Diversifying the transcriptome : Adenosine-to-inosine RNA editing in the mammalian brain

Sammanfattning: Metazoan transcriptomes are extensively modified by adenosine-to-inosine (A-to-I) RNA editing, a process by which adenosines within double-stranded RNA are enzymatically deaminated to form inosines. Inosines has base-pairing characteristics similar to guanosine, meaning that A-to-I RNA editing results in functional rewriting of transcripts, affecting many biological processes. The aim of this thesis was to investigate the regulation and the function of RNA editing in the mammalian brain. We applied a novel method, in situ sequencing (ISS), to detect editing substrates in the developing mouse brain, which revealed regional and cell type-specific editing patterns emerging during development. Additionally, we characterized the structural requirements of site-selective editing and discovered an editing inducer elements (EIEs) as a general feature of efficiently edited substrates. Also edited microRNAs (miRNAs) were found to have EIEs, in the form of neighboring primary miRNA (pri-miRNA) hairpin structures. Site-selective editing has previously been found to increase during brain development. To investigate this developmental increase, we analyzed the regulation of ADAR2, one of the two editing enzymes, in an in vitro model system for neuronal maturation. Here, we observed increased nuclear import of ADAR2 during neuronal maturation, correlating to increased editing levels. Increased expression of the proteins importin-ɑ4 and Pin1 were found to contribute to this increased nuclear import. Finally, we studied editing of members of the miR-379-410 cluster, and we found that in general pri-miRNAs are more frequently edited than the corresponding mature miRNAs, which possibly indicates an inhibitory role of ADAR enzymes on miRNA biogenesis.. However, the opposite trend was observed for miR-376b-3p, which displayed higher editing in its mature form. Studying the editing and biogenesis of miR-376b-3p, we found that ADAR1 edits the miRNA while ADAR2 inhibits its maturation. Furthermore, the two enzymes compete in binding the pri-miRNA. Subsequently, we identified neuronal target genes of the edited miR-376b-3p. These included the 4-aminobutyrate aminotransferase (Abat), the enzyme responsible for the catabolism of the neurotransmitter GABA, and our results indicate that editing of miR-376b-3p can regulate GABAergic signaling.