METAL-ION CATALYSIS DURING SPLICING OF PREMESSENGER RNA

The removal of intervening sequences from premessenger RNA is essentia l for the expression of most eukaryotic genes. The spliceosome ribonuc leoprotein complex catalyses intron removal by two sequential phosphot ransesterification reactions', but the catalytic mechanisms are unknow n. It has been proposed that two divalent metal ions may mediate catal ysis of both reaction steps, activating the 2'-or 3'-hydroxyl groups f or nucleophilic attack and stabilizing the 3'-oxyanion leaving groups by direct coordination(2). Here we show that in splicing reactions wit h a precursor RNA containing a 3'-sulphur substitution at the 5' splic e site, interaction between metal ion and leaving group is essential f or catalysis of the first reaction step. This establishes that the spl iceosome is a metalloenzyme and demonstrates a direct parallel with th e catalytic strategy used by the self-splicing group I intron from Tet rahymena(3). In contrast, 3'-sulphur substitution at the 3' splice sit e provides no evidence for a metal ion-leaving group interaction in th e second reaction step, suggesting that the two steps of splicing proc eed by different catalytic mechanisms and therefore in distinct active sites.