Date of Award
Bachelor of Science
Certain RNAs are proposed to have an internal ribosome entry site (IRES) in their 5’ untranslated region (UTR). RNAs with an IRES include the encephalomyocarditis virus (EMCV), poliovirus (PV), hepatitis C virus (HCV), cricket paralysis virus (CrPV) and the cellular myc mRNA. Using a traditional dual luciferase construct, we can determine the translation efficiency of IRES-containing RNAs in cells in the presence and absence of cellular proteins.
To identify cellular proteins important for IRES-mediated translation, such as ribosomal proteins and translation factors, we have established shRNA knockdowns in HeLa cells. We specifically depleted ribosomal proteins eS25 and RACK1, and translation initiation factors eIF4E, DAP5, and eIF3D to test their involvement in IRES-mediated translation. Based on the literature, we hypothesized that loss of RACK1, DAP5, eS25 and eIF3D may decrease IRES-mediated translation of specific IRESs, while eIF4E should not be required for IRES-mediated translation. First, we validated successful knockdown of these proteins in HeLa cells compared to a control by immunoblotting.
We next determined the translation efficiency of the EMCV, PV, HCV, and CrPV IRESs using dual luciferase constructs. As expected, the CrPV IRES only required eS25, while the other IRESs required eS25 and RACK1. We further found that the HCV IRES additionally required eIF3D, while only the EMCV IRES required all four proteins, but not eIF4E. Overall, my experiments established a system to dissect the protein requirements for cellular IRESs in the future.
Owusu Ampaw, Emmanuella, "Proteins Enhancing IRES-Mediated Translation" (2021). Biological Sciences. 69.