Figure 1.

Small RNA and Luc Reporter Expression by Three Human Type 3 Pol III Promoters (7SK, U6, and H1)

(A) Upper panel: P-N44 constructs encode Pol III promoters for transcription of an artificial 44-nt sequence, which terminates at the T6 signal. Middle panel: equimolar amounts of P-Luc constructs were transfected into HEK293T cells and total cellular RNA was harvested 36 hr post-transfection. The pBluescript (pBS) plasmid was used as a negative control. An equal amount of total RNA was subjected to northern blot for detection of the N44 RNA. M, RNA size marker (nt) is shown on the left. Ethidium bromide staining of rRNAs (5S and 5.8S) and tRNA serve as loading controls. Lower panel: quantitation of N44 RNA in middle panel. The N44 transcripts from the respective promoters were normalized to 5S RNA. 7SK-produced N44 was arbitrarily set at 1.0. The northern blot in (B) was repeated twice and very similar results were obtained. (B) Upper panel: design of the P-Luc constructs. Four promoters were inserted in the pGL3-basic backbone to drive Luc gene expression. The P(−) construct without any promoter was used as a negative control. Middle panel: Luc reporter activity of the respective promoters. Equimolar amounts of the P-Luc constructs and 1 ng Renilla plasmid to control for transfection efficiency were co-transfected into HEK293T cells. Dual-luciferase reporter assays were performed 36 hr after transfection, and the ratio of Firefly and Renilla luciferase was calculated to represent the Luc activity. The Luc activity measured for the SV40 promoter was arbitrarily set at 10. The results are presented as mean ± SD (n = 3). Lower panel: quantification of Luc mRNA made by different promoters. Total cellular RNA from P-Luc-transfected HEK293T cells was subjected to qPCR to quantitate the Luc mRNA level. The Luc RNA level for SV40 was arbitrarily set at 10. The GAPDH signal was used as an internal control. The data are shown as the mean ± SD (n = 3). (C) Luc expression in HCT116 and C33A cells and PBMCs. Luciferase assays for HCT116 and C33A cells were performed as in Figure 1B. The Luc activity measured for the SV40 promoter was arbitrarily set at 10. Equimolar amounts of P-Luc constructs were nucleotransfected into an equal number of PBMCs. After 24 hr, the firefly luciferase was measured and relative luminescence values were plotted.