Molecular characterization of the coding region determinant-binding protein interaction with KRAS oncogene mRNA

The coding region determinant binding protein (CRD-BP) plays a role in some cancers, regulating gene expression through mRNA trafficking, turnover, and translational repression. Recent evidence reveals CRD-BP binding to KRAS mRNA '" a proto-oncogene frequently mutated and/or overexpressed in cancer, leading to up-regulation. A CRD-BP-KRAS mRNA interaction therefore presents a novel therapeutic opportunity to target KRAS-driven cancers and is the aim of this thesis. Electrophoretic mobility and immunoprecipitation experiments presented here using GXXG-mutant CRD-BP variants show four K-homology domains are required to bind a 57-nucleotide minimum binding sequence in the coding region of KRAS RNA these mutations do not compromise protein folding as evidenced by circular dichroism spectroscopy. DNA-based anti-sense oligonucleotide inhibitors were developed based on the minimum binding sequence that successfully abrogate the CRD-BP-KRAS mRNA interaction in vitro. Finally, a CRD-BP KH1toKH4 construct purified using a novel '~on-column'-refolding scheme suggests a possible role for RRM-domains as negative regulators of RNA-binding. --Leaf i.