Daniel A. Lafontaine

Novel riboswitch ligand analogs as selective inhibitors of guanine-related metabolic pathways.

Riboswitches are regulatory elements modulating gene expression in response to specific metabolite binding. It has been recently reported that riboswitch agonists may exhibit antimicrobial properties by binding to the riboswitch domain. Guanine riboswitches are involved in the regulation of transport and biosynthesis of purine metabolites, which are critical for the nucleotides cellular pool. Upon guanine binding, these riboswitches stabilize a 5′-untranslated mRNA structure that causes transcription attenuation of the downstream open reading frame. In principle, any agonistic compound targeting a guanine riboswitch could cause gene repression even when the cell is starved for guanine. Antibiotics binding to riboswitches provide novel antimicrobial compounds that can be rationally designed from riboswitch crystal structures. Using this, we have identified a pyrimidine compound (PC1) binding guanine riboswitches that shows bactericidal activity against a subgroup of bacterial species including well-known nosocomial pathogens. This selective bacterial killing is only achieved when guaA, a gene coding for a GMP synthetase, is under the control of the riboswitch. Among the bacterial strains tested, several clinical strains exhibiting multiple drug resistance were inhibited suggesting that PC1 targets a different metabolic pathway. As a proof of principle, we have used a mouse model to show a direct correlation between the administration of PC1 and the reduction of Staphylococcus aureus infection in mammary glands. This work establishes the possibility of using existing structural knowledge to design novel guanine riboswitch-targeting antibiotics as powerful and selective antimicrobial compounds. Particularly, the finding of this new guanine riboswitch target is crucial as community-acquired bacterial infections have recently started to emerge.

Comparative Study between Transcriptionally- and Translationally-Acting Adenine Riboswitches Reveals Key Differences in Riboswitch Regulatory Mechanisms

Many bacterial mRNAs are regulated at the transcriptional or translational level by ligand-binding elements called riboswitches. Although they both bind adenine, the adenine riboswitches of Bacillus subtilis and Vibrio vulnificus differ by controlling transcription and translation, respectively. Here, we demonstrate that, beyond the obvious difference in transcriptional and translational modulation, both adenine riboswitches exhibit different ligand binding properties and appear to operate under different regulation regimes (kinetic versus thermodynamic). While the B. subtilis pbuE riboswitch fully depends on co-transcriptional binding of adenine to function, the V. vulnificus add riboswitch can bind to adenine after transcription is completed and still perform translation regulation. Further investigation demonstrates that the rate of transcription is critical for the B. subtilis pbuE riboswitch to perform efficiently, which is in agreement with a co-transcriptional regulation. Our results suggest that the nature of gene regulation control, that is transcription or translation, may have a high importance in riboswitch regulatory mechanisms.

Therapeutic applications of ribozymes and riboswitches.

Therapeutic approaches employing RNA as a tool or as a drug target have recently emerged and have been employed for various applications-ranging from cancer treatment to virus infection. Despite the paucity of its molecular groups compared to proteins, RNA has nevertheless proved to be an excellent choice for researchers who have aspired to develop therapeutic tools. Ribozymes and riboswitches are RNA-based therapeutic tools that are most often employed to knockdown gene expression and to inhibit bacterial infections, respectively. The aim of this review is to summarize recent advances observed in ribozyme- and riboswitch-based therapeutic applications that, in some cases, have reached clinical trials.

Molecular insights into the ligand-controlled organization of the SAM-I riboswitch

S-adenosylmethionine (SAM) riboswitches are widespread in bacteria, and up to five different SAM riboswitch families have been reported, highlighting the relevance of SAM regulation. On the basis of crystallographic and biochemical data, it has been postulated, but never demonstrated, that ligand recognition by SAM riboswitches involves key conformational changes in the RNA architecture. We show here that the aptamer follows a two-step hierarchical folding selectively induced by metal ions and ligand binding, each of them leading to the formation of one of the two helical stacks observed in the crystal structure. Moreover, we find that the anti-antiterminator P1 stem is rotated along its helical axis upon ligand binding, a mechanistic feature that could be common to other riboswitches. We also show that the nonconserved P4 helical domain is used as an auxiliary element to enhance the ligand-binding affinity. This work provides the first comprehensive characterization, to our knowledge, of a ligand-controlled riboswitch folding pathway.

Regulatory RNAs and target mRNA decay in prokaryotes.

