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ANNA MARIE PYLE

Research Description
RNA has many different important functions in the cell, from the transfer of genetic information to the catalysis of reactions that are essential for gene expression. Often it is the folded tertiary structure of an RNA, rather than its primary sequence, that is essential for function. Given the ubiquitous role of folded RNA in biology, we have set out to understand some basic principles: What are the building blocks for RNA tertiary structure and how is it energetically stabilized? What are pathways for reaching the folded state? How are RNA and ribonucleoprotein assemblies actively folded and unfolded by RNA remodeling enzymes such as helicases? How can computational and experimental tools be combined to better understand these problems?

Many of our studies focus on the self-splicing group II intron, which is a ribozyme that catalyzes its own excision from precursor mRNA and which has been shown to be a mobile genetic element that invades duplex DNA. The evolutionary impact, biological role and biotechnological applications of group II introns are of great interest. A rich source of information on RNA tertiary structure, these large ribozymes (~600 nts in size) assemble through complex networks of unusual tertiary interactions. They are excellent models for studying RNA folding because they fold directly to the native state through a series of structurally and energetically characterized intermediates. Using a combination of biophysical and chemogenetic techniques, we are identifying the molecular structure of group II introns and their folding intermediates, determining the energetic contribution of individual tertiary interactions, and monitoring the dynamic behavior of intermediates along the RNA folding pathway. In addition, we are examining the role of proteins in RNA tertiary folding and structural stabilization.

RNA metabolism requires that RNA folding be coordinated with RNA unfolding as a function of time or the presence of biochemical signals. We have been studying mechanisms of RNA unwinding and remodeling by a class of helicases that are involved in all aspects of RNA metabolism and in viral replication (the DExH/D subgroup of helicase superfamily 2). Our work has focused on two helicases that are essential for the replication of vaccinia and hepatitis C (HCV) viruses. These helicases serve as model systems for defining the behavior of DExH/D proteins in general, and more specifically, they are important drug targets in the effort to develop antiviral therapeutics. We have shown that members of this protein family are more than helicases: Some family members can unwind nucleic acids, but others serve to strip proteins from RNA, and in some cases, the proteins function as ATP-dependent conformational switches that specifically remodel RNP complexes. We are particularly interested in the NS3 helicase and its mechanistic function within the replication complex of HCV. NS3 is a uniquely valuable system for studying helicase function in the context of large macromolecular machines because it has inherent RNA unwinding activity that is modulated by allosteric domains and by other proteins within the replication machinery. Using techniques of transient kinetics, single molecule methods and structural biology, we are deducing the molecular mechanisms of NS3 helicase activity and attempting to understand its role in HCV replication.

Experimental approaches in our laboratory are complemented by computational studies. Our laboratory has independently developed robust methods for predicting macromolecular interactions from primary sequence data (a computational two-hybrid approach). We have also developed programs for describing the conformational states of RNA molecules in a manner analogous to the Ramachandran plot for protein conformation. Our programs are now widely used to analyze new RNA structures for the presence of new RNA structural motifs, to identify regions that require further refinement, and to provide a comprehensive analysis of motif composition. Finally, we collaborate with Barry Honig (HHMI, Columbia University) to develop and implement new methods for calculating the electrostatic properties of RNA, such as metal ion binding sites and pKa shifts.

Selected References
Serebrov, V. and Pyle, A.M. periodic cycles of RNA unwinding and pausing by hepatitis C virus NS3 helicase. Nature 430, 476-80 (2004)

de Lencastre, A., Hamill, S. and Pyle, A.M. A single active-site region for a group II intron. Nature Structural and Molecular Biology 7, 626-7 (2005)

Solem, A., Zingler, N. and Pyle, A.M. A DEAD protein that activates intron self-splicing without unwinding RNA. Molecular Cell 24, 611-617 (2006)

Waldsich, C., Pyle A.M. A folding control element for tertiary collapse of a group II intron ribozyme. Nature Structural and Molecular Biology (2006)

Hamill, S. and Pyle, A.M. The receptor for branch-site docking within a group II intron active site. Molecular Cell 23, 831-840 (2006)

Last Updated 12-18-06