RNA editing in the mammalian brain

One area that is known to require extensive protein diversity is the brain. It is well known that pre-mRNA processing such as alternative splicing is widespread and highly regulated in the nervous system. Another way to alter the mRNA is by RNA editing. Adenosine to inosine (A-to-I) deamination is the most common type of RNA editing found in mammals and it is catalyzed by adenosine deaminases that act on RNA (ADARs). There are two enzymes found to be active in adenosine deamination, ADAR1 and ADAR2. These enzymes convert A-to-I within double-stranded or highly structured RNA. Since inosine is recognized as guanosine by the cellular machinery, A-to-I editing has the potential to change the code for translation. Site-selective A-to-I editing is a mechanism used to fine-tune the transcriptome and increase the variety of expressed protein isoforms, mainly found in the central nervous system (CNS). Besides amino acid changes, editing has been found to be able to influence the outcome of splicing as well as other post-transcriptional events like 3’ end processing, stability, and transport (Figure 1). Thus, RNA editing by A-to-I modification has the power to affect the proteome in many different ways.

 

Figure 1: Ways that A-I editing can change the transcriptome

 

Finding novel substrates for A-to-I editing 

We believe that there are more substrates yet to be discovered that are subjected to RNA editing. This project has been focused on selective methods to find novel substrates of A-to-I editing. We have developed one method to detect novel sites of selective A-to-I editing using co-immunoprecipitations with an anti-ADAR2 antibody (Ohlson et al., 2005). By this method intrinsic ADAR2-RNA substrate complexes were extracted from mouse brain. Other methods that are used to identify new sites of A-to-I editing are computational analysis and high-throughput sequencing (RNA-Seq). By combining these methods the anticipation is to determine if there are other sites of editing essential for a functional mammalian brain.

GABA-A receptor editing 

One of the principal candidate genes that was found in our analysis searching for new editing substrates is coding for the gamma-aminobutyric acid type A (GABA-A) receptor subunit alpha3 (gabra-3). A short stem loop structure of 54 nucleotides in the Gabra-3 transcript turned out to be highly edited in the adult mouse brain (Ohlson et al., 2007). This site of editing is situated in exon 9 of the Gabra-3 transcript giving rise to an isoleucine to methionine (I/M) change. We have shown that editing of Gabra-3 vastly reduces the number of alpha3 containg GABA-A receptors on the cell surfacedue to reduced trafficking as well as facilitated lysosomal degradation (Daniel et al. 2011).

Developmental regulation of site selective editing 

We have used different high throughput sequencing technologies to determine the editing efficiency during development. Using this state of the art method it is possible to analyze editing in a large set of individual transcript (Silberberg & Öhman, 2011). The efficiency of editing within the coding sequence of all known mammalian substrates for editing have been analyzed using this technique. The results show that editing in general is regulated during development with low efficiency of editing during embryogenesis (Wahlstedt et al., 2009). In this project the mechanism of editing regulation is presently under investigation.