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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM050313-19
Application #
8538990
Study Section
Special Emphasis Panel (ZRG1-BCMB-U (02))
Program Officer
Preusch, Peter C
Project Start
1994-01-01
Project End
2016-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
19
Fiscal Year
2013
Total Cost
$321,345
Indirect Cost
$128,345
Name
Yale University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Zhao, Chen; Pyle, Anna Marie (2016) Crystal structures of a group II intron maturase reveal a missing link in spliceosome evolution. Nat Struct Mol Biol 23:558-65
Marcia, Marco (2016) Using Molecular Replacement Phasing to Study the Structure and Function of RNA. Methods Mol Biol 1320:233-57
Somarowthu, Srinivas (2016) Progress and Current Challenges in Modeling Large RNAs. J Mol Biol 428:736-47
Chillón, Isabel; Marcia, Marco; Legiewicz, Michal et al. (2015) Native Purification and Analysis of Long RNAs. Methods Enzymol 558:3-37
Somarowthu, Srinivas; Legiewicz, Michal; Chillón, Isabel et al. (2015) HOTAIR forms an intricate and modular secondary structure. Mol Cell 58:353-61
Zhao, Chen; Rajashankar, Kanagalaghatta R; Marcia, Marco et al. (2015) Crystal structure of group II intron domain 1 reveals a template for RNA assembly. Nat Chem Biol 11:967-72
Somarowthu, Srinivas; Legiewicz, Michal; Keating, Kevin S et al. (2014) Visualizing the ai5γ 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

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