Characterization of the Dicistroviridae intergenic region internal ribosome entry site

The IRES found in the intergenic (IGR) region of viruses belonging to the Dicistroviridae family is remarkable for its ability to bind directly to the ribosome with high affinity and initiate translation without the requirement for any initiation factors by mimicking a P/E hybrid tRNA. Here, we have conducted an in-depth biochemical characterization of the CrPV IGR IRES. We have found that the L1.1 region of the IRES is responsible for 80S assembly and reading frame maintenance, and may play an additional role downstream of ribosome binding. Additional studies on the modularity of the IRES showed that the two domains of the IGR IRES work independently, but in concert with one another to manipulate the ribosome. We then addressed the question of how the IGR IRES recruits ribosomes during periods of cellular stress, when inactive 80S couples accumulate in the cell. Here, we found that the IRES is able to bind directly to eEF2-associated 80S couples, providing a rationale as to how the IRES remains translated during these periods. Finally, we developed a new in vitro translation system to assess the functionality of specialized ribosomes, and used this system and the IGR IRES in order to ask questions about the pathology of dyskeratosis congenita. Though divergent from other viral IRESs, the simplicity of this tRNA-like IRES serves as a powerful model for understanding IRES functions in general, the role of tRNA/ribosome interactions that occur normally during translation, and how these processes are linked to the greater context of the cell.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat