RNA G-quadruplex Structures Research Articles

Identification of G-quadruplexes in long functional RNAs using 7-deazaguanine RNA.

RNA G-quadruplex (G4) structures are thought to affect biological processes, including translation and pre-mRNA splicing, but it is not possible at present to demonstrate that they form naturally at specific sequences within long functional RNA molecules. We have developed a novel strategy, footprinting of long 7-deazaguanine substituted RNAs (FOLDeR), that allows the formation of G4s to be confirmed in long RNAs and in functional conditions.

Visualization of NRAS RNA G-Quadruplex Structures in Cells with an Engineered Fluorogenic Hybridization Probe.

The RNA G-quadruplex is an important secondary structure formed by guanine-rich RNA sequences. However, its folding studies have mainly been studied in vitro. Accurate identification of RNA G-quadruplex formation within a sequence of interest remains difficult in cells. Herein, and based on the guanine-rich sequence in the 5'-UTR of NRAS mRNA, we designed and synthesized the first G-quadruplex-triggered fluorogenic hybridization (GTFH) probe, ISCH-nras1, for the unique visualization of the G-quadruplexes that form in this region. ISCH-nras1 is made up of two parts: The first is a fluorescent light-up moiety specific to G-quadruplex structures, and the second is a DNA molecule that can hybridize with a sequence that is adjacent to the guanine-rich sequence in the NRAS mRNA 5'-UTR. Further evaluation studies indicated that ISCH-nras1 could directly and precisely detect the targeted NRAS RNA G-quadruplex structures, both in vitro and in cells. Thus, this GTFH probe was a useful tool for directly investigating the folding of G-quadruplex structures within an RNA of interest and represents a new direction for the design of smart RNA G-quadruplex probes.

A Small Molecule That Represses Translation of G-Quadruplex-Containing mRNA.

The G-quadruplexes form highly stable nucleic acid structures, which are implicated in various biological processes in both DNA and RNA. Although DNA G-quadruplexes have been studied in great detail, biological roles of RNA G-quadruplexes have received less attention. Here, a screening of a chemical library permitted identification of a small-molecule tool that binds selectively to RNA G-quadruplex structures. The polyaromatic molecule, RGB-1, stabilizes RNA G-quadruplex, but not DNA versions or other RNA structures. RGB-1 intensified the G-quadruplex-mediated inhibition of RNA translation in mammalian cells, decreased expression of the NRAS proto-oncogene in breast cancer cells, and permitted identification of a novel sequence that forms G-quadruplex in NRAS mRNA. RGB-1 may serve as a unique tool for understanding cellular roles of RNA G-quadruplex structures.

Thioflavin T as an efficient fluorescence sensor for selective recognition of RNA G-quadruplexes.

RNA G-quadruplexes (G4s) play important roles in translational regulation, mRNA processing events and gene expression. Therefore, a fluorescent probe that is capable of efficiently recognizing RNA G-quadruplex structures among other RNA forms is highly desirable. In this study, a water-soluble fluorogenic dye (i.e., Thioflavin T (ThT)) was employed to recognize RNA G-quadruplex structures using UV–Vis absorption spectra, fluorescence spectra and emission lifetime experiments. By stacking on the G-tetrad, the ThT probe exhibited highly specific recognition of RNA G-quadruplex structures with striking fluorescence enhancement compared with other RNA forms. The specific binding demonstrates that ThT is an efficient fluorescence sensor that can distinguish G4 and non-G4 RNA structures.

