Splicing of nuclear pre-mRNA occurs via two steps of the transesterification reaction,
forming a lariat intermediate and product. The reactions are catalyzed by the spliceosome,
a large ribonucleoprotein complex composed of five small nuclear RNAs and numerous
protein factors. The spliceosome shares a similar catalytic core structure with that
of fungal group II introns, which can self-splice using the same chemical mechanism.
Like group II introns, both catalytic steps of pre-mRNA splicing can efficiently reverse
on the affinity-purified spliceosome. The spliceosome also catalyzes a hydrolytic
spliced-exon reopening reaction as observed in group II introns, indicating a strong
link in their evolutionary relationship. We show here that, by arresting splicing
after the first catalytic step, the purified spliceosome can catalyze debranching
of lariat-intron-exon 2. The debranching reaction, although not observed in group
II introns, has similar monovalent cation preferences as those for splicing catalysis
of group II introns. The debranching reaction is in competition with the reverse Step
1 reaction influenced by the ionic environment and the structure of components binding
near the catalytic center, suggesting that the catalytic center of the spliceosome
can switch between different conformations to direct different chemical reactions.