Date of Award

1-1-2019

Document Type

Thesis

Degree Name

M.S.

Department

Chemistry and Biochemistry

First Advisor

Erich G. Chapman

Keywords

decay-resistant, s. cerevisiae, XRN1-resistant, 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'-->3â?? exonuclease Xrn1. We show that these viral Xrn1-resistant structures (xrRNAs) can be used to protect specific homologously-expressed messenger RNAs from 5â??-->3â?? 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, β-galactosidase 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.

Provenance

Recieved from ProQuest

Rights holder

Ana Luisa Franklin

File size

75 p.

File format

application/pdf

Language

en

Discipline

Biochemistry, Molecular biology, Chemistry

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