Abstract B23: The 5 UTR of many oncogenes and transcription factors encodes a targetable dependence on the eIF4A RNA helicase

Abstract We report a mechanism of translational control that is determined by a requirement for eIF4A RNA helicase activity and underlies the anticancer effects of Silvestrol and related compounds. Briefly, activation of cap-dependent translation contributes to T-cell leukemia (T-ALL) development and maintenance. Accordingly, inhibition of translation initiation factor eIF4A with Silvestrol produces powerful therapeutic effects against T-ALL in vivo. We used transcriptome-scale ribosome footprinting on Silvestrol-treated T-ALL cells to identify Silvestrol-sensitive transcripts and the hallmark features of eIF4A-dependent translation. These include a long 5 UTR and a 12-mer sequence motif that encodes a guanine quartet (CGG)4. RNA folding algorithms as well as experimental evidences pinpoint the (CGG)4 motif as a common site of RNA G-quadruplex structures within the 5 UTR. In T-ALL these structures mark approximately eighty highly Silvestrol-sensitive transcripts that include key oncogenes and transcription factors and contribute to the drug's anti-leukemic action. Hence, the eIF4A-dependent translation of G-quadruplex containing transcripts emerges as a gene-specific and therapeutically targetable mechanism of translational control. Citation Format: Kamini Singh, Andrew L. Wolfe, Yi Zhong, Gunnar Rätsch, Hans-Guido Wendel. The 5 UTR of many oncogenes and transcription factors encodes a targetable dependence on the eIF4A RNA helicase. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B23.

RNA G-Quadruplex: The New Potential Targets for Therapy

Roles of RNA on transcriptional and post-transcriptional regulations have raised more attention since the new era of genomics. One of the secondary structures of RNA, the RNA Gquadruplex structure, is demonstrated a relatively stable existence in human tumor cells, virus, or other species relating to diseases. G-quadruplexes are special secondary structures formed by G-rich DNA and RNA sequences that fold into a four-stranded conformation. The G-quadruplexes formed in RNA are involved in many biological process including telomere elongation, transcription regulate, pre-mRNA splicing and translation. In this review, we will give a brief introduction to the structures of RNA G-quadruplexes, the biological roles and the potential to be as drug targets.

Human telomeric RNA G-quadruplex response to point mutation in the G-quartets.

Many putative G-quadruplex forming sequences have been predicted to exist in the human genome and transcriptome. As these sequences are subject to point mutations or SNPs (single nucleotide polymorphisms) during the course of evolution, we attempt to understand impact of these mutations in context of RNA G-quadruplex formation using human telomeric RNA (TERRA) as a model sequence. Our studies suggest that G-quadruplex stability is sensitive to substitution of the guanines comprising G-quartets. While central G-quartet plays a crucial role in maintaining the DNA G-quadruplex stability as evident in literature, there is equal importance of three G-quartets in the stability of RNA quadruplex structure. The work here highlights the alterations in the G-quartet are detrimental to the integrity of overall RNA G-quadruplex structure. Furthermore, TmPyP4 molecules are shown to exhibit similar binding behavior toward telomeric RNA G-quadruplex harboring base substitutions employing CD titrations and isothermal titration calorimetry; well indicating that mutation does not influence TmPyP4 recognition ability as it affects the stability of RNA G-quadruplex. Thus, our study implicates that mutation in G-quartets causes destabilization of RNA G-quadruplex without affecting its trans factor binding ability.

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

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.

Investigation of the Role Played by the RNA G-Quadruplex Structure in ALS/FTD Pathology

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in motor neuron loss in brain and spinal cord. Frontotemporal dementia (FTD) is one of the most common forms of young onset dementia and second most common form of dementia overall, after Alzheimer's, resulting in degeneration of temporal lobes along with personality changes and language impairment. ALS and FTD are now recognized as members of a broad continuum of neurodegenerative disorders, linked by similar pathology, mechanisms, and overlapping clinical symptoms. Two RNA-binding proteins of interest that link the two diseases are TAR DNA-binding protein 43 (TDP-43) and the fused in sarcoma/translocated in liposarcoma protein (FUS), which are the major protein components in over 90% of ALS and over 50% of FTD inclusions. We hypothesize that the G-quadruplex RNA structure might play an essential role in the pathogenic mechanisms of FUS in ALS and FTD. In this study, the G-quadruplex RNA binding properties of the wild type and C-terminal NLS mutant FUS protein implicated in ALS/FTD will be analyzed.

