| The spliceosome is a highly dynamic ribonucleoprotein machine that must assemble de novo on each pre-mRNA substrate before undergoing the two sequential chemical steps of splicing. Not only do the RNA components of the spliceosome aid in recognition and positioning of the pre-mRNA substrate, but they also likely promote the chemistry of splicing. To better understand how the snRNAs function during splicing, I have undertaken a molecular genetic approach to study the structure and dynamics of the snRNA components of the spliceosome during its fully assembled phase.;Using molecular genetics, we have demonstrated that the snRNA structure of the spliceosome looks strikingly similar during each catalytic step, as we have implicated a role for both U2 stem IIc and U2/U6 helix I during both of these stages. This conformation likely represents an arrangement of the snRNAs that efficiently carries out the phosphoryl transfer reactions of splicing. We have also demonstrated that the spliceosome is surprisingly dynamic between the two catalytic steps, as it forms an additional novel intermediate conformation between these steps. The intermediate state may promote substrate repositioning prior to exon ligation. Further, I have evidence that the catalytic core of the spliceosome contains a catalytic triple helix analogous to the catalytic triple helix of self-splicing group II introns. These data strengthen the evolutionary relationship between these two splicing systems, and strongly suggests that the spliceosome is indeed a ribozyme. Together, my work represents a significant advance in our understanding of snRNA structure, dynamics and function during pre-mRNA splicing. |
