256 research outputs found
RNA G-quadruplexes and their potential regulatory roles in translation
Abstract: DNA guanine (G)-rich 4-stranded helical nucleic acid structures called G-quadruplexes (G4), have been extensively studied during the last decades. However, emerging evidence reveals that 50- and
30-untranslated regions (50- and 30-UTRs) as well as open reading frames (ORFs) contain putative
RNA G-quadruplexes. These stable secondary structures play key roles in telomere homeostasis and
RNA metabolism including pre-mRNA splicing, polyadenylation, mRNA targeting and translation.
Interestingly, multiple RNA binding proteins such as nucleolin, FMRP, DHX36, and Aven were
identified to bind RNA G-quadruplexes. Moreover, accumulating reports suggest that RNA
G-quadruplexes regulate translation in cap-dependent and -independent manner. Herein, we
discuss potential roles of RNA G-quadruplexes and associated trans-acting factors in the regulation
of mRNA translation
Exploring mRNA 3'-UTR G-quadruplexes: evidence of roles in both alternative polyadenylation and mRNA shortening
Abstract: Guanine-rich RNA sequences can fold into noncanonical,
four stranded helical structures called
G-quadruplexes that have been shown to be widely
distributed within the mammalian transcriptome,
as well as being key regulatory elements in various
biological mechanisms. That said, their role
within the 30-untranslated region (UTR) of mRNA
remains to be elucidated and appreciated.
A bioinformatic analysis of the 30-UTRs of mRNAs
revealed enrichment in G-quadruplexes. To shed
light on the role(s) of these structures, those
found in the LRP5 and FXR1 genes were
characterized both in vitro and in cellulo. The 30-
UTR G-quadruplexes were found to increase
the efficiencies of alternative polyadenylation sites,
leading to the expression of shorter transcripts
and to possess the ability to interfere with the
miRNA regulatory network of a specific mRNA.
Clearly, G-quadruplexes located in the 30-UTRs of
mRNAs are cis-regulatory elements that have a
significant impact on gene expression
A conserved target site in HIV-1 Gag RNA is accessible to inhibition by both an HDV ribozyme and a short hairpin RNA
Abstract: Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1
infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on
the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting
a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described
candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible
to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different
modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is
sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the
modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA.
Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies
Unbiased in vitro selection reveals the unique character of the self-cleaving antigenomic HDV RNA sequence
In order to revisit the architecture of the catalytic center of the antigenomic hepatitis delta virus (HDV) ribozyme we developed an unbiased in vitro selection procedure that efficiently selected novel variants from a relatively small set of sequences. Using this procedure we examined all possible variants from a pool of HDV ribozymes that had been randomized at 25 positions (4(25)). The isolated set of sequences shows more variability than do the natural variants. Nucleotide variations were found at all randomized positions, even at positions when the general belief was that the specific base was absolutely required for catalytic activity. Covariation analysis supports the presence of several base pairs, although it failed to propose any new tertiary contacts. HDV ribozyme appears to possess a greater number of constraints, in terms of sequences capable of supporting the catalysed cleavage, than do other catalytic RNAs. This supports the idea that the appearance of this catalytic RNA structure has a low probability (i.e. is a rare event), which may explain why to date it has been found in nature only in the HDV. These contrasts with the hammerhead self-cleaving motif that is proposed to have multiple origins, and that is widespread among different organisms. Thus, just because a self-cleaving RNA motif is small does not imply that it occurs easily
The super-n-motifs model : a novel alignment-free approach for representing and comparing RNA secondary structures
Abstract : Motivation: Comparing ribonucleic acid (RNA) secondary structures of arbitrary size uncovers structural patterns that can provide a better understanding of RNA functions. However, performing fast and accurate secondary structure comparisons is challenging when we take into account the RNA configuration (i.e. linear or circular), the presence of pseudoknot and G-quadruplex (G4) motifs and the increasing number of secondary structures generated by high-throughput probing techniques. To address this challenge, we propose the super-n-motifs model based on a latent analysis of enhanced motifs comprising not only basic motifs but also adjacency relations. The super-n-motifs model computes a vector representation of secondary structures as linear combinations of these motifs. Results: We demonstrate the accuracy of our model for comparison of secondary structures from linear and circular RNA while also considering pseudoknot and G4 motifs. We show that the supern- motifs representation effectively captures the most important structural features of secondary structures, as compared to other representations such as ordered tree, arc-annotated and string representations. Finally, we demonstrate the time efficiency of our model, which is alignment free and capable of performing large-scale comparisons of 10 000 secondary structures with an efficiency up to 4 orders of magnitude faster than existing approaches
