G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential quadruplex sequences have been identified in G-rich eukaryotic telomeres, and more recently in non-telomeric genomic DNA, e.g. in nuclease-hypersensitive promoter regions. The natural role and biological validation of these structures is starting to be explored, and there is particular interest in them as targets for therapeutic intervention. This survey focuses on the folding and structural features on quadruplexes formed from telomeric and non-telomeric DNA sequences, and examines fundamental aspects of topology and the emerging relationships with sequence. Emphasis is placed on information from the high-resolution methods of X-ray crystallography and NMR, and their scope and current limitations are discussed. Such information, together with biological insights, will be important for the discovery of drugs targeting quadruplexes from particular genes.

Quadruplex DNA: sequence, topology and structure

G-quadruplexes are four-stranded DNA structures that are over-represented in gene promoter regions and are viewed as emerging therapeutic targets in oncology, as transcriptional repression of oncogenes through stabilization of these structures could be a novel anticancer strategy. Many gene promoter G-quadruplexes have physicochemical properties and structural characteristics that might make them druggable, and their structural diversity suggests that a high degree of selectivity might be possible. Here, we describe the evidence for G-quadruplexes in gene promoters and discuss their potential as therapeutic targets, as well as progress in the development of strategies to harness this potential through intervention with small-molecule ligands.

Targeting G-quadruplexes in gene promoters: a novel anticancer strategy?

'If G-quadruplexes form so readily in vitro, Nature will have found a way of using them in vivo' (Statement by Aaron Klug over 30 years ago).During the last decade, four-stranded helical structures called G-quadruplex (or G4) have emerged from being a structural curiosity observed in vitro, to being recognized as a possible nucleic acid based mechanism for regulating multiple biological processes in vivo. The sequencing of many genomes has revealed that they are rich in sequence motifs that have the potential to form G-quadruplexes and that their location is non-random, correlating with functionally important genomic regions. In this short review, we summarize recent evidence for the in vivo presence and function of DNA and RNA G-quadruplexes in various cellular pathways including DNA replication, gene expression and telomere maintenance. We also highlight remaining open questions that will have to be addressed in the future.

G-quadruplexes and their regulatory roles in biology.

mRNA polyadenylation is a critical cellular process in eukaryotes. It involves 3' end cleavage of nascent mRNAs and addition of the poly(A) tail, which plays important roles in many aspects of the cellular metabolism of mRNA. The process is controlled by various cis-acting elements surrounding the cleavage site, and their binding factors. In this study, we surveyed genome regions containing cleavage sites [herein called poly(A) sites], for 13,942 human and 11,155 mouse genes. We found that a great proportion of human and mouse genes have alternative polyadenylation ( approximately 54 and 32%, respectively). The conservation of alternative polyadenylation type or polyadenylation configuration between human and mouse orthologs is statistically significant, indicating that alternative polyadenylation is widely employed by these two species to produce alternative gene transcripts. Genes belonging to several functional groups, indicated by their Gene Ontology annotations, are biased with respect to polyadenylation configuration. Many poly(A) sites harbor multiple cleavage sites (51.25% human and 46.97% mouse sites), leading to heterogeneous 3' end formation for transcripts. This implies that the cleavage process of polyadenylation is largely imprecise. Different types of poly(A) sites, with regard to their relative locations in a gene, are found to have distinct nucleotide composition in surrounding genomic regions. This large-scale study provides important insights into the mechanism of polyadenylation in mammalian species and represents a genomic view of the regulation of gene expression by alternative polyadenylation.

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A large-scale analysis of mRNA polyadenylation of human and mouse genes

We report here the results of a systematic search for the existence and prevalence of potential intramolecular G-quadruplex forming sequences in the human genome. We have also examined the tendency for particular sequences of 'loop' regions to occur in particular positions with respect to the G-tracts in a quadruplex. Using arithmetic ratio and probability techniques we have discovered frequent and systematic occurrence of certain sequence types, the most prominent being a potential quadruplex containing CCTGT in the first 'loop' position. Being able to highlight types of potential quadruplex sequences in G-rich regions is an important step in searching for biologically relevant sequences and finding their function.

