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. 2012 Jul 30;40(19):9876–9886. doi: 10.1093/nar/gks691

Figure 2.

Figure 2.

Editing efficiency at the I/M site is lower in Gabra-3 transcripts in the absence of the intronic hairpin. (A) The editing levels in transcripts derived from transfected Gabra-3 constructs and edited by co-expressed ADAR1 or ADAR2 in HEK293 as well as endogenous editing in HeLa cells. Editing was determined by Sanger sequencing measuring the ratio of the A and G peak after RT-PCR and compared with known levels of editing using 454 high throughtput (HTP) sequencing (see ‘Materials and Methods’ section; Supplementary Figure S1). Reproducible triplicates were made for each sample shown in the figure. The Δ149 lane indicates a reporter construct where 149 nt and thereby the entire intronic stem loop was deleted. As a control 149 nt downstream of the intronic stem was deleted (Δ149 d.s. int). (B) The intronic duplex influences editing of an in vitro transcribed Gabra-3 RNA. A 532 nt long transcript including Gabra-3 exon 9 and part of intron 9 (WT) was used in an ADAR2 modification assay in vitro. This was compared with in vitro editing of a reporter lacking the intronic hairpin of 149 nt (Δ149). Editing was detected by Sanger sequencing after RT-PCR.