Pre-mRNA splicing is essential for gene expression in all eukaryotes, but our understanding of the molecular mechanisms of pre-mRNA splicing is limited. Splicing of introns is carried out through two transesterification reactions catalyzed by the spliceosome, a large RNA/protein complex composed of five snRNAs and over 100 protein factors. Many lines of evidence point to Prp8 as a key spliceosomal protein that interacts intimately with RNA at the catalytic core, potentially helping the formation and stabilization of the catalytic core. Prp8 is one of the largest and most conserved nuclear proteins known, but it does not have obvious sequence homology with any other known protein. Further structural and biochemical analyses would provide valuable insight into Prp8's function in splicing. However, these studies are hindered by difficulties in obtaining large quantities of full-length Prp8. Identifying, expressing, and purifying domains of Prp8 will provide a valuable alternative approach for characterizing Prp8. This project uses a unique high throughput approach to identify soluble domains of Prp8 and determine structures of these domains. Structures of these domains and comparison with other known structures can provide important information on the function of Prp8 in splicing, directing future mutational/genetic experiments. These soluble domains are also valuable resources for characterizing Prp8's biochemical properties. This research is a critical step toward generating an atomic picture of Prp8 that cannot be obtained otherwise, significantly advancing our understanding of the molecular mechanisms of pre-mRNA splicing.
This project focuses on understanding the molecular mechanism of pre-mRNA splicing, one of the most fundamental biological processes critical for all eukaryotic species including humans. In addition, a postdoctoral fellow, as well as Ph.D. and undergraduate students, including minority members, will participate in this work. Results from this project will be published in broad based scientific journals and presented at local and international meetings. This research not only advances the understanding of basic science but also helps train a new generation of scientists.
