Fig. 8. The 110 kDa protein is required for splicing in vitro. (A) Immunodepletion of the 110 kDa protein from HeLa nuclear extract inhibits splicing in vitro. The time course of splicing reactions was monitored in 110 kDa-depleted (lanes 5–8) and mock-depleted extracts (lanes 1–4). The bands corresponding to pre-mRNA, inter mediates and spliced products are indicated on the left. (B) Splicing activity of the depleted extract is restored by addition of the recombin ant 110 kDa protein expressed in E.coli. Splicing reactions containing the mock-depleted extract (lane 1), or 110 kDa-depleted extracts complemented with 0, 25, 50 and 100 ng of the recombinant protein (lane 2, 3, 4 and 5), were incubated for 60 min and analysed as described in Materials and methods. (C) The recombinant 110 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 the mock-depleted extract (lanes 1–4), the 110-kDa-depleted extract (lanes 5–8) and the 110 kDa-depleted extract complemented with 50 ng of the recombinant 110 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.