G-quadruplexes: the beginning and end of UTRs

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

Understanding the stability of DNA G-quadruplex units in long human telomeric strands

AbstractHuman telomeric DNA is composed of GGGTTA repeats. The presence of consecutive guanines makes the telomeric G-strand prone to fold into contiguous (or tandem) G-quadruplexes (G4s). The aim of this study was to provide a clarified picture of the stability of telomeric tandem G4 structures as a function of the number of G4 units and of boundary sequences, and an understanding of the diversity of their melting behaviors in terms of the single G4 units composing them. To this purpose we undertook an UV-spectroscopic investigation of the structure and stability of telomeric repeats potentially able to fold into up to four contiguous G4s, flanked or not by TTA sequences at their 5′ and 3′ extremities. We explain why the stability of (GGGTTA)4m−1GGG structures (m = 2, 3, 4 …) decreases with increasing the number m of G4 units, whereas the stability of TTA-(GGGTTA)4m−1GGG-TTA structures does not. Our results support that the inner G4 units have similar stabilities, whereas the stabilities of the terminal G4 units are modulated by their flanking nucleotides: in a TTA-(GGGTTA)4m−1GGG-TTA tandem context, the terminal G4 units are roughly as stable as the inner G4 units; while in a (GGGTTA)4m−1GGG tandem context, the G4 at the 5′ extremity is more stable than the G4 at the 3′ extremity, which in turn is more stable than an inner G4. Our study provides new information about the global and local stability of telomeric tandem G4 structures under near physiological conditions

A Sequence-Independent Study of the Influence of Short Loop Lengths on the Stability and Topology of Intramolecular DNA G-Quadruplexes†

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

Vers un processus de sélection in vitro à deux dimensions qui combine le Selex et la chimie combinatoire dynamyque (application à la sélection d'aptamères conjugués dirigés contre l'élément TAR du VIH-1)

En dépit de leurs remarquables propriétés, les aptamères ADN ou ARN identifiés par SELEX souffrent également de certaines limitations. Aussi, des méthodes ont été développées afin d'introduire des modifications chimiques pour augmenter leur potentiel. Elles consistent soit à utiliser une banque d'oligonucléotides modifiés soit à incorporer des modifications par synthèse chimique après sélection. Cependant, dans le premier cas, la variété des modifications utilisables est fortement limitée par la capacité des enzymes à incorporer des nucléotides modifiés, et, dans le deuxième cas, les modifications doivent respecter la structure de l'aptamère. Pour dépasser ces limitations et permettre l'exploration d'une plus grande diversité moléculaire, nous avons développé un processus de sélection in vitro à deux dimensions qui combine la chimie combinatoire dynamique et le SELEX. Cette technique a été appliquée à la sélection d'aptamères conjugués dirigés contre l'élément TAR du VIH.Despite their remarkable properties, DNA or RNA aptamers identified by SELEX also suffer from a number of limitations. Therefore, methods were developed to introduce chemical modifications that increase their potential. They consist in either using a library of modified oligonucleotides or incorporating modifications by chemical synthesis after selection. However, in the first case, the variety of the modifications usable is strongly limited by the capacity of the enzymes to incorporate modified nucleotides, and, in the second case, the modifications must respect the structure of the aptamer. To exceed these limitations and to allow the exploration of a greater molecular diversity, we developed double-level in vitro selection process which combines dynamic combinatorial chemistry and SELEX. This technique was applied to the selection of conjugated aptamers directed against the TAR element of the HIV.BORDEAUX2-BU Santé (330632101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

A Sequence-Independent Study of the Influence of Short Loop Lengths on the Stability and Topology of Intramolecular DNA G-Quadruplexes^†

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

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

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 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

Position and Stability Are Determining Factors for Translation Repression by an RNA G-Quadruplex-Forming Sequence within the 5′ UTR of the <i>NRAS</i> Proto-oncogene

Nucleic acid secondary structures in the 5′ untranslated regions (UTRs) of mRNAs have been shown to play a critical role in translation regulation. We recently demonstrated that a naturally occurring, conserved, and stable RNA G-quadruplex element (5′-GGGAGGGGCGGGUCUGGG-3′), located close to the 5′ cap within the 5′ UTR of the NRAS proto-oncogene mRNA, modulates gene expression at the translational level. Herein, we show that the translational effect of this G-quadruplex motif in NRAS 5′ UTR is not uniform, but rather depends on the location of the G-quadruplex-forming sequence. The RNA G-quadruplex-forming sequence represses translation when situated relatively proximal to the 5′ end, within the first 50 nt, in the 5′ UTR of the NRAS proto-oncogene, whereas it has no significant effect on translation if located comparatively away from the 5′ end. We have also demonstrated that the thermodynamic stability of the RNA G-quadruplex at its natural position within the NRAS 5′ UTR is an important factor contributing toward its ability to repress translation