Splicing complexes are assembled on all templates. RNA templates were derived from minigenes A, C, K, and L and lacked the terminal 5' spice site of exon 11. The RNAs were incubated in 40 % HeLa nuclear extract on ice or for increasing times at 30°C to allow the formation of splicing complexes. Reactions were loaded directly onto 2% low-melting agarose gels (Panel A) or 4% native polyacrylamide gels (Panel B) and complexes separated by electrophoresis. Gels were dried under vacuum and exposed to film. The non-specific hnRNP complex H is indicated as well as the pre-spliceosomal complexes E and A. In Panel B, the higher order complexes B and C are indicated. In Panel C, the nuclear extract was pretreated with RNase H and an oligonucleotide complementary to the first 14 nucleotides of the U2 snRNA or a control oligonucleotide before complex assembly. In Panel D, the assembly reactions were performed in the absence or presence of ATP.