Fig. 3.

Artificial GMD reporter mRNA harboring a GR-binding site is subject to efficient GMD in a way that depends on GR, PNRC2, and UPF1. (A) A schematic of GMD reporter RLuc constructs used in this study. See Results for details. A plasmid expressing FLuc served as a control of variation in transfection efficiency. C5′, CCL2 5′UTR; C3′, CCL2 3′UTR; B5′, BCL3 5′UTR; P5′, PHLDA1 5′UTR; SL, stem-loop structure; and Δ, a deletion of the GR-binding site. (B–E) The effects of a down-regulation of GR, UPF1, or PNRC2 on GMD reporter RLuc mRNAs. HeLa cells were transfected with the indicated siRNAs. One day later, the cells were transfected with a GMD reporter RLuc plasmid and a FLuc control plasmid. Two days later, the cells were either treated or not treated with Dex for 12 h before harvesting. (B and D) Western blots demonstrating specific down-regulation of GR, UPF1, or PNRC2. (C and E) RT-qPCR analysis of reporter RLuc mRNAs. The levels of GMD reporter RLuc mRNAs were normalized to the levels of FLuc mRNAs. The normalized levels of GMD reporter RLuc mRNAs without Dex treatment were arbitrarily set to 100%. (F) The effect of deletion of a GR-binding site and a positional effect of the GR-binding site. HeLa cells were transfected with the indicated GMD reporter RLuc plasmid and a FLuc control plasmid. Two days later, the cells were either treated or not treated with Dex for 12 h before harvesting. The levels of GMD reporter RLuc mRNAs were normalized as described in Fig. 3C. The columns and bars represent the mean and SD of three independent biological replicates. Two-tailed, equal-sample variance Student′s t tests were used to calculate the P values. **P < 0.01.