Faculty Bibliography

Recent studies have revealed that the majority of biological processes are controlled by noncoding RNAs. Among many classes of noncoding RNAs, metabolite-sensing segments of mRNAs called riboswitches are unique. Discovered over a decade ago in all three kingdoms of life, riboswitches specifically and directly interact with various metabolites and regulate expression of multiple genes, often associated with metabolism and transport of small molecules. Thus, riboswitches do not depend on proteins for binding to small molecules and play a role as both metabolite sensors and effectors of gene control. Riboswitches are typically located in the untranslated regions of mRNAs where they form alternative structures in the presence and absence of the ligand and modulate expression of genes through the formation of regulatory elements. To understand the mechanism of the riboswitch-driven gene control, it is important to elucidate how riboswitches interact with cognate and discriminate against non-cognate ligands. Here we outline the methodology to synthesize riboswitch RNAs and prepare riboswitch-ligand complexes for crystallographic and biochemical studies. The chapter describes how to design, prepare, and conduct crystallization screening of riboswitch-ligand complexes. The methodology was refined on crystallographic studies of several riboswitches and can be employed for other types of RNA molecules.

Naturally occurring L-glutamine riboswitches occur in cyanobacteria and marine metagenomes, where they reside upstream of genes involved in nitrogen metabolism. By combining X-ray, NMR, and MD, we characterized an L-glutamine-dependent conformational transition in the Synechococcus elongatus glutamine riboswitch from tuning fork to L-shaped alignment of stem segments. This transition generates an open ligand-binding pocket with L-glutamine selectivity enforced by Mg(2+)-mediated intermolecular interactions. The transition also stabilizes the P1 helix through a long-range "linchpin" Watson-Crick G-C pair-capping interaction, while melting a short helix below P1 potentially capable of modulating downstream readout. NMR data establish that the ligand-free glutamine riboswitch in Mg(2+) solution exists in a slow equilibrium between flexible tuning fork and a minor conformation, similar, but not identical, to the L-shaped bound conformation. We propose that an open ligand-binding pocket combined with a high conformational penalty for forming the ligand-bound state provide mechanisms for reducing binding affinity while retaining high selectivity.

Recent progress in identification and characterization of novel types of non-coding RNAs has proven that RNAs carry out a variety of cellular functions ranging from scaffolding to gene expression control. In both prokaryotic and eukaryotic cells, several classes of non-coding RNAs control expression of dozens of genes in response to specific cues. One of the most interesting and outstanding questions in the RNA field is whether regulatory RNAs are capable of employing basic biological concepts, such as allostery and cooperativity, previously attributed to the function of proteins. Aside from regulatory RNAs that form complementary base pairing with their nucleic acid targets, several RNA classes modulate gene expression via molecular mechanisms which can be paralleled to protein-mediated regulation. Among these RNAs are riboswitches, metabolite-sensing non-coding regulatory elements that adopt intrinsic three-dimensional structures and specifically bind various small molecule ligands. These characteristics of riboswitches make them well-suited for complex regulatory responses observed in allosteric and cooperative protein systems. Here we present an overview of the biochemical, genetic, and structural studies of riboswitches with a major focus on complex regulatory mechanisms and biological principles utilized by riboswitches for such genetic modulation.

Fragile X Mental Retardation Protein (FMRP) is a regulatory RNA binding protein that plays a central role in the development of several human disorders including Fragile X Syndrome (FXS) and autism. FMRP uses an arginine-glycine-rich (RGG) motif for specific interactions with guanine (G)-quadruplexes, mRNA elements implicated in the disease-associated regulation of specific mRNAs. Here we report the 2.8-A crystal structure of the complex between the human FMRP RGG peptide bound to the in vitro selected G-rich RNA. In this model system, the RNA adopts an intramolecular K+-stabilized G-quadruplex structure composed of three G-quartets and a mixed tetrad connected to an RNA duplex. The RGG peptide specifically binds to the duplex-quadruplex junction, the mixed tetrad, and the duplex region of the RNA through shape complementarity, cation-pi interactions, and multiple hydrogen bonds. Many of these interactions critically depend on a type I beta-turn, a secondary structure element whose formation was not previously recognized in the RGG motif of FMRP. RNA mutagenesis and footprinting experiments indicate that interactions of the peptide with the duplex-quadruplex junction and the duplex of RNA are equally important for affinity and specificity of the RGG-RNA complex formation. These results suggest that specific binding of cellular RNAs by FMRP may involve hydrogen bonding with RNA duplexes and that RNA duplex recognition can be a characteristic RNA binding feature for RGG motifs in other proteins.

This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5-3.2 A, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson-Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/.

5'-End-dependent RNA degradation impacts virulence, stress responses, and DNA repair in bacteria by controlling the decay of hundreds of mRNAs. The RNA pyrophosphohydrolase RppH, a member of the Nudix hydrolase superfamily, triggers this degradation pathway by removing pyrophosphate from the triphosphorylated RNA 5' terminus. Here, we report the x-ray structures of Escherichia coli RppH (EcRppH) in apo- and RNA-bound forms. These structures show distinct conformations of EcRppH.RNA complexes on the catalytic pathway and suggest a common catalytic mechanism for Nudix hydrolases. EcRppH interacts with RNA by a bipartite mechanism involving specific recognition of the 5'-terminal triphosphate and the second nucleotide, thus enabling discrimination against mononucleotides as substrates. The structures also reveal the molecular basis for the preference of the enzyme for RNA substrates bearing guanine in the second position by identifying a protein cleft in which guanine interacts with EcRppH side chains via cation-pi contacts and hydrogen bonds. These interactions explain the modest specificity of EcRppH at the 5' terminus and distinguish the enzyme from the highly selective RppH present in Bacillus subtilis. The divergent means by which RNA is recognized by these two functionally and structurally analogous enzymes have important implications for mRNA decay and the regulation of protein biosynthesis in bacteria.

