Metal ion catalysis during splicing of premessenger RNA

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Abstract
The removal of intervening sequences from premessenger RNA is essential for the expression of most eukaryotic genes. The spliceosome ribonucleoprotein complex catalyses intron removal by two sequential phosphotransesterification reactions1, but the catalytic mechanisms are unknown. It has been proposed that two divalent metal ions may mediate catalysis of both reaction steps, activating the 2′- or 3′-hydroxyl groups for nucleophilic attack and stabilizing the 3′-oxyanion leaving groups by direct coordination2. Here we show that in splicing reactions with a precursor RNA containing a 3′-sulphur substitution at the 5′ splice site, interaction between metal ion and leaving group is essential for catalysis of the first reaction step. This establishes that the spliceosome is a metalloenzyme and demonstrates a direct parallel with the catalytic strategy used by the self-splicing group I intron from Tetrahymena3. In contrast, 3′-sulphur substitution at the 3′ splice site provides no evidence for a metal ion–leaving group interaction in the second reaction step, suggesting that the two steps of splicing proceed by different catalytic mechanisms and therefore in distinct active sites.