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-to-I RNA editing can modify the messenger RNA
Figure 1: Ways that A-to-I RNA editing can modify the messenger RNA
 

  

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 is 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 sites of editing essential for a functional mammalian brain.

GABAA 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 (GABAA) receptor subunit α3. The encoded transcript (Gabra-3) is edited at one site, giving rise to a change from isoleucine to methionine (I/M) at one site upon translation (Ohlson et al., 2007). The site of modification is situated in a short stem loop structure of 54 nucleotides in Gabra-3 and is edited in more than 90% of all transcripts in the brain. We have shown that editing of Gabra-3 vastly reduces the number of α3 containing GABAA receptors on the cell surface, due to reduced trafficking as well as facilitated degradation (Daniel et al. 2011). In present investigations we are trying to understand how editing of α3 affects GABAA assembly and the importance of regulated editing at this site during brain development.

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 was 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). Furthermore, by analyzing the expression of non-coding RNAs such as miRNAs during brain development we have found that editing of miRNAs increase as the brain matures. Editing of miRNAs often occurs in the seed sequence influencing target recognition (Ekdahl et al., 2012). To understand the role of regulated editing during neuronal development we are analyzing expression and location of the edited miRNAs, as well as their potential targets. For this we us primary neuronal cells from mouse as a model system (Fig. 2). In another part of this project we are investigating how the activity of the ADAR editing enzymes are regulated during neuronal development.

 
Figure 2: Increased editing at the I/M site in the Gabra3 transcript upon maturation of primary cort
Figure 2: Increased editing at the I/M site in the Gabra3 transcript upon maturation of primary cort
 

 

ADAR substrate recognition

The ADAR enzymes recognize adenosines for deamination within duplexed RNA but little is known about other factors that determine the sites chosen for editing. We have discovered a large stem loop structure located in the intron several hundred nucleotides from the I/M edited site in the Gabra-3 transcript. This intronic stem loop is required for efficient editing in vivo (Daniel et al., 2012). In this project we are analyzing if large RNA stem loop structures are common as recruitment elements to attract the ADAR enzyme to selectively edited sites.

 

References

Daniel, C., Venö, M., Ekdahl, Y., Kjems, J., & Öhman, M. (2012). A distant cis acting intronic element induces site-selective RNA editing. Nucleic Acids Res. 40(19):9876-86

Ekdahl, Y., Farahani, H.S., Behm, M., Lagergren, J., & Öhman, M. (2012). A-to-I editing of microRNAs in the mammalian brain increases during development. Genome Res. 22(8):1477-87

Daniel, C., Wahlstedt, H., Ohlson, J., Björk, P., & Öhman, M. (2011). Adenosine-to-inosine RNA editing affects trafficking of the gamma-aminobutyric acid type A (GABA(A)) receptor. J Biol Chem. 286(3):2031-40

Silberberg, G., & Öhman, M. (2011). The edited transcriptome: novel high throughput approaches to detect nucleotide deamination. Curr. Opin. Genet. & Dev. 21:311-21

Wahlstedt, H., Daniel, C., Ensterö, M., & Öhman, M. (2009). Large-scale mRNA sequencing determines global regulation of RNA editing during brain development. Genome Research. 19:978-86

Ohlson, J., Skou Pedersen. J., Haussler, D., & Öhman, M. (2007). Editing modifies the GABA-A receptor subunit α3. RNA, 13:698-703

Ohlson, J., Ensterö, M., Sjöberg B-M., & Öhman, M. (2005). A method to find tissue specific novel sites of selective adenosine deamination. Nucleic Acids Res. 33:e167-73