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Using synthetic oligonucleotides to modify cellular IRES structures and control gene expression

Using synthetic oligonucleotides to modify cellular IRES structures and control gene expression
Using synthetic oligonucleotides to modify cellular IRES structures and control gene expression
There are different structures and sequences in the 5’ UTR of mRNAs that can control the levels of translation, such as G-quadruplexes, upstream open reading frames, hairpins or Internal Ribosome Entry Sites (IRESs). Among all of them, IRESs are particularly interesting as they have the ability to increase translation initiation. IRESs were first discovered in viruses and years later their existence in cellular mRNAs was verified. These elements have the ability to start translation when cap-mediated translation is compromised, such as in hypoxia, heat shock or nutrient depravation. These conditions are usually present in tumors, and it is not surprising IRES activity can be increased in cancer cells.

Researchers have tried to target both viral and cellular IRESs as a possible treatment for different viral infections and cancer. In this sense, antisense therapy could be of particular interest. Antisense therapy uses antisense oligonucleotides to control gene expression by inducing RNA degradation, changes in splicing or blocking the translation machinery. Since antisense therapy was developed, oligonucleotides have been modified in different ways to make them more stable in cellular environments and to increase their targeting or cell penetration abilities.

In this project we have designed oligonucleotides with different modifications to modify the secondary structure of the BAG1 IRES and in this way to control the expression of the IRES by either decreasing or increasing its activity. BAG1 is an anti-apoptotic gene implicated in the regulation of many cellular processes, and has been shown to be dysregulated in different cancers.

Being aware of the controversy surrounding the existence of cellular IRESs, we have first verified the presence of an IRES in the p36 BAG1 5’ UTR using stricter criteria than existed during its initial characterisation. We then designed oligonucleotides based on the proposed structure of the BAG1 IRES as well as a pool of oligonucleotides targeting different regions of the BAG1 IRES and generated a luciferase based method to quickly assay the effect of different oligonucleotides on IRES activity. The most promising oligonucleotides were modified with Locked Nucleic Acids, 2’-OMethyl RNAs and phosphorothioate bonds in an attempt to increase their activity and stability in cells.

We have shown to be able to increase the BAG1 IRES activity in a cell free system using different oligonucleotides and have different oligonucleotides that have shown promising results in cells.

Controlling translation initiation has a direct result on the efficiency of protein synthesis. Understanding the events in this process could lead to the discovery of new therapeutic targets, and thereby, the development of new therapies. In this way, these oligonucleotides could prove to be a treatment for diseases associated with an inappropriate amount of functional proteins and in this sense targeting cellular IRESs could be the key.
Gutierrez Aguirregabiria, Ane
71b077bd-2c6f-4960-a080-f4e47112456f
Gutierrez Aguirregabiria, Ane
71b077bd-2c6f-4960-a080-f4e47112456f
Stulz, Eugen
9a6c04cf-32ca-442b-9281-bbf3d23c622d

Gutierrez Aguirregabiria, Ane (2019) Using synthetic oligonucleotides to modify cellular IRES structures and control gene expression. Doctoral Thesis, 378pp.

Record type: Thesis (Doctoral)

Abstract

There are different structures and sequences in the 5’ UTR of mRNAs that can control the levels of translation, such as G-quadruplexes, upstream open reading frames, hairpins or Internal Ribosome Entry Sites (IRESs). Among all of them, IRESs are particularly interesting as they have the ability to increase translation initiation. IRESs were first discovered in viruses and years later their existence in cellular mRNAs was verified. These elements have the ability to start translation when cap-mediated translation is compromised, such as in hypoxia, heat shock or nutrient depravation. These conditions are usually present in tumors, and it is not surprising IRES activity can be increased in cancer cells.

Researchers have tried to target both viral and cellular IRESs as a possible treatment for different viral infections and cancer. In this sense, antisense therapy could be of particular interest. Antisense therapy uses antisense oligonucleotides to control gene expression by inducing RNA degradation, changes in splicing or blocking the translation machinery. Since antisense therapy was developed, oligonucleotides have been modified in different ways to make them more stable in cellular environments and to increase their targeting or cell penetration abilities.

In this project we have designed oligonucleotides with different modifications to modify the secondary structure of the BAG1 IRES and in this way to control the expression of the IRES by either decreasing or increasing its activity. BAG1 is an anti-apoptotic gene implicated in the regulation of many cellular processes, and has been shown to be dysregulated in different cancers.

Being aware of the controversy surrounding the existence of cellular IRESs, we have first verified the presence of an IRES in the p36 BAG1 5’ UTR using stricter criteria than existed during its initial characterisation. We then designed oligonucleotides based on the proposed structure of the BAG1 IRES as well as a pool of oligonucleotides targeting different regions of the BAG1 IRES and generated a luciferase based method to quickly assay the effect of different oligonucleotides on IRES activity. The most promising oligonucleotides were modified with Locked Nucleic Acids, 2’-OMethyl RNAs and phosphorothioate bonds in an attempt to increase their activity and stability in cells.

We have shown to be able to increase the BAG1 IRES activity in a cell free system using different oligonucleotides and have different oligonucleotides that have shown promising results in cells.

Controlling translation initiation has a direct result on the efficiency of protein synthesis. Understanding the events in this process could lead to the discovery of new therapeutic targets, and thereby, the development of new therapies. In this way, these oligonucleotides could prove to be a treatment for diseases associated with an inappropriate amount of functional proteins and in this sense targeting cellular IRESs could be the key.

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Restricted to Repository staff only until 26 April 2022.
Available under License University of Southampton Thesis Licence.
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More information

Published date: December 2019

Identifiers

Local EPrints ID: 447428
URI: http://eprints.soton.ac.uk/id/eprint/447428
PURE UUID: eb1f5b00-0cf3-4f46-8b89-094b15dd66c4
ORCID for Eugen Stulz: ORCID iD orcid.org/0000-0002-5302-2276

Catalogue record

Date deposited: 11 Mar 2021 17:34
Last modified: 12 Mar 2021 02:39

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Contributors

Author: Ane Gutierrez Aguirregabiria
Thesis advisor: Eugen Stulz ORCID iD

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