Changes in Human Spliceosome Interactions following Exon Ligation

Pre-mRNA splicing is an essential step in eukaryotic gene expression.  Splicing is catalyzed by the spliceosome, a large, dynamic macromolecular machine. Conformational changes in the spliceosome are known to occur between the first and second steps of splicing.  We have developed a system to investigate subsequent changes between the second step of chemistry and mRNA release.  Prp22 is a DEAxD/H box protein that promotes the second step of splicing and the release of mRNA.  Perturbing Prp22 binding in the 3' exon of a splicing substrate stalls the spliceosome following exon ligation.  Given this, we hypothesize numerous protein:RNA and RNA:RNA rearrangements occur within the post-catalytic spliceosome to promote mRNA release.  We compared C-complex, the catalytically active spliceosome, which is arrested prior to the second step of chemistry, to the post-catalytic spliceosome complex.   In the post-catalytic complex, both mRNA and cleaved lariat intron are associated with the U5, U2 and U6 snRNPs.  Proteomics and structural analysis by electron microscopy demonstrate that the composition and global architecture of the post-catalytic complex is similar to C-complex.  Both complexes show protections against DNA oligo-mediated RNase H cleavage ~30 nt upstream of the 5' splice site.   However, we also see protection across the exon-exon junction and extending ~15 nt into the 3' exon of the unreleased mRNA that are not present in C-complex.  We are further investigating the RNA:RNA and protein:RNA interactions in the post-catalytic complex with C-complex to characterize the structural transitions that occur in the spliceosome after the second step of splicing.