Figure 1
The 3′ UTRs of XIAP and MDM2 transcripts are targeted by ADAR1-mediated RNA editing. (a and b) Top panel depicts the UCSC Genome Browser-based representation of the distribution of the annotated editing sites in the XIAP (a) and MDM2 (b) 3′ UTR. The editing status was independently monitored by Sanger sequencing, which was performed on cDNAs derived from 293 or WI38 cells with either control (siCtrl) or ADAR1-targeting (siADAR1) siRNAs, as indicated. The Sanger sequencing chromatograms corresponding to selected editing regions of the XIAP and MDM2 transcripts are shown in the lower panel, with positions of A-to-G conversion being marked by red stars. The estimated editing ratios in the indicated samples are noted in the boxes. (c) RNA-immunoprecipitation (RIP) assay was performed to examine the binding of XIAP and MDM2 transcripts by ADAR1. The RNA precipitated by the control IgG or ADAR1 (IP-ADAR1) antibodies was subjected to real-time RT-PCR with primers specific to 3′ UTR of XIAP and MDM2. Fold of binding enrichment was normalized to the value of IgG, and shown with mean±S.D. GAPDH levels in the immunoprecipitates served as control. (d) Sanger sequencing chromatograms for selected editing regions of XIAP (top) and MDM2 (bottom) transcripts derived from different transfectants of 293 cells: pSUPER control vector (shCtrl) and shADAR1 (shA1) for the knockdown experiments; and pcDNA control vector (over-Ctrl), ADAR1 (over-A1) and dominant-negative ADAR1 (over-A1DN) for the ectopic expression experiments. Red stars mark the positions of RNA editing sites (for statistical analyses shown in this figure: NS, not significant or P>0.05; *P<0.05; **P<0.01; ***P<0.001)
