Dissertations, Theses, and Capstone Projects

Date of Degree


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Degree Name





Nancy L. Greenbaum

Subject Categories



Fluorine, interconversion, NMR, splicing, U2-U6


Pre-mRNA splicing, the process by which non-coding sequences are removed and coding sequences are ligated, plays an important role in the maturation of precursor messenger RNA molecules prior to their translation into proteins. In eukaryotes, splicing is catalyzed by the spliceosome, a large and dynamic nuclear macromolecular "machine" that comprises five small nuclear (sn)RNA molecules and more than one hundred proteins. Among the RNA components, the complex formed between the U2 and U6 snRNA molecules is implicated in catalytic activity. In this dissertation, I have used 19F NMR techniques to characterize the conformation ("fold") and dynamics of a protein-free biophysically tractable paired construct representing the human U2-U6 snRNA complex in which a single 19F-substituted nucleotide is incorporated in the U2 snRNA sequence. 19F spectra of the complex in the absence of Mg2+ are consistent with formation of a four-helix junction structure as the predominant fold. However, spectra also identify a lesser fraction (up to 14% at 25°C) of conformation that suggesting a collection of folds comprising three helices. In the presence of 5 mM Mg2+, the fraction of the three-helix conformation increased to ~17%, suggesting a slight shift to the alternative conformation. The low energy cost of interconversion between different conformers (delta G is approximately -4.6 kJ/mol for the interconversion) suggests the complex is very flexible and may be capable of undergoing spontaneous conformational rearrangement between conformers. Supporting this, 19F-19F EXSY data provide the evidence of the chemical exchange between different conformations in a sub-second timescale. Taken together, these results indicate conformational heterogeneity and interconversion in the protein-free human U2-U6 snRNA complex, consistent with a model in which the RNA has sufficient conformational flexibility to facilitate rearrangements of the spliceosome between steps of splicing.

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