Effects of Pan3 isoform knockdowns on individual mRNA decay pathways in vivo. (A–C) Northern blots showing the decay of the β-globin mRNA (BBB; A), β-globin mRNA carrying the c-fos ARE (BBB+ARE; B), or β-globin mRNA carrying three consecutive let-7 binding sites (BBB+3xlet7; C) in human BEAS-2B cells transfected with control siRNA, Pan3L-specific siRNA, Pan3S-specific siRNA, or both Pan3 siRNAs (Pan3S+L). The tTA-expressing BEAS-2B-19 cells were first transfected with the indicated siRNAs and then cotransfected with plasmids encoding the indicated reporter mRNA and a constitutively expressed α-globin/GAPDH hybrid mRNA that served as a control (ctrl) mRNA. Transcription of the reporter mRNA was transiently induced for 2 h by tetracycline removal. RNA samples were prepared at the indicated time points after tetracycline was replenished. Poly(A)− RNA samples were prepared in vitro by treating RNA samples from early time points with oligo(dT) and RNase H. RT-PCR and agarose gel electrophoresis were performed to assess the Pan3 knockdown efficiency; GAPDH served as the loading control. Semi-log plots illustrate decay kinetics of BBB+ARE (B) and BBB+3xlet7 (C) mRNAs after knocking down the Pan3 isoforms. Decay curves and RNA half-lives (t1/2) were obtained by least squares regression of the fraction of mRNA remaining as a function of time, using data from two separate time-course experiments with reproducible results. (D) Dual luciferase assay showing the effects of Pan3 isoform knockdowns on miRNA-mediated gene silencing. (Upper left) Schematic diagram of renilla luciferase (RL) mRNAs carrying three copies of the WT or mutant let-7 binding sites in the 3′UTR (RL+3xlet7 or RL+3xlet7mut). (Lower left) RT-PCR and agarose gel electrophoresis were performed to assess the Pan3 knockdown efficiency; GAPDH served as the loading control. (Right) Results of dual luciferase assays. Firefly luciferase (FL) activity, derived from the same plasmid carrying the RL reporter gene, was used for normalization. The relative fold changes were measured by comparing the RL/FL activity detected under different knockdown (KD) conditions as indicated with that detected in cells expressing the corresponding reporter mRNA under control KD (set as 1). All data represent the average ±SD (n = 3).
