Stringency of start codon selection modulates autoregulation of translation initiation factor eIF5

Abstract

An AUG in an optimal nucleotide context is the preferred translation initiation site in eukaryotic cells. Interactions among translation initiation factors, including eIF1 and eIF5, govern start codon selection. Experiments described here showed that high intracellular eIF5 levels reduced the stringency of start codon selection in human cells. In contrast, high intracellular eIF1 levels increased stringency. High levels of eIF5 induced translation of inhibitory upstream open reading frames (uORFs) in eIF5 mRNA that initiate with AUG codons in conserved poor contexts. This resulted in reduced translation from the downstream eIF5 start codon, indicating that eIF5 autoregulates its own synthesis. As with eIF1, which is also autoregulated through translation initiation, features contributing to eIF5 autoregulation show deep evolutionary conservation. The results obtained provide the basis for a model in which auto- and cross-regulation of eIF5 and eIF1 translation establish a regulatory feedback loop that would stabilize the stringency of start codon selection.

Upregulation of eIF5B controls cell-cycle arrest and specific developmental stages

“…1F), indicating tight control over eIF5B levels. Similar control is also observed with other translation initiation factors, eIF5 and eIF1, in mammalian cells (30). Limited overexpression of eIF5B in immature oocytes led to increased trans- lation (Fig.…”

Coordinated Movements of Eukaryotic Translation Initiation Factors eIF1, eIF1A, and eIF5 Trigger Phosphate Release from eIF2 in Response to Start Codon Recognition by the Ribosomal Preinitiation Complex*

“…Consistent with this observation, overexpressing eIF5 in yeast cells elevates utilization of nearcognate UUG start codons (16), whereas overexpressing eIF1 has the opposite effect and suppresses UUG initiation (6,29,30). Similarly, it was shown that overexpression of eIF5 in mammalian cells increases use of near-cognate codons and AUG codons in suboptimal sequence contexts as start sites, and this effect is suppressed by co-overexpression of eIF1, consistent with the notion that high concentrations of eIF5 reduce the fidelity of start codon recognition in vivo by promoting release of eIF1 from the PIC (31).…”

“…In this model, one of the domains of eIF5 would move into part of the binding site for eIF1 upon start codon recognition, promoting the latter factor's irreversible release from the PIC (16,22). Consistent with an important role for competition between eIF5 and eIF1 in start codon recognition, recent work has shown that overexpression of eIF5 in mammalian cells also reduces the fidelity of start codon recognition, in a manner that can be suppressed by overexpression of eIF1 (31). To further explore the molecular basis of the interaction between these two factors and establish which domain of eIF5 is responsible for antagonizing eIF1 binding to the PIC, we expressed and purified the isolated NTD and CTD of eIF5 as separate proteins.…”

Translation of the human erythropoietin transcript is regulated by an upstream open reading frame in response to hypoxia

“…In addition, in the pGL2-WT vector we mutated the context of the uAUG codon (gggAUGa→gccAUGg) to obtain the pGL2-optimal_uAUG construct ( Fig. 3A) with a uAUG sequence context shown by Kozak to yield maximum initiation frequency in higher eukaryotes (Kozak 1997;Wang 2004;Loughran et al 2011). In this case, the majority of the ′ -leader sequence encompassing its uORF (open box) with the intact initiation (uAUG) and termination (UGA) codons was cloned into the empty vector (pGL2-Luc), upstream of the firefly luciferase coding region (FLuc; gray boxes) to create the pGL2-WT construct.…”