Poster abstracts

Poster number 144 submitted by Julia Widom

Dissecting the functions of RNA helicases in splicing by single-molecule FRET

Julia R. Widom (Department of Chemistry, University of Michigan), Matthew L. Kahlscheuer (Department of Chemistry, University of Michigan), Nils G. Walter (Department of Chemistry, University of Michigan)

Abstract:
The process of splicing is a critical step in gene expression, and defects in the splicing pathway are associated with many human genetic diseases. The spliceosome is unlike many macromolecular machines in that it lacks a pre-formed catalytic core and is built through sequential steps of assembly and rearrangement on the template of the pre-mRNA. I will present recent and ongoing work in which single-molecule fluorescence resonance energy transfer (smFRET) was applied to the spliceosome, allowing conformational changes in the pre-mRNA to be monitored in real time. I will describe previous experiments that used the technique of single-molecule pulldown FRET (SiMPull-FRET) to isolate complexes stalled before the first chemical reaction of splicing. I will also describe ongoing work investigating the roles of RNA helicases and ATPases in structural rearrangements of the spliceosome. Prp22 is an RNA helicase found in yeast (with a human homologue, HRH1) that is required for the release of the mRNA product after the second chemical reaction of splicing is complete. I will present experiments in which smFRET is used to investigate the unwinding activity of Prp22 on model substrates, with the goal of understanding how this unwinding activity is functionally utilized in the spliceosome. The study of Prp22 will be extended to the spliceosome using SiMPull-FRET, allowing us to isolate from yeast extract spliceosomal complexes that have been stalled after the second step of splicing by a dominant negative mutation in Prp22. This will permit investigation of the effects of Prp22 on mRNA dynamics after both steps of splicing and during mRNA release.

References:
1. Krishnan, R., Blanco, M. R., Kahlscheuer, M. L., Abelson, J., Guthrie, C. & Walter, N. G. Biased Brownian racheting leads to pre-mRNA remodeling and capture prior to first-step splicing. Nat. Struct. Mol. Biol. 20, 1450–1457 (2013).
2. Schwer, B. & Gross, C. H. Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing. EMBO J. 17, 2086–2094 (1998).

Keywords: splicing, helicase, single-molecule