An internal ribosomal entry site mediates redox-sensitive translation of Nrf2

Abstract

Nrf2 plays pivotal roles in coordinating the antioxidant response and maintaining redox homeostasis. Nrf2 expression is exquisitely regulated; Nrf2 expression is suppressed under unstressed conditions but strikingly induced under oxidative stress. Previous studies showed that stress-induced Nrf2 up-regulation results from both the inhibition of Nrf2 degradation and enhanced Nrf2 translation. In the present study, we elucidate the mechanism underlying translational control of Nrf2. An internal ribosomal entry site (IRES) was identified within the 5' untranslated region of human Nrf2 mRNA. The IRES(Nrf2) contains a highly conserved 18S rRNA binding site (RBS) that is required for internal initiation. This IRES(Nrf2) also contains a hairpin structured inhibitory element (IE) located upstream of the RBS. Deletion of this IE remarkably enhanced translation. Significantly, treatment of cells with hydrogen peroxide (H(2)O(2)) and phyto-oxidant sulforaphane further stimulated IRES(Nrf2)-mediated translation initiation despite the attenuation of global protein synthesis. Polyribosomal profile assay confirmed that endogenous Nrf2 mRNAs were recruited into polysomal fractions under oxidative stress conditions. Collectively, these data demonstrate that Nrf2 translation is suppressed under normal conditions and specifically enhanced upon oxidant exposure by internal initiation, and provide a mechanistic explanation for translational control of Nrf2 by oxidative stress.

Figure 1.

Schematic drawing of the structure of the 5′-UTR of human Nrf2 mRNA and plasmid constructs. Wild-type (WT) or various deleted mutants of 5′-UTR of Nrf2 mRNA were subcloned into the intercistronic region of a bicistronic vector containing a Rluc and a Fluc. 5′-UTR: 5′-untranslated region; (A)_n: poly(A) tail; IE: inhibitory element; ORF: open reading frame; RBS: ribosomal binding site; TSS: transcription start site.

Figure 2.

The 5′-UTR of hNrf2 mRNA possesses a functional IRES. (A) Bicistronic analysis of the 5′-UTR of Nrf2 mRNA. When pRluc-Nrf2-Fluc vector was expressed in HeLa, HEK and HepG2 cells, robust Fluc activities were observed, comparable in magnitude to IRES_p53 activities. The Fluc and Rluc activities of pRluc-null-Fluc were arbitrarily set as 1. In all figures, asterisks indicate statistical significance (t-test) of P < 0.05. (B) RT-PCR confirmation of the integrity of the bicistronic reporter mRNA (C) Examination of Nrf2 5′-UTR in the pGL3 basic vector rules out the possibility that Nrf2 5′-UTR possesses cryptic promoter activity. (D) Addition of a HP structure upstream of Nrf2 5′-UTR rules out the possibility that the observed Fluc activities result from ribosomal read through.

Figure 3.

The 5′-UTR of Nrf2 mRNA contains a putative 18S rRNA binding site (RBS). (A, B) Sequence homology of the Nrf2 5′-UTR within the RBS region among different species, and complementation between Nrf2 RBS1 and RBS2 and 18S rRNA. Open circles represent mismatches. (C–E) Expression of wild-type (WT) and deletion mutants in HeLa, HEK and HepG2 cells. Deletion of RBS1 (construct ΔN2) remarkably abrogated the Nrf2 IRES activity, while the deletion of RBS2 (construct ΔC) had only minimal effect on the activity of Nrf2 IRES.

Figure 4.

The 5′-UTR of Nrf2 contains an inhibitory element. (A) The mFold algorithm predicted the existence of a hairpin structure (boxed) upstream of the RBS1. The positions of RBS1 and RBS2 are indicated by green and yellow fonts on black box, respectively. (B) The IRES_Nrf2 secondary structure deduced by NMIA probing. The arrows indicate NMIA reactive sites. The RBS1 and RBS2 motifs are highlighted (C) Deletion of the IE segment (ΔN1) robustly enhanced Nrf2 IRES activity.

Figure 5.

IRES_Nrf2-dependent translation initiation is redox sensitive. (A) Two hours treatment of H₂O₂ (200 µM) and sulforaphane (SFN, 50 µM) significantly enhanced Nrf2 IRES activity. Treatment with reducing compounds glutathione (GSH, 1 mM) and N-acetyl-cysteine (NAC, 1 mM) virtually had no effect. (B) H₂O₂ and SFN treatment enhanced Nrf2 immunoreactivities and eIF-2α phosphorylation. In addition, H₂O₂ and SFN had opposite effect on eIF-4E phosphorylation. (C) Real-time PCR showed that H₂O₂ and SFN treatments failed to change the mRNA level of hNrf2. (D–F) Redox-sensitivity remained in the ΔN1 (D) and ΔC mutants (F) but completely diminished in the ΔN1 mutant (E).

Figure 6.

Polyribosomal profile assay of HEK cells treated with H₂O (control), 200 µM H₂O₂ or 50 µM SFN for 2 h. (A–C) Typical polyribome profiles after sucrose gradient ultracentrifugation monitored at 254 nm from the top (7% sucrose, left) to the bottom (47% sucrose, right) are shown. Each sucrose gradient was separated into 11 fractions as indicated. (D, E) RT-PCR data shows that Nrf2 mRNAs were recruited into polysomal fractions in response to H₂O₂ and SFN treatment as verified by real time PCR measurement (F, G). Results are representative of three independent experiments.