Structurexplor : a platform for the exploration of structural features of RNA secondary structures
Abstract : Summary: Discovering function-related structural features, such as the cloverleaf shape of transfer
RNA secondary structures, is essential to understand RNA function. With this aim, we have developed
a platform, named Structurexplor, to facilitate the exploration of structural features in
populations of RNA secondary structures. It has been designed and developed to help biologists
interactively search for, evaluate and select interesting structural features that can potentially explain
RNA functions
Target-dependent on/off switch increases ribozyme fidelity
Ribozymes, RNA molecules that catalyze the cleavage of RNA substrates, provide an interesting alternative to the RNA interference (RNAi) approach to gene inactivation, especially given the fact that RNAi seems to trigger an immunological response. Unfortunately, the limited substrate specificity of ribozymes is considered to be a significant hurdle in their development as molecular tools. Here, we report the molecular engineering of a ribozyme possessing a new biosensor module that switches the cleavage activity from ‘off’ (a ‘safety lock’) to ‘on’ solely in the presence of the appropriate RNA target substrate. Both proof-of-concept and the mechanism of action of this man-made riboswitch are demonstrated using hepatitis delta virus ribozymes that cleave RNA transcripts derived from the hepatitis B and C viruses. To our knowledge, this is the first report of a ribozyme bearing a target-dependent module that is activated by its RNA substrate, an arrangement which greatly diminishes non-specific effects. This new approach provides a highly specific and improved tool with significant potential for application in the fields of both functional genomics and gene therapy
Motif independent identification of potential RNA G-quadruplexes by G4RNA screener
Abstract: Motivation: G-quadruplex structures in RNA molecules are known to have regulatory impacts in
cells but are difficult to locate in the genome. The minimal requirements for G-quadruplex folding
in RNA (G 3N1-7G 3N1-7G 3N1-7G 3) is being challenged by observations made on specific examples
in recent years. The definition of potential G-quadruplex sequences has major repercussions
on the observation of the structure since it introduces a bias. The canonical motif only
describes a sub-population of the reported G-quadruplexes. To address these issues, we propose
an RNA G-quadruplex prediction strategy that does not rely on a motif definition.
Results: We trained an artificial neural network with sequences of experimentally validated
G-quadruplexes from the G4RNA database encoded using an abstract definition of their sequence.
This artificial neural network, G4NN, evaluates the similarity of a given sequence to known G-quadruplexes
and reports it as a score. G4NN has a predictive power comparable to the reported G richness
and G/C skewness evaluations that are the current state-of-the-art for the identification of
potential RNA G-quadruplexes. We combined these approaches in the G4RNA screener, a program
designed to manage and evaluate the sequences to identify potential G-quadruplexes
In-line probing of RNA G-quadruplexes
Abstract: Although the majority of the initial G-quadruplex studies were performed on DNA molecules, there currently
exists a rapidly growing interest in the investigation of those formed in RNA molecules that possess
high potential of acting as gene expression regulatory elements. Indeed, G-quadruplexes found in the
50-untranslated regions of mRNAs have been reported to be widespread within the human transcriptome
and to act as general translational repressors. In addition to translation regulation, several other mRNA
maturation steps and events, including mRNA splicing, polyadenylation and localization, have been
shown to be influenced by the presence of these RNA G-quadruplexes. Bioinformatic approaches have
identified thousands of potential RNA G-quadruplex sequences in the human transcriptome. Clearly there
is a need for the development of rapid, simple and informative techniques and methodologies with which
the ability of these sequences, and of any potential new regulatory elements, to fold into G-quadruplexes
in vitro can be examined. This report describes an integrated methodology for monitoring RNA G-quadruplexes
formation that combines bioinformatic algorithms, secondary structure prediction, in-line probing
with semi-quantification analysis and structural representation software. The power of this approach
is illustrated, step-by-step, with the determination of the structure adopted by a potential G-quadruplex
sequence found in the 50-untranslated region of the cAMP responsive element modulator (CREM) mRNA.
The results unambiguously show that the CREM sequence folds into a G-quadruplex structure in the presence
of a physiological concentration of potassium ions. This in-line probing-based method is easy to use,
robust, reproducible and informative in the study of RNA G-quadruplex formation
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