All ETDs from UAB

Advisory Committee Chair

Sunnie R Thompson

Advisory Committee Members

David Bedwell

Kim Keeling

Elliot Lefkowitz

David Schneider

Document Type

Dissertation

Date of Award

2017

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Most messenger RNA (mRNA) are translated in a cap-dependent manner, requir-ing the recognition of a 5’ cap structure by a set of eukaryotic initiation factors (eIFs) in-cluding the 40S ribosomal subunit. The subunit scans the mRNA until the start codon is recognized, triggering the release of eIFs and recruitment of the 60S subunit. Cellular stress and viral infection down-regulate cap-dependent translation, therefore, cellular and viral messages rely on a cap-independent mechanism. One mechanism requires an inter-nal ribosome entry site (IRES). Viral IRESs were discovered over 25 years ago, with cellular IRESs to follow. The study of viral IRESs has brought forth what little understanding we have of the mechanisms employed by IRESs to initiate translation. Here we discuss the importance of properly identifying an IRES by eliminating the possibility of: cryptic promoter activi-ty, cryptic splicing, and ribosome read-through. The increasing identification of cellular IRESs warrants a better understanding of their mechanisms. Remarkably, ribosomal protein S25 (eS25) is required for both viral and cellular IRES-mediated translation. eS25 has been implicated in binding of the cricket paralysis intergenic region (CrPV IGR) IRES and 40S subunit. However, little else is understood on the roles of eS25 and how it may link diverse IRESs. To elucidate the function of eS25 in IRES-mediated translation, we explore its roles in CrPV IGR IRES-mediated translation. Through genetic, biochemical, and kinetic analyses we demonstrate that the binding of the CrPV IGR IRES and 40S subunit is a two-step binding process mediated by eS25 with the first step being initial binding, fol-lowed by a conformational change in the complex. Mutational analysis of eS25 identi-fied residues of importance for IRES activity. Our findings were recapitulated in the presence of the hepatitis C virus (HCV) IRES. Our data suggests a model in which eS25 is required for a conformational change in the IRES-40S complex, which is shared amongst diverse IRESs.

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