Fig. 7. The 65 kDa protein is required for splicing in vitro. (A) Immunodepletion of the 65 kDa protein from HeLa nuclear extract inhibits splicing in vitro. The time course of splicing reactions was monitored in 65 kDa-depleted (lanes 5–8) and mock-depleted extracts (lanes 1–4). The bands corresponding to pre-mRNA, intermediates and spliced products are indicated on the left. (B) Splicing activity of the depleted extract is restored by addition of the recombinant 65 kDa protein produced in E.coli. Splicing reactions containing the mock-depleted extract (lane 1), or 65 kDa-depleted extracts complemented with 0, 34 and 68 ng of the recombinant protein (lanes 2, 3, and 4), were incubated for 60 min and analysed as described in Materials and methods. (C) Recombinant 65 kDa protein promotes the transition from the A to B complex in the depleted extracts. The spliceosome assembly was analysed by native gel electrophoresis in mock-depleted extracts (lanes 1–4), 65 kDa-depleted extracts (lanes 5–8) and 65 kDa-depleted extracts complemented with 34 ng of recombinant 65 kDa protein (lanes 9–12). The bands corresponding to the H, A and B complexes as well as the gel origin are indicated on the left.