Evidence for two active sites in the spliceosome provided by stereochemistry of pre-mRNA splicing

The exchange of a 3'-5'- for 2'-5' bond in the first step of lariat formation and the exchange of one 3'-5' phosphodiester for another in the second step of exon ligation are the two different reactions for subtracting the introns from nuclear precursors to messenger RNAs (pre-mRNAs) by the spliceosome. A study conducted to detect the stereochemical course for each step, utilizing the splicing substrate containing phosphothioate, showed that both steps occur as a single 'in-line' Sn2 nucleophilic substitution reaction, similar to the mechanism of group I self splicing introns. The reaction sites for second step of spliceosomal processing is similar to that of group I self splicing introns.