Protection of the 5' and 3' splice sites and the branch point sequence. An RNaseH protection assay was used to probe occupancy of the splice sites on the IR templates. The four templates Anat, Cnat, Knat, and Lnat containing the natural terminal 5' splice site were incubated on ice or at 30°C for 30 min. Oligonucleotides complementary to the 5' splice site, the 3' splice site, or the branch point sequence (10 μM) were added followed by 2U RNaseH. Reactions were digested for 5 min at 37°C, the RNA extracted and separated on 5% sequencing gels. The time of incubation at 30°C is indicated above each panel along with the oligo added. Open arrowheads indicate RNase cleavage products for each oligonucleotide. RNase protection results in a decrease in cleavage products and an increase in uncleaved RNA. Spliced product and 5' exon are visible in some reactions that have been incubated at 30°C. The small fragment from cleavage of probes Anat, Cnat and Knat at the 3' splice site has run off the gel in this experiment. The two cleavage products from the 5' splice site oligo on template Lnat co-migrate.