Length-dependence of poly(A)- and SRE-effects assayed in a series of RNAs based on nanoluciferase. (A) Scheme of the RNAs used. All contained an m7G cap and the nLuc ORF. They had poly(A) tails and/or SREs at their 3′ end as shown. All SRE-containing RNAs were synthesized in an SRE+ and an SRE− version as indicated. The two RNAs at the bottom were extended by the ORF encoding MBP (orange line) inserted between the nLuc stop codon and the SREs. “N” indicates the number of independent RNA preparations assayed. Each of the three preparations of each RNA was tested in two to four independent batches of Drosophila embryo extract, resulting in nine 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) stretch or not (P ≤ 3.8 × 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 ≤ 5.8 × 10−4). Both stimulation and repression are shown with a correction for translational efficiency in RRL (red bars) and without correction (blue bars). Correction factors in individual experiments were typically between 1 and 1.5 and up to 3 in a few cases. Error bars indicate the standard deviation based on the numbers of independent experiments reported in A. The statistical significance of the pairwise comparisons indicated by brackets is indicated by (*) (P < 0.05), (**) (P < 0.01), and ns (nonsignificant).
