Figure 2.
Artificial tethering of human Ago2 at the 3′UTR or loading of endogenous miRISC onto 3′UTR abrogates nonsense-mediated mRNA decay. (A) Schematic representations of the NMD reporter constructs. (B–E) Cos-7 cells were transiently transfected with (i) 1 μg of either pCI-λN-HA or pCI-λN-HA-Ago2, (ii) 0.1 μg of either phRL-Gl-5BoxB Norm or Ter, and (iii) 0.1 μg of pCI-F. (B) Western blotting of λN-HA-Ago2 using HA antibody. (C) sqRT–PCR of Gl-5BoxB mRNAs and FLuc mRNAs. The levels of Gl-5BoxB mRNAs were normalized to the levels of FLuc mRNAs. The normalized level of Gl-5BoxB Norm mRNA in the presence of λN-HA was set to 100% (upper numbers). Alternatively, the normalized levels of Gl-5BoxB Norm mRNA in the presence of each effector were set to 100% (lower numbers). (D) Quantitative real-time PCR of Gl-5BoxB mRNAs and FLuc mRNAs. The levels of Gl-5BoxB mRNAs were normalized to the levels of FLuc mRNAs. The normalized level of Gl-5BoxB Norm mRNA in the presence of λN-HA was set to 100%. (E) Translational efficiency of Gl-5BoxB Norm mRNAs. The relative RLuc activity (RLuc activity/FLuc activity) was normalized to the relative amount of Gl-5BoxB Norm mRNA (Gl-5BoxB Norm mRNA/FLuc mRNA). The normalized translation efficiency of Gl-5BoxB Norm mRNA in the presence of λN-HA was set to 100%. (F–H) HeLa cells (2 × 106) were transiently co-transfected with (i) miRNA-targeted NMD reporter plasmid, either pRL-Gl-CXCR4 Norm or Ter, (ii) the pCI-F, and (iii) either CXCR4 miRNA mimetic or non-specific control siRNA. (F,G) The abundance of Gl-CXCR4 Norm or Ter mRNAs and (H) translational efficiency of Gl-CXCR4 Norm mRNAs were analysed by (F) sqRT–PCR, (G) quantitative real-time PCR and (H) dual luciferase assay, respectively. 3′UTR, 3′-untranslated region; Ago2, Argonaute 2; HA, haemagglutinin; miRISC, microRNA-induced silencing complex; mRNA, messenger RNA; NMD, nonsense-mediated mRNA decay; siRNA, small interfering RNA; sqRT–PCR, semi-quantitative reverse transcriptase PCR.