Translation Reinitiation Promoting Elements Figure 5. The extreme NTD of a/TIF32 contains two distal regions that promote efficient REI in the 59 enhancer-dependent manner. (A) Schematic representation of the first 200 amino acid residues of a/TIF32 shown as numbered circles (Boxes 1–20), each of them composed of 10 consecutive residues that were substituted with a stretch of 10 alanines. The sequence of Boxes 6, 8, and 17 is given below the schematic. (B) The a/tif32-Boxes 6, 8, and 17 impart a strong Gcn- phenotype. YBS52 (GCN2 a/tif32D) was transformed with individual YCplac111-based plasmids carrying the indicated a/TIF32 alleles and the resident YCpTIF32-His-U plasmid was evicted on 5-FOA. The resulting strains, together with isogenic strains H2880 (GCN2 a/TIF32; row 1) and H2881 (gcn2D a/TIF32; row 2), were then spotted in five serial 10-fold dilutions on SD (left panel) or SD containing 30 mM 3-AT (right panel) and incubated at 30uC for 3 and 6 days, respectively. (C) The failure of the a/tif32-NTD-Box mutations to derepress GCN4 is caused by a defect in resumption of scanning of post-termination 40S ribosomes on uORF1. Selected strains described in section B were introduced with the GCN4-lacZ constructs p180 (i.), pG67 (ii.), pM199 (iii.), and p209 (iv.), respectively, and analyzed as in Figure 2B. To induce the GCN4-lacZ expression (section i.), the transformants grown at the minimal media for 2 hrs after dilution were treated with 10 mM 3-AT for 6 hrs (a/TIF32) or overnight (Box mutants). An asterisk indicates data taken from [10] for comparison purposes. (D) Indicated strains described in section B were introduced with the GCN4-lacZ deletion constructs described in Figure 1D and Figure 4A–4C and analyzed as in Figure 2B. The RPEs affected by individual mutations are indicated above the bar diagram. wt, construct 1111; bg*, construct 4411. doi:10.1371/journal.pgen.1002137.g005 grossly unexplored, cis-regulatory function in translational control of gene expression [2]. Indeed, the first examples of aberrant protein expression leading to pathophysiological mechanisms in the etiology of human diseases that are connected to defective uORF-mediated translational control have already been described [2,24–29]. Translational control of yeast GCN4 transcriptional activator is unarguably the best studied example of the REI mechanism [8]. Particularly intriguing is the fact that only one of its four short uORFs (uORF1) promotes efficient REI thanks to the presence of two specific enhancing sequences (designated here as ‘‘enhancers’’) flanking its coding region. Importantly, both of these enhancing sequences were previously demonstrated to be transferable [19]; i.e. to function independently of the sequence context of uORF1 indicating that their activity is directly imprinted in their sequence and/or structure. Whereas the mode of action of the 39 enhancer is not known, the 59 sequences encompassing a rather long (,160) stretch of nucleotides were shown to co-operate with the NTD of the a/TIF32 subunit of eIF3 in stabilizing the post-termination 40S subunit on the mRNA [10]. In this study, experiments are described that (i) identify individual stimulatory elements within the 59 sequences of uORF1, making up the 59 enhancer, as well as in the NTD of a/TIF32, and (ii) allow us to evaluate their functional importance for the REI mechanism. In addition, our analysis of the 59 leader of yet another transcriptional activator YAP1 demonstrates that (iii) the functional interaction between the a/TIF32-NTD and the specifically folded sequences 59 of a REI- permissive uORF represent a generally applicable requirement for efficient REI at least in yeast. Modeling the events that, following termination of translation of uORF1, are required for subsequent resumption of scanning We first tested the individual contributions of both uORF1’s enhancers on efficiency of REI by their individual replacements with the corresponding sequences of the REI-nonpermissive uORF4. Previously, a similar cassette replacement mutagenesis was carried out [19]; however, it did not include the uORF1’s 59 sequences. In accordance with Grant et al. [14], the uORF1’s 39 enhancer alone (Figure 1D; construct 4411) was still capable to allow some REI on GCN4-lacZ (,4-fold higher than the background control in uORF4; construct 4444), albeit the overall REI activity was strongly reduced by ,80% when compared to wt (construct 1111). On the contrary, the REI activity of the 59 enhancer alone containing all four RPEs (construct 1144) dropped to the background levels of uORF4. Thus rather than making additive contributions to the uORF1’s ability to support a high frequency of REI at GCN4, as originally proposed, it seems that the 39 enhancer acts first and its action is a prerequisite for the subsequent contribution of the 59 enhancer. We propose the following model of the sequence of events on the uORF1 that follow termination of its translation and that, in the light of our YAP1 data, could be applicable to short uORFs with the REI- permissive character in general (Figure 1A and 1B). Upon stop codon recognition, the 39 enhancer, buried for its most part in the mRNA binding channel, interacts somehow with the ribosome and ensures that the 40S subunit remains attached to the mRNA during the first ribosomal recycling reaction that removes the large ribosomal subunit and is thought to be catalyzed by RLI1/ABCE1 [30]. This alone suffices for a certain level of elevated efficiency of REI. In the meantime, the 59 enhancer that has gradually emerged from the mRNA exit channel progressively folds into its secondary/tertiary structure and contacts the a/ TIF32-NTD, previously shown to interact with RPS0A occurring near the mRNA exit pore [10,15], to further stabilize the 40S subunit on the GCN4 mRNA. This second step considerably boosts the efficiency of REI as it prevents recycling of at least 50% of small subunits [14]. Consistent with our model, mammalian eIF3a was shown to interact with mRNA in the 48S PIC in a way extending the mRNA binding channel beyond the exit site [31]. In addition to a/TIF32, the g/TIF35 subunit of eIF3 also promotes this process, however, by an unknown mechanism that does not depend on the 59 enhancer and awaits a detailed investigation [17]. Interestingly, plant eIF3g together with eIF3h were similarly shown to support efficient REI [32,33], however, their mechanistic contributions also remain to be explored. Once the mRNA-40S complex is sufficiently stabilized, eIF3 most probably facilitates recruitment of scanning-promoting factors namely eIF1 and eIF1A. These factors were shown to trigger conformational changes of the 40S head region resulting in the open/scanning conducive conformation that is required for linear scanning from the mRNA9s 59 cap [34]. It is very likely that similar conformational changes are also needed for the mRNA-bound post-termination 40S subunit in order to resume scanning. How the 39 enhancer performs its initial task is currently under investigation in our laboratory. Previous work suggested that its AU-rich content (,60%) rather than a particular sequence could be critical for its function. In fact, it was proposed that the AU-rich sequence would not form strong base-pairing interactions with the 40S subunit and would allow it to promptly resume scanning [13]. However, with the exception of uORF4 (AU-content ,40%), the sequences corresponding to the 39 enhancer of other two GCN49s uORFs (2 and 3) have even higher AU-content (,85% and ,70%, respectively), yet they do not promote REI as uORF1. Besides, our model posits that the ribosome terminating on uORF1 spends longer than usual time on the termination/ recycling steps to allow the 59 enhancer to fold and interact with the a/TIF32-NTD. Hence we think that a simple enrichment in A and U nt is unlikely to be the key to this puzzle, and it is still possible that the 39 enhancer contains a less stringent sequential PLoS Genetics | www.plosgenetics.org 12 July 2011 | Volume 7 | Issue 7 | e1002137