a, Schematic representation of the ACT1 pre-mRNA tagged with three MS2-binding sites (M3–ACT1) used for E complex assembly and purification. Boxes represent exon 1 (E1) and truncated exon 2 (E2). The 5′ SS (GU) and BPS (UACUAAC) are also shown. The red line represents the DNA oligo complementary to a region 5 nt upstream of the BPS for the RNase H cleavage experiment. b, RNA components of the assembled E complex (with or without DNA oligo and RNase H treatment) after proteinase K digestion are shown on a denaturing urea gel or native agarose gel. These results demonstrate that RNase treatment cleaved M3–ACT1 into two fragments. Note that the sizes of RNA on the native gel do not match their linear length, possibly owing to the existence of secondary structures. This experiment was repeated two additional times with similar results.