Faculty Bibliography

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.

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.

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!

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.

Riboswitches were discovered in 2002 in bacteria as RNA-based intracellular sensors of vitamin derivatives. During the last decade, naturally occurring RNA sensor elements have been found to bind a range of small metabolites and ions and to exert regulatory control of transcription, translation, splicing, and RNA stability. Extensive biochemical, structural, and genetic studies have established the basic principles underpinning riboswitch function in all three kingdoms of life with implications for developing antibiotics, designing new molecular sensors, and integrating riboswitches into synthetic circuits.

We report the results of a first, collective, blind experiment in RNA three-dimensional (3D) structure prediction, encompassing three prediction puzzles. The goals are to assess the leading edge of RNA structure prediction techniques; compare existing methods and tools; and evaluate their relative strengths, weaknesses, and limitations in terms of sequence length and structural complexity. The results should give potential users insight into the suitability of available methods for different applications and facilitate efforts in the RNA structure prediction community in ongoing efforts to improve prediction tools. We also report the creation of an automated evaluation pipeline to facilitate the analysis of future RNA structure prediction exercises.

Coenzyme B(12) has a key role in various enzymatic reactions and controls expression of bacterial genes through riboswitches. Here we report the crystal structure of the Symbiobacterium thermophilum B(12) riboswitch bound to its ligand adenosylcobalamin. The riboswitch forms a unique junctional structure with a large ligand-binding pocket tailored for specific recognition of the adenosyl moiety and flanked by structural elements that stabilize the regulatory region and enable control of gene expression.

Regulatory mRNAs elements termed riboswitches respond to elevated concentrations of cellular metabolites by modulating expression of associated genes. Riboswitches attain their high metabolite selectivity by capitalizing on the intrinsic tertiary structures of their sensor domains. Over the years, riboswitch structure and folding have been amongst the most researched topics in the RNA field. Most recently, novel structures of single-ligand and cooperative double-ligand sensors have broadened our knowledge of architectural and molecular recognition principles exploited by riboswitches. The structural information has been complemented by extensive folding studies, which have provided several important clues on the formation of ligand-competent conformations and mechanisms of ligand discrimination. These studies have greatly improved our understanding of molecular events in riboswitch-mediated gene expression control and provided the molecular basis for intervention into riboswitch-controlled genetic circuits.

Riboswitches are mRNA elements capable of modulating gene expression in response to specific binding by cellular metabolites. Riboswitches exert their function through the interplay of alternative ligand-free and ligand-bound conformations of the metabolite-sensing domain, which in turn modulate the formation of adjacent gene expression controlling elements. X-ray crystallography and NMR spectroscopy have determined three-dimensional structures of virtually all the major riboswitch classes in the ligand-bound state and, for several riboswitches, in the ligand-free state. The resulting spatial topologies have demonstrated the wide diversity of riboswitch folds and revealed structural principles for specific recognition by cognate metabolites. The available three-dimensional information, supplemented by structure-guided biophysical and biochemical experimentation, has led to an improved understanding of how riboswitches fold, what RNA conformations are required for ligand recognition, and how ligand binding can be transduced into gene expression modulation. These studies have greatly facilitated the dissection of molecular mechanisms underlying riboswitch action and should in turn guide the anticipated development of tools for manipulating gene regulatory circuits.

We have determined the solution structure of the complex between an arginine-glycine-rich RGG peptide from the human fragile X mental retardation protein (FMRP) and an in vitro-selected guanine-rich (G-rich) sc1 RNA. The bound RNA forms a newly discovered G-quadruplex separated from the flanking duplex stem by a mixed junctional tetrad. The RGG peptide is positioned along the major groove of the RNA duplex, with the G-quadruplex forcing a sharp turn of R(10)GGGGR(15) at the duplex-quadruplex junction. Arg10 and Arg15 form cross-strand specificity-determining intermolecular hydrogen bonds with the major-groove edges of guanines of adjacent Watson-Crick G*C pairs. Filter-binding assays on RNA and peptide mutations identify and validate contributions of peptide-RNA intermolecular contacts and shape complementarity to molecular recognition. These findings on FMRP RGG domain recognition by a combination of G-quadruplex and surrounding RNA sequences have implications for the recognition of other genomic G-rich RNAs.