Talk abstracts

Talk on Friday 04:10-04:30pm submitted by Mario Blanco

Single molecule observations of pre-mRNA dynamics during splicing

Mario R. Blanco (Cellular and Molecular Biology, University of Michigan), Mark Ditzler (Biophysics, University of Michigan), Frank Fuller (Biophysics, University of Michigan), Mona Wood (Chemistry, University of Michigan), John Abelson (Biochemistry and Biophysics, University of California San Francisco), Nils Walter (Chemistry and Biophysics, University of Michigan)

Abstract:
In the maturation process of a eukaryotic messenger RNA (mRNA) several steps need to be completed if proper gene expression and fidelity are desired. A particularly important step in this pathway is splicing, which involves the removal of intervening sequences (introns) from the precursor messenger RNA (pre-mRNA). This process involves two transesterification reactions catalyzed by a multimegadalton RNA-protein complex termed the spliceosome1. The spliceosome is composed of 5 ribonucleoproteins (RNPs) which contain a total of over 100 different proteins, and 5 small nuclear RNAs (snRNAs)2. The architectural complexity of the spliceosome is only overshadowed by the network of protein-protein, protein-RNA, and RNA-RNA interactions which the spliceosome utilizes to catalyze the splicing reaction. To fully understand the complexity of the interactions between spliceosomal components and pre-messenger RNAs, it is necessary to observe the process in a manner that preserves dynamics without disrupting the transient interactions that are crucial to splicing. Working in collaboration with John Abelson (UCSF) we have developed a single molecule FRET (sm-FRET) splicing assay. By using pre-mRNAs fluorescently labeled with a FRET pair, and Total Internal Reflection Fluorescence (TIRF) microscopy we have tracked the conformational states through which the spliceosome takes a single pre-mRNA. With the ability to track all the steps in splicing, from recognition to catalysis, we can start asking questions that have been difficult to answer with traditional biochemical techniques. Preliminary FRET data has shown us that at every step of the reaction the pre-mRNA substrate is taken through a variety of reversible conformational states. The use of temperature sensitive spliceosomal components and pre-mRNAs with mutations at conserved sites will allow us to assign particular conformational states to the different steps in splicing and ultimately determine the kinetics of spliceosome assembly and catalysis.

References:
(1) E. Brody, J. Abelson. Science. 1985 May 24;228(4702):963-7
(2) JP. Staley, C. Guthrie. Cell. 1998 Feb 6;92(3):315-26.

Keywords: single molecule, splicing, FRET