The spliceosome is a protein-rich ribonucleoprotein (RNP) machine that catalyses intron removal from pre-mRNA in a two-step splicing reaction. By these means it leads to formation of mRNA that can be translated into a protein. The spliceosome undergoes a series of profound structural and compositional rearrangements during its de novo assembly on each nascent pre-mRNA and during splicing catalysis. Here, we capture the spliceosome just before the first step of splicing, purify it and investigate thoroughly its proteomic composition. We then by combining purified spliceosomes with recombinant proteins reconstitute both steps of splicing in vitro, showing which proteins are necessary for catalysis. Finally, using the in vitro reconstitution approach we test in detail requirements and outcomes of the activity of a spliceosomal helicase protein Prp2 on the structure and composition of this macromolecular complex.weiterlesen