Figure 3.
Effects of nNOS 5′-UTR sequences on translational efficiency. (A) Schematic diagram of the chimeric reporter constructs. Various nNOS 5′-UTR sequences were inserted between the Sp6 viral RNA polymerase promoter and the firefly luciferase reporter ORF. (B) In vitro transcribed and capped RNAs were quantified and translated in rabbit reticulocyte lysates. In these experiments vector luciferase activity averaged 4.2 ± 0.2 × 106 relative light units (RLU). (C) Effects of the various nNOS leader sequences on the translational efficiency of in vitro transcribed, capped, chimeric nNOS 5′-UTR/luciferase reporter RNAs that were transiently expressed in cultured cells. Activity profiles of nNOS 5′-UTR sequences in C2C12 cells [mouse skeletal muscle, nNOS(+)], NT2/D1 cells [human neuroblastoma, nNOS(+)], and HeLa cells [human squamous carcinoma, nNOS(−)] were normalized with β-galactosidase activity and protein content. In these experiments, vector luciferase activity averaged (5.6 ± 0.4) × 105 RLU for nondifferentiated C2C12 cells, (4.6 ± 0.9) × 105 RLU for NT2/D1 cells, and (5.1 ± 0.9) × 105 RLU for HeLa cells. (D) Translational efficiency of selected nNOS 5′-UTR sequences examined by using liposome-mediated DNA transfections. Expression of RNA molecules from Sp6 polymerase sequences employed infection of nondifferentiated C2C12 cells with recombinant vaccinia vectors expressing Sp6 viral RNA polymerase. The translational efficiency profile is similar to that derived from RNA transfection assay shown in C. (E) Effects of selected nNOS 5′-UTR sequences on translational efficiency assessed with a transient DNA transfection assay using SV40 promoter-directed luciferase reporter constructs. Luciferase activity was normalized to β-galactosidase activity and protein content. The translational efficiency profile is again similar to that derived from RNA transfection assay shown in C. Shown for B, C, D, and E are results from three or four identical experiments, each in duplicate (mean ± SE).