Date of Award

Fall 12-2018

Document Type

Honors Thesis

Degree Name

Bachelor of Science

Department

Anthropology

Advisor/Committee Chair

Dr. Gabriele Fuchs, Ph.D.

Abstract

Ribosomes, the cellular machinery that translates mRNA sequences into protein sequences, are surprisingly heterogeneous molecules. More and more ribosomal proteins have been shown to facilitate translation of mRNA subsets. These mRNA subsets include mRNAs that can initiate translation using non-canonical pathways, for example using an internal ribosome entry site (IRES). IRESs are RNA structures that facilitate translation with fewer translation initiation factors than are required for canonical cap-dependent translation initiation. The ribosomal protein Receptor for Activated C Kinase 1 (RACK1) has been previously shown to be required for translation of the hepatitis C virus (HCV) IRES, but not required for translation of the intergenic IRES of cricket paralysis virus (CrPV).

We tested if RACK1 is generally required for translation of IRES-containing mRNAs by employing dual-luciferase constructs. These constructs allow us to measure cap-dependent and IRES-dependent translation from the same sample, even from the same mRNA. Using haploid 1 wildtype, RACK1 knockout cells generated by CRISPR-Cas9 mediated genome editing, and RACK1 add-back cell lines, we investigated if RACK1 is also required for translation of other viral IRESs, specifically encephalomyocarditis Virus (EMCV) and poliovirus (PV). Indeed, PV and EMCV also require RACK1 for efficient IRES translation.

Further, certain cellular mRNAs also contain IRESs, which allow these mRNAs to be translated under conditions of stress. We next tested if the cellular IRESs myb, L-myc, Bag-1, cyclin D, c-myc, and Set7 also require RACK1 for translation. Interestingly, we found that translation of all cellular IRESs we tested was also decreased in cells lacking RACK1. Overall, in cells lacking RACK1 translation of all tested viral and cellular IRESs is decreased and translation efficiency can be mostly partially or fully rescued in RACK1 knockout cells that express RACK1.

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