The adenosine deaminase acting on RNA (ADAR) enzymes catalyse the hydrolytic deamination of adenosine (A) to inosine (I) in double stranded (ds) RNA. Mutations in ADAR1 underlie the autoimmune disorder Aicardi Goutiѐres syndrome (AGS). Patients with AGS display heightened levels of type I interferon (IFN) and IFN stimulated genes (ISGs). The first aim of my thesis was to determine whether the mutations found in the human ADAR1 gene affected RNA editing. I found that the ADAR1 mutants identified in the AGS patients have reduced editing activity. Interestingly, the mutations have a greater effect on the IFN-inducible cytoplasmic isoform, ADAR1p150 than on the constitutive ADAR1p110 isoform. These results imply that A-to-I editing plays a role in regulating the type I IFN response. The Adar1 null mouse dies by E12.5 with a type I IFN signature similar to that observed in the AGS patients. The second aim of my thesis was to characterize the immune signalling pathway aberrantly activated in the absence of Adar1. A colleague in our research group rescued the Adar1 null mouse to birth by blocking the cellular response to cytoplasmic dsRNA by generating a double mutant with the mitochondrial antiviral signalling adaptor, Mavs. In the Adar1-/-; Mavs-/- mutant I found that the aberrant immune response is rescued at E11.5. This indicates that MAVS is the downstream adaptor in the aberrant immune response that underlies the embryonic lethality in the Adar1-/- mouse. The third aim of my thesis was to determine if the lack of inosine modification within cellular RNA was triggering the aberrant immune response in the Adar1-/- mouse. To study this, Adar1-/-; p53 -/- mouse embryonic fibroblasts (MEFs) were generated. By reintroducing various ADAR isoforms into the Adar1-/-; p53 -/- MEFs I found that to rescue the aberrant immune response requires both catalytic activity and the location of an ADAR protein within the cytoplasm. Moreover, I demonstrated that transfecting inosine-containing dsRNA oligonucleotides into Adar1-/-; p53 -/- MEFs suppresses the aberrant immune response. Overall my results suggest that A-to-I editing by ADAR1 is an essential RNA modification that is required by the cell to distinguish between ‘self’ and ‘non-self’ RNA. Editing of cellular RNAs prevents an autoimmune response whereas editing of viral RNA may act to suppress a heightened antiviral immune response and prevent long-term damage to the cell.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:700057 |
| Date | January 2015 |
| Creators | Mannion, Niamh |
| Contributors | O'Connell, Mary ; Ditzel, Mark |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/17887 |
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