Добірка наукової літератури з теми "RNA G4 structures"

G-quadruplexes (G4) are the most actively studied non-canonical secondary structures formed by contiguous repeats of guanines in DNA or RNA strands. Small molecule mediated targeting of G-quadruplexes has emerged as an attractive tool for visualization and stabilization of these structures inside the cell. Limited number of DNA and RNA G4-selective assays have been reported for primary ligand screening. A combination of fluorescence spectroscopy, AFM, CD, PAGE, and confocal microscopy have been used to assess a dimeric carbocyanine dye B6,5 for screening G4-binding ligands in vitro and in cellulo. The dye B6,5 interacts with physiologically relevant DNA and RNA G4 structures, resulting in fluorescence enhancement of the molecule as an in vitro readout for G4 selectivity. Interaction of the dye with G4 is accompanied by quadruplex stabilization that extends its use in primary screening of G4 specific ligands. The molecule is cell permeable and enables visualization of quadruplex dominated cellular regions of nucleoli using confocal microscopy. The dye is displaced by quarfloxin in live cells. The dye B6,5 shows remarkable duplex to quadruplex selectivity in vitro along with ligand-like stabilization of DNA G4 structures. Cell permeability and response to RNA G4 structures project the dye with interesting theranostic potential. Our results validate that B6,5 can serve the dual purpose of visualization of DNA and RNA G4 structures and screening of G4 specific ligands, and adds to the limited number of probes with such potential.

The role of G-quadruplex (G4) RNA structures is multifaceted and controversial. Here, we have used as a model the EBV-encoded EBNA1 and the Kaposi’s sarcoma-associated herpesvirus (KSHV)-encoded LANA1 mRNAs. We have compared the G4s in these two messages in terms of nucleolin binding, nuclear mRNA retention, and mRNA translation inhibition and their effects on immune evasion. The G4s in the EBNA1 message are clustered in one repeat sequence and the G4 ligand PhenDH2 prevents all G4-associated activities. The RNA G4s in the LANA1 message take part in similar multiple mRNA functions but are spread throughout the message. The different G4 activities depend on flanking coding and non-coding sequences and, interestingly, can be separated individually. Together, the results illustrate the multifunctional, dynamic and context-dependent nature of G4 RNAs and highlight the possibility to develop ligands targeting specific RNA G4 functions. The data also suggest a common multifunctional repertoire of viral G4 RNA activities for immune evasion.

The most common form of DNA is a right-handed helix or the B-form DNA. DNA can also adopt a variety of alternative conformations, non-B-form DNA secondary structures, including the DNA G-quadruplex (DNA-G4). Furthermore, besides stem-loops that yield A-form double-stranded RNA, non-canonical RNA G-quadruplex (RNA-G4) secondary structures are also observed. Recent bioinformatics analysis of the whole-genome and transcriptome obtained using G-quadruplex–specific antibodies and ligands, revealed genomic positions of G-quadruplexes. In addition, accumulating evidence pointed to the existence of these structures under physiologically- and pathologically-relevant conditions, with functional roles in vivo. In this review, we focused on DNA-G4 and RNA-G4, which may have important roles in neuronal function, and reveal mechanisms underlying neurological disorders related to synaptic dysfunction. In addition, we mention the potential of G-quadruplexes as therapeutic targets for neurological diseases.

Abstract The identification of G-quadruplex (G4) binding proteins and insights into their mechanism of action are important for understanding the regulatory functions of G4 structures. Here, we performed an unbiased affinity-purification assay coupled with mass spectrometry and identified 30 putative G4 binding proteins from the fission yeast Schizosaccharomyces pombe. Gene ontology analysis of the molecular functions enriched in this pull-down assay included mRNA binding, RNA helicase activity, and translation regulator activity. We focused this study on three of the identified proteins that possessed putative arginine-glycine-glycine (RGG) domains, namely the Stm1 homolog Oga1 and the DEAD box RNA helicases Dbp2 and Ded1. We found that Oga1, Dbp2, and Ded1 bound to both DNA and RNA G4s in vitro. Both Dbp2 and Ded1 bound to G4 structures through the RGG domain located in the C-terminal region of the helicases, and point mutations in this domain weakened the G4 binding properties of the helicases. Dbp2 and Ded1 destabilized less thermostable G4 RNA and DNA structures, and this ability was independent of ATP but dependent on the RGG domain. Our study provides the first evidence that the RGG motifs in DEAD box helicases are necessary for both G4 binding and G4 destabilization.

