Editing modifies the GABA(A) receptor subunit alpha3

Abstract

Adenosine to inosine (A-to-I) pre-mRNA editing by the ADAR enzyme family has the potential to increase the variety of the proteome. This editing by adenosine deamination is essential in mammals for a functional brain. To detect novel substrates for A-to-I editing we have used an experimental method to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA(A) receptor, is a substrate for editing by both ADAR1 and ADAR2. Editing of the Gabra-3 mRNA recodes an isoleucine to a methionine. The extent of editing is low at birth but increases with age, reaching close to 100% in the adult brain. We therefore propose that editing of the Gabra-3 mRNA is important for normal brain development.

FIGURE 1.

Subunit-specific sequences of the gabra genes. The predicted structure of the stem–loop within exon 9 of Gabra-3. The edited A is printed in bold and circled in gray. The differences between Gabra-3 and other Gabra sequences are shown in blue for Gabra-1, red for Gabra-2, and green for Gabra-5.

FIGURE 2.

Editing of the Gabra-3 transcript. (A) The editing of Gabra-3 was demonstrated by DNA sequencing. The chromatogram of the genomic DNA sequence shows an adenosine at the I/M-site, indicated by an arrow. Total brain RNA from the same NMRI adult mouse was reverse transcribed (cDNA) and sequenced after PCR amplification. A guanosine was present at the I/M site in the cDNA. The cDNA from an ADAR2^−/− adult mouse was amplified by PCR and sequenced. A dual A and G peak appeared with the majority of the transcripts showing an A at the I/M site. (B) PCR products from wild-type and ADAR2^−/− cDNA were cloned and sequenced. The sequences of 10 wild-type and 22 ADAR2^−/− clones were determined. (C) Wild-type Gabra-3 and Gabra-3 with a mutated ECS (ECS-G) were transfected into HEK 293 cells with an ADAR1 or ADAR2 expression vector. An empty expression vector (labeled “C” for control) was used as a control for endogenous editing. In wild-type Gabra-3 (left), no editing was observed in the control since a single A peak is seen at the I/M site. During cotransfection with ADAR1 or ADAR2 dual G/A peaks were seen with the majority in the G peak. Adenosine was present at the editing site in the ECS-G mutant in all transfection experiments (right).

FIGURE 3.

The position of the edited site in the α3 subunit and evolutionary conservation. (A) The protein sequence of Gabra-3 consists of four transmembrane domains (TM1–TM4). The edited site was located within TM3 at amino acid 5 (of 22). (B) The I/M sites of Gabra-3 in frog (Xenopus tropicalis) and pufferfish (Tetraodon nigroviridis) have a genomically encoded methionine where all other organisms have an isoleucine (Hinrichs et al. 2006). (C) An evolutionary tree showing the phylogenetic relationship between the species in B (Meyer and Zardoya 2003; Kriegs et al. 2006).

FIGURE 4.

Editing of the α3 subunit at different stages during development. Editing of the Gabra-3 transcript extracted from mouse brain at different developmental stages. The RT-PCR products from mice at day 2, day 7, day 12, and adult mouse were cloned and editing at the I/M site was analyzed by sequence determination. The number (n) of clones analyzed at each developmental stage is indicated. Below, the chromatogram from the sequence at the I/M site is based on the population derived from the RT-PCR product.