Date of Award

1-1-2019

Document Type

Masters Thesis

Degree Name

M.S.

Organizational Unit

College of Natual Science and Mathematics, Chemistry and Biochemistry

First Advisor

Erich G. Chapman, Ph.D.

Keywords

Decay-resistant, S. cerevisiae, XRN1-resistant, 5'-3' exoribonuclease 1, xrRNAs, Yeast

Abstract

Flaviviruses are positive-strand single-stranded RNA viruses that are known to form pseudo-knot RNA structures that halt the progression of 5’→3’ exonuclease Xrn1. We show that these viral Xrn1-resistant structures (xrRNAs) can be used to protect specific homologously-expressed messenger RNAs from 5’→3’ degradation. We investigated the effects of addition of xrRNAs, artificially-installed into the intergenic region of bicistronic mRNA reporters, in the observed levels of protein expression in yeast. The reporters also contain an internal ribosome entry site from the cricket paralysis virus (CrPV IRES) to allow for cap-independent translation of the decay-protected gene, LacZ, encoding the enzyme β-galactosidase. Through the use of primer extension, βgalactosidase assay, and western blots, the results indicate that the partially-decayed RNAs are successfully translated, and that addition of xrRNAs results in a 30-50 fold increase in measured enzymatic activity and an accumulation of decay-resistant transcripts in the cell.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Rights Holder

Ana Luisa Franklin

Provenance

Received from ProQuest

File Format

application/pdf

Language

en

File Size

75 p.

Discipline

Biochemistry, Molecular biology, Chemistry

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