Accessibility of the pre-mRNA in the spliceosomal C complex, as revealed by DNA oligonucleotide-directed RNase H digestion. (A) Cleavage of the pre-mRNA in the C complex by RNase H using oligodeoxynucleotides complementary to the pre-mRNA sequences close to the cleaved 5′ splice site and the branch point. C complexes were assembled on 32P-labeled PM5 pre-mRNA in HeLa nuclear extract, DNA oligonucleotides complementary to selected pre-mRNA regions were added to the splicing reaction, and RNA bound by DNA was then cleaved by the RNase H present in the extract. The cleavage products were separated by gel electrophoresis and visualized by autoradiography. The hybridization positions of the oligonucleotides used here are shown for illustration in B. The cleavage products of the lariat are not sharply resolved but run as diffuse bands in a region above the pre-mRNA. (B) Summary of accessible regions of the splicing intermediates in the C complex as determined by RNase H cleavage. Accessible regions are shown in green and those protected from enzymatic cleavage are shown in red. The stretches that hybridize with the oligodeoxyribonucleotides used in A and with the longer oligoribonucleotides used for EM localization are indicated by black lines. The biotin positions of the three 2′-O-methyl-RNA oligonucleotides used for EM localization (“label intron anchoring site,” “label intron 5′ splice site,” and “label exon 1”) are each indicated with a green asterisk. The individual results of all the RNase H cleavage experiments performed with ∼100 oligonucleotides are shown in Supplemental Figure S1.
