Date of Award

1-1-2021

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Biological Sciences

Content Description

1 online resource (vii, 48 pages) : illustrations.

Dissertation/Thesis Chair

Gabriele Fuchs

Committee Members

Cara T Pager, Andrew Berglund

Keywords

circular RNA, IRES, translation, Proteins, Gene expression, Genetic translation, Genetic regulation, RNA splicing, Ribosomes, Messenger RNA

Subject Categories

Biology | Molecular Biology

Abstract

Translation initiation is a critical step in the process of protein synthesis. The canonical way of translation initiation involves ribosomes being recruited to the 7-methyl guanosine cap present at the 5’end of the untranslated region (5’ UTR) of the RNAs. However, viral RNAs and some cellular mRNAs lack this 5’ cap structure and thus deploy an alternate non-canonical translation initiation mechanism. In non-canonical translation initiation, ribosome recruitment is facilitated by the RNA secondary structures called Internal Ribosome Entry Site (IRES) present most often in the 5’ UTR. To measure IRES-mediated translation, the dual luciferase assay has been the gold standard. As the name suggests, this assay consists of a construct with two cistrons namely, Renilla luciferase and Firefly luciferase. The first cistron is initiates via cap-dependent translation initiation and measures the amount of protein made through the canonical translation initiation pathway. The sequence to be tested for IRES activity is inserted in between the cistrons and thus the second cistron which encodes the Firefly luciferase is synthesized via IRES-mediated translation. Thus, the ratio of Firefly to Renilla luciferase is considered a measure of IRES activity. However, under certain circumstances this assay which can give a false indication of IRES activity. The presence of a cryptic promoter in between the two cistrons, the presence of cryptic splice sites within the construct or in the event of ribosome readthrough, firefly luciferase expression will be elevated resulting in a false indication of IRES activity. Therefore, we designed a construct to overcome these shortcomings of the existing assay.

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