Fig. 1.

Exonic binding of the intronic activator, U2AF65, inhibits splicing. (A) SELEX motifs were mapped to a dataset of 312,275 human splice site regions and plotted on an amalgamated exon. (B) The synthetic polypyrimidine tract returned by the SELEX consensus U2AF65 motifs and a genomic polypyrimidine tract were ligated into an exon and tested for U2AF65 binding by UV cross-linking in extract without antibody (lane 1, 3, and 5) or in extract that was blocked by an anti-U2AF65 antibody (lane 2 and 4). The radiolabel transferred to several products of differing mobility—a 65 kD interaction that was sensitive to preincubation with antiU2AF65 antibody is indicated with an arrow. (C) The sizes of RT-PCR products reflecting varying degrees of splicing are shown by the arrows. The disruptive effects of ligating the synthetic and natural PPT into the test exon of pZW4 is shown by RT-PCR in lane 7 and 8.