Figure 1. Presence of LREs destabilizes reporter mRNA. (A) Schematic diagram of firefly luciferase reporter construct, with blue box representing coding region and thin blue line representing 3′-UTR where a LRE was inserted. (B) Results of reporter gene assays. Parental reporter plasmid (FFL) or plasmids containing individual wild-type (WT) or mutant (MUT) LREs were each transfected into HEK293 cells, followed by measurement of luciferase activities (top panel) and mRNA levels (bottom panel) 18 h post-transfection. Top, relative luciferase activities plotted with FFL luciferase activity arbitrarily set as 1. Bottom, relative luciferase mRNA levels with FFL mRNA level arbitrarily set as 1. Numbers are mean ± SD (n = 3). ** p < 0.01 (compared with FFL levels). (C) Schematics of secondary structures of three LREs predicted by mFOLD.¹² The characteristic “A” bulges are depicted in red with corresponding point mutations (“A” replaced by “U”) labeled in blue. The point mutations were predicted not to affect the secondary structures of the LREs.¹² The sizes of the LREs Oct4–95, Hmga1–129 and Rps19–106 in nucleotides are 95, 129 and 106, respectively. (D) Results of RNA stability analysis. HEK293 cells were transfected with each indicated reporter construct. Actinomycin D was added to the cells to a final concentration of 5 ug/ml to stop transcription 18 h post-transfection. Total RNAs were isolated at 0, 2 and 4 h time points after ActD treatment and luciferase mRNA levels determined by RT-qPCR analysis. Results are presented after normalization against internal control β-actin mRNA levels with 0 time point luciferase mRNA levels arbitrarily set as 1. Numbers are mean ± SD (n = 3). * p < 0.05; ** p < 0.01 (compared with corresponding time points in the WT group). T1/2, half-life of indicated mRNA.