Figure 2
ADAR1 targets the 3′ UTR of XIAP and MDM2 for post-transcriptional expression regulation. (a and b) Following transfection with control (siCtrl) or ADAR1-targeting (siADAR1) siRNAs, total RNAs were isolated from WI38 or 293 cells and subjected to real-time RT-PCR to quantify the expression levels of XIAP (a) and MDM2 (b) mRNA. Relative RNA levels (to GAPDH) were normalized to the control group, and shown as mean±S.D. (c) WI38 and 293 cells were transfected with siRNAs targeting ADAR1 (siADAR1) or control siRNAs (siCtrl) prior to western blot analysis for XIAP, MDM2 and ADAR1 expression. ACTIN serves as the internal control. (d) Putative secondary structure of the XIAP 3′ UTR, as predicted by the RNAfold Webserver. Hyperediting region is denoted by a red arrow, with a magnified view of the boxed area shown below. Colors of the nucleotides correspond to base-pair probabilities, based on the color scale bar. Sequences corresponding to this particular stem loop, with the embedded IRAlus, were subcloned into the 3′ UTR reporter plasmid. (e) 3′ UTR reporter assay was conducted on cells transfected with control empty vector (pMIR vector) or the construct containing IRAlus derived from XIAP 3′ UTR (pMIR-XP-Alu). Luciferase activity was detected after 48 h and normalized to the co-expressed β-gal levels, with controls being represented as 1. (f and g) 3′ UTR reporter assay was done as in (e), except with the additional co-transfection of expression plasmids for ADAR1-targeting shRNAs (shA1), the wild-type form of ADAR1 (A1), or the dominant-negative mutant (A1DN). (h) The construct encoding a 3′ UTR-free Myc-tagged XIAP and control vector (Ctrl) were ectopically co-expressed with pSUPER vector (shCtrl) or ADAR1-targeting (shADAR1) shRNAs, as indicated. Protein expression of XIAP, Myc and ADAR1 in different transfectants of HeLa cells was subsequently detected by immunoblotting, with GAPDH as the loading control (for statistical analyses shown in this figure: NS, not significant or P>0.05; *P<0.05; **P<0.01; ***P<0.001)
