RNA-metal ion interactions and metal ion-induced conformational change in the spliceosomal U2-U6 snRNA complex studied by lanthanide ion luminescnece and resonance energy transfer techniques

RNA splicing is an integral step of gene expression. In eukaryotic cells, it is carried out by a dynamic RNA-protein assembly called the spliceosome. Among its RNA components, U2 and U6 snRNAs are most conserved and form a complex with extensive base pairs. This complex is able to catalyze splicing-related reactions in the absence of proteins. Metal ions are required for the activity both in vivo and in vitro. The goal of this dissertation is to study the metal ion binding properties of the U2-U6 snRNA complex and elucidate the impact of metal ions on the complex structure. Spectroscopic informative Tb(III) is used to study RNA and metal ion binding properties. Specific metal ion binding sites and their pH-dependence on the complex are mapped out by using Tb(III) bound on RNA as FRET donors. The results not only confirm the metal ion binding sites suggested by other methods, but also reveal a new pH-dependent metal ion bindings site. Proper experimental conditions of using Tb(III) as FRET donors are suggested. By using FRET and NSET methods, the structural impact of metal ions on the U2-U6 snRNA complex is monitored. Under the conditions tested in the study, no conformational changes have been observed upon the addition of metal ions. Our results introduce the use of Tb(III) bound on RNA as FRET donor for the first time; suggest proper conditions of how to use Tb(III) as FRET donor; and show that there is no conformational changes observed by the discussed experimental designs and methods.