Initiation context modulates autoregulation of eukaryotic translation initiation factor 1 (eIF1)

Abstract

The central feature of standard eukaryotic translation initiation is small ribosome subunit loading at the 5' cap followed by its 5' to 3' scanning for a start codon. The preferred start is an AUG codon in an optimal context. Elaborate cellular machinery exists to ensure the fidelity of start codon selection. Eukaryotic initiation factor 1 (eIF1) plays a central role in this process. Here we show that the translation of eIF1 homologs in eukaryotes from diverse taxa involves initiation from an AUG codon in a poor context. Using human eIF1 as a model, we show that this poor context is necessary for an autoregulatory negative feedback loop in which a high level of eIF1 inhibits its own translation, establishing that variability in the stringency of start codon selection is used for gene regulation in eukaryotes. We show that the stringency of start codon selection (preferential utilization of optimal start sites) is increased to a surprising degree by overexpressing eIF1. The capacity for the cellular level of eIF1 to impact initiation through the variable stringency of initiation codon selection likely has significant consequences for the proteome in eukaryotes.

Fig. 1.

Starting codon context of eIF1 genes is poor, which leads to inefficient initiation. (A) Logo representation of the initiation context of 233 metazoan eIF1 homologs. (B) Representation of the initiation context of 91 fungal eIF1 homologs. (C) Representation of the initiation context of 220 plant eIF1 homologs (for discussion of excluded plant paralogs, see legend for Fig. S3). Letter heights are proportional to the frequency of conservation. The consensus conservation is shown at the top of each logo; the position relative to the A of the AUG start codon is indicated below (crucial positions −3 and +4 are in red). Alignments of the sequences used to generate the logogram are shown in Fig. S1. (D) Relative initiation efficiency of consensus mammalian initiation context and human EIF1 initiation context (arbitrary units). The results are derived from dual luciferase transfection experiments as described in Materials and Methods.

Fig. 2.

Overexpression of eIF1 is autoregulatory. (A) Schematic of the triple transfections used in this experiment. The initiation context for each expressed exogenous mRNA is indicated. (B) Western blot of protein lysates from cells transfected with vectors expressing human eIF1. The type of initiation context of each eIF1 construct is indicated above the actual sequence. Black type: EIF1 initiated by the native initiation context; red type: EIF1 initiated by perfect Kozak consensus context; maroon type: EIF1 initiated by near-consensus context that preserves the wild-type EIF1 coding sequence. In each case, the context is bracketed upstream by an in-frame stop codon blocking potential translation initiation further 5′. In lanes marked “10×”, 10-fold more vector with insert was transfected compared with lanes marked “1×,” where the difference is made up with inert vector. Anti-β-actin antibody is used to control for loading differences. The control cells are transfected with the same amount of inert vector. (C) Ratio from dual luciferase measurements from the same cells for which Western analysis is shown in B. The firefly reporter in all cotransfections is initiated with an AUG in the native (wild-type) human EIF1 context. Renilla luciferase is initiated by an AUG in the consensus (good) context. The fold reduction of firefly reporter activity (normalized to Renilla reporter activity) relative to firefly reporter expression in the empty vector control cells is shown above each column.

Fig. 3.

eIF1 overexpression inhibits initiation on poorly recognized codons. The results are from dual luciferase measurements. Renilla luciferase is initiated by an AUG in a consensus (good) context and is used to normalize firefly luciferase values. The firefly reporter is initiated by the codon, and the context, indicated on the left. (A and B) Firefly luciferase measurements from cells cotransfected with (i) empty vector (control)—gray-blue bars; (ii) “10×” vector expressing eIF1 in its native initiation context—maroon bars; or (iii) “10×” vector expressing eIF1 in the near consensus (good*) context—yellow bars. Right-facing bars show relative expression of firefly and Renilla luciferase. Left-facing bars show the ratio of normalized firefly luciferase expression (fold repression) calculated by comparing the values of (i) cells transfected with empty vector (control) and cells transfected with vector containing eIF1 initiated with the wild-type context (green bars); or (ii) cells transfected with empty vector and cells transfected with vector containing eIF1 initiated with the good* context (orange bars). (A) Results from experiments varying the context at positions −3 and +4. Varied nucleotides are shown in green or red, depending on whether they are predicted to provide a good or a poor context for initiation, respectively. (B) Results from experiments changing the start codon to one of the nine possible near cognate non-AUG codons. The fold repression marked by “◆” is probably an underestimate because the firefly activity is close to background level even under control conditions. The results in A and B represent independent experiments accounting for the slight difference in the values from firefly luciferases starting with “best” and “eIF1” contexts.