Figure 1. U1 snRNP/5′ splice site interaction is altered during splicing repression.

A) Schematic maps of the wildtype and mutant N1 exon containing pre-mRNAs. B) CU-elements flanking the N1 exon are essential for splicing repression. In vitro splicing of the wildtype and mutant BS713 transcripts was carried out in HeLa (lanes 1–3) and WERI-1 (lanes 4–6) extract. The RNA splicing products and intermediates are shown to the right. B) C-src transcripts containing the N1 exon were site specifically ³²P labeled at the N1 exon 5′ splice site. The labeled wildtype and PTB binding site mutant RNAs were incubated in Buffer DG (lanes 1–3 and 10–12), HeLa extract (lanes 5–7 and 13–15), and WERI extract (lanes 7–9 and 16–18). After incubation, reactions were treated with micrococcal nuclease (lanes 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17 and 18). Extract were either mock treated or pre-incubated with oligonucleotide U1_1–15 prior to addition of the labeled N1 RNA. The protected fragments were extracted, using PCA, ethanol precipitated, separated using urea-PAGE, and visualized by phosphorimaging. The presence of a downstream 3′ splice site did not alter the MNase protection pattern in HeLa extract (see Figure S1).