RNA G-Quadruplexes as Key Motifs of the Transcriptome.

G-Quadruplexes are non-canonical secondary structures that can be adopted under physiological conditions by guanine-rich DNA and RNA molecules. They have been reported to occur, and to perform multiple biological functions, in the genomes and transcriptomes of many species, including humans. This chapter focuses specifically on RNA G-quadruplexes and reviews the most recent discoveries in the field, as well as addresses the upcoming challenges researchers studying these structures face.

G-quadruplex located in the 5'UTR of the BAG-1 mRNA affects both its cap-dependent and cap-independent translation through global secondary structure maintenance.

The anti-apoptotic BAG-1 protein isoforms are known to be overexpressed in colorectal tumors and are considered to be potential therapeutic targets. The isoforms are derived from alternative translation initiations occuring at four in-frame start codons of a single mRNA transcript. Its 5'UTR also contains an internal ribosome entry site (IRES) regulating the cap-independent translation of the transcript. An RNA G-quadruplex (rG4) is located at the 5'end of the BAG-1 5'UTR, upstream of the known cis-regulatory elements. Herein, we observed that the expression of BAG-1 isoforms is post-transcriptionally regulated in colorectal cancer cells and tumors, and that stabilisation of the rG4 by small molecules ligands reduces the expression of endogenous BAG-1 isoforms. We demonstrated a critical role for the rG4 in the control of both cap-dependent and independent translation of the BAG-1 mRNA in colorectal cancer cells. Additionally, we found an upstream ORF that also represses BAG-1 mRNA translation. The structural probing of the complete 5'UTR showed that the rG4 acts as a steric block which controls the initiation of translation at each start codon of the transcript and also maintains the global 5'UTR secondary structure required for IRES-dependent translation.

G-quadruplexes formation in the 5'UTRs of mRNAs associated with colorectal cancer pathways.

RNA G-quadruplexes (rG4) are stable non-canonical secondary structures composed of G-rich sequences. Many rG4 structures located in the 5'UTRs of mRNAs act as translation repressors due to their high stability which is thought to impede ribosomal scanning. That said, it is not known if these are mRNA-specific examples, or if they are indicative of a global expression regulation mechanism of the mRNAs involved in a common pathway based on structure folding recognition. Gene-ontology analysis of mRNAs bearing a predicted rG4 motif in their 5'UTRs revealed an enrichment for mRNAs associated with the colorectal cancer pathway. Bioinformatic tools for rG4 prediction, and experimental in vitro validations were used to confirm and compare the folding of the predicted rG4s of the mRNAs associated with dysregulated pathways in colorectal cancer. The rG4 folding was confirmed for the first time for 9 mRNAs. A repressive effect of 3 rG4 candidates on the expression of a reporter gene was also measured in colorectal cancer cell lines. This work highlights the fact that rG4 prediction is not yet accurate, and that experimental characterization is still essential in order to identify the precise rG4 folding sequences and the possible common features shared between the rG4 overrepresented in important biological pathways.

Methods to monitor monocytes-mediated amyloid-beta uptake and phagocytosis in the context of adjuvanted immunotherapies.

Antibody-mediated capture of amyloid-beta (Aß) in peripheral blood was identified as an attractive strategy to eliminate cerebral toxic amyloid in Alzheimer's disease (AD) patients and murine models. Alternatively, defective capacity of peripheral monocytes to engulf Aß was reported in individuals with AD. In this report, we developed different approaches to investigate cellular uptake and phagocytosis of Aß, and to examine how two immunological devices--an immunostimulatory Adjuvant System and different amyloid specific antibodies--may affect these biological events. Between one and thirteen months of age, APPswe X PS1.M146V (TASTPM) AD model mice had decreasing concentrations of Aß in their plasma. In contrast, the proportion of blood monocytes containing Aß tended to increase with age. Importantly, the TLR-agonist containing Adjuvant System AS01B primed monocytes to promote de novo Aß uptake capacity, particularly in the presence of anti-Aß antibodies. Biochemical experiments demonstrated that cells achieved Aß uptake and internalization followed by Aß degradation via mechanisms that required effective actin polymerization and proteolytic enzymes such as insulin-degrading enzyme. We further demonstrated that both Aß-specific monoclonal antibodies and plasma from Aß-immunized mice enhanced the phagocytosis of 1 µm Aß-coated particles. Together, our data highlight a new biomarker testing to follow amyloid clearance within the blood and a mechanism of Aß uptake by peripheral monocytes in the context of active or passive immunization, and emphasize on novel approaches to investigate this phenomenon.

The folding of 5'-UTR human G-quadruplexes possessing a long central loop.

