FIGURE 3.

mTOR 5′UTR induces firefly luciferase expression from a proper full-length mRNA transcribed from the transfected reporter plasmid and from transfected reporter mRNAs. (A) Representation of the pR_x_F and hpR_x_F sets of constructs. A stable hairpin was cloned either downstream or upstream of the Renilla luciferase (RLuc) open reading frame (ORF), originating the pR_x_F (described in Fig. 2B) or the hpR_x_F subset of constructs, respectively. The “x” represents a linker sequence (hpR_F or pR_F), the human β-globin 5′ untranslated region (HBB 5′UTR) (hpR_HBB_F or pR_HBB_F), the human mTOR 5′UTR (hpR_mTOR_F or pR_mTOR_F), or the EMCV IRES (hpR_EMCV_F or pR_EMCV_F), cloned upstream of the firefly luciferase (FLuc) ORF. (B) Reduction of RLuc by a stable hairpin does not change FLuc induction by mTOR 5′UTR. HEK293T cells were transiently cotransfected with the dicistronic plasmids depicted in A and the pSV-β-Galactosidase control vector (β-gal). Luciferase and β-gal activities were measured 24 h post-transfection. The values (RLU) are shown as the RLuc (RLuc levels), or FLuc (FLuc levels) luminescence percentage relative to that obtained from the pR_EMCV_F construct, arbitrarily set to 100%. Histograms show mean ± SD from three independent experiments. Statistical analysis was performed using the Student's t-test (unpaired, two-tailed); (NS) nonsignificant; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (C) Representation of the pR_x_F constructs, in which “x” represents a linker sequence (pR_F), the human β-globin 5′ untranslated region (HBB 5′UTR) (pR_HBB_F), or the human mTOR 5′UTR (pR_mTOR_F), cloned upstream of the firefly luciferase (FLuc) ORF. Positions of the short-interfering RNA (siRNA) oligonucleotides targeting RLuc or FLuc are represented below. HEK293T cells were cotransfected with the constructs depicted in C and FLuc or RLuc siRNAs. Alternatively, an unspecific siRNA targeting the green fluorescent protein (GFP) was used as a control. Luciferase activity and total protein content was measured 24 h post-transfection. (D) RLuc activity is decreased to residual values by siRNA-mediated depletion of RLuc and FLuc, whereas FLuc activity is reduced to background levels by siRNA-mediated depletion of RLuc and to residual values by siRNAs targeting FLuc. The luciferase values (RLU) are shown as the RLuc per µg of total protein (RLuc/µg) (RLuc levels), or FLuc per µg of total protein (FLuc/µg) (FLuc levels), relative to that obtained in the pR_mTOR_F construct expressed in GFP siRNA-treated cells, arbitrarily set to 100%. Histograms show mean ± SD from three independent experiments. Statistical analysis was performed using the Student's t-test (unpaired, two-tailed); (NS) nonsignificant. (E) Representation of the pR_x_F constructs, in which “x” represents a linker sequence (pR_F), the human mTOR 5′UTR (pR_mTOR_F), or the human MutL homolog 1 (MLH1) 5′UTR, cloned upstream of the FLuc ORF, in plasmids with or without SV40 promoter. MLH1 5′UTR was used as a positive control for cryptic promoter activity. HEK293T cells were transiently cotransfected with each of the constructs described in E, along with the pSV-β-Galactosidase control vector (β-gal). Luciferase and β-gal activities were measured 24 h post-transfection. (F) FLuc activity induced by mTOR 5′UTR is not a result of cryptic promoter activity. The values (RLU) are shown as the luminescence ratio between RLuc and β-gal (RLuc/β-gal), or FLuc and β-gal (FLuc/β-gal), compared to the RLuc/β-gal ratio from the empty construct with promoter, arbitrarily set to 1. Histogram shows mean ± SD from three independent experiments. Statistical analysis was performed using the Student's t-test (unpaired, two-tailed); (NS) nonsignificant, (***) P < 0.001. (G) Schematic illustration of the in vitro transcribed, m⁷GpppG-capped (m⁷G-capped; 5′G) (left panel) or GpppA-capped (A-capped; 5′A) (right panel), and polyadenylated monocistronic reporter constructs. (H) In vitro transcribed monocistronic mRNAs containing mTOR 5′UTR undergo cap-independent translation after transient transfection. The 5′G-capped and 5′A-capped polyadenylated monocistronic mRNAs were transiently transfected into HEK293T cells, along with the pSV-β-Galactosidase control vector (β-gal), and FLuc and β-gal enzymatic activity were measured 4 h post-transfection. The values (RLU) are shown as the luminescence ratio between 5′A and 5′G, normalized to that of the F mRNA (empty vector), which was arbitrarily set to 1. Histogram shows means from three independent experiments. Statistical analysis was performed using the Student's t-test (unpaired, two-tailed); (*) P < 0.05, (**) P < 0.01.