Biochemical Characterization Of The Rbfox 3 Protein

The Rbfox proteins are a family of homologous splicing regulators composed of RbFox 1, RbFox 2, and RbFox 3. Specifically, Rbfox 3 is expressed only in neuronal cells and its protein expression in regions of the brain like the cerebellum might play a role during neuronal development in mammals. The study of these Rbfox proteins is important to understand the types of interactions they may have with Ataxin proteins whose mutations lead to neurodegenerative disorders. The Rbfox proteins contain a highly conserved RNA recognition motif which binds specifically to the sequence UGCAUG. The Rbfox proteins act as splicing activators when bound downstream and splicing inhibitors when bound upstream of regulated exons. In lieu of characterizing these Rbfox complexes, a recombinant protein clone was constructed and expressed. Recombinant protein was purified and assayed in vitro for binding to RNA containing a UGCAUG element. A mutant downstream control sequence (DCS) with the UGCAUG mutated to UCAGUC was also assayed and did not show any RNA-protein complex with Rbfox 3. This confirmed that the UGCAUG hexanucleotide is responsible for mediating the interaction between the Rbfox 3 protein and the DCS RNA. Binding is also being assayed with RNA binding proteins hnRNP H, F and M which have been shown to interact with Rbfox proteins in other studies. Recombinant clone expressing only the RNA binding domain of Rbfox 1, Rbfox 2, and Rbfox 3 is under construction to see how RNA-protein interaction is effected.