ATPγS blocks spliceosome assembly before catalytic activation. (A) In vitro splicing of 32P-labeled MINX pre-mRNA was performed for the indicated times in untreated HeLa nuclear extract (lanes 22–24) or extract depleted of ATP by incubating with 2 mM glucose (lanes 1–21). Splicing was carried out in the presence of 2 mM ATP and creatine phosphate ATP/CP (lanes 19–24) or in the presence of increasing concentrations of ATPγS (in the absence of ATP) as indicated above (lanes 1–18). Splicing was analyzed by denaturing PAGE and the positions of the pre-mRNA substrate and intermediates/products of the splicing reaction are shown on the right. The spliced out lariat has been debranched and thus is not visible above the unspliced pre-mRNA. (B) In vitro splicing was performed as in A, and the formation of splicing complexes was analyzed on a native agarose gel. The positions of the splicing complexes (A, B, Bact, and C), as well as the H complex, are indicated on the left. (C) In vitro splicing as in A, performed in ATP-depleted extract (lanes 1–3) or ATP-depleted extract supplemented with 2 mM ATP and CP (lanes 4–6), 2 mM ATPγS (lanes 7–9), or 2 mM AMP-PNP (lanes 10–12) for 0–180 min as indicated above. (D) Analysis of splicing complex formation (as in B) in the absence of ATP (lanes 1–5) or in its presence (ATP/CP, lanes 6–10) or in the presence of 2 mM ATPγS (lanes 11–15) or AMP-PNP (lanes 16–20). Splicing was performed for 0–60 min as indicated above each lane.
