Characterizing the CRD-BP-RNA interaction in-vitro and in cells

The highly conserved family of RNA-binding proteins known as the VICKZ RNA-binding proteins play an integral role in the formation of cytoplasmic RNPs which leads to the stabilization, localization and translational control of many mRNA transcripts in the cell. The key investigation of this thesis was to analyse the binding ability of the VICKZ protein family member, the coding region determinant-binding protein (CRD-BP), both in-vitro and in cells. CRD-BP has four K-homology (KH) domains and two RNA-recognition motif (RRM) domains. Deletion studies in CRD-BP orthologs have shown that the KH domains, and not the RRM domains, are predominantly responsible for binding to RNA substrates. However, it is still unclear to what extent each of the KH domains play in their physical interaction with RNA molecules, nor is it known if each of the KH domains an play equal role in interacting with different RNA substrates. In an effort to address the above questions, we used site-directed mutagenesis to mutate the first glycine of the G-X-X-G motif in each KH domain separately, and in combinations. We mutated the glycine to an aspartate to introduce both physical and electrostatic hindrance for binding at the G-X-X-G motif. The goal was to determine if such a mutation can disrupt CRD-BP's ability to bind its RNA substrates both in-vitro and in cells. Our results showed that KH single mutants KH2, KH3 and KH4 did not disrupt the CRD-BP-c-myc CRD RNA interaction in-vitro . CRD-BP KH1 single mutant exhibited a modest reduction in binding to the c-myc CRD RNA substrate in-vitro. However, double KH domain mutations (KHl-2, KHl-3, and KH2-4) resulted in a complete abrogation of CRD-BP's ability to bind the c-myc CRD RNA substrate, suggesting these KH domains work in tandem to bind to the c-myc CRD RNA substrate in-vitro. Interestingly, the CRD-BP KH domain double mutant, KH3-4, showed only a modest reduction in the c-myc CRD RNA substrate binding, suggesting that the first glycine in the G-X-X-G motif of KH3 and KH4 does not play a significant role in binding the c-myc CRD RNA substrate in-vitro.;A secondary goal of this thesis was to test the ability of specific oligonucleotides and antibiotics to break the CRD-BP interaction in cells. Previous research showed that DD7 oligonucleotide and neomycin small molecule antibiotic can disrupt the CRD-BP-CD44 RNA interaction in-vitro. Results from this thesis showed that neither DD7 oligonucleotide nor neomycin had ability to disrupt CRD-BP-CD44 mRNA interaction in HeLa cells. Such findings have important implications for future studies aimed at developing potential anti-cancer compounds that act by breaking CRD-BP-RNA interactions.;The mRNA-binding ability of the CRD-BP KH single mutants, KHI, KH2, KH3 and KH4, were then analyzed in HeLa cells. Our results showed that KHI, KH2 and KH4 single mutants did not affect CRD-BP binding to β-actin mRNA in cells, but the KH3 single mutant showed a significant decrease in the ability to bind β-actin mRNA in cells. CRD-BP KHI, KH2, and KH3 single mutants showed a significant decrease in the ability to bind c-myc mRNA in cells, but the KH4 single mutant did not affect the CRD-BP-c-myc mRNA interaction in cells. Finally, all four CRD-BP KH single mutants showed a significant decrease in the ability to bind CD44 mRNA in cells. These results demonstrated, for the first time, that the KH domains of CRD-BP KH play unequal roles in binding different mRNAs in the cells. Such findings have important implications for future studies aimed at developing potential anti-cancer compounds that act by breaking CRD-BP-RNA interactions.