Vala´ sˇek1* Abstract Translation Reinitiation Relies on the Interaction between eIF3a/TIF32 and Progressively Folded cis -Acting mRNA Elements Preceding Short uORFs Vanda Munzarova´ 1, Josef Pa´ nek2, Stanislava Guni sˇova´ 1, Istva´ n Da´ nyi1, Be´ la Szamecz1, Leosˇ Shivaya 1 Laboratory of Regulation of Gene Expression, Institute of Microbiology AS CR, Prague, Czech Republic, 2 Laboratory of Bioinformatics, Institute of Microbiology AS CR, Prague, Czech Republic Reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream uORFs to retain post-termination 40S subunits on mRNA. Its efficiency depends on surrounding cis-acting sequences, uORF elongation rates, various initiation factors, and the intercistronic distance. To unravel effects of cis-acting sequences, we investigated previously unconsidered structural properties of one such a cis-enhancer in the mRNA leader of GCN4 using yeast genetics and biochemistry. This leader contains four uORFs but only uORF1, flanked by two transferrable 59 and 39 cis- acting sequences, and allows efficient reinitiation. Recently we showed that the 59 cis-acting sequences stimulate reinitiation by interacting with the N-terminal domain (NTD) of the eIF3a/TIF32 subunit of the initiation factor eIF3 to stabilize post-termination 40S subunits on uORF1 to resume scanning downstream. Here we identify four discernible reinitiation-promoting elements (RPEs) within the 59 sequences making up the 59 enhancer. Genetic epistasis experiments revealed that two of these RPEs operate in the eIF3a/TIF32-dependent manner. Likewise, two separate regions in the eIF3a/ TIF32-NTD were identified that stimulate reinitiation in concert with the 59 enhancer. Computational modeling supported by experimental data suggests that, in order to act, the 59 enhancer must progressively fold into a specific secondary structure while the ribosome scans through it prior uORF1 translation. Finally, we demonstrate that the 59 enhancer’s stimulatory activity is strictly dependent on and thus follows the 39 enhancer’s activity. These findings allow us to propose for the first time a model of events required for efficient post-termination resumption of scanning. Strikingly, structurally similar RPE was predicted and identified also in the 59 leader of reinitiation-permissive uORF of yeast YAP1. The fact that it likewise operates in the eIF3a/TIF32-dependent manner strongly suggests that at least in yeasts the underlying mechanism of reinitiation on short uORFs is conserved. Citation: Munzarova´ V, Pa´nek J, Gunisˇova´ S, Da´nyi I, Szamecz B, et al. (2011) Translation Reinitiation Relies on the Interaction between eIF3a/TIF32 and Progressively Folded cis-Acting mRNA Elements Preceding Short uORFs. PLoS Genet 7(7): e1002137. doi:10.1371/journal.pgen.1002137 Editor: Hideko Kaji, Thomas Jefferson University, United States of America Received February 22, 2011; Accepted May 3, 2011; Published July 7, 2011 Copyright: 2011 Munzarova´ et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported for the most part by The Wellcome Trusts Grants 076456/Z/05/Z and 090812/B/09/Z, and partly also by the Howard Hughes Medical Institute and Inst. Research Concept AV0Z50200510 to LSV and by Czech Science Foundation 303/09/0475 to JP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: valasekl@biomed.cas.cz Introduction Translation of the majority of eukaryotic mRNAs encoding almost exclusively only a single large open reading frame (ORF) is initiated by the canonical mechanism involving formation of the 48S pre-initiation complex (PIC) at the mRNA’s 59 cap structure followed by scanning through the 59 untranslated region (UTR) for usually the nearest AUG start codon (reviewed in [1]). According to recent reports, however, in approximately 13% of yeast and 50% of human transcripts the main ORF is preceded by one or more short upstream ORFs (uORFs) [1,2], consisting of the AUG start codon and at least one additional coding triplet. Presence of a short uORF in mRNA’s 59 UTR generally leads to significant reduction in expression of a main ORF [2], the degree of which depends on the ‘‘strength’’ of the nucleotide context surrounding the uORF’s initiating AUG (called the Kozak consensus sequence) [3]. Short uORFs with a relatively poor initiation context can be skipped by at least some 48S PICs via leaky scanning, which decreases their inhibitory impact. On the other hand, there is growing evidence that there are many non- AUG-initiating short uORFs that, if in a good context, may serve as very potent inhibitors [4,5]. Short uORFs may also down- regulate expression of a main ORF by their special ability to mediate ribosome stalling at coding or termination codons, or by influencing the mRNA stability through the Nonsense Mediated Decay (NMD) pathway (reviewed in [6]). On the other side of the spectrum of short regulatory uORFs are those that permit the small ribosomal subunit to stay mRNA-bound post-termination and resume scanning for efficient reinitiation (REI) downstream. It has been shown that the ability of some uORFs to retain the 40S subunit on the same mRNA molecule after it has terminated translation at the uORF’s stop codon depends on: (i) cis–acting mRNA features, (ii) the time required for the uORF translation, which is determined by the relative length of a short uORF and the translation elongation rates, and (iii) on various initiation factors (for review see [6–8]). The last two requirements are united PLoS Genetics | www.plosgenetics.org 1 July 2011 | Volume 7 | Issue 7 | e1002137