Translation initiation on the 5′ leader from yeast GCN4 in Arabidopsis, and microarray meta-analysis. (A) The schematic illustrates the predictions of two contrasting hypotheses concerning the molecular function of eIF3h (after Hinnebusch 2005). uORF4 is shaded (gray) for emphasis. If eIF3h supported resumption of scanning, few ribosomes would scan the intercistronic spacer in the eif3h mutant, but those that do would easily acquire a fresh ternary complex (TC) and initiate at the inhibitory uORF4. Hence, adding uORF4 to uORF1 will reduce FLUC translation to similar degrees in wild type and eif3h (bars on the right symbolize predicted FLUC expression levels). In contrast, if the eif3h mutation delayed TC acquisition, some mutant ribosomes would leaky-scan past uORF4 and thus reach the main FLUC ORF. Meanwhile, wild-type ribosomes, having acquired their TC early, will be preferentially intercepted by uORF4, resulting in a reversal of the eIF3h dependence (bars on right). (B) GCN4 5′ leader sequences tested. The version with four uORFs is the original. (*) uAUGs in a strong context. (C) Expression data for the constructs shown in panel B. Error bars, standard error (n > 4). (*) Statistical significance at P < 0.05. Where shown, GCN4-FLUC and endogenous EF1α mRNA levels were estimated by RT-PCR. (D) The translation state of Arabidopsis mRNAs corresponding to the scheme outlined at the top was mined from polysome microarray data (Kim et al. 2007) and displayed for wild type and eif3h mutant. Translation state is defined as the log(2) of the ratio of polysomal (PL) to nonpolysomal (NP) mRNA signal. Note that the translation state is equal or lower in eif3h than in wild type, as predicted by the resumption-of-scanning hypothesis.
