Length-dependence of poly(A)- and SRE-effects assayed in a series of RNAs based on firefly luciferase. (A) Scheme of the RNAs used: All contained an m7G cap and the fLuc ORF (green lines). They had poly(A) tails and/or SREs at their 3′ ends as shown. All SRE-containing RNAs were synthesized in an SRE+ and an SRE− version as indicated. The fLuc-MBP RNAs were extended by the ORF encoding MBP (orange lines) inserted between the fLuc stop codon and the SREs. For the fLuc-2xMBP RNAs, a second MPB ORF was inserted in the middle of the first MBP ORF (blue lines). “N” indicates the number of independent RNA preparations assayed. Each of the two preparations of each RNA was tested in three independent batches of Drosophila embryo extract, resulting in six independent experiments per RNA (“n”). (B) Summary of translation experiments carried out with the RNAs depicted above. Poly(A) stimulation, shown on the left, was calculated from the comparison of equivalent SRE− RNAs carrying a poly(A) tail or not (P ≤ 1.3 × 10−5). SRE-dependent repression, shown on the right, was calculated from the comparison of SRE+ and SRE− RNAs that also carried a poly(A) tail (P ≤ 1.1 × 10−5). Both stimulation and repression are shown with a correction for translational efficiency in RRL (red bars) and without correction (blue bars). Correction factors were similar as in Figure 7. Error bars and indications of statistical significance of the comparisons indicated by brackets are as in Figure 7.
