Poster abstracts

Poster number 17 submitted by Rajan Lamichhane

Alternative Splicing Regulation Through RNA looping

Rajan Lamichhane (Chemistry, Wayne State University, Detroit, MI 48202), Gerrit Daubner, Judith Thomas-Crusells, Sigrid Auweter, Cristina Manatchal (Molecular Biology and Biophysics, ETH, Zurich, Switzerland), Keyunna Austin, Oksana Valniuk (Chemistry, Wayne State University, Detroit, MI 48202), Frederic Allain (Molecular Biology and Biophysics, ETH, Zurich, Switzerland), David Rueda (Chemistry, Wayne State University, Detroit, MI 48202)

Abstract:
Polypyrimidine Tract Binding protein (PTB) is a key alternative splicing factor involved in exon repression and Fox-1 (feminizing on X) is a splicing enhancer. It has been proposed that PTB acts by looping out exons flanked by pyrimidine tracts. Fox-1 has been proposed to bind a UGCAUG element to activate exon inclusion, but the mechanism of Fox binding to RNA and its implications for alternative splicing regulation are still poorly understood. Here, we present fluorescence, NMR and in vivo splicing data that directly support a looping mechanism for PTB. We show that PTB loops the RNA by binding two distant pyrimidine tracts with RNA binding domains (RBD) 3 and 4 of PTB to bring the 5’ and 3’ ends in close proximity. Looping efficiency depends on the length of the intervening sequence with preference for a spacer at least 15 nucleotides between the pyrimidine tracts. Furthermore, RBDs 3 and 4 work synergistically for efficient RNA looping in vivo. Finally, our preliminary data suggest that Fox-1 acts as a splicing enhancer by displacing one of PTB’s RBDs from the RNA to abrogate looping. This work makes significant progress in understanding the mechanisms of alternative splicing regulation by PTB and Fox-1.

Keywords: Alternative Splicing, FRET, PTB