Folding and persistence times of intramolecular G-quadruplexes transiently embedded in a DNA duplex.

G-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA metabolic transactions. While many in vitro studies have focused on the kinetics of G4 formation within DNA single-strands, G4 are found in vivo in double-stranded DNA regions, where their formation is challenged by the complementary strand. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on G4 persistence. To address this, we designed a single-molecule assay allowing to measure G4 folding and persistence times in the presence of the complementary strand. We quantified both folding and unfolding rates of biologically relevant G4 sequences, such as the cMYC and cKIT oncogene promoters, human telomeres and an avian replication origin. We confirmed that G4s are found much more stable in tested replication origin and promoters than in human telomere repeats. In addition, we characterized how G4 dynamics was affected by G4 ligands and showed that both folding rate and persistence time increased. Our assay opens new perspectives for the measurement of G4 dynamics in double-stranded DNA mimicking a replication fork, which is important to understand their role in DNA replication and gene regulation at a mechanistic level.

Investigating the Effect of Mono- and Dimeric 360A G-Quadruplex Ligands on Telomere Stability by Single Telomere Length Analysis (STELA).

Telomeres are nucleoprotein structures that cap and protect the natural ends of chromosomes. Telomeric DNA G-rich strands can form G-quadruplex (or G4) structures. Ligands that bind to and stabilize G4 structures can lead to telomere dysfunctions by displacing shelterin proteins and/or by interfering with the replication of telomeres. We previously reported that two pyridine dicarboxamide G4 ligands, 360A and its dimeric analogue (360A)2A, were able to displace in vitro hRPA (a single-stranded DNA-binding protein of the replication machinery) from telomeric DNA by stabilizing the G4 structures. In this paper, we perform for the first time single telomere length analysis (STELA) to investigate the effect of G4 ligands on telomere length and stability. We used the unique ability of STELA to reveal the full spectrum of telomere lengths at a chromosome terminus in cancer cells treated with 360A and (360A)2A. Upon treatment with these ligands, we readily detected an increase of ultrashort telomeres, whose lengths are significantly shorter than the mean telomere length, and that could not have been detected by other methods.

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

Human 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.

An RNA hairpin to G-quadruplex conformational transition.

RNA molecules can fold into noncanonical structures such as the four-stranded structures known as G-quadruplexes. G-quadruplexes in the transcriptome have recently emerged as relevant regulatory elements of gene expression. Conformational transitions in RNA molecules offer an important way to regulate their biological functions. Here we report on the competition between a canonical hairpin structure and a G-quadruplex structure within an RNA molecule. We show that the conformational preference strongly depends on the relative amounts of mono- and divalent metal ions present in solution. In our system, the G-quadruplex, whose formation is not predicted by available predictive RNA folding programs, is the major conformer at physiologically relevant K(+) and Mg(2+) concentrations. Furthermore, we show that a synthetic small molecule can displace the structural dynamic equilibrium in favor of the hairpin conformer. This work highlights a new and important level of complexity in RNA folding that could be relevant to the biological functions and targeting of RNAs comprising G-quadruplex motifs.

5'-UTR RNA G-quadruplexes: translation regulation and targeting.

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.

A LIN28-dependent structural change in pre-let-7g directly inhibits dicer processing.

Several recent studies have provided evidence that LIN28, a cytoplasmic RNA-binding protein, inhibits the biogenesis of members of the let-7 microRNA family at the Dicer step in both mammals and Caenorhabditis elegans. However, the precise mechanism of inhibition is still poorly understood. Here we report on an in vitro study, which combined RNase footprinting, gel shift binding assays, and processing assays, to investigate the molecular basis and function of the interaction between the native let-7g precursor (pre-let-7g) and LIN28. We have mapped the structure of pre-let-7g and identified some regions of the terminal loop of pre-let-7g that physically interact with LIN28. We have also identified a conformational change upon LIN28 binding that results in the unwinding of an otherwise double-stranded region at the Dicer processing site of pre-let-7g. Furthermore, we showed that a mutant pre-let-7g that displays an open upper stem inhibited pre-let-7g Dicer processing to the same extent as LIN28. The data support a mechanism by which LIN28 can directly inhibit let-7g biogenesis at the Dicer processing step.

