Translation of chimeric RNAs is stimulated by poly(A) tails and repressed by SREs. (A) Scheme of chimeric constructs. All nLuc RNAs (blue) had a 5′ m7G cap and a 3′ biotin. Regulatory RNAs had either a 5′ biotin combined with an unmodified 3′ end (forward constructs) or a 5′ A cap combined with a 3′ biotin (flipped constructs). The green RNA section in the forward-long-SRE±p(A) construct represents the firefly luciferase stuffer fragment (see text). Regulatory RNAs attached to the right of the central divalent streptavidin were as described in Figure 2 and the text. All SRE-containing RNAs were synthesized as SRE+ and SRE− variants as indicated. “N” indicates the number of independent chimeric RNA preparations tested in translation. The number of independent translation experiments (each carried out as three technical replicates) is represented by “n.” Each type of RNA was tested in at least three batches of embryo extract except the flipped-p(A) construct, which was tested in two batches. (B) Translation assays of nLuc and chimeric RNAs containing poly(A) segments in forward and flipped orientations. RNAs were assayed in parallel for translation in Drosophila embryo extract as described in Materials and Methods. The pairwise comparisons used to calculate poly(A)-dependent stimulation are indicated at the top. (C) Assays as in B were carried out with the chimeric constructs containing poly(A) segments plus SREs. The pairwise comparisons used to calculate poly(A)-dependent stimulation or SRE-dependent repression, respectively, are indicated at the top. (D,E) The same RNA preparations as in B and C were translated in parallel in rabbit reticulocyte lysate (RRL). It was assumed that differences in luciferase activities represented variable qualities of the RNA preparations rather than SRE- or poly(A)-effects. Numbers from such assays were therefore used to correct luciferase yields obtained in embryo extract in Figures 4, 7, and 8. Panels B through E show the results of single representative experiments with error bars representing the standard deviations of three technical replicates.
