Structural Studies of U6 snRNA

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DESCRIPTION (provided by applicant): Accurate and efficient nuclear pre-mRNA splicing is essential to eukaryotic life. Aberrant pre-mRNA splicing has been linked to a number of diseases. These include breast, colorectal, epithelial and ovarian cancer, as well as neurodegenerative diseases such as Parkinson's and Alzheimer's. Our objective is to develop an atomic-level understanding of the structure and function of a key component of the pre-mRNA splicing machinery, that of U6 snRNA. Currently, there is a profound lack of structural information about U6 snRNA. We hypothesize that the U6 snRNA structure functions to coordinate a magnesium ion cofactor during splicing, and that proteins and other RNAs are not required for magnesium binding. We will use NMR spectroscopy to investigate the structure, function, dynamics and metal binding properties of U6 snRNA. We anticipate that this research will provide a basis for the interpretation of genetic and biochemical data compiled on this region of U6 snRNA over the past decade. Our specific aims are: 1. Define metal ion interactions with the U6 snRNA stem-loop. We will use NMR to test our hypothesis that the U6 stem-loop is sufficient for coordination of the catalytically essential metal ion. 2. Determine the structure and investigate the dynamics of the S. cerevisiae U6 snRNA intramolecular stem-loop by NMR. The corresponding human U6 and U6atac structures will also be investigated. 3. Investigate the structural basis for U6 mutant phenotypes. We will correlate structural effects with well studied S. cerevisiae mutant phenotypes. 4. Investigate the influence of surrounding U6 snRNA sequences and the splicoesomal protein Prp24 on the U6 snRNA sem-loop structure.