Rationally induced RNA:DNA G-quadruplex structures elicit an anticancer effect by inhibiting endogenous eIF-4E expression.

RNA G-quadruplex (GQ) structures act as regulators of a diverse array of cellular processes including translation, pre-mRNA processing, and mRNA targeting. We report here a strategy of harnessing the natural ability of RNA GQs to inhibit translation by rationally inducing a GQ on a targeted mRNA to knockdown endogenous gene expression. We chose to target eIF-4E because of its key role in translation initiation and overexpression in multiple cancers and with the expectation that downregulation of eIF-4E would result in antiproliferation of cancer cells. Targeted hybrid (RNA:DNA) GQ structures were induced at the 5'-untranslated region (UTR) and the protein coding region of the eIF-4E mRNA by rationally designed and partially modified extraneous DNA sequences and their effect on eIF-4E expression was determined. The formation of a stable induced G-quadruplex was established by biophysical and biochemical methods. Thermodynamic parameters calculated from CD melting indicate formation of a stable induced GQ at a physiologically relevant salt concentration. We established the specificity and efficacy of the induced GQ formation by monitoring the targeted repression of a reporter gene. Most importantly we have demonstrated that inducing GQ in the 5'-UTR and the protein coding region of eIF-4E mRNA in human cancer cells results in 30% and 60% inhibition of the endogenous protein expression, respectively. Treating with the GQ inducing oligonucleotide sequences resulted in a decrease in the viability of human cancer cells in a dose-dependent manner. The above concept opens up a new strategy for targeted modulation of endogenous gene expression.

RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer.

The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A/DDX2 RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of Silvestrol and related compounds. For example, eIF4A promotes T-ALL development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with Silvestrol has powerful therapeutic effects in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5′UTR sequences such as the 12-mer guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and Silvestrol-sensitive transcripts are a number of oncogenes, super-enhancer associated transcription factors, and epigenetic regulators. Hence, the 5′UTRs of selected cancer genes harbour a targetable requirement for the eIF4A RNA helicase.

Finding a human telomere DNA–RNA hybrid G-quadruplex formed by human telomeric 6-mer RNA and 16-mer DNA using click chemistry: A protective structure for telomere end

Telomeric repeat-containing RNA is a non-coding RNA molecule newly found in mammalian cells. The telomere RNA has been found to localize to the telomere DNA, but how the newly discovered RNA molecule interacts with telomere DNA is less known. In this study, using the click chemistry we successfully found that a 6-mer human telomere RNA and 16-mer human telomere DNA sequence can form a DNA–RNA hybrid type G-quadruplex structure. Detection of the click-reaction products directly probes DNA–RNA G-quadruplex structures in a complicated solution, whereas traditional methods such as NMR and crystallography may not be suitable. Importantly, we found that formation of DNA–RNA G-quadruplex induced an exonuclease resistance for telomere DNA, indicating that such structures might be important for protecting telomeric DNA from enzyme digestion to avoid telomere DNA shortening. These results provide the direct evidence for formation of DNA–RNA hybrid G-quadruplex structure by human telomere DNA and RNA sequence, suggesting DNA–RNA hybrid G-quadruplex structure associated between telomere DNA and RNA may respond to chromosome end protection and/or present a valuable target for drug design.