A sequence-independent analysis of the loop length dependence of intramolecular RNA G-quadruplex stability and topology.

G-Quadruplexes are noncanonical nucleic acid secondary structures based on guanine association that are readily adopted by G-rich RNA and DNA sequences. Naturally occurring genomic G-quadruplex-forming sequences have functional roles in biology that are mediated through structure. To appreciate how this is achieved, an understanding of the likelihood of G-quadruplex formation and the structural features of the folded species under a defined set of conditions is informative. We previously systematically investigated the thermodynamic stability and folding topology of DNA G-quadruplexes and found a strong dependence of these properties on loop length and loop arrangement [Bugaut, A., and Balasubramanian, S. (2008) Biochemistry 47, 689-697]. Here we report on a complementary analysis of RNA G-quadruplexes using UV melting and circular dichroism spectroscopy that also serves as a comparison to the equivalent DNA G-quadruplex-forming sequences. We found that the thermodynamic stability of G-quadruplex RNA can be modulated by loop length while the overall structure is largely unaffected. The systematic design of our study also revealed subtle loop length dependencies in RNA G-quadruplex structure.

Distinct functions of maternal and somatic Pat1 protein paralogs.

We previously identified Xenopus Pat1a (P100) as a member of the maternal CPEB RNP complex, whose components resemble those of P-(rocessing) bodies, and which is implicated in translational control in Xenopus oocytes. Database searches have identified Pat1a proteins in other vertebrates, as well as paralogous Pat1b proteins. Here we characterize Pat1 proteins, which have no readily discernable sequence features, in Xenopus oocytes, eggs, and early embryos and in human tissue culture cells. xPat1a and 1b have essentially mutually exclusive expression patterns in oogenesis and embryogenesis. xPat1a is degraded during meiotic maturation, via PEST-like regions, while xPat1b mRNA is translationally activated at GVBD by cytoplasmic polyadenylation. Pat1 proteins bind RNA in vitro, via a central domain, with a preference for G-rich sequences, including the NRAS 5' UTR G-quadruplex-forming sequence. When tethered to reporter mRNA, both Pat proteins repress translation in oocytes. Indeed, both epitope-tagged proteins interact with the same components of the CPEB RNP complex, including CPEB, Xp54, eIF4E1b, Rap55B, and ePAB. However, examining endogenous protein interactions, we find that in oocytes only xPat1a is a bona fide component of the CPEB RNP, and that xPat1b resides in a separate large complex. In tissue culture cells, hPat1b localizes to P-bodies, while mPat1a-GFP is either found weakly in P-bodies or disperses P-bodies in a dominant-negative fashion. Altogether we conclude that Pat1a and Pat1b proteins have distinct functions, mediated in separate complexes. Pat1a is a translational repressor in oocytes in a CPEB-containing complex, and Pat1b is a component of P-bodies in somatic cells.

The BCL-2 5' untranslated region contains an RNA G-quadruplex-forming motif that modulates protein expression.

The BCL-2 gene encodes a 25 kDa membrane protein that plays critical roles in the control of apoptosis. The regulation of BCL-2 gene expression is highly complex and occurs both transcriptionally and posttranscriptionally. In particular, the 5' upstream region of BCL-2 contains a number of elements that control its expression. We have identified a highly conserved 25-nucleotide G-rich sequence (BCL2Q), with potential to fold into a RNA G-quadruplex structure, located 42 nucleotides upstream of the translation start site of human BCL-2. In this study, we used a series of biophysical experiments to show that the BCL2Q sequence folds into a stable RNA G-quadruplex in vitro, and we conducted functional luciferase reporter-based assays, in a cell-free lysate and in three types of human cell lines, to demonstrate that the BCL2Q sequence modulates protein expression in the context of the 493-nucleotide native 5' untranslated region of BCL-2.

Small molecule-mediated inhibition of translation by targeting a native RNA G-quadruplex.

Herein, we show that a naturally occurring RNA G-quadruplex element within the 5' UTR of the human NRAS proto-oncogene is a target for a small molecule that inhibits translation in vitro. The present study provides a first demonstration that natural 5' UTR mRNA G-quadruplexes have potential as molecular targets for small molecules that modulate translation.

LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing in Caenorhabditis elegans.

The let-7 microRNA (miRNA) is an ultraconserved regulator of stem cell differentiation and developmental timing and a candidate tumor suppressor. Here we show that LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 processing in Caenorhabditis elegans. We demonstrate that lin-28 is necessary and sufficient to block let-7 activity in vivo; LIN-28 directly binds let-7 pre-miRNA to prevent Dicer processing. Moreover, we have identified a poly(U) polymerase, PUP-2, which regulates the stability of LIN-28-blockaded let-7 pre-miRNA and contributes to LIN-28-dependent regulation of let-7 during development. We show that PUP-2 and LIN-28 interact directly, and that LIN-28 stimulates uridylation of let-7 pre-miRNA by PUP-2 in vitro. Our results demonstrate that LIN-28 and let-7 form an ancient regulatory switch, conserved from nematodes to humans, and provide insight into the mechanism of LIN-28 action in vivo. Uridylation by a PUP-2 ortholog might regulate let-7 and additional miRNAs in other species. Given the roles of Lin28 and let-7 in stem cell and cancer biology, we propose that such poly(U) polymerases are potential therapeutic targets.

A G-rich sequence within the c-kit oncogene promoter forms a parallel G-quadruplex having asymmetric G-tetrad dynamics.

Guanine-rich DNA sequences with the ability to form quadruplex structures are enriched in the promoter regions of protein-coding genes, particularly those of proto-oncogenes. G-quadruplexes are structurally polymorphic and their folding topologies can depend on the sample conditions. We report here on a structural study using solution state NMR spectroscopy of a second G-quadruplex-forming motif (c-kit2) that has been recently identified in the promoter region of the c-kit oncogene. In the presence of potassium ions, c-kit2 exists as an ensemble of structures that share the same parallel-stranded propeller-type conformations. Subtle differences in structural dynamics have been identified using hydrogen-deuterium exchange experiments by NMR spectroscopy, suggesting the coexistence of at least two structurally similar but dynamically distinct substates, which undergo slow interconversion on the NMR timescale.

Aptamers targeting RNA molecules.

Oligonucleotides complementary to RNA sequences interact poorly with folded target regions. In vitro selection of oligonucleotides carried out against RNA structures have led to aptamers that frequently differ from antisense sequences, but rather take advantage of non-double-stranded peculiarities of the target. Studies along this line provide information about tertiary RNA architectures as well as their interaction with ligand of interest. We describe here a genomic SELEX approach and its application to the recognition of stem-loop structures prone to the formation of kissing complexes. We also provide technical details for running a procedure termed 2D-SELEX that takes advantage of both in vitro selection and dynamic combinatorial chemistry. This allows selecting aptamer derivatives containing modified nucleotides that cannot be incorporated by polymerases. Last we present in vitro transcription conditions under which large amounts of RNA, suitable for NMR structural studies, can be obtained. These different aspects of the SELEX technology have been applied to the trans-activating responsive element of the human immunodeficiency virus type 1, which is crucial for the transcription of the retroviral genome.

Position and stability are determining factors for translation repression by an RNA G-quadruplex-forming sequence within the 5' UTR of the NRAS 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.

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.

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)-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.

SELEX and dynamic combinatorial chemistry interplay for the selection of conjugated RNA aptamers.

SELEX (for Systematic Evolution of Ligands by Exponential enrichment) has proven to be extraordinarily powerful for the isolation of DNA or RNA aptamers that bind with high affinity and specificity to a wide range of molecular targets. However, the modest chemical functionality of nucleic acids poses some limits on the versatility of aptamers as binders and catalysts. To further improve the properties of aptamers, additional chemical diversity must be introduced. The design of chemical modifications is not a trivial task. Recently, dynamic combinatorial chemistry (DCC) has been introduced as an alternative to traditional combinatorial chemistry. DCC employs equilibrium shifting to effect molecular evolution of a dynamic combinatorial library of molecules. Herein, we describe an original process that combines DCC and SELEX for the in vitro selection of modified aptamers which are conjugated to chemically diverse small-molecules. Its successful application for the selection of small-molecule conjugated RNA aptamers that bind tightly to the transactivation-response (TAR) element of HIV-1 is presented.