In vitro reconstitution of both steps of splicing and investigation of the spliceosome dynamics

The spliceosome is a protein-rich ribonucleoprotein (RNP) machine that catalyses intron removal from pre-mRNA in a two step reaction. It comprises over 80 proteins and 5 snRNAs in yeast (S. cerevisiae). To investigate structural and compositional dynamics of the spliceosome, and the requirements and kinetics of the catalytic steps of splicing we established an in vitro splicing reconstitution system. The system comprised highly purified yeast spliceosomes stalled before step 1 of splicing, and recombinant proteins. The composition of the purified spliceosomes was defined by mass spectrometry and RNA analyses. Using the splicing reconstitution system we showed that Prp2, a DEAH box helicase, structurally and compositionally rearranges the spliceosome by destabilization of a subset of proteins (SF3a/b U2 snRNP proteins). This Prp2 NTP-driven action likely exposes the branchsite sequence of the pre-mRNA, and thus makes step 1 catalysis possible. We also identified a novel splicing factor, Cwc25, which binds to the spliceosome after Prp2 has acted, and promotes step 1 catalysis in an ATP-independent manner. By addition of further 4 proteins we were able to reconstitute step 2 of splicing. Our approach paves the way for future ultrastructural and biophysical studies to dissect spliceosome activation and catalysis.