UC San Diego

The Encephalomyocarditis Virus-Internal Ribosome Entry Site (EMCV-IRES) is a specialized viral mRNA sequence that facilitates translation initiation through an alternative mechanism. Unlike the canonical translation initiation process, which requires a 5’-7-methyl guanosine cap and numerous initiation factors, EMCV-IRES can initiate translation with reduced initiation factor requirements. This unique capability makes EMCV-IRES an important tool in cell-free translation systems, where it is widely used to enhance protein production.Despite its utility, the mechanism and structural details of EMCV-IRES-mediated translation initiation remain poorly understood. One of the main challenges in studying EMCV-IRES is its dependence on multiple protein factors, as well as the transient nature of the IRES initiation complex. These factors complicate efforts to capture and analyze the structure and function of the EMCV-IRES initiation complex. To address these challenges and gain deeper insights into the mechanisms and structure of EMCV-IRES, we have developed a novel method for purifying the ribosome-bound EMCV-IRES complex from rabbit reticulocyte lysate (RRL). Our approach involves a pulldown method that applies the cleavage activity of Csy4, a Cas6f nuclease. The Csy4 specifically binds and cleaves a 20-nucleotide hairpin sequence, which was incorporated into the 3’-end of the EMCV-IRES mRNA. Using this method, we can isolate the EMCV-IRES complex in a ribosome-bound state, which is crucial for subsequent structural analyses. The purified EMCV-IRES complex obtained through this method can be subjected to advanced structural analysis techniques, including mass spectrometry and Cryo-EM. These techniques will enable us to dissect the architecture of the EMCV-IRES initiation complex at high resolution. Ultimately, our work aims to provide a comprehensive understanding of how EMCV-IRES functions at a molecular level, which could inform the development of improved translation systems and therapeutic strategies.