Poster abstracts

Poster number 36 submitted by Krishanthi Karunatilaka

Single-Molecule Studies of Human Spliceosomal snRNAs

Krishanthi Karunatilaka (Department of Chemistry, Wayne State University), David Rueda (Department of Chemistry, Wayne State University)

Abstract:
Splicing is an essential and highly complex process in eukaryotic gene expression. It is catalyzed by the spliceosome, which is a dynamic assembly of five small nuclear RNAs (snRNAs) and a large number of proteins. Out of five snRNAs, the highly conserved U2 and U6 snRNAs are required in both steps of splicing. The U2-U6 snRNA complex forms the active site of the spliceosome and has been shown to undergo splicing-related catalysis in the absence of proteins. Our single-molecule data of yeast U2-U6 snRNAs has shown a Mg²⁺-induced conformational change consisting of a two-step process, which may be involved in spliceosomal activation in vivo. In contrast to yeast, human U2 and U6 snRNAs contain a large number of post-transcriptional modifications and CA–GU wobble base pairs as opposed to the UU wobble base pairs found in yeast. According to recent studies, these differences make human snRNAs more stable than that of yeast lending to the possibility of different activation mechanisms between the two species.
In order to understand and compare the catalytic mechanisms, we use single molecule florescence to characterize the conformational changes of the human U2-U6 complex in the presence and absence of modifications using Mg²⁺ as a divalent metal ion. Our FRET data clearly shows a Mg²⁺-induced conformational change of the unmodified human U2-U6 complex as we previously observed in yeast. We are currently studying the effects of post-transcriptional modifications of the human snRNAs on these dynamics.

Keywords: Single-molecule, Spliceosome, U2-U6 snRNAs