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Wang, Xinying

University of British Columbia

The dicistrovirus intergenic region internal ribosome entry site (IGR IRES) adopts a triple-pseudoknot (PK) structure to directly bind to the conserved core of the ribosome and drive translation from a non-AUG codon. The origin of this IRES mechanism is not known. In this thesis, I describe two studies that attempt to examine how the IGR IRES may have come about. In the first study, I characterized an IGR IRES from a 700-year-old dicistrovirus, named ancient Northwest territories cripavirus (aNCV). From structural prediction of the aNCV IGR sequence and filter binding assays, we showed that the aNCV IGR secondary structure is similar to contemporary IGR IRES structures and could tightly bind to purified human ribosomes. However, there are differences including 105 nucleotides upstream of the IRES of unknown function. We also demonstrated that the aNCV IGR IRES can direct internal ribosome entry in vitro. Lastly, we generated a chimeric virus clone by swapping the aNCV IRES into the cricket paralysis virus (CrPV) infectious clone. The chimeric infectious clone with an aNCV IGR IRES supported translation and virus infection. The characterization and resurrection of a functional IGR IRES from a divergent 700-year-old virus provides a historical framework of the importance of this viral translational mechanism. In the second study, I have examined candidate RNAs that may have IGR IRES-like properties from the Drosophila genome. Previously, we adapted a selective evolution approach to identify RNA elements in the Drosophila genome which have IGR IRES-like properties. From the potential candidate RNAs, RNA3, RNA5 and RNA7 showed tight binding to purified human ribosomes. However, in a competition assay only RNA5 could compete with excess wild-type but not mutant CrPV IGR IRESs for ribosomes. However, we demonstrated that RNA5 did not bind to ribosomal core, as it was accessible by RNase I. Structural predictions were used to identify stemloop (SL) structures of RNA5. Mutations at SL2 altered RNA5 binding affinity, suggesting a potential interaction region. Finally, incubation of RNA5 in rabbit reticulocyte lysate (RRL) did not affect translation in vitro.

Text

2022-05-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/78482

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/