Figure 4

Editing of the DNA repair enzyme NEIL1 in vivo and co-transfected in HEK293 cells. (a) Intron 5 and exon 6 of human NEIL1 pre-mRNA. Two inverted Alu repeats located 200 nucleotides from the K/R stem are illustrated when it is folded using Mfold. The −1, K/R, +1 site found at the 5' end of exon 6 is highlighted. (b) Sanger sequencing chromatograms after RT-PCR of NEIL1 transcripts from human, mouse and rhesus brains. Editing was detected at the −1, K/R and +1 site in human and rhesus brains as a dual A and G peak. No editing was detected in the RNA from a mouse brain. (c) Top: Sequencing chromatograms after RT-PCR on RNA from co-transfections of ADAR1 with the human NEIL1 construct including the inverted Alu repeats (hNEIL1), inverted Alu repeats deleted (hNEIL1 ΔAlu), mouse NEIL1 (mNEIL1) and mNEIL1 where the inverted Alus from the human sequence were fused into the mouse sequence 200 nucleotides upstream of the K/R site (mNEIL1 + Alu). Bottom: Quantification of editing efficiency of the different NEIL1 constructs co-transfected with ADAR1 in HEK293 cells. Editing efficiency was calculated at the −1, K/R and +1 site. The mean value of the ratio between the A and G peak heights from at least three individual experiments were calculated as percentage editing. 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). K/R, lysine-to-arginine; nt, nucleotide; RT-PCR, reverse transcription polymerase chain reaction.