Intellectual merit. Most human protein-coding genes contain the primary protein-coding regions (exons) interrupted by non-coding regions (introns). In the process of precursor-mRNA splicing, the introns must be excised and the exons spliced together to generate an mRNA transcript suitable for translation into protein. Precursor-mRNA splicing is catalyzed by a remarkably large and highly dynamic "machine", the spliceosome. The spliceosome is formed by the assembly onto a precursor-mRNA transcript of five RNA-protein complexes or U snRNPs. We lack a clear understanding of how the U snRNPs initiate spliceosome assembly and organize the spliceosome for catalysis. This gap in knowledge is due in large part to experimental challenges posed by the spliceosome's large size, dynamic properties, and necessity to use crude cell extract for its study. The aim of this project is to elucidate how the initial events in the assembly of the U snRNPs onto a precursor-mRNA transcript contribute to formation of an active spliceosome that exhibits high fidelity. To accomplish this long-term goal, this project will utilize new and innovative methods designed to "trap" the spliceosome at early intermediate states of its reaction cycle and to aid the visualization of its structure.
Broader impact. A long-term goal of this project is to lay the foundation for an educational initiative aimed at motivating students to engage in science and be exposed to how understanding structure provides insight into function. Using new media, this project aims to create an educational outreach that will engage high school students in the sciences and provide their science teachers with training and professional development on integration of new media and macromolecular structures into their classrooms.
