The debranching reaction occurs on the Prp16-associated and post-Prp16 spliceosomes. (A) A splicing reaction was carried out in Prp16-depleted extracts in the presence of recombinant HA-tagged Prp16 mutant D473A (lane 1), and the spliceosome was precipitated with unconjugated (lane 2) or anti-HA antibody-conjugated protein A-Sepharose (lanes 3–5). The precipitated spliceosome was incubated in 10 mM Tris-HCl, pH 8.8, 4 mM MgCl2, in the absence (lane 4) or presence of 150 mM KCl (lane 5). (B) A splicing reaction was carried out with ACAC pre-mRNA in Prp22-4V5 extracts (lane 1). The spliceosome was precipitated with anti-V5 antibody (lane 2), and the purified spliceosome was incubated at 25°C for 1 h in 10 mM Tris-HCl, pH 8.8, without (lane 3) or with 0.01 mM, 0.1 mM, 0.5 mM, 1 mM, or 2 mM MgCl2 in the absence (lanes 3–8) or presence of 150 mM KCl (lanes 9–13) or 150 mM NaCl (lanes 14–18). (R) 1/10 of reaction mixture, (P) protein A-Sepharose, (SP) spliceosome.
