Stabilization of the G-quadruplex at the VEGF IRES represses cap-independent translation

Abstract

The activation of translation contributes to malignant transformation and is an emerging target for cancer therapies. RNA G-quadruplex structures are general inhibitors of cap-dependent mRNA translation and were recently shown to be targeted for oncoprotein translational activation. In contrast however, the G-quadruplex within the 5'UTR of the human vascular endothelial growth factor A (VEGF) has been shown to be essential for IRES-mediated translation. Since VEGF has a pivotal role in tumor angiogenesis and is a major target of anti-tumoral therapies, we investigated the structure/function relationship of the VEGF G-quadruplex and defined whether it could have a therapeutic potential. We found that the G-quadruplex within the VEGF IRES is dispensable for cap-independent function and activation in stress conditions. However, stabilization of the VEGF G-quadruplex by increasing the G-stretches length or by replacing it with the one of NRAS results in strong inhibition of IRES-mediated translation of VEGF. We also demonstrate that G-quadruplex ligands stabilize the VEGF G-quadruplex and inhibit cap-independent translation in vitro. Importantly, the amount of human VEGF mRNA associated with polysomes decreases in the presence of a highly selective stabilizing G-quadruplex ligand, resulting in reduced VEGF protein expression. Together, our results uncover the existence of functionally silent G-quadruplex structures that are susceptible to conversion into efficient repressors of cap-independent mRNA translation. These findings have implications for the in vivo applications of G-quadruplex-targeting compounds and for anti-angiogenic therapies.

Keywords: G; G-quadruplex; IRES; UTR; VEGF; cap-independent; guanine; ligands; mRNA translation; untranslated region.

Figure 1.

Structure/function relationship of the IRES-A-associated G-quadruplex in the full length VEGF 5’ UTR. (A) Illustration of the bicistronic vectors in which the VEGF 5’ UTR (nts 1-1038) has been cloned in between the Renilla (RLuc) and the firefly (FLuc) luciferase open reading frames. Individual clones carrying the G-quadruplex forming sequence wild-type (WT) or mutated, in which the Gs at positions 774, 777, 781, and 783 (in gray) of the full length VEGF 5’ UTR (with the first nucleotide being the transcription initiation site) are changed to Us (G>U), to As (G>A), or to Cs (G>C), are depicted. CMV, cytomegalovirus promoter. (B) VEGF cap-independent activity measured by the ratio of Firefly/Renilla luciferase activities (FLuc/RLuc) was determined using HeLa cells transfected with the WT, G>U, G>A and G>C reporter plasmids depicted in A. Statistical analysis of the mutant reporter plasmids was calculated relatively to the WT reporter plasmid (Anova test with Dunnett's multiple comparison test. ***, P <0.001). (C) Western blot analysis of total and phosphorylated eIF2α (Eukaryotic Initiation Factor 2 α) in HeLa cells treated or not with DTT (upper panel). RT-PCR analysis of XBP1 (X-box binding protein 1) mRNA splicing after DTT-induced ER stress (lower pannel). US, unspliced XBP1; S, spliced XBP1. (D) Contribution of the G-quadruplex to VEGF cap-independent activation by ER stress measured by transfection of the indicated plasmids in HeLa cells treated or not with the ER stress inducer DTT, followed by measurement of the FLuc/RLuc ratio. (E) Cation-dependent pausing of reverse transcription at VEGF RNA WT or mutated (G>U, G>A or G>C). Strong pauses of reverse transcriptase are indicated by arrowheads, with their positions within the VEGF 5’ UTR. Full-length extension products are indicated by a triangle.

Figure 2.

Characterization of the G>U mutation in IRES-mediated translation. (A) Illustration of the bicistronic vectors containing the full length VEGF 5’ UTR with the G-quadruplex WT, G>U (G774,777,781,783U; in gray) and 2 additional variants of the G>U construct: the G>U AUG/AAG, containing the G777A mutation to abolish formation of an AUG initiation codon (underlined), and the G>U STOP, containing a A791U mutation to insert a stop codon (boxed), resulting in a smaller open reading frame. (B) IRES activity of the aforementioned plasmids measured after transfections in HeLa cells. Statistical analysis of the mutant reporter plasmids were calculated relatively to the WT reporter plasmid (Anova test with Dunnett's multiple comparison test. ***, P <0.001).

