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Title: Development of an antisense oligonucleotide based method to manipulate RNA editing of AMPAR subunits
Author: Chaytow, Helena Anne
ISNI:       0000 0004 8499 4099
Awarding Body: Royal Holloway, University of London
Current Institution: Royal Holloway, University of London
Date of Award: 2017
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AMPA receptors (AMPARs) are a subset of ionotropic glutamate receptor composed of one or more of four subunits (GluA1-4) and are essential for normal synaptic function. The GluA2 subunit undergoes RNA editing at a specific base, converting the amino acid from glutamine to arginine, which is critical for regulating calcium permeability. RNA editing is performed by Adenosine Deaminases Acting on RNAs (ADARs). ADAR2 exists as multiple alternatively-spliced variants within mammalian cells and some have been shown to reduce their editing efficiency. RNA editing in AMPARs is inefficient in patients with Amyotrophic Lateral Sclerosis and manipulating this process could be therapeutic against AMPAR-triggered neuronal cell death. Antisense oligonucleotides (ASOs) are bases with chemically altered backbones used to manipulate DNA or RNA processing through complementary base pairing. ASOs were used to alter the GluA2 RNA editing event, either by disrupting the GluA2 double-stranded RNA structure essential for editing or by affecting the alternative splicing of ADAR2. The effects of specific ASOs on RNA editing were assessed by transfection into cell lines. Editing was quantified by an RT-PCR-based assay on RNA extracts then densitometric analysis of BbvI digestion products. ASOs targeting the secondary structure of the GluA2 subunit disrupted editing at this site to 50% of control in HeLa cells, and further disrupted editing to less than 10% in SH-SY5Y cells endogenously expressing the GluA2 subunit. ASOs targeting the ADAR2 transcript successfully inhibited the expression of a less efficient isoform, which led to increased editing of 30% compared to control HeLa cells. Further work will include building on attempts to introduce the antisense transcripts into primary cortical cultures to analyse downstream effects of reduced editing. This is the first example of increasing RNA editing using ASOs and provides a number of potential tools to investigate associated cellular processes and implications of RNA editing.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available