G-quadruplex (G4) is one of the most important secondary structures in nucleic acids. Until recently, G4 RNAs have not been reported in any ribovirus, such as the hepatitis C virus. Our bioinformatics analysis reveals highly conserved guanine-rich consensus sequences within the core gene of hepatitis C despite the high genetic variability of this ribovirus; we further show using various methods that such consensus sequences can fold into unimolecular G4 RNA structures, both in vitro and under physiological conditions. Furthermore, we provide direct evidences that small molecules specifically targeting G4 can stabilize this structure to reduce RNA replication and inhibit protein translation of intracellular hepatitis C. Ultimately, the stabilization of G4 RNA in the genome of hepatitis C represents a promising new strategy for anti–hepatitis C drug development.

G-quadruplex (G4) structures are highly stable four-stranded DNA and RNA secondary structures held together by non-canonical guanine base pairs. G4 sequence motifs are enriched at specific sites in eukaryotic genomes, suggesting regulatory functions of G4 structures during different biological processes. Considering the high thermodynamic stability of G4 structures, various proteins are necessary for G4 structure formation and unwinding. In a yeast one-hybrid screen, we identified Slx9 as a novel G4-binding protein. We confirmed that Slx9 binds to G4 DNA structures in vitro. Despite these findings, Slx9 binds only insignificantly to G-rich/G4 regions in Saccharomyces cerevisiae as demonstrated by genome-wide ChIP-seq analysis. However, Slx9 binding to G4s is significantly increased in the absence of Sgs1, a RecQ helicase that regulates G4 structures. Different genetic and molecular analyses allowed us to propose a model in which Slx9 recognizes and protects stabilized G4 structures in vivo.

Guanine quadruplexes (G4s) are highly polymorphic four-stranded structures formed within guanine-rich DNA and RNA sequences that play a crucial role in biological processes. The recent discovery of the first G4 structures within mitochondrial DNA has led to a small revolution in the field. In particular, the G-rich conserved sequence block II (CSB II) can form different types of G4s that are thought to play a crucial role in replication. In this study, we decipher the most relevant G4 structures that can be formed within CSB II: RNA G4 at the RNA transcript, DNA G4 within the non-transcribed strand and DNA:RNA hybrid between the RNA transcript and the non-transcribed strand. We show that the more abundant, but unexplored, G6AG7 (37%) and G6AG8 (35%) sequences in CSB II yield more stable G4s than the less profuse G5AG7 sequence. Moreover, the existence of a guanine located 1 bp upstream promotes G4 formation. In all cases, parallel G4s are formed, but their topology changes from a less ordered to a highly ordered G4 when adding small amounts of potassium or sodium cations. Circular dichroism was used due to discriminate different conformations and topologies of nucleic acids and was complemented with gel electrophoresis and fluorescence spectroscopy studies.

Some DNA or RNA sequences rich in guanine (G) nucleotides can adopt noncanonical conformations known as G-quadruplexes (G4). In the nuclear genome, G4 motifs have been associated with genome instability and gene expression defects, but they are increasingly recognized to be regulatory structures. Recent studies have revealed that G4 structures can form in the mitochondrial genome (mtDNA) and potential G4 forming sequences are associated with the origin of mtDNA deletions. However, little is known about the regulatory role of G4 structures in mitochondria. In this short review, we will explore the potential for G4 structures to regulate mitochondrial function, based on evidence from the nucleus.

Guanine (G)-quadruplexes (G4s) are unique nucleic acid structures that are formed by stacked G-tetrads in G-rich DNA or RNA sequences. G4s have been reported to play significant roles in various cellular events in both macro- and micro-organisms. The identification and characterization of G4s can help to understand their different biological roles and potential applications in diagnosis and therapy. In addition to biophysical and biochemical methods to interrogate G4 formation, G4 fluorescent turn-on ligands can be used to target and visualize G4 formation both in vitro and in cells. Here, we review several representative classes of G4 fluorescent turn-on ligands in terms of their interaction mechanism and application perspectives. Interestingly, G4 structures are commonly identified in DNA and RNA aptamers against targets that include proteins and small molecules, which can be utilized as G4 tools for diverse applications. We therefore also summarize the recent development of G4-containing aptamers and highlight their applications in biosensing, bioimaging, and therapy. Moreover, we discuss the current challenges and future perspectives of G4 fluorescent turn-on ligands and G4-containing aptamers.

