Identification of epigenetic regulators of RNA editing mechanisms

Thesis

This thesis looks to identify up-regulating mechanisms of ADAR 2 (Adenosine deaminases acting on RNA 2) through investigation of 52 novel small-inhibitor small molecules provided by the Structural Genomics Consortium (SGC, 2022). These small molecules were incubated with neuronal-2A mouse cells containing a CRISPR engineered construct, this doxycycline-induced construct was formed of a stem loop with a point mutation with mCherry and eGFP proteins respectively either side of the stem loop. The point mutation meant that the stem loop, along with the eGFP, was not translated, therefore levels of RNA repair could be measured by quantifying the ratio of eGFP/mCherry fluorescence. Upon identification of five significant up-regulators of RNA editing activity, and eight significant down-regulators, we repeated the treatment but this time extracted RNA and performed a qPCR which revealed that these identified treatments significantly effected either the expression of ADAR1, ADAR2 or both. It was interesting to note that bromodomain inhibitors appeared to have the greatest transcriptional effect upon ADARs. Next we used siRNA to knock-out ADAR 1 and ADAR 2, then repeating the fluorescence experiment. It is interesting to see that whilst ADAR 1 was seen to be up-regulated, inhibition of ADAR 1 did not inhibit transcriptional effects of treatment. On the other hand, inhibition of ADAR 2 led to decrease in efficacy of small molecule treatment. This thesis goes on to present the results of bromodomain 7 inhibition and it's effect upon ADAR, arguing that bromodomains are a key regulator of ADAR activity, as well as identifying potential targets in the treatment of neurogenetic diseases.

Authors: Buss, G

• DOI: 10.5287/ora-1r2o1yr5z
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English
• Keywords: bromodomain; pharmacology; genetic; ADAR; neurogenetics
• Subjects: Neurosciences; Molecular Neuroscience; Neurogenetics; Genetics