Elucidation of the function of proteins at the second catalytic step of the pre-mRNA splicing reaction

The spliceosome is a highly dynamic ribonucleoprotein (RNP) machine that catalyses the excision of non-coding regions (introns) from a nascent pre-mRNA transcript and the subsequent ligation of coding sequences (exons). Before catalysis of the first and the second step can take place, the spliceosome undergoes dramatic changes in its RNA and protein composition as well as in its RNA-RNA and RNA-protein network. The second step of splicing as a relatively late event in the spliceosomal cycle is substantially less well characterized than the first step or other earlier steps in the assembly pathway (Smith et al., 2008). Therefore, the aim of this study was to get a comprehensive picture of the function of proteins during the second catalytic step as well as how the spliceosome’s protein inventory is remodeled during the second step of splicing. In detail, one goal of this study was to define the minimal protein factor requirements for the second catalytic step in vitro using actin wt and actin7 as pre-mRNA substrates. Via singly depleting the protein factors Prp16, Slu7, Prp18 and Prp22 it has been shown earlier in yeast whole cell extract that these proteins are required for catalyzing step-2. Single depletion of Slu7, Prp18 or Prp22, however, had no effect on the second step on actin7 as a pre-mRNA substrate (Brys and Schwer, 1996; Zhang and Schwer, 1997; Schwer and Gross, 1998). The question remained whether step-2-catalysis on actin7 would be accomplished in the absence of all three protein factors. We set out to answer this question, thereby gaining more insights to the nature of the spliceosome’s catalytic centre for step-2. A pre-requisite to perform these studies was the establishment of an in vitro reconstitution system with biochemically defined components. We set out to purify spliceosomes on actin wt pre-mRNA, which are stalled before step-2-catalysis. In detail, we made use of the temperature sensitive yeast strain prp16-2, in which the helicase Prp16 could be heatinactivated.