Author Summary Protein synthesis is a fundamental mechanism capturing the rejuvenation of DNA–encoded genetic information by its translation into molecular effectors—proteins. Its regulation can be used to change the protein content and thus to adapt a cell to changing environmental conditions. Translation requires mRNAs delivering genetic information of corresponding genes, tRNAs carrying amino-acids, ribosomes as the molecular translators, and accessory proteins/factors facilitating the entire process. There are numerous regulatory mechanisms that modulate translation at its various stages. Here we describe one such a translational control mechanism called reinitiation. Most eukaryotic mRNAs contain only a single translatable gene (ORF); however, in many of them this gene is preceded by a short coding sequence (uORF) that is in some cases translated first. In order to reinitiate translation on the downstream main ORF, a ribosome has to stay bound to mRNA after it has terminated short uORF translation. This requires a concerted action of specific mRNA elements surrounding the uORF and selected initiation factors. Our results delineate how these key players interact with each other and suggest a sequence of general events that ought to take place on short uORF to enable the ribosome to reach and translate the main ORF downstream. in the idea that eIFs important for promoting reinitiation remain at least transiently associated with the elongating ribosome, and that increasing the uORF length or the ribosome transit time increases the likelihood that these factors are dropped off [9]. There is now genetic evidence for this hypothesis showing that in yeast S. cerevisiae eIF3 remains 80S-bound for several rounds of elongation and critically enhances the REI capacity of post- termination 40S ribosomes [10] (see also below). With respect to cis-acting features, with the exception of the uORF-mediated translational control of the budding yeast GCN4 described below, there is virtually nothing known about what other REI-promoting mRNA features are required. Finally, REI efficiency is also directly dependent on (iv) the distance between the uORF termination codon and a downstream initiation codon owing to the fact that the rescanning PICs require a certain time for de novo recruitment of the eIF2NGTPNMet-tRNAiMet ternary complex (TC) to be able to decode the next AUG start site [11]. The GCN4 mRNA encodes a transcriptional activator of mainly amino acid biosynthetic genes and its leader sequence contains four short uORFs (Figure 1A). Independent of amino acid availability, most ribosomes translate the first REI- permissive uORF (uORF1) and, following termination, about a half of them resumes scanning downstream. When amino acid levels are high, re-scanning ribosomes reacquire the TC relatively rapidly afterward and preferentially reinitiate at one of the last three uORFs, none of which supports efficient REI (see our model in Figure 1A). When amino acid levels are low, deacylated tRNAs accumulate, activating the eIF2a kinase GCN2. As a result, the TC levels are decreased and the re- scanning ribosomes must travel for a longer period till they have rebound the TC. This significantly increases the likelihood of bypassing all three REI-nonpermissive uORFs to reach the GCN49s start codon. Thus, whereas the global protein synthesis is significantly down-regulated, translational expression of GCN4 is concurrently induced (derepressed). A failure to derepress GCN4 expression is called the Gcn- (general control nonder- epressible) phenotype. A similar regulatory mechanism has been also shown to govern expression of for example the mammalian Translation Reinitiation Promoting Elements functional homologue of GCN4, the ATF4 transcription factor [12]. The pressing question of why ribosomes readily reinitiate after translation of uORF1 but not the other uORFs has baffled the translational field for many years. Mutational analyses indicated that AU-rich sequences surrounding the stop codon of uORF1 (dubbed the 39 enhancer herein) might favor resumption of scanning and REI [13] (Figure 1B). In addition, sequences 59 of uORF1 were also shown to be critical for efficient REI [14] (Figure 1B). In contrast to the 39 enhancer, the molecular mechanism of which remains to be elucidated, the molecular contribution of the 59 sequences has been recently proposed on the basis of our characterization of the N-terminal truncation of the a/ TIF32 subunit of eIF3 [10]. The N-terminal domain (NTD) of a/ TIF32 was previously shown to interact with the small ribosomal protein RPS0A in vitro [15], and we subsequently found that the N- terminal truncation in a/tif32-D8 severely reduced association of eIF3 and its associated eIFs with the small ribosomal subunit in vivo [10]. (RPS0A is positioned near the mRNA exit pore on the solvent side of the 40S subunit [16]). Unexpectedly, however, a/ tif32-D8 also produced a severe Gcn- phenotype as it failed to up- regulate GCN4 expression under starvation conditions by prevent- ing the post-termination ribosomes from resuming scanning downstream of the uORF1’s stop codon. Detailed genetic analysis suggested that besides RPS0A, the a/TIF32-NTD also interacts with a yet to be identified element(s) within the uORF1’s 59 sequences. Together our findings led to a working model in which wild-type eIF3 remains at least transiently associated with the translating 80S ribosome, and if it does not drop off prior to termination, the a/TIF32-NTD interacts with the 59 sequences to permit ribosomal recycling of only the large 60S subunit while aiding to preserve the small subunit on the GCN4 mRNA [10] (Figure 1A and 1B). This last step serves as a critical prerequisite for subsequent resumption of scanning by the 40S subunit for REI downstream. Interestingly, we have only recently showed that the eIF3g/TIF35 subunit of yeast eIF3 also critically contributes to this process, but the mechanism of its involvement seems to differ from that of a/TIF32 [17]. Besides the uORF1 of GCN4, there is another well described example of a REI-permissive uORF in yeast represented by uORF of the YAP1 gene, an AP1-like transcription factor [18]. The intriguing question is whether the molecular aspects of its reinitiation mechanism are similar to that of GCN4’s uORF1, which could indicate a broad mechanistic conservation of reinitiation on short uORFs. In this study we have subjected the ,220-nt long 59 sequences of uORF1 as well as the first 200 amino acid residues of the a/ TIF32-NTD to an in-depth mutational analysis to identify specific elements/regions required for their common REI-promoting activity. Four such elements designated REI-promoting elements (RPEs) are described that together make up what we now call the 59 enhancer. In addition, two distal regions within the NTD of a/ TIF32 were identified and shown to promote REI in the 59 enhancer-dependent manner. Enhanced computer modeling taking into account a progressive character of mRNA folding combined with classical enzymatic probing surprisingly revealed that the 59 enhancer contains only two well-defined structural features in a 9-nt long stem and a double-circle hairpin representing the RPEs ii. and iv., respectively. Strikingly, a similar structural motif working in concert with the a/TIF32-NTD was also found upstream of the REI-permissive uORF of YAP1. These findings thus strongly suggest existence of a conserved short uORF-mediated mechanism of reinitiation, whereby the a/ TIF32-NTD of the post-termination 80S-bound eIF3 must contact the specifically folded cis-acting REI-promoting elements 59 of PLoS Genetics | www.plosgenetics.org 2 July 2011 | Volume 7 | Issue 7 | e1002137