Highly prevalent putative quadruplex sequence motifs in human DNA

The quadruplex structures formed by guanine-rich nucleic acid sequences have received significant attention recently because of growing evidence for their role in important biological processes and as therapeutic targets. G-quadruplex DNA has been suggested to regulate DNA replication and may control cellular proliferation. Sequences capable of forming G-quadruplexes in the RNA have been shown to play significant roles in regulation of polyadenylation and splicing events in mammalian transcripts. Whether quadruplex structure directly plays a role in regulating RNA processing requires investigation. Computational approaches to study G-quadruplexes allow detailed analysis of mammalian genomes. There are no known easily accessible user-friendly tools that can compute G-quadruplexes in the nucleotide sequences. We have developed a web-based server, QGRS Mapper, that predicts quadruplex forming G-rich sequences (QGRS) in nucleotide sequences. It is a user-friendly application that provides many options for defining and studying G-quadruplexes. It performs analysis of the user provided genomic sequences, e.g. promoter and telomeric regions, as well as RNA sequences. It is also useful for predicting G-quadruplex structures in oligonucleotides. The program provides options to search and retrieve desired gene/nucleotide sequence entries from NCBI databases for mapping G-quadruplexes in the context of RNA processing sites. This feature is very useful for investigating the functional relevance of G-quadruplex structure, in particular its role in regulating the gene expression by alternative processing. In addition to providing data on composition and locations of QGRS relative to the processing sites in the pre-mRNA sequence, QGRS Mapper features interactive graphic representation of the data. The user can also use the graphics module to visualize QGRS distribution patterns among all the alternative RNA products of a gene simultaneously on a single screen. QGRS Mapper can be accessed at http://bioinformatics.ramapo.edu/QGRS/.

QGRS Mapper: a web-based server for predicting G-quadruplexes in nucleotide sequences

G-quadruplex motifs in the RNA play significant roles in key cellular processes and human disease. While sequences capable of forming G-quadruplexes in the pre-mRNA are involved in regulation of polyadenylation and splicing events in mammalian transcripts, the G-quadruplex motifs in the UTRs may help regulate mRNA expression. GRSDB2 is a second-generation database containing information on the composition and distribution of putative Quadruplex-forming G-Rich Sequences (QGRS) mapped in approximately 29 000 eukaryotic pre-mRNA sequences, many of which are alternatively processed. The data stored in the GRSDB2 is based on computational analysis of NCBI Entrez Gene entries with the help of an improved version of the QGRS Mapper program. The database allows complex queries with a wide variety of parameters, including Gene Ontology terms. The data is displayed in a variety of formats with several additional computational capabilities. We have also developed a new database, GRS_UTRdb, containing information on the composition and distribution patterns of putative QGRS in the 5'- and 3'-UTRs of eukaryotic mRNA sequences. The goal of these experiments has been to build freely accessible resources for exploring the role of G-quadruplex structure in regulation of gene expression at post-transcriptional level. The databases can be accessed at the G-Quadruplex Resource Site at: http://bioinformatics.ramapo.edu/GQRS/.

GRSDB2 and GRS_UTRdb: databases of quadruplex forming G-rich sequences in pre-mRNAs and mRNAs.

Auxiliary factors likely play an important role in determining the polyadenylation efficiency of mammalian pre-mRNAs We previously identified an auxiliary factor, hnRNP H/H', which stimulates 3'-end processing through an interaction with sequences downstream of the core elements of the SV40 late polyadenylation signal Using in vitro reconstitution assays we have demonstrated that hnRNP H/H' can stimulate processing of two additional model polyadenylation signals by binding at similar relative downstream locations but with significantly different affinities A short tract of G residues was determined to be a common property of all three hnRNP H/H' binding sites A survey of mammalian polyadenylation signals identified potential G-rich hnRNP H/H' binding sites at similar downstream locations in approximately 34% of these signals All of the novel G-rich elements tested were found to bind hnRNP H/H' protein and the processing of selected signals identified in the survey was stimulated by the protein both in vivo and in vitro Downstream G-rich tracts, therefore, are a common auxiliary element in mammalian polyadenylation signals Sequences capable of binding hnRNP H protein with varying affinities may play a role in determining the processing efficiency of a significant number of mammalian polyadenylation signals

