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.

Figure 1.

The mRNAs of eIF5 homologs from eukaryotes have one or more uAUGs in poor contexts preceding the AUG for the main ORF. (A) Schematic representation of the eIF5 mRNA from mammals. The position of the uAUGs and the AUG of the main ORF are indicated. The uORFs initiated by the uAUGs are shown as yellow-hued rectangles. The main ORF is shown as a blue rectangle. Representation of the 3′-end of the eIF ORF and mRNA 3′-UTR is omitted. (B) Weblogo representation of initiation contexts of the uAUGs in eIF5 mRNAs from diverse eukaryotes. Letter heights are proportional to the frequency of conservation of each nucleotide at each position. Each line represents a different eukaryotic branch. (i–iv Animalia): (i) Vertebrata; (ii) Arthropoda; (iii) Nematoda; (iv) Mollusca. (v–vii Fungi): (v) Pezizomycotina; (vi) Basidiomycota; (vii) Zygomycota, Glomeromycota, Neocallimastigomycota and Chytridiomycota. (viii) Plantae. The column on the left represents the contexts of uAUG1 which almost invariably initiates the longest uORF. The column on the right represents the contexts of all uAUGs. The number of AUGs used to generate each representation is indicated in parentheses on its right. The nucleotide position relative to the A of the AUG start codon is indicated below (crucial positions −3 and +4 are in red). The AUG is boxed. Alignments of the sequences used to generate the logogram are shown in Supplementary Figure S1.

Figure 2.

Overexpression of eIF5 is autoregulatory. (A) Schematic representation of the triple transfections used in these experiments. The firefly luciferase reporter is fused downstream of the wild-type 5′-UTR of human eIF5 mRNA. Its initiation context matches the context of the eIF5 start codon, which is near optimal (see Figure 3A for its sequence). The translation of the Renilla luciferase reporter is initiated with an AUG codon in optimal ‘Kozak’ context. The third plasmid used in the triple transfection encoded one of the four eIF5 or eIF1 expression constructs shown in (B). (B) Schematic representation of the constructs used to overexpress eIF5 or eIF1 (see text for details). (C) Western blots of protein lysates from cells transfected with the overexpression eIF5 or eIF1 constructs indicated in (B). The eIF1 overexpression construct is the same as ‘eIF1 good*’ described previously (5). In lanes marked ‘10×’, 10-fold more vector with insert was transfected compared with lanes marked ‘1×,’ where the difference in the amount of transfecting DNA is made up with the inert plasmid pcDNA3. The control cells are transfected with ‘10×’ amount of pcDNA3. The blot shown was probed with anti-eIF5 and anti-β-actin antibodies and separately with anti-eIF1 antibodies. The corresponding detected proteins are indicated by arrows. Anti-β-actin antibody is used to control for loading differences. (D) Fold repression of firefly luciferase activity in response to eIF5 or eIF1 overexpression. The ratio from dual luciferase measurements from the same cells for which western blots were performed in (C) was calculated. The firefly luciferase measurements were normalized to those from Renilla luciferase. The Renilla and firefly reporters are those illustrated in (A). The ratios in test cells were then compared to the luciferase ratio in control cells transfected with pcDNA3 and the fold-repression was calculated from this comparison. Negative ‘repression’ values indicate stimulation.

Figure 3.

eIF5 and eIF1 have opposing effects on the stringency of start codon selection. (A) Comparative effects of eIF1 and eIF5 overexpression on initiation at a reporter starting with AUG in the following different contexts: human eIF5 uAUG1, uAUG2, uAUG3 or the main eIF5 AUG. (B) The effects of eIF1 and eIF5 overexpression on AUG start codons with varied context at positions −3 and +4. All other positions between −6 and −1 contain the least favorable nucleotide U. (C) The effects of eIF1 and eIF5 overexpression on initiation at non-AUG start codons. In (B) and (C), the results are displayed in descending order with the most efficient initiation contexts (B) or codons (C) toward the top and the least efficient toward the bottom. In (A–C), ‘10×’ eIF1-overexpression, eIF5-overexpression or control vectors were co-transfected with the reporter vectors. Co-transfected Renilla luciferase was used for normalizing reporter activity; the fold-stimulation in response to overexpression of eIF1 or eIF5 was determined as in Figure 2. Negative ‘stimulation’ values indicate repression. In each case the firefly reporter was initiated by the codon and the context indicated on the left. In (B), the firefly luciferase reporters starting with contexts in which −3 is a pyrimidine and the +4 position is not G, are highlighted in yellow. In all cases, nucleotides matching the preferred initiation consensus in humans are indicated in green. Nucleotides deviating from the preferred context are in red. The fold-difference in translation of the reporter co-transfected with eIF1 compared to co-transfection with eIF5 is indicated on the right. The percentage of normalized firefly reporter activity is given relative to reporter activity from the construct whose AUG start is in optimal ‘Kozak’ context in the parentheses following this value. (D) Results from co-overexpressing eIF1 and eIF5. Schematic representations of the firefly luciferase reporter constructs used are on the left. (A–C): fold-stimulation of normalized firefly luciferase activity in cells co-transfected with (i) ‘10×’ vector expressing eIF1 in a near consensus context (eIF1 good*)—orange bars; or (ii) ‘10×’ vector expressing eIF5 in which its 5′-UTR is altered so that all uAUG codons are eliminated by substitution with AAA codons (eIF5 AAA)—blue bars. (D)—fold translation stimulation in cells co-transfected with (iii) ‘5×’ ‘eIF1 good*’ and ‘5×’ pcDNA3 vectors—orange bars; (iv) ‘5×’ ‘eIF1 good*’ and ‘5×’ ‘eIF5 AAA’ green bars; (v) ‘5×’ ‘eIF5 AAA’ and ‘5×’ pcDNA3—blue bars. The results in (A–D) each represent two independent experiments done it triplicate. Error bars or ‘±’ values represent SDs.

Figure 4.

Model for auto- and cross-regulation of eIF1 and eIF5 translation. eIF1 overexpression increases the stringency of start codon selection (upper half of the figure), resulting in reduced initiation at the mAUG of eIF1 and at the uAUGs of the eIF5. As a consequence, eIF1 translation decreases and eIF5 translation increases. eIF5 overexpression decreases the stringency of start codon selection (lower half of the figure), resulting in increased initiation at the mAUG of eIF1 and at the uAUGs of eIF5. As a consequence, eIF1 translation increases and eIF5 translation decreases.