FIGURE 3.

Prp16 mediates the release of Yju2 and Cwc25. Splicing was carried out for 10 min with wild-type actin transcript in Prp16-depleted extracts, and ATP was depleted by incubation for 5 min upon the addition of 10 mM glucose. After addition or no addition of recombinant Prp16, the reaction mixtures were precipitated with antibody against Ntc20 (lanes 2–6), Yju2 (lanes 8–12,14–18) or Cwc25 (lanes 20–24,26–30). The precipitated spliceosome was reincubated in the presence or absence of ATP, and the pellet and supernatant fractions were separated. The amount of RNA was quantified by PhosphoImager and plotted in a bar graph with the sum of supernatant and pellet as 100%. The x-axis indicates lane numbers of the gel. R: 1/10 of the reaction mixture; SP: spliceosome; T: total precipitate; P: pellet; S: supernatant.