Poster abstracts

Poster number 5 submitted by Mario Blanco

Monitoring pre-mRNA Splicing by Single-Molecule Fluorescence Resonance Energy Transfer

Mario R. Blanco (Uniersity of Michigan), Mark Ditzler, Mona Wood, Jesse Sinanan (University of Michigan), Nils Walter (University of Michigan), John Abelson (University of California San Francisco)

Abstract:
The spliceosome is composed of 5 ribonucleoproteins (RNPs) which contain a total of over 100 different proteins, and 5 small nuclear RNAs (snRNAs). By acting in an ATP dependent manner, this complex is able to precisely excise diverse introns from their precursor-mRNAs (pre-mRNAs) to give mature mRNA products. Although the spliceosome has been studied extensively, there is still little known about the timing, coordination and magnitude of pre-mRNA conformational changes upon spliceosome assembly and splicing. Ensemble techniques have been unable to provide detailed kinetic information about the complicated series of events associated with splicing, and thus we chose to apply single-molecule Fluorescence Resonance Energy Transfer (sm-FRET) to a small yeast intron to gain better insight into these processes.
In our system, a pre-mRNA substrate encoding the yeast ubiquitin-conjugating protein 4 (UBC4) is labeled with Cy3 Donor (D) fluorophore in the 5’-exon and Cy5 Acceptor (A) fluorophore in the 3’-exon. The FRET efficiency between fluorophores is thus related to the distance between the two exons, allowing us to monitor the prevalence of distinct pre-mRNA conformations and transitions between them during assembly and catalytic action of the spliceosome in yeast cell extract. Our data in an ATP deficient yeast cell extract show that our pre-mRNA exhibits several low, interchanging FRET states. Upon addition of ATP we observe a clear change in mRNA structural dynamics toward higher FRET efficiencies, indicating ATP-dependent spliceosomal activity.

Keywords: splicing, mRNA, FRET