Translation Reinitiation Promoting Elements Figure 6. The 59 sequences of the REI-permissive uORF of YAP1 contain structurally similar features to the RPEs of GCN4’s uORF1 and promote REI in co-operation with the a/TIF32-NTD. (A) In silico prediction of secondary structures of the 59 sequences of uORF1 of GCN4 (nt 2131 through 210), uORF of YAP1 (nt 281 through 21), and uORF1 of YAP2 (nt 2101 through 24) carried out with the RNA fold software [21]. Pair wise structural similarities of 59 sequences of GCN4 with 59 sequences of YAP1 and YAP2 were computed using the RNA distance program [21]. Numbered nucleotides in the YAP1 sequence indicate mutated positions as illustrated in panel D. (B) Schematic showing the GCN4-lacZ construct containing solitary uORF1 (G4-uORF1), whose 59 sequences past the trailing edge of the post-termination 40S ribosome (mRNA exit pore) were replaced by the corresponding 5’ sequences of either uORF of YAP1 (Y1-uORF1) or uORF1 of YAP2 (Y2-uORF1). YAP1 and YAP2 constructs where the individual genes were fused with lacZ while their 59 UTRs were kept intact are also shown (Y1-lacZ and Y2-lacZ, respectively). (C) The YBS47 and YBS53 strains were introduced with the lacZ constructs described in panel B and analyzed as in Figure 2B. (D) The YBS47 and YBS53 strains were introduced with two structural mutants in the 59 sequences of YAP1 (specified in panel A) inserted into Y1-uORF1 and analyzed as in Figure 2B. doi:10.1371/journal.pgen.1002137.g006 motif that contacts some component of the post-termination complex, presumably 18S rRNA (Figure 1B). If true, this mechanism would bear a significant resemblance to the termination/reinitiation mechanism that is the best described for the polycistronic mRNA of feline calicivirus [35,36]. A specific 87-nt element (called TURBS) preceding the overlapping termination/initiation site of two long ORFs 2 and 3 folds into a specific secondary structure that in fact resembles our double- circle hairpin. A part of this structure interacts with a complementary segment of 18S rRNA and also with eIF3 via several subunits including eIF3a and eIF3g to prevent dissociation of the mRNA/eIF3/40S complex in order to allow efficient REI on ORF3. Even though this system operates on long ORFs, its mechanistic likeness with the short uORF-mediated REI does not seem to be accidental from the evolutionary point of view. The 59 enhancer of uORF1 contains four REI-promoting elements, two of which act in synergy in the a/TIF32- NTD–dependent manner The original data by the Hinnebusch9s group suggested that the ,160 nt-long 59 sequences may contain two critical REI- promoting motifs [14]. In agreement, we identified not only two but together four individual elements denoted RPEs that together account for the stimulatory effect of the uORF1’s 59 enhancer on REI. Individual mutations of the unstructured RPE i. and the structured RPE iv., as well as the combination of these mutations were found to be epistatic with the a/tif32-D8 mutant. These results clearly suggest that both elements are needed to contact the a/TIF32-NTD and thus it seems conceivable that they might fold together in a higher-order structure. The fact that the RPE iv. is structurally conserved among various yeasts (JP and LV, unpublished observations) may suggest that the RPE iv. provides a structural basis for the 59 enhancer–a/TIF32 interaction, whereas the RPE i. lends a specificity to it. Whether it is a direct interaction is currently being explored in our laboratory in the living cells. In addition to that, our genetic epistasis experiments revealed that the RPEs i. and iv. interact with the a/TIF32-NTD via its two relatively distal REI-promoting regions represented by Boxes 6 and 8, and by Box17, respectively (Figure 5). Importantly, neither of these regions mediates a direct contact of a/TIF32-NTD with RPS0A to facilitate eIF3 binding to 40S ribosomal subunits in vivo (Figure S3) clearly suggesting that they promote efficient REI solely in the 59 enhancer-dependent manner. Interestingly, unlike in the case of RPEs i. and iv., combination of mutations in both of these REI-promoting regions exacerbated the effect of the individual mutations. Hence it seems likely that even though each region may contact the 59 enhancer individually, their mutual co- operation is required to establish a strong interaction. Mutations in RPEs ii. and iii. showed additive effects when combined with a/tif32-D8 indicating that the molecular mecha- nism of their involvement in REI differs from that of RPEs i. and iv. The model structure predicts that the RPE ii. forms a 9 nt-long stem loop whose sequence and less likely also the structure are crucial for its stimulatory activity. At present we can only speculate about the molecular nature of the roles of these two RPEs. They could either contact other eIF3 subunits or other eIFs, or act independently, for example by interacting directly with the ribosomal components. Is the critical involvement of the a/TIF32 subunit of eIF3 and the sequences upstream of a short uORF a general requirement for the efficient REI? Even though there is an increasing number of short uORFs demonstrated to permit efficient REI after their translation [12,28,37,38], perhaps none of them, besides uORF1 of GCN4 [8,10], has been studied deeply enough to draw any general conclusions regarding the molecular details of the short uORF- mediated REI mechanism. Until now, this has also applied on the only other well defined REI-permissive uORF in yeast occuring in the mRNA leader of the transcription factor YAP1 [18]. Here we showed that its 59 sequences share significant structural similarity predictions with GCN49s uORF1 and, most importantly, stimulate REI on YAP1 in a strict dependency on the NTD of a/TIF32 (Figure 6). Hence the functional if not direct interaction between the a/TIF32-NTD and the specifically folded sequences upstream of a short REI-permissive uORF represents the first generally applicable requirement of this type of a regulatory mechanism described to date, at least in yeast. Considering the remarkable similarity with the aforementioned termination/reinitiation mech- anism utilized by viruses, it is very likely that the analogous principles apply also to uORFs promoting efficient REI in higher eukaryotes. Future work exploring the mechanistic details of some of these uORFs, especially those connected with pathophysiolog- ical mechanisms, will certainly tell us more about the evolutionary conservation of this important translational control process. Materials and Methods Yeast strains, plasmids, RNA structure probing, and other biochemical methods Lists of strains (Table S1), plasmids (Table S2), and PCR primers (Table S3) used in this study and details of their construction can be found in the Text S1. Commercially synthesized 79-mer RNA (East Port) was 59-end- labeled using c-32P-ATP and T4 Polynucleotide Kinase (Fermen- tas). Free radioactive nucleotides were removed by NucAway Spin Columns (Ambion). The RNA was then subjected to limited digestion with RNase T1 (cleaves after single-stranded G residues) or RNase V1 (cleaves within double-stranded RNA). RNase T1 was used in RNA Sequencing buffer or in RNA Structure buffer to induce denaturing (denatur) or folding-promoting (fold) conditions, respectively. Alkaline hydrolysis of the RNA was used to generate appropriate reference landmarks. (All enzymes, buffers and protocols were provided by Ambion). The digested products were PLoS Genetics | www.plosgenetics.org 14 July 2011 | Volume 7 | Issue 7 | e1002137