Translational repression of the disintegrin and metalloprotease ADAM10 by a stable G-quadruplex secondary structure in its 5'-untranslated region - PubMed (original) (raw)

Translational repression of the disintegrin and metalloprotease ADAM10 by a stable G-quadruplex secondary structure in its 5'-untranslated region

Sven Lammich et al. J Biol Chem. 2011.

Abstract

Anti-amyloidogenic processing of the amyloid precursor protein APP by α-secretase prevents formation of the amyloid-β peptide, which accumulates in senile plaques of Alzheimer disease patients. α-Secretase belongs to the family of a disintegrin and metalloproteases (ADAMs), and ADAM10 is the primary candidate for this anti-amyloidogenic activity. We recently demonstrated that ADAM10 translation is repressed by its 5'-UTR and that in particular the first half of ADAM10 5'-UTR is responsible for translational repression. Here, we asked whether specific sequence motifs exist in the ADAM10 5'-UTR that are able to form complex secondary structures and thus potentially inhibit ADAM10 translation. Using circular dichroism spectroscopy, we demonstrate that a G-rich region between nucleotides 66 and 94 of the ADAM10 5'-UTR forms a highly stable, intramolecular, parallel G-quadruplex secondary structure under physiological conditions. Mutation of guanines in this sequence abrogates the formation of the G-quadruplex structure. Although the G-quadruplex structure efficiently inhibits translation of a luciferase reporter in in vitro translation assays and in living cells, inhibition of G-quadruplex formation fails to do so. Moreover, expression of ADAM10 was similarly repressed by the G-quadruplex. Mutation of the G-quadruplex motif results in a significant increase of ADAM10 levels and consequently APPsα secretion. Thus, we identified a critical RNA secondary structure within the 5'-UTR, which contributes to the translational repression of ADAM10.

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Figures

FIGURE 1.

FIGURE 1.

The 5′-UTR of ADAM10 contains a G-quadruplex motif. A, representation of the human ADAM10 5′-UTR-RNA sequence. The predicted G-quadruplex sequence located between nucleotides 66 and 94 of the ADAM10 5′-UTR is underlined. The guanines predicted to be involved in the formation of the potential G-quadruplex secondary structure are highlighted in red. B, model of the parallel ADAM10 5′-UTR G-quadruplex secondary structure. Guanines (red circles) of the canonical repeats of the G-rich stretches involved in G-quadruplex formation are located at the four edges of each plane marked in light red. C, sequences of RNA oligonucleotides used for CD spectroscopy measurements in this study. Guanines potentially involved in G-quadruplex formation are marked in red, and substitutions to adenines are highlighted in green.

FIGURE 2.

FIGURE 2.

Biophysical analysis of the ADAM10 G-quadruplex motif. A, CD spectra of 5 μ

m

ADAM10GQ-WT oligonucleotide in the absence (blue) or presence of different monovalent cations (green, LiCl; red, NaCl; black, KCl; 1 m

m

each) in 10 m

m

Tris/HCl (pH 7.4), 0.1 m

m

EDTA. Note that the formation of a stable G-quadruplex structure was strongly induced in the presence of 1 m

m

KCl. B, CD melting experiments of 5 μ

m

ADAM10GQ-WT in the presence of 1 m

m

KCl. Melting (black) and annealing (red) curves are almost identical and show a Tm of 60 ± 1 °C. In contrast, at 50 m

m

KCl (green), the folded G-quadruplex could not be unfolded at higher temperatures. C, plot of Tm values for ADAM10GQ-WT at various strand concentrations. All experiments were performed in the presence of 10 m

m

Tris/HCl (pH 7.4), 0.1 m

m

EDTA, and 1 m

m

KCl. Results are expressed as the mean ± S.D. of at least three different measurements. D, CD spectra in the presence of 1 m

m

KCl of ADAM10GQ-WT (black) and mutated variants thereof (red, ADAM10GQ-mut1; green, ADAM10GQ-mut2).

FIGURE 3.

FIGURE 3.

Translational repression of a luciferase reporter by the ADAM10 G-quadruplex motif. A, Schematic representation of the plasmids used for reporter gene assays. The wild-type G-quadruplex sequence (GQ-WT) of the ADAM10 5′-UTR or mutated variants thereof were cloned directly in front of the Renilla coding region. B, 24 h after transfection of the indicated plasmids in HEK293 cells dual-luciferase assays were performed and mRNA was isolated. Renilla luciferase activity was normalized to Firefly luciferase activity and the value for GQ-WT was set to 100%. CT values for Renilla and Firefly luciferase mRNA were determined by quantitative RT-PCR and the ratio of CT Renilla/CT Firefly was calculated as described (47). Results are expressed as means ± S.D. of at least three independent experiments made in triplicates.

FIGURE 4.

FIGURE 4.

ADAM10 G-quadruplex motif in context of entire 5′-UTR inhibits translation of firefly luciferase reporter. Equal amounts of in vitro_-transcribed firefly luciferase mRNAs with the full-length 5′-UTR of ADAM10 containing the wild-type G-quadruplex sequence (5′-UTR-GQ-WT-Luc) or the indicated mutations as depicted in Fig. 1_C (5′-UTR-GQ-mut1-Luc, 5′-UTR-GQ-mut2-Luc) were subjected to in vitro translation using nuclease-treated rabbit reticulocyte lysates. Results are expressed as means ± S.D. of three independent experiments made in triplicate.

FIGURE 5.

FIGURE 5.

ADAM10 expression is repressed by the G-quadruplex motif. A, HEK293 cells were transiently transfected with the indicated ADAM10 cDNA constructs, and lysates were analyzed by immunoblotting for V5-tagged ADAM10, endogenous APP, β-actin as loading control, and GFP as transfection control. Supernatants were analyzed for APPsα secretion using antibody 2D8. Cellular APP is present in low molecular weight immature forms (im) and high molecular weight mature form (m). ADAM10 is present as a mature (m) form and predominantly as an immature (im) form. B, quantification of ADAM10 protein (black bars) and mRNA levels (white bars) from cells transfected with ADAM10 cDNA constructs shown in A. ADAM10 protein levels were normalized to GFP and actin levels. The signal for ADAM10 with the wild-type G-quadruplex GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments made in triplicate. ADAM10 mRNA was normalized to glycerolaldehyde-3-phosphate-dehydrogenase mRNA levels, and the signal for GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments. C, quantification of secreted APPsα from cells transfected with the indicated ADAM10 variants were shown in A. The signal for APPsα from GQ-WT ADAM10 transfected cells was set to 100%. Results are expressed as the means ± S.D. from three experiments. Asterisks indicate statistical significance (one-way analysis of variance with Dunnett's post test) relative to GQ-WT ADAM10 transfected cells (*, p < 0.05; **, p < 0.01).

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