Figure 1

Analysis of editing efficiency at the I/M site in Gabra-3 editing reporters in HeLa cells. (a) Mouse Gabra-3 mutants used to analyze editing efficiency depending on the location of the inducer element. The I/M site is located in exon 9 of the Gabra-3 transcript and the dsRNA structure and editing site is illustrated as a line and a dot. The reverse arrows illustrate the position of the IE in the different mutants. In the WT construct the IE is positioned 150 nucleotides from the I/M site and illustrated as a dotted line. In the ΔIE mutant the IE is deleted. In the DDS IE mutant the IE is moved 300 nucleotides downstream of the I/M site and in the US IE the IE is moved 150 nucleotides upstream of the I/M site. In the Alu-IE, the native IE is replaced by the human inverted Alu found in the 3' UTR of the PSMB2 gene. (b) Example Sanger sequence chromatograms of the I/M site after RT-PCR from transfections with Gabra-3 mutants. Editing is seen as a dual A and G peak. Below, reproducible triplicates were compared with known levels of I/M site editing using 454 high-throughput sequencing (see Materials and methods and [11]) and classified into different levels of editing from non to full. (c) Quantification of editing efficiency of the different Gabra-3 mutants. All mutants were tested at least in triplicate. The amount of edited transcript was determined by measuring the ratio between the A and G peak heights and represented as a percentage. The bars represent the mean value of the ratio between the A and G peak heights. Error bars are standard deviation. Significance: *P = 0.05, **P < 0.05 (two-tailed Student’s t-test) (for details see Materials and methods section). DDS IE, downstream inducer element; ΔIE, deleted inducer element; IE, inducer element; I/M, isoleucine to methionine; nt nucleotide; RT-PCR, reverse transcription polymerase chain reaction; US IE, upstream inducer element; WT, wild-type.