (lanes 2 to 4, 9 to 11, 17, and 18) or not containing (lanes 5 to 7, 12 to 14, 19, and 20) ATP, and supernatant and pellet fractions were separated. The released materials (lanes 22 and 26, as from lanes 4 and 11) were incubated for 20 min following the addition (lanes 24 and 28) or no addition (lanes 23 and 27) of micrococcal nuclease-treated extracts. R, 1/10 of the reaction mixture used for immunoprecipitation; T, total precipitates; P, pellet; S, supernatant; ExtMN, micrococcal nuclease-treated extracts. (E) The spliceosome formed with biotinylated ACAC pre-mRNA in wild-type (lane 1) or Δprp2 extracts without (lane 2) or with (lane 3) the addition of the Prp2 protein was isolated by precipitation with streptavidin-Sepharose, and the components were analyzed by Western blotting.