Translation Reinitiation Relies on the Interaction between eIF3a/TIF32 and Progressively Folded cis-Acting mRNA Elements Preceding Short uORFs

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.

Eukaryote-specific extensions in ribosomal proteins of the small subunit: Structure and function

“…[52][53][54] Further, eIF3g was found to interact with uS3 and uS10 located in the 40S subunit beak and head, respectively. 55 RpuS10 harbors an eukaryote-specific Nterminal extension that occupies part of the head region where eIF3g is believed to reside.…”

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…This aberrant termination/ribosome-release cycle permits reinitiation to occur at a subsequent downstream AUG codon. It is likely that only the 40S ribosome remains attached to the mRNA following termination at uORF1 and that the 40S retains some bound translation factors, including eIF3 (Szamecz et al 2008;Munzarova et al 2011;Peter et al 2015). The 40S lacks eIF2, as this factor was released at AUG recognition ( Figure 9B, step iia) and prior to 60S joining ( Figure 9B, step iib).…”

“…The 59 reinitiation enhancer region (59 RER) was shown to interact with eIF3a bound to the 40S (Szamecz et al 2008). The 59 RER contains four distinct elements, two of which are important for eIF3a binding (Munzarova et al 2011). Based on its ribosome-binding properties, eIF3a bridges the mRNA interaction to the 40S protein Rps0A located near the 40S mRNA exit channel (Szamecz et al 2008).…”

In-depth analysis of cis-determinants that either promote or inhibit reinitiation on GCN4 mRNA after translation of its four short uORFs

“…This repressive effect of uORFs can be, however, alleviated under specific conditions, such as various types of stress, in order to boost expression of some regulatory uORFcontaining mRNAs that help the cell to cope with sudden environmental changes. It has been shown that the efficiency of REI depends on four main factors: (i) time required for uORF translation, which is determined by the relative length of uORF and the translation elongation rate; (ii) its 5 ′ and 3 ′ flanking sequences, which contain specific cis-acting features with poorly understood molecular roles; (iii) translation initiation factors (eIFs) involved in the primary initiation event such as the eIF3 and eIF4F complexes, which are believed to remain associated with the ribosome throughout the short elongation as well as termination and recycling phases; and (iv) its distance to the next open reading frame, which determines the likelihood of acquisition of the new TC by the posttermination 40S ribosome that has resumed scanning (Kozak 1987;Dever et al 1992;Pöyry et al 2004;Szamecz et al 2008;Cuchalová et al 2010;Roy et al 2010;Munzarová et al 2011). …”