This review is focused on the structural and physicochemical aspects of metal cation coordination to G-Quadruplexes (GQ) and their effects on GQ stability and conformation. G-quadruplex structures are non-canonical secondary structures formed by both DNA and RNA. G-quadruplexes regulate a wide range of important biochemical processes. Besides the sequence requirements, the coordination of monovalent cations in the GQ is essential for its formation and determines the stability and polymorphism of GQ structures. The nature, location, and dynamics of the cation coordination and their impact on the overall GQ stability are dependent on several factors such as the ionic radii, hydration energy, and the bonding strength to the O6 of guanines. The intracellular monovalent cation concentration and the localized ion concentrations determine the formation of GQs and can potentially dictate their regulatory roles. A wide range of biochemical and biophysical studies on an array of GQ enabling sequences have generated at a minimum the knowledge base that allows us to often predict the stability of GQs in the presence of the physiologically relevant metal ions, however, prediction of conformation of such GQs is still out of the realm.

MicroRNAs (miRNAs) are an important set of oligonucleotide sequences with a biogenesis that involves Dicer-mediated cleavage as a critical step. Dicer cleaves the precursor miRNA (pre-miRNA) stem-loop structure to produce the mature miRNA. Using bioinformatics analysis, we discovered the presence of putative G-quadruplex (GQ)-forming sequences in about 16% of pre-miRNAs. We report the existence of a GQ as an alternative to the canonical stem-loop structure in the clinically important human pre-miRNA 92b. GQ formation led to unwinding of the stem-loop structure imparting resistance to Dicer-mediated cleavage both in vitro and in vivo. A potential K(+) ion-dependent equilibrium between GQ and the stem-loop structure has the ability to regulate the Dicer-mediated maturation of pre-miRNA 92b, which consequently affects target gene silencing. These findings unravel a new mechanism of regulation in pre-miRNA maturation, albeit at the RNA structure level.

BackgroundStructured noncoding RNAs (ncRNAs) play essential roles in many biological processes such as gene regulation, signaling, RNA processing, and protein synthesis. Among the most common groups of ncRNAs in bacteria are riboswitches. These cis-regulatory, metabolite-binding RNAs are present in many species where they regulate various metabolic and signaling pathways. Collectively, there are likely to be hundreds of novel riboswitch classes that remain hidden in the bacterial genomes that have already been sequenced, and potentially thousands of classes distributed among various other species in the biosphere. The vast majority of these undiscovered classes are proposed to be exceedingly rare, and so current bioinformatics search techniques are reaching their limits for differentiating between true riboswitch candidates and false positives.ResultsHerein, we exploit a computational search pipeline that can efficiently identify intergenic regions most likely to encode structured ncRNAs. Application of this method to five bacterial genomes yielded nearly 70 novel genetic elements including 30 novel candidate ncRNA motifs. Among the riboswitch candidates identified is an RNA motif involved in the regulation of thiamin biosynthesis.ConclusionsAnalysis of other genomes will undoubtedly lead to the discovery of many additional novel structured ncRNAs, and provide insight into the range of riboswitches and other kinds of ncRNAs remaining to be discovered in bacteria and archaea.Electronic supplementary materialThe online version of this article (10.1186/s12866-019-1433-7) contains supplementary material, which is available to authorized users.

Five distinct riboswitch classes that regulate gene expression in response to the cofactor S-adenosylmethionine (SAM) or its metabolic breakdown product S-adenosylhomocysteine (SAH) have been reported previously. Collectively, these SAM- or SAH-sensing RNAs constitute the most abundant collection of riboswitches, and are found in nearly every major bacterial lineage. Here, we report a potential sixth member of this pervasive riboswitch family, called SAM-VI, which is predominantly found in Bifidobacterium species. SAM-VI aptamers selectively bind the cofactor SAM and strongly discriminate against SAH. The consensus sequence and structural model for SAM-VI share some features with the consensus model for the SAM-III riboswitch class, whose members are mainly found in lactic acid bacteria. However, there are sufficient differences between the two classes such that current bioinformatics methods separately cluster representatives of the two motifs. These findings highlight the abundance of RNA structures that can form to selectively recognize SAM, and showcase the ability of RNA to utilize diverse strategies to perform similar biological functions.

