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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular and Cellular Biophysics Study Section (BBCA)
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Lewis, Catherine D
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University of Wisconsin Madison
Schools of Earth Sciences/Natur
United States
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Montemayor, Eric J; Didychuk, Allison L; Yake, Allyson D et al. (2018) Architecture of the U6 snRNP reveals specific recognition of 3'-end processed U6 snRNA. Nat Commun 9:1749
Didychuk, Allison L; Montemayor, Eric J; Carrocci, Tucker J et al. (2017) Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities. Nat Commun 8:497
Montemayor, Eric J; Didychuk, Allison L; Liao, Honghong et al. (2017) Structure and conformational plasticity of the U6 small nuclear ribonucleoprotein core. Acta Crystallogr D Struct Biol 73:1-8
Didychuk, Allison L; Montemayor, Eric J; Brow, David A et al. (2016) Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly. Nucleic Acids Res 44:1398-410
Cornilescu, Gabriel; Didychuk, Allison L; Rodgers, Margaret L et al. (2016) Structural Analysis of Multi-Helical RNAs by NMR-SAXS/WAXS: Application to the U4/U6 di-snRNA. J Mol Biol 428:777-789
Rodgers, Margaret L; Didychuk, Allison L; Butcher, Samuel E et al. (2016) A multi-step model for facilitated unwinding of the yeast U4/U6 RNA duplex. Nucleic Acids Res 44:10912-10928
Vander Meulen, Kirk A; Horowitz, Scott; Trievel, Raymond C et al. (2016) Measuring the Kinetics of Molecular Association by Isothermal Titration Calorimetry. Methods Enzymol 567:181-213
Burke, Jordan E; Butcher, Samuel E; Brow, David A (2015) Spliceosome assembly in the absence of stable U4/U6 RNA pairing. RNA 21:923-34
Price, Argenta M; Görnemann, Janina; Guthrie, Christine et al. (2014) An unanticipated early function of DEAD-box ATPase Prp28 during commitment to splicing is modulated by U5 snRNP protein Prp8. RNA 20:46-60
Montemayor, Eric J; Curran, Elizabeth C; Liao, Hong Hong et al. (2014) Core structure of the U6 small nuclear ribonucleoprotein at 1.7-Å resolution. Nat Struct Mol Biol 21:544-51

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