Novel sites of A-to-I RNA editing in the mammalian brain

Abstract: The number of protein-coding genes are likely not sufficient to account for the complexity of higher organisms. It is plausible that the proteome is responsible for the complexity of an organism.An important mechanism that increases the protein variability is post-transcriptional modifications that alter the pre-mRNA sequence from that encoded in the genome. In this thesis work I have been focusing on a post-transcriptional process where adenosine (A) is deaminated to inosine (I), A-to-I RNA editing. Inosine is read as a guanosine (G) by the translation machinery, editing within coding regions can therefore give rise to more than one protein isoform from a single gene. A-to-I RNA editing is catalyzed by members of the ADAR enzyme family. ADARs have been found in all metazoans tested and two active ADAR proteins, ADAR1 and ADAR2, have been found in mammals. However, recoding by A-to-I editing is a rarely found event in mammals.To detect novel substrates for A-to-I editing we developed an experimental approach to pull down ADAR2 substrates using immunoprecipitations. The captured RNAs were identified by microarray analysis. In this thesis two novel substrates for A-to-I editing are presented that were found using our IP-array approach, in combination with bioinformatic techniques.The transcript coding for the GABAA receptor subunit α3 (Gabra-3) was found to be selectively edited by both ADAR1 and ADAR2. Editing of Gabra-3 recodes an isoleucine to a methionine and it was found to have a negative effect on the Gabra-3 assembly into the receptor. Moreover, the mouse specific CTN-RNA that codes for the CAT2 Transcribed Nuclear-RNA was shown to be hyper-edited by ADAR2.In conclusion, this thesis work has resulted in an experimental method that extracts ADAR substrates. Two novel editing substrates were discovered. Our data adds additional evidence to the fact that RNA editing is of principal significance for a functional brain.