Guanine-rich nucleic acid sequences can adopt noncanonical four-stranded secondary structures called guanine (G)-quadruplexes1. Bioinformatics analysis suggests that G-quadruplex motifs are prevalent in genomes2, which raises the need to elucidate their function. There is now evidence for the existence of DNA G-quadruplexes at telomeres with associated biological function3. A recent hypothesis supports the notion that gene promoter elements contain DNA G-quadruplex motifs that control gene expression at the transcriptional level4. 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 Gquadruplex. 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.The existence of RNA G-quadruplexes in vivo is more inevitable than the existence of DNA G-quadruplexes, given that (i) the former are generally more thermodynamically stable in the folded form than their DNA counterparts5, and (ii) RNA is single-stranded, which implies that quadruplex formation does not have to compete with hybridization to a complementary strand. In this study we have focused on the 5′ UTRs of mRNA, which are known to be involved in translational regulation, particularly for growth factors, transcription factors and oncoproteins6. The neuroblastoma RAS viral oncogene homolog (NRAS)-encoded protein p21 mediates both signal transduction across the plasma membrane and the intracellular signaling pathways responsible for cell proliferation and differentiation7. Activating mutations in the coding region of NRAS are responsible for increased cell proliferation8. The suppression of oncogenic NRAS by small interfering RNA causes apoptosis of tumor cells9, which suggests that inhibiting the expression of oncogenic NRAS is a potential therapeutic strategy. Using a computational search algorithm we developed for locating quadruplex sequence motifs2, we identified a putative G-quadruplex Fig. 1 online). This motif is highly conserved, in both its sequence and its position relative to the translation start site, across the 5′ UTRs of human, chimpanzee, macaque, mouse, rat and dog genes orthologous to NRAS (Table 1 and Supplementary Table 1 online).To confirm that the putative RNA G-quadruplex NRQ folds into a stable quadruplex, we carried out biophysical experiments on the synthetic oligonucleotide 5′-UGUGGGAGGGGCGGGUCUGGG-3′. Circular dichroism (CD) spectroscopy has been widely used to characterize the structure of folded nucleic acid quadruplexes11. At pH 7.4, 100 mM KCl, the CD spectrum of NRQ showed a positive peak at 263 nm and a negative peak at 241 nm ( Fig. 1a), which is the ch...

RNA structures in the untranslated regions (UTRs) of mRNAs influence post-transcriptional regulation of gene expression. Much of the knowledge in this area depends on canonical double-stranded RNA elements. There has been considerable recent advancement of our understanding of guanine(G)-rich nucleic acids sequences that form four-stranded structures, called G-quadruplexes. While much of the research has been focused on DNA G-quadruplexes, there has recently been a rapid emergence of interest in RNA G-quadruplexes, particularly in the 5′-UTRs of mRNAs. Collectively, these studies suggest that RNA G-quadruplexes exist in the 5′-UTRs of many genes, including genes of clinical interest, and that such structural elements can influence translation. This review features the progresses in the study of 5′-UTR RNA G-quadruplex-mediated translational control. It covers computational analysis, cell-free, cell-based and chemical biology studies that have sought to elucidate the roles of RNA G-quadruplexes in both cap-dependent and -independent regulation of mRNA translation. We also discuss protein trans-acting factors that have been implicated and the evidence that such RNA motifs have potential as small molecule target. Finally, we close the review with a perspective on the future challenges in the field of 5′-UTR RNA G-quadruplex-mediated translation regulation.

Molecular mechanisms that regulate gene expression can occur either before or after transcription. The information for post-transcriptional regulation can lie within the sequence or structure of the RNA transcript and it has been proposed that G-quadruplex nucleic acid sequence motifs may regulate translation as well as transcription. Here, we have explored the incidence of G-quadruplex motifs in and around the untranslated regions (UTRs) of mRNA. We observed a significant strand asymmetry, consistent with a general depletion of G-quadruplex-forming RNA. We also observed a positional bias in two distinct regions, each suggestive of a specific function. We observed an excess of G-quadruplex motifs towards the 5′-ends of 5′-UTRs, supportive of a hypothesis linking 5′-UTR RNA G-quadruplexes to translational control. We then analysed the vicinity of 3′-UTRs and observed an over-representation of G-quadruplex motifs immediately after the 3′-end of genes, especially in those cases where another gene is in close proximity, suggesting that G-quadruplexes may be involved in the termination of gene transcription.

G-Rich sequences found within biologically important regions of the genome have been shown to form intramolecular G-quadruplexes with varied loop lengths and sequences. Many of these quadruplexes will be distinguishable from each other on the basis of their thermodynamic stabilities and folded conformations. It has been proposed that loop lengths can strongly influence the topology and stability of intramolecular G-quadruplexes. Previous studies have been limited to the analysis of quadruplex sequences with particular loop sequences, making it difficult to make generalizations. Here, we describe an original study that aimed to elucidate the effect of loop length on the biophysical properties of G-quadruplexes in a sequence-independent context. We employed UV melting and circular dichroism spectroscopy to examine and compare the properties of 21 DNA quadruplex libraries, each comprising partially randomized loop sequences with lengths ranging from one to three nucleotides. Our work supports a number of general predictions that can be made solely on the basis of loop lengths. In particular, the results emphasize the strong influence of single-nucleotide loops on quadruplex properties. This study provides a predictive framework that may help identify or classify biologically relevant G-quadruplex-forming sequences.