Poster abstracts

Poster number 15 submitted by Zhuojun Guo

Single Molecule Studies of spliceosomal snRNAs U2-U6

Zhuojun Guo (Department of Chemistry, Wayne State University), David Rueda (Department of Chemistry, Wayne State University)

Abstract:
Splicing is an essential step in the maturation of eukaryotic pre-mRNA in which intervening sequences (introns) are removed from the coding sequences (exons). The spliceosome is a dynamic assembly of five snRNAs and a large number of proteins- that catalyzes the splicing process. U2 and U6 are the only two spliceosomal snRNAs strictly required for both steps of splicing, and they form RNA complex using base pairing. Major conformational changes are expected to take place during the assembly and catalysis of the spliceosome.
Here, we have used Fluorescence Resonance Energy Transfer and Single Molecule Spectroscopy to study the structural dynamics of a protein free U2-U6 complex from S. cerevisiae. Our FRET data clearly shows a Mg²⁺ induced large amplitude conformation change of the U2-U6 complex. In the absence of Mg²⁺ helix I and the U6-ISL are in close proximity, while in the presence of Mg²⁺ these two helices are far from each other. Our single molecule data shows that the conformational change consists of a two-step process, with an obligatory folding intermediate. We propose that the observed conformational change corresponds to the activation process expected to occur during spliceosomal assembly and catalysis and this hypothesis is confirmed by the recent data from mutant U2-U6 complex which contains point mutations in highly conserved regions.

Keywords: spliceosome, U2-U6 complex, Single Molecule Spectroscopy