Efficient and accurate RNA splicing is fundamentally important for proper gene expression. Group II introns provide a valuable window into the molecular mechanisms of RNA splicing and retrotransposition, and they are being harnessed as powerful tools for site-specific gene manipulation. Because of their large size and structural complexity, group II introns have provided new insights into RNA tertiary organization and folding, while also serving as a proving ground for the development of tools used in the biochemical and structural analysis of large RNA molecules. To advance all of these goals, we study the molecular structure and catalytic mechanism of group II introns. We recently solved the structure of a small, highly reactive group II intron in the spliced product state. This work revealed an elaborate architectural frame that surrounds a conserved active-site motif for binding catalytic metal ions. Our continuing projects will build on these findings by revealing the multiple conformational states that are adopted during the different stages of splicing and by revealing the structural features of group II intron-maturase complexes. At each phase of the project, structural information will be used to guide functional studies on molecular mechanisms of splicing and retrotransposition. There are three complementary aims of this project.
In AIM 1, we will crystallographically characterize the conformational states of the Oceanobacillus iheyensis group IIC intron, enabling us to visualize its function as a molecular machine during splicing. We will also characterize the folding pathway of this intron and identify the molecular determinants for transition- state stabilization during catalysis.
In AIM 2, we will structurally characterize the more elaborate group II intron subtype that maintains high levels of sequence specificity and which contains novel architectural motifs.
In AIM 3, we will solve the structure of a group II intron-maturase RNP, which is the natural form of the intron that typically functions during intron mobility and splicing. Using a combination of x-ray crystallography, solution biochemical structure mapping and mechanistic enzymology, we will reveal the respective contributions of RNA and protein to the processes of group II intron splicing and mobility, providing information that is essential for our understanding of molecular evolution and for the development of group II introns as tools and potential therapeutics.

Public Health Relevance

Efficient and accurate RNA splicing is essential for human gene expression and group II introns are among the most important systems for understanding this central aspect of our metabolism. Structural studies on group II introns continue to provide basic insights into the molecular mechanisms and sequence specificity of RNA splicing. These studies are also important for the development of group II introns as gene targeting agents for use in biotechnology and medicine.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-U (02))
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Preusch, Peter C
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Yale University
Schools of Arts and Sciences
New Haven
United States
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Somarowthu, Srinivas; Legiewicz, Michal; Keating, Kevin S et al. (2014) Visualizing the ai5? group IIB intron. Nucleic Acids Res 42:1947-58
Marcia, Marco; Pyle, Anna Marie (2014) Principles of ion recognition in RNA: insights from the group II intron structures. RNA 20:516-27
Marcia, Marco; Humphris-Narayanan, Elisabeth; Keating, Kevin S et al. (2013) Solving nucleic acid structures by molecular replacement: examples from group II intron studies. Acta Crystallogr D Biol Crystallogr 69:2174-85
Nagy, Vivien; Pirakitikulr, Nathan; Zhou, Katherine Ismei et al. (2013) Predicted group II intron lineages E and F comprise catalytically active ribozymes. RNA 19:1266-78
Keating, Kevin S; Humphris, Elisabeth L; Pyle, Anna Marie (2011) A new way to see RNA. Q Rev Biophys 44:433-66
Roitzsch, Michael; Fedorova, Olga; Pyle, Anna Marie (2010) The 2'-OH group at the group II intron terminus acts as a proton shuttle. Nat Chem Biol 6:218-224
Pyle, Anna Marie (2010) The tertiary structure of group II introns: implications for biological function and evolution. Crit Rev Biochem Mol Biol 45:215-32
Toor, Navtej; Keating, Kevin S; Fedorova, Olga et al. (2010) Tertiary architecture of the Oceanobacillus iheyensis group II intron. RNA 16:57-69
Fedorova, Olga; Solem, Amanda; Pyle, Anna Marie (2010) Protein-facilitated folding of group II intron ribozymes. J Mol Biol 397:799-813
Keating, Kevin S; Toor, Navtej; Perlman, Philip S et al. (2010) A structural analysis of the group II intron active site and implications for the spliceosome. RNA 16:1-9

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