Recent advances in prokaryote genetics have highlighted the important and complex roles of small regulatory RNAs (sRNAs). Although blocking mRNA translation is often the main function of sRNAs, these molecules can also induce the degradation of target mRNAs using a mechanism that drastically differs from eukaryotic RNA interference (RNAi). Whereas RNAi relies on RNase III-like machinery that is specific to double-strand RNAs, sRNA-mediated mRNA degradation in Escherichia coli and Samonella typhimurium depends on RNase E, a single-strand specific endoribonuclease. Surprisingly, the latest descriptions of sRNA-mediated mRNA degradation in various bacteria suggest a variety of previously unsuspected mechanisms. In this review, we focus on recently characterized mechanisms in which sRNAs can bind to target mRNAs to induce decay. These new mechanisms illustrate how sRNAs and mRNA structures, including riboswitches, act cooperatively with protein partners to initiate the decay of mRNAs. This article is part of a Special Issue entitled: RNA Decay mechanisms.

Dual-acting riboswitch control of translation initiation and mRNA decay

Riboswitches are mRNA regulatory elements that control gene expression by altering their structure in response to specific metabolite binding. In bacteria, riboswitches consist of an aptamer that performs ligand recognition and an expression platform that regulates either transcription termination or translation initiation. Here, we describe a dual-acting riboswitch from Escherichia coli that, in addition to modulating translation initiation, also is directly involved in the control of initial mRNA decay. Upon lysine binding, the lysC riboswitch adopts a conformation that not only inhibits translation initiation but also exposes RNase E cleavage sites located in the riboswitch expression platform. However, in the absence of lysine, the riboswitch folds into an alternative conformation that simultaneously allows translation initiation and sequesters RNase E cleavage sites. Both regulatory activities can be individually inhibited, indicating that translation initiation and mRNA decay can be modulated independently using the same conformational switch. Because RNase E cleavage sites are located in the riboswitch sequence, this riboswitch provides a unique means for the riboswitch to modulate RNase E cleavage activity directly as a function of lysine. This dual inhibition is in contrast to other riboswitches, such as the thiamin pyrophosphate-sensing thiM riboswitch, which triggers mRNA decay only as a consequence of translation inhibition. The riboswitch control of RNase E cleavage activity is an example of a mechanism by which metabolite sensing is used to regulate gene expression of single genes or even large polycistronic mRNAs as a function of environmental changes.

Riboswitches: Ancient and Promising Genetic Regulators

Bait and switch: Metabolite‐sensing riboswitches make use of RNA structural modulation to regulate gene expression, as illustrated in the scheme, in response to subtle changes in metabolite concentrations. This review describes the current knowledge about naturally occurring riboswitches and their growing potential as antibacterial cellular targets and as molecular biosensors.

Folding of the SAM aptamer is determined by the formation of a K-turn-dependent pseudoknot.

The S-adenosylmethionine (SAM) riboswitch is one of the most recurrent riboswitches found in bacteria and has three known different natural aptamers. The Bacillus subtilis yitJ SAM riboswitch aptamer is organized around a four-way junction which is characterized by the presence of a pseudoknot and a K-turn motif. By replacing the adenine involved in a Watson-Crick base pair at position 138 in the core region of the aptamer with the fluorescent analogue 2-aminopurine (2AP), we show that the ligand-induced reorganization of the aptamer strongly attenuates 2AP fluorescence. The fluorescence quenching process is specific to SAM on the basis of the observation that the structural analogue S-adenosylhomocysteine does not promote a similar effect. We find that the pseudoknot is important for the reorganization of the core domain and that the K-turn motif also has a marked influence on the core domain reorganization, most probably through its important role in pseudoknot formation. Finally, we show that SAM riboswitch ligand binding is facilitated by the L7Ae K-turn binding protein, which suggests that K-turn motifs may be protein anchor sites used by riboswitches to promote RNA folding.

Small RNA-mediated regulation at the level of transcript stability

In recent years, bacterial small regulatory RNAs (sRNAs) have been demonstrated to be powerful modulators of gene expression. Whether it is by modulating mRNA functions or protein activities, sRNAs often employ unexpected and extremely diverse mechanisms to modify the genetic output. Although the first sRNAs were characterized as molecules blocking translation of specific target mRNAs, this review will focus on an emerging subset of sRNAs that promote the decay of their target mRNAs. While the outcome resembles the RNAi silencing described in eukaryotes, the mechanism of bacterial sRNAs differs fundamentally. These sRNAs are the subject of intensive studies, which makes them the best characterized sRNAs to date.

New insights into riboswitch regulation mechanisms

Riboswitches are genetic elements located in non‐coding regions of some messenger RNAs (mRNAs) that are present in all three domains of life. The binding of ligands to riboswitches induces conformational changes in the mRNA molecule, resulting in modulation of gene transcription, or RNA splicing, translation or stability. This mechanism of regulation is particularly widespread in bacteria and allows a direct response to various metabolic changes. A large number of riboswitches have been discovered in the last few years, suggesting the existence of a huge diversity of regulatory ligands and genetic mechanisms of regulation. This review focuses on recent discoveries in riboswitch regulatory mechanisms as well as current outstanding challenges.