Contribution of individual uAUGs to translational repression and eIF3h-dependent translation of AtbZip11-FLUC. (A) Single uAUGs were eliminated by site-directed mutagenesis. Note the anti-inhibitory effect of uORF 1 (also see C). Error bars represent standard error (n = 8–11). Data on the original and uORF-less leaders are included from Figure 2A for comparison. (*) Statistical significance at P < 0.01, (**) P < 0.002. (B) The combination of uORFs 2 and 3 caused a robust dependence on eIF3h, in contrast to uORFs 1 and 4 together (n = 8–12). eIF3h dependence of uORF2 alone was also significant but that of uORF3 alone was not. (*) Statistical significance at P < 0.01. (Right panel) AtbZip-FLUC and endogenous EF1α mRNA levels were estimated by RT-PCR. (C) A pairwise comparison demonstrating the anti-inhibitory effect of uORF1 in the AtbZip11 leader. Note that leaders harboring uORF1 are still eIF3h-dependent. Bars, standard error (n = 9–22). (*) Statistical significance at P < 0.04, (**) P < 0.01.
