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? 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.
Extensive Research Description
Our research focuses on exploring the underlying principles that govern folding, dynamic rearrangement, and stability of RNA molecules and RNA-protein interfaces. To address these issues, we focus on three major problems: 1) Group II introns, which are large self-splicing ribozymes and mobile genetic elements. We are elucidating the folding pathway and tertiary structure of these highly structured RNA molecules, while also examining their interactions with proteins involved in transposition. 2) DExH/D proteins, which are a diverse family of enzymes that manipulate RNA conformation in an ATP-dependent manner. We are studying the motor function and mechanism of the NPH-II helicase from Vaccinia and the NS3/4B/5A replication complex from Hepatitis C Virus. 3) This experimental work is complemented by the development of new computational approaches for analyzing RNA conformation.
- Dumont, S., et al. (2006). RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 439:105-108.
- Solem, A., Zingler, N., and Pyle, A.M. (2006). A DEAD protein that activates intron self-splicing without unwinding RNA. Mol. Cell 24:611-617.
- Toor, N., Keating, K. S., Taylor, S. D. and Pyle, A. M. Crystal structure of a self-spliced group II intron. Science 320, 77-82 (2008)
- Pyle, A. M. Translocation and unwinding mechanisms of RNA and DNA helicases. Ann. Rev. Biophys. 37, 317-336 (2008)
- Group II Introns: Catalysis for splicing, genomic change and evolution. Anna Marie Pyle (2008) in Ribozymes and RNA Catalysis (ed. D. M. J. Lilley and F. Eckstein) pp. 201-228. Royal Society of Chemistry, Cambridge