Natural RNA molecules can have a high degree of structural complexity but even the most complexly folded RNAs are assembled from simple structural building blocks. Among the simplest RNA elements are double-stranded helices that participate in the formation of different folding topologies and constitute the major fraction of RNA structures. One common folding motif of RNA is a pseudoknot, defined as a bipartite helical structure formed by base-pairing of the apical loop in the stem-loop structure with an outside sequence. Pseudoknots constitute integral parts of the RNA structures essential for various cellular activities. Among many functions of pseudoknotted RNAs is feedback regulation of gene expression, carried out through specific recognition of various molecules. Pseudoknotted RNAs autoregulate ribosomal and phage protein genes in response to downstream encoded proteins, while many metabolic and transport genes are controlled by cellular metabolites interacting with pseudoknotted RNA elements from the riboswitch family. Modulation of some genes also depends on metabolite-induced messenger RNA (mRNA) cleavage performed by pseudoknotted ribozymes. Several regulatory pseudoknots have been characterized biochemically and structurally in great detail. These studies have demonstrated a plethora of pseudoknot-based folds and have begun uncovering diverse molecular principles of the ligand-dependent gene expression control. The pseudoknot-mediated mechanisms of gene control and many unexpected and interesting features of the regulatory pseudoknots have significantly advanced our understanding of the genetic circuits and laid the foundation for modulation of their outcomes. WIREs RNA 2014, 5:803-822. doi: 10.1002/wrna.1247 For further resources related to this article, please visit the WIREs website. CONFLICT OF INTEREST: The authors have declared no conflicts of interest for this article.

The complexity of gene expression control by non-coding RNA has been highlighted by the recent progress in the field of riboswitches. Discovered a decade ago, riboswitches represent a diverse group of non-coding mRNA regions that possess a unique ability to directly sense cellular metabolites and modulate gene expression through formation of alternative metabolite-free and metabolite-bound conformations. Such protein-free metabolite sensing domains utilize sophisticated three-dimensional folding of RNA molecules to discriminate between a cognate ligand from related compounds so that only the right ligand would trigger a genetic response. Given the variety of riboswitch ligands ranging from small cations to large coenzymes, riboswitches adopt a great diversity of structures. Although many riboswitches share structural principles to build metabolite-competent folds, form precise ligand-binding pockets, and communicate a ligand-binding event to downstream regulatory regions, virtually all riboswitch classes possess unique features for ligand recognition, even those tuned to recognize the same metabolites. Here we present an overview of the biochemical and structural research on riboswitches with a major focus on common principles and individual characteristics adopted by these regulatory RNA elements during evolution to specifically target small molecules and exert genetic responses. This article is part of a Special Issue entitled: Riboswitches.

Bacterial second messenger cyclic di-AMP (c-di-AMP) is implicated in signaling DNA damage and cell wall stress through interactions with several protein receptors and a widespread ydaO-type riboswitch. We report the crystal structures of c-di-AMP riboswitches from Thermoanaerobacter pseudethanolicus and Thermovirga lienii determined at approximately 3.0-A resolution. In both species, the RNA adopts an unforeseen 'square'-shaped pseudosymmetrical architecture that features two three-way junctions, a turn and a pseudoknot, positioned in the square corners. Uncharacteristically for riboswitches, the structure is stapled by two ligand molecules that span the interior of the structure and employ similar noncanonical interactions for RNA recognition. Mutations in either ligand-binding site negatively affect c-di-AMP binding, suggesting that the riboswitch-triggered genetic response requires contribution of both ligands. Our data provide what are to our knowledge the first insights into specific sensing of c-di-AMP and a molecular mechanism underlying the common c-di-AMP-dependent control of essential cellular processes in bacteria.

RNA-Puzzles is a CASP-like collective blind experiment for the evaluation of RNA 3-dimensional structure prediction. The primary aims of RNA-Puzzles are to determine the capabilities and limitations of current methods of 3D RNA structure prediction based on sequence, to find whether and how progress has been made, and to illustrate whether there are specific bottlenecks that hold back the field. Ten puzzles have been set up and three assessments are published. Nine groups of modelers around the world participate in this collective effort. We now report a second round focusing on the prediction of two large riboswitches, the adenosylcobalamin and the T-box bound to a tRNA. No homologous structures existed in the databases at the time of the experiment. Although only two targets were selected, these targets provide a wealth of sub-domains (around 10), including both well-known modules like K-turns as well as new ones. The 168nt adenosylcobalamin riboswitch consists of a ligandbound structured core and a bent peripheral domain. Although the RMSDs of the prediction models range from 11.7 to 37.5 A, the topology of the top ranked models are quite similar to the native structure. Top ranked models show much better scores in Deformation Index (DI) and non-Watson-Crick interaction network fidelity (nwc INF) than others, but surprisingly have worse clash scores. The T-box and tRNA, 96 and 75nt in length respectively, form a large complex. The difficulty in prediction lies mainly in (i) the lack of homologous model for T-box and (ii) the interaction between T-box and tRNA. The RMSD range of the predictions is 6.8-17.4 A and the top ranked models also have better DI score with worse clash scores. The Das group performed best in both problems with their models ranked #1 at 14.5 and 7.6 A, respectively. The Bujnicki group performed well in the second problem with the model ranked #1 at 10.2 A and excellent clash scores with nwc INF around 0.5 like the models of the Das group. Further, the less well predicte!