Identification of a signature motif for the eIF4a3-SECIS interaction

Abstract

eIF4a3, a DEAD-box protein family member, is a component of the exon junction complex which assembles on spliced mRNAs. The protein also acts as a transcript-selective translational repressor of selenoprotein synthesis during selenium deficiency. Selenocysteine (Sec) incorporation into selenoproteins requires a Sec Insertion Sequence (SECIS) element in the 3' untranslated region. During selenium deficiency, eIF4a3 binds SECIS elements from non-essential selenoproteins, preventing Sec insertion. We identified a molecular signature for the eIF4a3-SECIS interaction using RNA gel shifts, surface plasmon resonance and enzymatic foot printing. Our results support a two-site interaction model, where eIF4a3 binds the internal and apical loops of the SECIS. Additionally, the stability of the complex requires uridine in the SECIS core. In terms of protein requirements, the two globular domains of eIF4a3, which are connected by a linker, are both critical for SECIS binding. Compared to full-length eIF4a3, the two domains in trans bind with a lower association rate but notably, the uridine is no longer important for complex stability. These results provide insight into how eIF4a3 discriminates among SECIS elements and represses translation.

Figure 1.

eIF4a3 binds to SECIS elements with a large apical loop. (A) Illustration of the structures of various type 1 and 2 SECIS elements. (B) REMSA analysis. The ³²P-labeled SelN, Dio1, SelW or SelT SECIS elements were incubated with increasing amounts of eIF4a3 as indicated. Samples were analyzed by native gel electrophoresis and autoradiography. (C) In vitro UGA-recoding assays. Luciferase reporter mRNAs containing a UGA codon in the open reading frame and the indicated SECIS element in the 3′-UTR were in vitro translated in the presence of varying amounts of eIF4a3. The luciferase results were expressed relative to reactions that were performed in the absence of eIF4a3. Statistically significant differences (P < 0.005) are denoted by an asterisk.

Figure 2.

eIF4a3 binding requires both loops of the SECIS element. (A) Schematic representation of the wild-type GPx1 SECIS, the internal loop mutant (mutant A) and the apical loop mutant (mutant B). eIF4a3–SECIS interactions were analyzed by SPR and representative sensorgrams for the wild-type GPx1 SECIS (B), mutant A (C) or mutant B (D) RNAs are shown. Varying concentrations of eIF4a3 (ranging from 3.125 to 100 nM for the wild-type GPx1, from 100 nM to 3.2 μM for mutant A and from 25 to 800 nM for mutant B) were injected. Kinetic parameters were derived as described in ‘Material and Methods’ section.

Figure 3.

Enzymatic footprinting of eIF4a3–SECIS complexes. (A) The 5′-end-labeled GPx1 SECIS RNA, alone or in complex with eIF4a3, was partially cleaved with RNase A or V1. The products were analyzed by denaturing gel electrophoresis. The U+C and G ladders are shown. Guanine positions indicated on the left correspond to the numbering in Figure 3D. Boxes indicate changes in nucleotide sensitivity in the presence of eIF4a3. Identical experiments were performed with the MsrB1 (B) and SelN SECIS RNAs (C). Bands resulted from the RNAse A and V1 cleavage in the footprinting lanes migrate slightly slower compared to the sequencing lanes (D). Summary of the RNase cleavage sites affected by eIF4a3. Protections are indicated by open circle for the RNase A and open square for the RNase V1. Nucleotides that became sensitive to RNase A cleavage in the presence of eIF4a3 are indicated by open triangle.

Figure 4.

Uridine base in the SECIS core is required for stable eIF4a3 binding. (A) A representative sensorgram for the eIF4a3–U54 → C mutant GPx1 SECIS interaction is shown. Various concentrations of eIF4a3 (from 25 to 800 nM) were passed over a sensor chip immobilized with U54 → C mutant GPx1 SECIS. (B) Recoding assays were performed as described in Figure 1C using reporter constructs containing the wild-type GPx1 SECIS and the U54 → C mutant. Statistically significant differences (P < 0.005) are represented by an asterisk.

Figure 5.

The two domains of eIF4a3 bind the GPx1 SECIS in trans. SPR analysis between the two domains of eIF4a3 in trans with the wild-type (A) and the U54 → C mutant (B) GPx1 SECIS are shown. eIF4a3 (from 25 to 800 nM) was injected to immobilized SECIS RNAs to determine the binding parameters. (C) In vitro recoding assays using reporter mRNAs containing a UGA codon in the open reading frame and a wild-type or U54 → C mutant GPx1 SECIS element in the 3′-UTR. The two domains of eIF4a3 were added to the assay in trans as indicated. Reporter mRNA harboring the wild-type PHGPx SECIS element was used as a control for selective inhibition. The luciferase results were expressed relative to reactions that were performed in the absence of eIF4a3. Statistically significant differences (P < 0.005) are denoted by an asterisk.