TMPyP4 Porphyrin Distorts RNA G-quadruplex Structures of the Disease-associated r(GGGGCC)n Repeat of the C9orf72 Gene and Blocks Interaction of RNA-binding Proteins

Certain DNA and RNA sequences can form G-quadruplexes, which can affect genetic instability, promoter activity, RNA splicing, RNA stability, and neurite mRNA localization. Amyotrophic lateral sclerosis and frontotemporal dementia can be caused by expansion of a (GGGGCC)n repeat in the C9orf72 gene. Mutant r(GGGGCC)n- and r(GGCCCC)n-containing transcripts aggregate in nuclear foci, possibly sequestering repeat-binding proteins such as ASF/SF2 and hnRNPA1, suggesting a toxic RNA pathogenesis, as occurs in myotonic dystrophy. Furthermore, the C9orf72 repeat RNA was recently demonstrated to undergo the noncanonical repeat-associated non-AUG translation (RAN translation) into pathologic dipeptide repeats in patient brains, a process that is thought to depend upon RNA structure. We previously demonstrated that the r(GGGGCC)n RNA forms repeat tract length-dependent G-quadruplex structures that bind the ASF/SF2 protein. Here we show that the cationic porphyrin (5,10,15,20-tetra(N-methyl-4-pyridyl) porphyrin (TMPyP4)), which can bind some G-quadruplex-forming sequences, can bind and distort the G-quadruplex formed by r(GGGGCC)8, and this ablates the interaction of either hnRNPA1 or ASF/SF2 with the repeat. These findings provide proof of concept that nucleic acid binding small molecules, such as TMPyP4, can distort the secondary structure of the C9orf72 repeat, which may beneficially disrupt protein interactions, which may ablate either protein sequestration and/or RAN translation into potentially toxic dipeptides. Disruption of secondary structure formation of the C9orf72 RNA repeats may be a viable therapeutic avenue, as well as a means to test the role of RNA structure upon RAN translation.

Visualization and selective chemical targeting of RNA G-quadruplex structures in the cytoplasm of human cells

Following extensive evidence for the formation of four-stranded DNA G-quadruplex structures in vitro, DNA G-quadruplexes have been observed within human cells. Although chemically distinct, RNA can also fold in vitro into G-quadruplex structures that are highly stable because of the 2'-hydroxyl group. However, RNA G-quadruplexes have not yet been reported in cells. Here, we demonstrate the visualization of RNA G-quadruplex structures within the cytoplasm of human cells using a G-quadruplex structure-specific antibody. We also demonstrate that small molecules that bind to G-quadruplexes in vitro can trap endogenous RNA G-quadruplexes when applied to cells. Furthermore, a small molecule that exhibits a preference for RNA G-quadruplexes rather than DNA G-quadruplexes in biophysical experiments also shows the same selectivity within a cellular context. Our findings provide substantive evidence for RNA G-quadruplex formation in the human transcriptome, and corroborate the selectivity and application of stabilizing ligands that target G-quadruplexes within a cellular context.

Unusual −1 Ribosomal Frameshift Caused by Stable RNA G-Quadruplex in Open Reading Frame

Tertiary structures formed by mRNAs impact the efficiency of the translation reaction. Ribosomal frameshift is a well-characterized recoding process that occurs during translation elongation. Pseudoknot and stem-loop structures may stimulate frameshifting by causing a translational halt at a slippery sequence. In this study, we evaluated the efficiency of an unusual -1 frameshift caused by a noncanonical RNA G-quadruplex structure in mammalian cells. The reporter gene construct consisting of a fluorescent protein and Luciferase enabled evaluation of apparent and absolute values of the -1 frameshift efficiency and revealed significant increase of the efficiency by G-quadrupex forming potential sequence. In addition, berberine, a small molecule that binds to and stabilizes G-quadruplex structures, further increased the frameshift efficiency. These results indicate that the stable G-quadruplex structure stimulates the unusual -1 frameshift and has a potential to regulate the frameshift with its ligand.

Stimulation of ribosomal frameshifting by RNA G-quadruplex structures

Guanine-rich sequences can fold into four-stranded structures of stacked guanine-tetrads, so-called G-quadruplexes (G4). These unique motifs have been extensively studied on the DNA level; however, exploration of the biological roles of G4s at the RNA level is just emerging. Here we show that G4 RNA when introduced within coding regions are capable of stimulating −1 ribosomal frameshifting (−1 FS) in vitro and in cultured cells. Systematic manipulation of the loop length between each G-tract revealed that the −1 FS efficiency positively correlates with G4 stability. Addition of a G4-stabilizing ligand, PhenDC3, resulted in higher −1 FS. Further, we demonstrated that the G4s can stimulate +1 FS and stop codon readthrough as well. These results suggest a potentially novel translational gene regulation mechanism mediated by G4 RNA.

