FIG. 2.

CE9 inhibits in vitro splicing. (A) The structures of pre-mRNA B and BΔ, as well as derivatives containing multiple copies of CE9 or complementary sequences, are shown at the top. The distance of the insertion point to the 3′ splice site (ss) is indicated in nucleotides. Note that the CE6 element is absent from all pre-mRNAs. Labeled pre-mRNAs were incubated in HeLa nuclear extracts for the times indicated for BΔ and B RNAs or for 2 h for multiple inserts. Splicing products were run on an 11% acrylamide–8 M urea gel. Mixtures containing two different RNAs were analyzed to rule out the presence of a nonspecific inhibitor in the B2x and B3x RNA preparations (lanes 13 and 14). (B) Copies of CE9 or complementary sequences were inserted into a pre-mRNA substrate derived from the adenovirus (Ad) major late transcription unit (A RNA). The labeled splicing products were resolved on a 7% acrylamide–8 M urea gel. Because mRNA products were obscured by the degradation of the pre-mRNAs, only the portion of the gels that indicates the pre-mRNAs and lariat products is shown.