A library of 52 distyryl and 9 mono-styryl cationic dyes was synthesized and investigated with respect to their optical properties, propensity to aggregation in aqueous medium, and capacity to serve as fluorescence “light-up” probes for G-quadruplex (G4) DNA and RNA structures. Among the 61 compounds, 57 dyes showed preferential enhancement of fluorescence intensity in the presence of one or another G4-DNA or RNA structure, while no dye displayed preferential response to double-stranded DNA or single-stranded RNA analytes employed at equivalent nucleotide concentration. Thus, preferential fluorimetric response towards G4 structures appears to be a common feature of mono- and distyryl dyes, including long-known mono-styryl dyes used as mitochondrial probes or protein stains. However, the magnitude of the G4-induced “light-up” effect varies drastically, as a function of both the molecular structure of the dyes and the nature or topology of G4 analytes. Although our results do not allow to formulate comprehensive structure–properties relationships, we identified several structural motifs, such as indole- or pyrrole-substituted distyryl dyes, as well as simple mono-stryryl dyes such as DASPMI [2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide] or its 4-isomer, as optimal fluorescent light-up probes characterized by high fluorimetric response (I/I 0 of up to 550-fold), excellent selectivity with respect to double-stranded DNA or single-stranded RNA controls, high quantum yield in the presence of G4 analytes (up to 0.32), large Stokes shift (up to 150 nm) and, in certain cases, structural selectivity with respect to one or another G4 folding topology. These dyes can be considered as promising G4-responsive sensors for in vitro or imaging applications. As a possible application, we implemented a simple two-dye fluorimetric assay allowing rapid topological classification of G4-DNA structures.

