GROUP-II INTRON SPLICING IN-VIVO BY FIRST-STEP HYDROLYSIS

Group I, group II and spliceosomal introns splice by two sequential tr ansesterification reactions(1). For both spliceosomal and group II int rons, the first-step reaction occurs by nucleophilic attack on the 5' splice junction by the 2' hydroxyl of an internal adenosine, forming a 2'-5' phosphodiester branch in the intron. The second reaction joins the two exons with a 3'-5' phosphodiester bond and releases intron lar iat. In vitro, group II introns can self-splice by an efficient altern ative pathway in which the first-step reaction occurs by hydrolysis. T he resulting linear splicing intermediate participates in normal secon d-step reactions, forming spliced exon and linear intron RNAs2,3. Here we show that the group II intron first-step hydrolysis reaction occur s in vivo in place of transesterification in the mitochondria of yeast strains containing branch-site mutations. As expected, the mutations block branching, but surprisingly still allow accurate splicing. This hydrolysis pathway may have been a step in the evolution of splicing m echanisms.