Characterization of RNA-protein crosslinks in yeast spliceosomes

The spliceosome is a macromolecular machinery comprised of RNA and proteins. It assembles on its substrate pre-mRNA in a stepwise manner. Both during assembly and catalysis different components of the spliceosome interact with functionally important regions of the pre-mRNA in precise temporal and spatial coordination. In this study the proteins interacting with the pre-mRNA upstream and downstream of the branch site were investigated in purified yeast spliceosomal complexes. The region directly upstream of the branch site is a major interaction platform for the U2 snRNP proteins in yeast activated spliceosomes. This is similar to human spliceosomes, where this region of the pre-mRNA is called the “anchoring sequence”. These protein interactions persist through the first step of splicing. Furthermore a number of formerly unknown protein-pre-mRNA interactions downstream of the branch site are reported.