La traduction des ARNm et la production de protéines sont des phénomènes étroitement contrôlés dans la cellule. La séquence de l'ARNm et sa structure, auxquelles sont associées des protéines se liant à l'ARN, ainsi que la qualité de la protéine produite à partir de cet ARNm, évaluée notamment par la voie de contrôle qualité associée aux ribosomes, sont des éléments impliqués dans ce processus majeur pour la cellule. Dans ce domaine, le contrôle de la production de protéine EBNA1 (Epstein-Barr Nuclear Antigen 1) du virus d'Epstein -Barr est un exemple intéressant. La protéine EBNA1 est essentielle pour la survie du virus dans les cellules hôtes. Les niveaux cellulaires de protéine EBNA1 sont très faibles, bien que la protéine soit présente dans toutes les cellules infectées. Cette dernière est aussi extrêmement antigénique. Il est aujourd'hui admis que la quantité de protéine EBNA1 présente dans les cellules est suffisante pour assurer le maintien du virus dans la cellule, mais assez basse pour lui permettre d'échapper au système immunitaire de l'hôte. Un contrôle de sa production est nécessaire à cet équilibre. Des études précédentes ont montré que le domaine GAr (répétitions de glycine et alanine), présent dans la partie N-terminale de la protéine, déclenche un mécanisme conduisant à l'inhibition de l'initiation de la traduction de l'ARNm d'EBNA1 en cis, sans affecter la traduction des autres ARNm présents dans la cellule. L'équipe a montré précédemment que les structures G4s (G-quadruplex) peuvent être formées dans l'ARNm codant le GAr. De nombreuses études ont montré l'importance de ces structures secondaires de l'ARN dans la régulation de la traduction de l'ARNm d'EBNA1. La nucléoline, un facteur nucléaire, peut se lier aux G4s de l'ARNm du GAr. Cependant, il a aussi été montré que le peptide GAr, et non l'ARNm associé, est nécessaire au contrôle de la traduction de l'ARNm du GAr en cis. L'objectif principal de ma thèse est de mieux comprendre le mécanisme déclenché par l'ARNm et le polypeptide naissant conduisant au contrôle de la traduction de l'ARNm d'EBNA1 en cis. En accord avec le fait que les structures G4s de l'ARN sont extrêmement dynamiques, nous avons montré dans un premier temps que les fonctions associées au G4s de l'ARNm du GAr, à savoir la localisation de l'ARNm, sa traduction et sa capacité à se lier à certaines protéines, dépendent du contexte dans lesquelles ces structures se trouvent. Nous montrons ensuite que la traduction de l'ARNm d'EBNA1 est nécessaire à l'interaction nucléoline-ARNm, signifiant que la traduction de l'ARNm induit des changements dans les propriétés de l'ARNm. En parallèle, nous avons étudié le NACA, une sous-unité du complexe chaperon NAC (nascent polypeptide-associated complex). NACA se détache du ribosome lors de la synthèse du GAr et interagit avec le GAr. NACA est aussi capable de se lier aux ARN et est déterminant dans la suite des évènements liés à l'ARNm codant le GAr. Enfin, et de façon assez surprenante, les facteurs d'initiation de la traduction sont aussi des éléments clés dans l'inhibition de la traduction de l'ARNm d'EBNA1. Le facteur le plus impactant identifié jusqu'à maintenant est le facteur eIF4A1. Ces résultats indiquent que la séquence et structure de l'ARNm et le polypeptide naissant correspondant sont impliqués dans l'inhibition de l'initiation de la traduction de l'ARNm d'EBNA1. Cependant, cela n'enlève pas la possibilité que l'ARNm et le polypeptide naissant déclenchent chacun une voie d'inhibition de la synthèse d'EBNA1 distincte l'une de l'autre. Les virus utilisent des éléments déjà présents dans la cellule pour assurer leur maintien dans la cellule hôte. Ainsi, les principes de biologie cellulaire décrits ici peuvent apporter des indications importantes pour une meilleure compréhension d'autres pathologies en plus de celles liées au virus d'Epstein-Barr
MRNA translation and protein synthesis are tightly regulated events in the cell. Mechanisms describing these key cellular events involve the mRNA sequence and its structure with the association of RNA-binding protein to it, as well as the quality of the translation product encoded by the mRNA, assessed notably through ribosome-associated quality control. In this context, the Epstein-Barr virus EBNA1 (Epstein-Barr Nuclear Antigen 1) mRNA translation regulation is an interesting example. EBNA1 is known to be an essential protein for the virus survival in the host cells. Even though EBNA1 is present in every infected cell, its protein level is remarkably low. As EBNA1 is highly antigenic, it has been suggested that EBNA1 levels in the cells are low enough to escape the immune system of the host, but sufficient to maintain EBV infection. This balance requires a tightly controlled EBNA1 production. Further studies showed that the GAr (glycine-alanine repeat) domain, located in the N-terminal part of EBNA1, triggers an in cis mechanism leading to the inhibition of the translation initiation of its own mRNA, without affecting translation of other mRNAs in the cell. Thus, the GAr domain of EBNA1 is a unique tool to study selective mRNA translation control without affecting general protein synthesis. It was previously shown that RNA G4 (G-quadruplex) structures can be folded in the GAr-encoding mRNA. Numerous studies underlined the importance of these RNA structures in the regulation of EBNA1 mRNA translation, and the team previously showed that nucleolin can interact with these RNA G4 structures, interaction which can be competed by some G4 ligands. However, it was also formerly shown that the GAr peptide itself plays a role in controlling in cis the translation of EBNA1-encoding mRNA, rather than just the RNA sequence. The main focus of the study presented here is to shed light on how this translation event and the fate of the encoding mRNA are regulated in cis by the mRNA and the encoded nascent polypeptide. In line with the fact that RNA G4 structures are highly dynamic, we first showed that GAr RNA G4-associated functions, namely mRNA localisation, translation and ability to bind RNA-binding proteins, are dependent on the context they are in, i.e. their position in the mRNA, the structures in their surrounding or the factors binding the mRNA, such as G4 ligands. We next demonstrated that translation of the EBNA1 mRNA is necessary for nucleolin-binding to it, meaning that the translation event modifies some properties of the EBNA1 mRNA. In parallel, we showed that the NACA, a subunit of the NAC chaperone complex, is detached from the ribosome and interacts with the GAr polypeptide. Interestingly, the NACA is also an RNA binding protein in addition to its chaperone function, and is determinant for the future processing of the EBNA1 mRNA. Finally, and unexpectedly, we show that translation initiation factors are also key players in the downregulation of the EBNA1 mRNA translation, affecting also the mRNA nucleolin-binding capacity, the most effective translation initiation factor in the downregulation of EBNA1 mRNA translation identified so far being eIF4A1. These results support the idea that both the RNA sequence and structure and the corresponding nascent polypeptide are involved in the downregulation of EBNA1 mRNA translation. However, it does not rule out the possibility that both the RNA structure and the polypeptide sequence trigger also their own separated inhibitory pathway. As viruses use components already present in the cells to maintain themselves, the cellular biology elements brought out here can provide insights on many other pathologies in addition to EBV-associated diseases

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Mar. 26, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum in Genetics and Molecular Biology." Discipline: Genetics and Molecular Biology; Department/School: Medicine.