Poster abstracts

Poster number 80 submitted by Aaron Kim

Analysis of the catalytically essential interactions in protein-free snRNA-mediated splicing

Aaron Kim (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106), Yasaman Jaladat (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106), Saba Valadkhan (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106)

Abstract:
We have previously shown that two of the spliceosomal snRNAs, U6 and U2, form a basepaired complex that can catalyze a two step splicing reaction on short RNA substrates (Valadkhan et al., 2009). This reaction not only resembles group II intron splicing and spliceosomal catalysis in its chemistry, but also occurrs in the vicinity of an invariant sequence in U6 which is known to be close to the 5' splice site during the catalysis of the first step of spliceosomal splicing (Lee et al., 2010). Mutations in this invariant sequence that block spliceosomal splicing are also incompatible with the snRNA-mediated catalysis. Further, we have shown that an intramolecular stemloop (ISL) in U6, which closely resembles the catalytically critical domain V of group II introns, is required for the snRNA-mediated splicing.
Numerous mechanistic and structural similarities between the spliceosomal snRNAs and group II introns have led to the hypothesis that the snRNAs are descendants of group II-like introns. Recent structural studies have elucidated the presence of a base triplet interaction in the catalytic core of group II introns (Toor et al., 2008). This interaction, which formed between the base of domain V and the linker sequence between domains II and III in group II introns (J2/3), led to the juxtaposition of catalytically essential residues. Interestingly, J2/3 is thought to be the functional counterpart of the evolutionarily invariant ACAGAGA sequence of U6. To determine whether an analogous interaction is formed in the folded structure of the catalytically active U6/U2 complex, we have made mutations at the base of the ISL and in the ACAGAGA sequence and experiments are under way to determine the catalytic activity of the resulting constructs.

Keywords: snRNA, Catalysis, Group II introns