Orphan riboswitch candidates are noncoding RNA motifs whose representatives are believed to function as genetic regulatory elements, but whose target ligands have yet to be identified. The study of certain orphans, particularly classes that have resisted experimental validation for many years, has led to the discovery of important biological pathways and processes once their ligands were identified. Previously, we highlighted details for four of the most common and intriguing orphan riboswitch candidates. This facilitated the validation of riboswitches for the signaling molecules c-di-AMP, ZTP, and ppGpp, the metal ion Mn, and the metabolites guanidine and PRPP. Such studies also yield useful linkages between the ligands sensed by the riboswitches and numerous biochemical pathways. In the current report, we describe the known characteristics of 30 distinct classes of orphan riboswitch candidates - some of which have remained unsolved for over a decade. We also discuss the prospects for uncovering novel biological insights via focused studies on these RNAs. Lastly, we make recommendations for experimental objectives along the path to finding ligands for these mysterious RNAs.

We recently implemented a bioinformatics pipeline that can uncover novel, but rare, riboswitch candidates as well as other noncoding RNA structures in bacteria. A prominent candidate revealed by our initial search efforts was called the ‘thiS motif’ because of its frequent association with a gene coding for the ThiS protein, which delivers sulfur to form the thiazole moiety of the thiamin precursor HET-P. In the current report, we describe biochemical and genetic data demonstrating that thiS motif RNAs function as sensors of the thiamin precursor HMP-PP, which is fused with HET-P ultimately to form the final active coenzyme thiamin pyrophosphate (TPP). HMP-PP riboswitches exhibit a distinctive architecture wherein an unusually small ligand-sensing aptamer is almost entirely embedded within an otherwise classic intrinsic transcription terminator stem. This arrangement yields remarkably compact genetic switches that bacteria use to tune the levels of thiamin precursors during the biosynthesis of this universally distributed coenzyme.

We previously reported a large collection of structured noncoding RNAs (ncRNAs) that includes many riboswitch candidates identified through comparative sequence analysis of bacterial intergenic regions. One of these candidates, initially named the "folE motif," adopts a simple architecture commonly found upstream of folE genes. FolE enzymes catalyze the first enzyme in the de novo folate biosynthesis pathway. Herein, we demonstrate that folE motif RNAs selectively bind the enzyme cofactor tetrahydrofolate (THF) and several of its close derivatives. These aptamers, commonly found in Gramnegative bacteria, are distinct from aptamers of the previous validated THF riboswitch class found in Gram-positive bacteria. Our findings indicate that folE motif RNAs are aptamer domains for a second THF riboswitch class, named THF-II. The biochemical validation of THF-II riboswitches further highlights the ability of bacteria to utilize diverse RNA structures to sense universal enzyme cofactors that are predicted to be of ancient origin.

Since the elevated levels of microRNAs (miRNAs) often cause various diseases, selective inhibition of miRNA maturation is an important therapeutic strategy. Commonly used anti-miRNA strategies are limited to targeting of mature miRNAs, as the upstream targeting of miRNA maturation with an oligonucleotide is challenging due to the presence of a stable pre-miRNA stem-loop structure. Previously, we reported that about 16% of known human pre-miRNAs have the potential to adopt G-quadruplex (GQ) structures alternatively to canonical stem-loops. Herein, we showed that a rationally designed locked nucleic acid (LNA) binds specifically the GQ conformation of pre-miRNA 92b and inhibits pre-miRNA maturation. Further, we showed that the LNA treatment rescues PTEN expression in non-small-cell lung cancer (NSCLC) cells, which is suppressed by the elevated level of miRNA 92b. Treatment of LNA significantly decreases the IC of doxorubicin for NSCLC cells. This strategy can be developed as a novel anti-miRNA therapeutic approach to target GQ harboring miRNAs. This can potentially be a more powerful approach than targeting of the mature miRNA, as it is an upstream targeting and can reduce both 3' and the 5' mature miRNA levels at once.