Translation Reinitiation Promoting Elements Figure 4. Identification of the forth REI-promoting element within the 59 enhancer that acts in synergy with the RPE i. in the a/TIF32-NTD–dependent manner. (A–C) Schematics showing the solitary uORF1 GCN4-lacZ construct with the battery of substitutions and/or deletions in the RPE i. (A), RPE ii. (B), and RPE iv. (C) that are used in the next three panels, respectively. (D) The RPE i. acts in the a/TIF32-NTD- dependent manner. The YBS47 and YBS53 strains were introduced with the GCN4-lacZ substitution constructs described in panel A and Figure 1D, and analyzed as in Figure 2B. wt, construct 1111; bg*, construct 4411. (E) The RPE ii. acts in the a/TIF32-NTD-independent manner. The strains as in panel D were introduced with the GCN4-lacZ deletion or substitution constructs described in panel B and analyzed as in Figure 2B. (F) The RPE iv. acts in synergy with the RPE i. in the a/TIF32-NTD-dependent manner. The strains as in panel D were introduced with the GCN4-lacZ deletion and/or substitution constructs described in panel C and analyzed as in Figure 2B. doi:10.1371/journal.pgen.1002137.g004 other by Box17. Importantly, in a striking analogy with the a/tif32- D8 mutation [10], all three Boxes as well as their combinations decreased b-galactosidase activities measured from constructs carrying only uORF1 at three different positions relative to GCN4-lacZ by a similar number (,50–80%) (Figure 5C, constructs ii. – vi.) strongly indicating that the failure to induce GCN4 expression emanates from the inability of 40S subunits to resume scanning after translating uORF1. Remarkably, in contrast to a/ tif32-D8, neither of the Boxes either alone or in pair wise combinations affected the overall eIF3 affinity for 40S subunits in vivo (Figure S3A and data not shown). Furthermore, binding of the in vitro synthesized a/TIF32-NTD to GST-fused RPS0A was also not affected by these mutations (Figure S3B). Together these findings strongly suggest that the a/tif32-Boxes impact REI specifically by impairing the a/TIF32-NTD interaction with the 59 enhancer. To demonstrate directly that the amino acid regions represented by the latter Boxes mediate the REI-promoting interaction between the a/TIF32-NTD and the 59 enhancer, we analyzed b- galactosidase activities of the selected GCN4-lacZ constructs described in Figure 2 and Figure 4 eliminating the key RPEs in the background of the Box6+17 and Box8+17 mutations (Figure 5D and data not shown). Whereas neither DEL36, SUB40 and SUB49 (impairing RPE i.) nor AA-C and DEL36+AA-C (impairing RPE iv. either alone or together with RPE i.) significantly exacerbated deleterious effects of the double-Box mutations on REI efficiency in the mutant cells, CAAII impairing eIF3-independent RPE ii. showed an additive effect when combined with either of the double- Box mutations. These results thus clearly corroborate identification of the two critical 59 enhancer-dependent regions that together account for the REI-promoting activity of the a/TIF32-NTD independently of its 40S-binding activity. The 59 sequences of the REI-permissive uORF of YAP1 contain structurally similar features to the RPEs of GCN4’s uORF1 and analogously promote efficient REI in concert with the a/TIF32-NTD Next we asked whether the just described mRNA and protein features required for efficient REI on the GCN4 mRNA are unique to its uORF1. We took advantage of two genes, YAP1 and YAP2, both encoding stress related transcription factors, the mRNA leaders of which contain short uORF(s) with well described regulatory roles. Whereas the YAP1’s uORF permits post- termination 40S ribosomes to efficiently resume scanning for REI on the main ORF (similar to GCN4’s uORF1), the uORFs 1 and 2 of YAP2 act to block ribosomal scanning after their translation by promoting efficient termination followed by rapid mRNA decay [18]. To our knowledge the uORF of YAP1 is the only short uORF in yeast experimentally proven to promote efficient REI besides GCN4’s uORF1; however, in contrast to GCN4, the exact link between its REI-mediated translational control mechanism and its stress-protective cellular role(s) is still not fully understood. We first computationally predicted potential secondary struc- tures of the 59 sequences of YAP1’s uORF (281 to 21) and of YAP2’s uORF1 (2101 to 24) occuring behind the trailing edge (the mRNA exit channel) of the post-termination 40S ribosome, using an analogous folding model as that described for the GCN4 59 sequences above. The predicted secondary structures were compared with that occurring in the corresponding region of GCN49s uORF1 (2131 to 210) (Figure 6A). The structure similarities, computed using the RNA distance program [21], revealed a remarkable resemblance between predicted secondary structures of 59 sequences of YAP1’s uORF and RPEs of GCN4’s uORF1; the similarity score reached the value of 35 (compared to 46 for YAP2 versus GCN4; the higher the number, the lower the similarity), which is highly significant considering that the compared sequences are fairly short (,90 nt). It mainly arises from (i) the occurrence of a double-circle hairpin and (ii) similar lengths of unstructured sequences indicating congruent positioning of the structured elements in the overall folds. It is worth noting that no significant sequence similarities were observed (data not shown) suggesting that these particular structural features might truly play an important role in the REI mechanism. To examine that, we replaced the entire 59 leader of uORF1 of GCN4 excluding the promoter region with the corresponding sequences from both YAP genes in our GCN4-lacZ construct containing solitary uORF1 (Figure 6B) and measured b-galacto- sidase activities in wt as well as a/tif32-D8 cells. Whereas the 59 leader of uORF1 of YAP2 (in Y2-uORF1) showed background levels in both strains, as expected, the 59 sequences of uORF of YAP1 (in Y1-uORF1) stimulated the GCN4-lacZ expression by ,2- fold over the background in wt cells (Figure 6C). Strikingly, this activity dropped by ,80% in a/tif32-D8. The similar reduction was also obtained when we fused the YAP1 gene with its intact 59 leader with lacZ (in Y1-lacZ). In contrast, a lacZ fusion with the YAP2 gene containing its natural 59 leader (in Y2-lacZ) showed no b-galactosidase activity at all in accord with previous observations implicating both uORFs of the YAP2 mRNA in promoting its rapid degradation [18]. Importantly, point mutations designed to disrupt the conserved double-circle hairpin (in Y1-uORF1- hairpin_G-45U C-57A) reduced the Y1-uORF1 activity by ,30% in wt cells and showed the epistatic interaction with a/ tif32-D8 (Figure 6D), in good agreement with the data presented in Figure 4F. In contrast, mutations disrupting the predicted non- conserved bulged-stem (in Y1-uORF1-‘‘stem’’_C-32G G-33C) showed no reduction in either of the strains indicating that it is either not functionally important or not affected by our mutations. Taken together these results clearly demonstrate that the specifically structured 59 enhancers of REI-permissive uORFs of GCN4 and YAP1 are at least partially functionally interchangeable and critically require the NTD of a/TIF32 for their function. Hence a possibility for a common mechanism of translational control operating on short REI-permissive uORFs seems highly likely. Discussion The widespread prevalence of uORFs in mammalian tran- scriptomes (up to 50%) suggests that REI after translation of a short ORF represents a comprehensive, yet underestimated and PLoS Genetics | www.plosgenetics.org 10 July 2011 | Volume 7 | Issue 7 | e1002137