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Downstream sequence elements with different affinities for the hnRNP H/H′ protein influence the processing efficiency of mammalian polyadenylation signals

Guanine-rich nucleic acids are known to form highly stable G-quadruplex structures, also known as G-quartets. Recently, there has been a tremendous amount of interest in studying G-quadruplexes owing to the realization of their biological importance. G-rich sequences (GRSs) capable of forming G-quadruplexes are found in the vicinity of polyadenylation regions and are involved in regulating 3' end processing of mammalian pre-mRNAs. G-rich motifs are also known to play an important role in alternative, tissue-specific splicing by interacting with hnRNP H protein subfamily. Whether quadruplex structure directly plays a role in regulating RNA processing events requires further investigation. To date there has not been a comprehensive effort to study G-quadruplexes near RNA processing sites. We have applied a computational approach to map putative Quadruplex forming GRSs within the transcribed regions of a large number of alternatively processed human and mouse gene sequences that were obtained as fully annotated entries from GenBank and RefSeq. We have used the computed data to build the GRSDB database that provides a unique avenue for studying G-quadruplexes in the context of RNA processing sites. GRSDB website offers visual comparison of G-quadruplex distribution patterns among all the alternative RNA products of a gene with the help of dynamic graphics. At present, GRSDB contains data from 1310 human and mouse genes, of which 1188 are alternatively processed. It has a total of 379 223 predicted G-quadruplexes, of which 54 252 are near RNA processing sites. GRSDB is a good resource for researchers interested in investigating the functional relevance of G-quadruplexes, especially in the context of alternative RNA processing. It can be accessed at http://bioinformatics.ramapo.edu/grsdb/.

GRSDB: a database of quadruplex forming G-rich sequences in alternatively processed mammalian pre-mRNA sequences

Nucleic acids containing guanine tracts can form quadruplex structures via non-Watson-Crick base pairing. Formation of G-quadruplexes is associated with the regulation of important biological functions such as transcription, genetic instability, DNA repair, DNA replication, epigenetic mechanisms, regulation of translation, and alternative splicing. G-quadruplexes play important roles in human diseases and are being considered as targets for a variety of therapies. Identification of functional G-quadruplexes and the study of their overall distribution in genomes and transcriptomes is an important pursuit. Traditional computational methods map sequence motifs capable of forming G-quadruplexes but have difficulty in distinguishing motifs that occur by chance from ones which fold into G-quadruplexes. We present Quadruplex forming ‘G’-rich sequences (QGRS)-Conserve, a computational method for calculating motif conservation across exomes and supports filtering to provide researchers with more precise methods of studying G-quadruplex distribution patterns. Our method quantitatively evaluates conservation between quadruplexes found in homologous nucleotide sequences based on several motif structural characteristics. QGRS-Conserve also efficiently manages overlapping G-quadruplex sequences such that the resulting datasets can be analyzed effectively. We have applied QGRS-Conserve to identify a large number of G-quadruplex motifs in the human exome conserved across several mammalian and non-mammalian species. We have successfully identified multiple homologs of many previously published G-quadruplexes that play post-transcriptional regulatory roles in human genes. Preliminary large-scale analysis identified many homologous G-quadruplexes in the 5′- and 3′-untranslated regions of mammalian species. An expectedly smaller set of G-quadruplex motifs was found to be conserved across larger phylogenetic distances. QGRS-Conserve provides means to build datasets that can be filtered and categorized in a variety of biological dimensions for more targeted studies in order to better understand the roles that G-quadruplexes play.

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Paramjeet Singh Bagga

Papers

QGRS Mapper: a web-based server for predicting G-quadruplexes in nucleotide sequences

GRSDB2 and GRS_UTRdb: databases of quadruplex forming G-rich sequences in pre-mRNAs and mRNAs.

Downstream sequence elements with different affinities for the hnRNP H/H′ protein influence the processing efficiency of mammalian polyadenylation signals

GRSDB: a database of quadruplex forming G-rich sequences in alternatively processed mammalian pre-mRNA sequences

QGRS-Conserve: a computational method for discovering evolutionarily conserved G-quadruplex motifs