Poster abstracts

Poster number 18 submitted by Zhuojun Guo

Single Molecule Studies of spliceosomal RNAs U2 and U6

Zhuojun Guo (Chemistry, Wayne State University), Chandani Warnasooriya (Chemistry, Wayne State University), David Rueda (Chemistry, Wayne State University)

Abstract:
Splicing is an essential step in the maturation reaction of eukaryotic pre-mRNA, in which intervening sequences (introns) are removed from the coding sequences (exons). The spliceosome is a dynamic assembly of five small nuclear RNAs and a large number of proteins that catalyzes splicing. U2 and U6 are the only two spliceosomal snRNAs strictly required for both steps of splicing, and they form an RNA complex using base pairing. Major conformational changes are expected to take place during the assembly and catalysis of the spliceosome.
We have developed a Single-Molecule Fluorescence Resonance Energy Transfer (smFRET) assay to study the structural dynamics of a protein-free U2/U6 complex from S. cerevisiae. Our previous data have revealed a two-step large amplitude conformational change of the U2-U6 complex. The first step is a Mg2+-induced conformational change where helix III and the U6-ISL are in close proximity in low [Mg2+] and separated in high [Mg2+]. The second rearrangement corresponds to the formation of the highly conserved Helix IB.
According to the recent crystal structure of the group II intron and given the similarity between the group II intron and the spliceosome, we hypothesize that similar base triple interaction may exist in the U2/U6 complex. Here, we present new experiments that examine the role of the highly conserved base U80 and the ACAGAGA in formation of base triple with the AGC triad. The data show that both U80 and the ACAGAGA loop play an important role in stabilizing the interactions maintaining the high FRET structure. Mutations that disrupt these interactions prevent helix III and the U6-ISL from being in proximity of each other. We hypothesize that in physiological [Mg2+], U80, G52and A53 form base triples with the AGC triad to pull helix III and the U6-ISL together. Our results raise the interesting possibility that this base triple interaction plays an important role in bringing the 5’ splice site and the branched A into close proximity of U80, which may be critical for catalysis.

Keywords: splicing, U2U6, base triples