(from the application): Rearrangement of both RNA and DNA structure is critical to many basic biological processes, including pre-mRNA splicing. Upon interaction with a pre-mRNA substrate, the spliceosome undergoes a series of dramatic RNA rearrangements that configure the spliceosome for catalysis. Strikingly, these rearrangements reflect the disruption and formation of mutually exclusive structures. This mutually excusive design is a hallmark of splicing and likely ensures that the spliceosome assembles in a highly coordinated and carefully regulated fashion. Although these rearrangements play a critical role in governing spliceosome activation, it is not clear how these rearrangements are catalyzed. RNA-stimulated ATPases of the ubiquitous DEAD-box family have been implicated in orchestrating these RNA rearrangements, but no target, either RNA or protein, has been proved. Moreover, to establish specificity and prevent spurious RNA rearrangements, the activity of these DEAD-box proteins must be tightly controlled, but mechanisms for regulating these ATPases have not yet been identified. Finally, the RNA folding pathway for the spliceosome likely reflects a hierarchy of RNA rearrangements carefully designed to regulate spliceosome activation, but this hierarchy remains to be defined. The long-term goal of this proposal is to address these issues by focusing on a critical rearrangement required for 5' splice site recognition, the switch of U1 for U6. A major aim is to determine how Prp28, a DEAD-box ATPase, catalyzes the switch.
A second aim i s to determine how the switch of U1 for U6 triggers unwinding of the U4/U6 duplex, a key step in the catalytic activation of the spliceosome.
A final aim i s to establish the hierarchy between the switch of U1 for U6 and unwinding of the 5' stem-loop of U2, another critical rearrangement required to catalytically activate the spliceosome. To pursue these aims, a combined approach of genetics and biochemistry will be employed in the yeast S. cerevisiae. These studies will likely have broad implications for understanding (i) the function of DEAD-box proteins in their biologically relevant contexts, (ii) the mechanisms for regulating the timing and specificity of these ATPases, and (iii) the rationale underlying the dynamic design of the spliceosome. Since coupling of spliceosome activation with substrate recognition likely serves to ensure fidelity in splicing, these studies should also contribute insights into the mechanisms for preventing splicing errors that lead to human disorders, such as cancer and heart disease.
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