Figure 3.

Engineered three-G-quartets IRES-A G-quadruplex inhibits cap-independent translation. (A) Illustration of the bicistronic vectors containing only the IRES-A (nts 746-1038) with the G-quadruplex sequence either wild-type (WT) or mutated to increase the G-stretches length (G4 3p, mutations underlined) or replaced by the NRAS G-quadruplex sequence (in gray) wild-type (NRAS) or mutated (NRAS mut; mutations underlined). (B) IRES activity of the aforementioned plasmids measured after transfections in HeLa cells. Statistical analysis of the mutant reporter plasmids were calculated relatively to the WT reporter plasmid (Anova test with Dunnett's multiple comparison test. ***, P <0.001). (C) Cation-dependent pausing of reverse transcription at VEGF RNA WT, G4 3p, NRAS and NRAS mut. The strong pause at position +790 is shown.

Figure 4.

Stabilization of the G-quadruplex at VEGF IRES-A inhibits cap-independent translation in vitro. (A) Cation-dependent pausing of reverse transcription at VEGF RNA WT in the presence of increasing concentrations of the G-quadruplex ligands 360A (16 nM, 80 nM and 400 nM), Phen-DC(3) (1,6 nM, 8 nM and 40 nM), and Phen-DC(6) (1,6 nM, 8 nM and 40 nM). (B) Illustration of the in vitro transcribed, capped and polyadenylated (50 nts poly-A tail) bicistronic mRNAs containing only the IRES-A with the G-quadruplex sequence either wild-type (WT) or mutated (G>A). (C) VEGF IRES activity was determined using in vitro translation in RRL of in vitro transcribed bicistronic reporter RNAs depicted in B with increasing amounts of the ligands 360A, Phen-DC(3) and Phen-DC(6), as indicated. Statistical analysis of the G>A bicistronic mRNAs were calculated relatively to the WT bicistronic mRNAs for each G4-ligand concentration (2 tailed, Student's t-test *, P <0.05; **, P<0.01; ***, P<0.001).

Figure 5.

G-quadruplex ligands inhibit VEGF mRNA translation in living cells. (A) Representative polysome distribution profile obtained after centrifugation of cytoplasmic lysates over sucrose gradients and measurement of the UV-absorbance (254 nm). Lysates were prepared from either DMSO (Ctrl)- or Phen-DC(6)- treated HeLa cells (20μM). Fractions were collected and divided in 2 groups: non-polysome (NP, contain the ribonucleoproteins, the 40S, 60S and 80S ribosomal subunits) and polysome (P, contain mRNAs engaged in translation). (B) Quantitative RT-PCR was performed on each NP and P fractions using specific primers for TRF2 (Telomeric Repeat-binding Factor 2), VEGF and HPRT (Hypoxanthine Phosphoribosyltransferase 1) mRNAs. The mRNA levels in P out of NP in Phen-DC(6)- treated HeLa cells were plotted relatively to DMSO (Ctrl)- treated HeLa cells +/- SEM of two independent experiments. (C) In cellulo VEGF IRES activity was determined by transfecting HeLa cells with in vitro transcribed, capped and polyadenylated bicistronic mRNAs containing the IRES-A WT or mutated (G>A) (depicted in Fig. 4B) followed by treatment with Phen-DC(6). Statistical analysis of the G>A bicistronic mRNAs were calculated relatively to the WT bicistronic mRNAs for each Phen-DC(6) concentration (2 tailed, Student's t-test *, P <0.05; **, P<0.01). (D) Quantification of VEGF protein expression after treatment of HeLa cells with Phen-DC(6) (20μM) by using VEGF ELISA. Statistical analysis of the Phen-DC(6)-treated condition was calculated relatively to the DMSO treated condition (2 tailed, Student's t-test **, P<0.01).

Figure 6.

Model for the role of the IRES-A G-quadruplex in VEGF expression. The translationally silent G-quadruplex at the VEGF IRES-A can be converted into an efficient repressor of cap-independent translation by increasing the stability of the structure either by increasing the number of G-quartets or by adding a G-quadruplex ligand (depicted as a star).