G-quadruplexes are widespread four-stranded structures that are adopted by G-rich regions of both DNA and RNA and are involved in essential biological processes such as mRNA translation. They are formed by the stacking of two or more G-quartets that are linked together by three loops. Although the maximal loop length is usually fixed to 7 nt in most G-quadruplex-predicting software, it has already been demonstrated that artificial DNA G-quadruplexes containing two distal loops that are limited to 1 nt each and a central loop up to 30 nt long are likely to form in vitro. This report demonstrates that such structures possessing a long central loop are actually found in the 5'-UTRs of human mRNAs. Firstly, 1453 potential G-quadruplex-forming sequences (PG4s) were identified through a bioinformatic survey that searched for sequences respecting the requirement for two 1-nt long distal loops and a long central loop of 2-90 nt in length. Secondly, in vitro in-line probing experiments confirmed and characterized the folding of eight candidates possessing central loops of 10-70 nt long. Finally, the biological effect of several G-quadruplexes with a long central loop on mRNA expression was studied in cellulo using a luciferase gene reporter assay. Clearly, the actual definition of G-quadruplex-forming sequences is too conservative and must be expanded to include the long central loop. This greatly expands the number of expected PG4s in the transcriptome. Consideration of these new candidates might aid in elucidating the potentially important biological implications of the G-quadruplex structure.

New scoring system to identify RNA G-quadruplex folding.

G-quadruplexes (G4s) are non-canonical structures involved in many important cellular processes. To date, the prediction of potential G-quadruplex structures (PG4s) has been based almost exclusively on the sequence of interest agreeing with the algorithm Gx-N-1-7-Gx-N1-7-Gx-N1-7-Gx (where x ≥ 3 and N = A, U, G or C). However, many sequences agreeing with this algorithm do not form G4s and are considered false-positive predictions. Here we show the RNA PG4 candidate in the 3'-untranslated region (UTR) of the TTYH1 gene to be one such false positive. Specifically, G4 folding was observed to be inhibited by the presence of multiple-cytosine tracks, located in the candidate's genomic context, that adopted a Watson-Crick base-paired structure. Clearly, the neighbouring sequences of a PG4 may influence its folding. The secondary structure of 12 PG4 motifs along with either 15 or 50 nucleotides of their upstream and downstream genomic contexts were evaluated by in-line probing. Data permitted the development of a scoring system for the prediction of PG4s taking into account the effect of the neighbouring sequences. The accuracy of this scoring system was assessed by probing 14 other novel PG4 candidates retrieved in human 5'-UTRs. This new scoring system can be used, in combination with the standard algorithm, to better predict the folding of RNA G4s.

In-line probing of RNA G-quadruplexes.

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 5'-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 5'-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.

Toll-like receptor 4 stimulation with the detoxified ligand monophosphoryl lipid A improves Alzheimer's disease-related pathology.

Alzheimer's disease (AD) is the most common cause of dementia worldwide. The pathogenesis of this neurodegenerative disease, currently without curative treatment, is associated with the accumulation of amyloid ß (Aß) in brain parenchyma and cerebral vasculature. AD patients are unable to clear this toxic peptide, leading to Aß accumulation in their brains and, presumably, the pathology associated with this devastating disease. Compounds that stimulate the immune system to clear Aß may therefore have great therapeutic potential in AD patients. Monophosphoryl lipid A (MPL) is an LPS-derived Toll-like receptor 4 agonist that exhibits unique immunomodulatory properties at doses that are nonpyrogenic. We show here that repeated systemic injections of MPL, but not LPS, significantly improved AD-related pathology in APP(swe)/PS1 mice. MPL treatment led to a significant reduction in Aß load in the brain of these mice, as well as enhanced cognitive function. MPL induced a potent phagocytic response by microglia while triggering a moderate inflammatory reaction. Our data suggest that the Toll-like receptor 4 agonist MPL may be a treatment for AD.

Programming a highly structured ribozyme into complex allostery using RNA oligonucleotides.

RNA possesses great potential for expanding the toolbox currently available to synthetic biologists. Here, the modulation of the Hepatitis Delta Virus ribozyme's activity with a series of rationally designed aptamers and effector RNA oligonucleotides is described. The ribozyme was initially fused with an 18-nucleotide hairpin structure that abolished its self-cleaving activity. The binding of a 14-mer oligonucleotide to the hairpin rescued the self-cleavage in a concentration-dependent manner. This modified ribozyme was inserted into the 5' UTR of a reporter gene, and the resulting construct was used to demonstrate that it is possible to modulate the ribozyme activity in cellulo with the oligonucleotide. Subsequently, ribozymes possessing specialized aptamers respecting other logic gates were also successfully designed and found to be functional in vitro. To our knowledge, this is the first example of HDV ribozyme regulation by oligonucleotides, as well as the first allosteric regulation of HDV ribozyme in mammalian cells.