Poster abstracts

Poster number 26 submitted by Yasaman Jaladat

Characterization of the functional group in human U6/U2 complex

Yasaman Jaladat (RNA Center, Case Western Reserve University)

Abstract:
We have shown that human spliceosomal snRNAs U6 & U2- can perform a reaction similar to the splicing reaction. To gain insight into the splicing reaction, we have embarked on a detailed functional characterization of this minimal U6&U2 “Spliceosomeâ€?. Specifically, we are interested in defining the functional groups needed for catalysis, including those important in Mg⁺⁺ coordination.

To this end, we use Nucleotide Analogue Interference Mapping to determine the functional groups needed for catalytic activity in this minimal system. To reach these means, we have tightened the binding of U6&U2 and one of the splicing substrates to ensure the formed product remains associated to the U6/U2 that has catalyzed its formation. The other splicing substrate has a biotin at its 5' end, which will allow us to separate reactive U6/U2 in complex with the product from unreactive U6/U2s. Further, in parallel experiments, we are in the process of determining the metal binding sites in the U6U2 complex using Terbium cleavage assay.

Taken together, the above experiments will allow us to define several functional aspects of the active site of the human spliceosome.

Keywords: Minimal Splicing Reaction, U6/U2, Metal Ions