The Disease-associated r(GGGGCC)n Repeat from the C9orf72 Gene Forms Tract Length-dependent Uni- and Multimolecular RNA G-quadruplex Structures

Certain DNA and RNA sequences can form G-quadruplexes, which can affect promoter activity, genetic instability, RNA splicing, translation, and neurite mRNA localization. Amyotrophic lateral sclerosis and frontotemporal dementia were recently shown to be caused by expansion of a (GGGGCC)n·(GGCCCC)n repeat in the C9orf72 gene. Mutant r(GGGGCC)n-containing transcripts aggregate in nuclear foci possibly sequestering repeat-binding proteins, suggesting a toxic RNA pathogenesis. We demonstrate that the r(GGGGCC)n RNA but not the C-rich r(GGCCCC)n RNA forms extremely stable uni- and multimolecular parallel G-quadruplex structures (up to 95 °C). Multimolecular G-quadruplex formation is influenced by repeat number and RNA concentration. MBNL1, a splicing factor that is sequestered in myotonic dystrophy patients by binding to expanded r(CUG)n repeat hairpins, does not bind the C9orf72 repeats, but the splicing factor ASF/SF2 can bind the r(GGGGCC)n repeat. Because multimolecular G-quadruplexes are enhanced by repeat length, RNA-RNA interactions facilitated by G-quadruplex formation at expanded repeats might influence transcript aggregation and foci formation in amyotrophic lateral sclerosis-frontotemporal dementia cells. Tract length-dependent G-quadruplex formation by the C9orf72 RNA should be considered when assessing the role of this repeat in C9orf72 gene activity, protein binding, transcript foci formation, and translation of the C9orf72 product, including the noncanonical repeat-associated non-ATG translation (RAN translation) into pathologic dipeptide repeats, as well as any oligonucleotide repeat-based therapy.

Thermal stabilisation of RNA·RNA duplexes and G-quadruplexes by phosphorothiolate linkages

The effect of 3'-S-phosphorothiolate linkages on the stability of RNA·RNA duplexes and G-quadruplex structures has been studied. 3'-Thio-2'-deoxyuridine was incorporated into RNA duplexes and thermal melting studies revealed that the resulting 3'-S-phosphorothiolate linkages increased the stability of the duplex to thermal denaturation. Additionally, and contrary to expectation, a similar effect on duplex stability was observed when the same thionucleoside was incorporated into the RNA strand of a RNA·DNA duplex. A suitably protected derivative of 3'-thio-2'-deoxyguanosine was prepared using an oxidation-reduction strategy and this residue also increased the thermal stability the [d(TGGGGT)](4) G-quadruplex when positioned centrally. The results are discussed in terms of the influence that the sulfur atom has on the conformation of the furanose ring and imply that the previously noted high thermal stability of parallel RNA quadruplexes is not derived from H-bonding interactions of the 2'-hydroxyl group, but can be attributed to conformational effects.

An RNA G-quadruplex in the 3' UTR of the proto-oncogene PIM1 represses translation

We have identified a conserved G-quadruplex forming sequence in the 3′ untranslated region (UTR) of the proto-oncogene PIM1. Circular dichroism and thermal denaturation studies revealed that the PIM1 mRNA guanine-rich sequence forms a highly stable intramolecular G-quadruplex structure. Reporter gene assay demonstrated that the PIM1 RNA G-quadruplex represses translation. It is the first experimental evidence of an RNA G-quadruplex structure located in the 3' UTR that may act as posttranscriptional regulator.

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.