Abstract
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 5′ and 3′ cis-acting sequences, and allows efficient reinitiation. Recently we showed that the 5′ 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 5′ sequences making up the 5′ 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 5′ enhancer. Computational modeling supported by experimental data suggests that, in order to act, the 5′ enhancer must progressively fold into a specific secondary structure while the ribosome scans through it prior uORF1 translation. Finally, we demonstrate that the 5′ enhancer's stimulatory activity is strictly dependent on and thus follows the 3′ 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 5′ 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.
Highlights
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
Most eukaryotic mRNAs contain only a single translatable gene (ORF); in many of them this gene is preceded by a short coding sequence 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 upstream ORFs (uORFs) translation. This requires a concerted action of specific mRNA elements surrounding the uORF and selected initiation factors
Summary
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]). 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 may downregulate 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
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