Poster abstracts

Poster number 30 submitted by Rajan Lamichhane

Probing the Mechanism of Alternative Splicing Regulation by PTB using Fluorescence Resonance Energy Transfer

Rajan Lamichhane (Department of Chemistry, Wayne State University, Detroit, MI 48202), Oksana Valniuk (Department of Chemistry, Wayne State University, Detroit, MI 48202), Sigrid D. Auweter, Frederic Allain (Institute for Molecular Biology and Biophysics, Swiss Federal Institute of Technology ETH-Honggerberg, Zurich, Switzerland), David Rueda (Department of Chemistry, Wayne State University, Detroit, MI 48202)

Abstract:
Polypyrimidine-tract-binding protein (PTB) is a highly conserved protein containing four RNA recognition motifs (RRM1-4), and tightly binds CU rich elements in precursor mRNAs. PTB plays an important role in the negative regulation of alternative splicing. Alternative splic-ing amplifies proteomic diversity from animal genomes. Misregulation of alternative splicing is frequently observed in human diseases involving the nervous system and cancer. However, the mechanisms that control alternative splicing regulation are complex and poorly understood. It has been proposed that PTB bridges the binding sites flanking a repressed exon, thus looping out the intervening RNA and sequestering it from the splicing machinery. Recent NMR structures of the individual RRMs support this hypothesis. [Oberstrass F.C. et al. (2005), Science 309:2054]

In order to further investigate the looping mechanism, we have prepared a series of ssRNA’s with the sequence 5’-CUCUCU(A)NCUCUCU-3’ (N = 5-30), and labeled their 5’ and 3’ ends with a fluorophore pair to monitor the conformational changes induced upon binding by PTB’s RRM3 and 4 using FRET. Our results clearly show that RRM3 and 4 binding brings the 5’ and 3’ ends of the ssRNA in close proximity supporting a mechanism where PTB loops out the intervening sequence. Furthermore, looping efficiency depends on the length of the interven-ing sequence, with optimal looping for loops 15-20 nucleotides long. Distance measurements using time resolved FRET yield donor-acceptor distances in agreement with the NMR structures. We are currently using single molecule FRET to further characterize the structural dynamics of the PTB-RNA complex and its role in alternative splicing regulation.

Keywords: PTB, Alternative Splicing, FRET