An RNA G-quadruplex in the 5' UTR of the NRAS proto-oncogene modulates translation

Abstract

Guanine-rich nucleic acid sequences can adopt noncanonical four-stranded secondary structures called guanine (G)-quadruplexes. Bioinformatics analysis suggests that G-quadruplex motifs are prevalent in genomes, which raises the need to elucidate their function. There is now evidence for the existence of DNA G-quadruplexes at telomeres with associated biological function. A recent hypothesis supports the notion that gene promoter elements contain DNA G-quadruplex motifs that control gene expression at the transcriptional level. We discovered a highly conserved, thermodynamically stable RNA G-quadruplex in the 5' untranslated region (UTR) of the gene transcript of the human NRAS proto-oncogene. Using a cell-free translation system coupled to a reporter gene assay, we have demonstrated that this NRAS RNA G-quadruplex modulates translation. This is the first example of translational repression by an RNA G-quadruplex. Bioinformatics analysis has revealed 2,922 other 5' UTR RNA G-quadruplex elements in the human genome. We propose that RNA G-quadruplexes in the 5' UTR modulate gene expression at the translational level.

Figure 1

Biophysical analysis of the NRQ RNA G-quadruplex. (a) CD spectrum of NRQ at 4 μM strand concentration in 10 mM Tris-HCl, pH 7.4, and 100 mM KCl, at 20 °C. (b) Melting and annealing UV profiles of NRQ at 4 μM strand concentration in 10 mM Tris-HCl, pH 7.4, and 1 mM KCl.

Figure 2

Effect of the NRAS 5′ UTR on the translational efficiency of chimeric RNA. (a) Schematic representation of mRNAs of luciferase reporter constructs: UTRQ, full-length (254 nucleotides) NRAS 5′ UTR; DelQ, in which the first 29 nucleotides of UTRQ, containing the G-quadruplex sequence, are deleted; and MutQ, full-length NRAS 5′ UTR with a GGG-to-AAA mutation (shown in blue) at nucleotides 15-17 to disrupt the RNA G-quadruplex formation. (b) Relative translation efficiency of the three constructs, as judged by quantitation of luciferase enzyme activity. Results were normalized relative to data for the UTRQ system. Error bars represent the s.d. of three independent experiments.