The long-term goal of this project is to understand key aspects of the post-transcriptional modification process of pre-ribosomal RNA (pre-rRNA) at the molecular level, including the basis of molecule specific RNA recognition by modification enzymes and the functional basis for modifications within RNA molecules. This research focuses on pseudouridine modification sites within eukaryotic rRNA and the small nucleolar ribonucleoprotein particles (snoRNPs) that introduce them. The project will explore the interaction of snoRNP proteins with their RNA ligand, and examine the thermodynamic and structural effects of snoRNA-mediated selection of pseudouridylation sites in functionally important regions of the eukaryotic large subunit rRNA using biochemical methods and high resolution NMR spectroscopy as the principal tools. Specifically, the following topics will be addressed: (1) structural and thermodynamic studies of snoRNA-rRNA complexes and (2) comparative structural and thermodynamic studies of the unmodified and pseudouridyl-modified forms of the eukaryotic large subunit rRNA. The results of these studies are directly relevant to understanding the specificity and affinity of protein-RNA interactions and the mechanisms used by RNA molecules to tweak local conformation and stability. These investigations complement genetic studies of the pseudouridylation reactions catalyzed by the snoRNPs and together provide a route to understanding the functional roles of the most common and phylogenetically conserved RNA base modification. The methods developed during these studies will serve as tools for structural biologists and chemical biologists.
