In vivoevidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation

Abstract

Translation reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream ORFs to prevent recycling of the post-termination 40S subunit in order to resume scanning for reinitiation downstream. Its efficiency decreases with the increasing uORF length, or by the presence of secondary structures, suggesting that the time taken to translate a uORF is more critical than its length. This led to a hypothesis that some initiation factors needed for reinitiation are preserved on the 80S ribosome during early elongation. Here, using the GCN4 mRNA containing four short uORFs, we developed a novel in vivo RNA–protein Ni2+-pull down assay to demonstrate for the first time that one of these initiation factors is eIF3. eIF3 but not eIF2 preferentially associates with RNA segments encompassing two GCN4 reinitiation-permissive uORFs, uORF1 and uORF2, containing cis-acting 5΄ reinitiation-promoting elements (RPEs). We show that the preferred association of eIF3 with these uORFs is dependent on intact RPEs and the eIF3a/TIF32 subunit and sharply declines with the extended length of uORFs. Our data thus imply that eIF3 travels with early elongating ribosomes and that the RPEs interact with eIF3 in order to stabilize the mRNA-eIF3-40S post-termination complex to stimulate efficient reinitiation downstream.

A Unique ISR Program Determines Cellular Responses to Chronic Stress

“…There are several examples of a highly specific role of the multisubunit eIF3 complex in mRNA recruitment to the 40S subunits, often occurring under various stresses (Mohammad et al, 2017). Almost 500 mRNAs with supposedly related structural elements in their 5’ UTR were PAR-CLIPed to eIF3a, b, d and g subunits in human 293T cells and proposed to be regulated in an eIF3-specific, cap-dependent manner (Lee et al, 2015).…”

Crystal Structure of the Human Ribosome in Complex with DENR-MCT-1

“…Though the mechanism of translation reinitiation is still an open question, experimental data suggest that reinitiation-competent 40S subunits are those that remain bound to mRNA after termination and to which some eIFs (eIF4F, eIF3) remain bound even after completing translation of the first uORF. According to recent data, among the initiation factors that promote reinitiation (eIF1, eIF1A, eIF2, eIF3), eIF3 plays a key role in the retention of mRNA on the post-TC 40S subunit and even alone is able to mediate reinitiation on some mRNAs (Jackson et al, 2012; Kozak, 1987; Mohammad et al, 2017; Poyry et al, 2004; Skabkin et al, 2013). In the case of bicistronic calicivirus mRNAs, a cis -acting mRNA element TURBS (“termination upstream ribosomal binding site”), located upstream of the restart AUG, interacts with the helix h26 of the 18S rRNA (Zinoviev et al, 2015).…”

Recent Discoveries on the Role of TOR (Target of Rapamycin) Signaling in Translation in Plants

“…Where the 59-UTR of mRNA contains a short uORF (typically , 30-50 codons), a fraction of ribosomes (usually , 50%) can resume scanning after translation of this uORF and reinitiate at a further downstream ORF (Kozak, 2001). According to the current model, the short translation elongation event leaves eIFs that have been recruited during the cap-dependent initiation event bound to the 80S ribosome, making the ribosome competent for reinitiation (Kozak, 1987;Mohammad et al, 2017). The temporary retention of these reinitiation-promoting factors (RPFs) can assist the terminating ribosome to resume scanning, rapidly reacquire the ternary complex, eIF2-GTP-MettRNA, and the 60S ribosomal subunit de novo.…”