Catalysis of the first step of pre-mRNA splicing leads to the formation of the spliceosomal C complex, which contains the
pre-mRNA intermediates—the cleaved 5′ exon and the intron-3′ exon lariat. To topographically locate the catalytic center of
the human C complex, the authors first determined, by DNA oligonucleotide-directed RNAse H digestions, accessible pre-mRNA
regions closest to nucleotides of the cleaved 5′ splice site (i.e., the 3′ end of exon 1 and the 5′ end of the intron) and
the intron lariat branch point, which are expected to be at/near the catalytic center in complex C. For electron microscopy
(EM) localization studies, C complexes were allowed to form and biotinylated 2′-OMe RNA oligonucleotides were annealed to
these accessible regions. To allow localization by EM of the bound oligonucleotide, first antibiotin antibodies and then protein
A-coated colloidal gold were additionally bound. EM analyses allowed mapping the position of exon and intron nucleotides near
the cleaved 5′ splice site, as well as close to the anchoring site just upstream of the branch adenosine. The identified positions
in the C complex EM map give first hints as to the path of the pre-mRNA splicing intermediates in an active spliceosomal C
complex and further define a possible location for its catalytic center.
