Our long-term goal is to understand the strategies used by biology to control the specificity and fidelity of RNA processing reactions. Over the past 15 years we have exploited the powerful tool of yeast genetics to analyze the RNA and protein components of the spliceosome. In particular, compensatory base pair analysis has revealed critical RNA-RNA interactions at the catalytic core, in striking agreement with data of others obtained by photochemical crosslinking. Taken together, these findings have allowed the conclusion that the spliceosome is a spectacularly dynamic macromolecular machine. Future goals are centered on our continuing efforts to understand the mechanism of these ATP-driven structural rearrangements and their regulation. Additionally, we will explore the prediction that the hand-off of spliced mRNA to the export apparatus, and subsequent release from the cytoplasmic face of the pore, are also steps which are coordinated by eIF4A-like ATPases, ensuring directionality and selectivity. Using a blend of molecular genetic, biochemical and cell biological approaches, our experiments will address four broad questions: I. How do DEAD-box proteins link ATP-driven RNA rearrangements with fidelity? II. What is the role of protein at the spliceosomal catalytic core? III. How are snRNPs recycled for new rounds of splicing? IV. How is mRNA export coupled to RNA processing? We predict that the proposed experiments will shed much-needed insight into fundamental questions about RNP-mediated catalysis, RNA folding and RNA transport. In several cases, these findings also promise to impact on health- related problems, including retroviral (HIV) RNA metabolism, and Spinal Muscular Atrophy, the most common genetic cause of childhood mortality.
Showing the most recent 10 out of 102 publications
