Narasimhan Sudarsan

Riboswitches in Eubacteria Sense the Second Messenger Cyclic Di-GMP

Cyclic di-guanosine monophosphate (di-GMP) is a circular RNA dinucleotide that functions as a second messenger in diverse species of bacteria to trigger wide-ranging physiological changes, including cell differentiation, conversion between motile and biofilm lifestyles, and virulence gene expression. However, the mechanisms by which cyclic di-GMP regulates gene expression have remained a mystery. We found that cyclic di-GMP in many bacterial species is sensed by a riboswitch class in messenger RNA that controls the expression of genes involved in numerous fundamental cellular processes. A variety of cyclic di-GMP regulons are revealed, including some riboswitches associated with virulence gene expression, pilus formation, and flagellum biosynthesis. In addition, sequences matching the consensus for cyclic di-GMP riboswitches are present in the genome of a bacteriophage.

An mRNA structure that controls gene expression by binding S-adenosylmethionine.

Riboswitches are metabolite-binding RNA structures that serve as genetic control elements for certain messenger RNAs. These RNA switches have been identified in all three kingdoms of life and are typically responsible for the control of genes whose protein products are involved in the biosynthesis, transport or utilization of the target metabolite. Herein, we report that a highly conserved RNA domain found in bacteria serves as a riboswitch that responds to the coenzyme S-adenosylmethionine (SAM) with remarkably high affinity and specificity. SAM riboswitches undergo structural reorganization upon introduction of SAM, and these allosteric changes regulate the expression of 26 genes in Bacillus subtilis. This and related findings indicate that direct interaction between small metabolites and allosteric mRNAs is an important and widespread form of genetic regulation in bacteria.

Control of alternative RNA splicing and gene expression by eukaryotic riboswitches

Bacteria make extensive use of riboswitches to sense metabolites and control gene expression, and typically do so by modulating premature transcription termination or translation initiation. The most widespread riboswitch class known in bacteria responds to the coenzyme thiamine pyrophosphate (TPP), which is a derivative of vitamin B1. Representatives of this class have also been identified in fungi and plants, where they are predicted to control messenger RNA splicing or processing. We examined three TPP riboswitches in the filamentous fungus Neurospora crassa, and found that one activates and two repress gene expression by controlling mRNA splicing. A detailed mechanism involving riboswitch-mediated base-pairing changes and alternative splicing control was elucidated for precursor NMT1 mRNAs, which code for a protein involved in TPP metabolism. These results demonstrate that eukaryotic cells employ metabolite-binding RNAs to regulate RNA splicing events that are important for the control of key biochemical processes.

An Allosteric Self-Splicing Ribozyme Triggered by a Bacterial Second Messenger

Riboswitch Revealed Short regulatory regions—riboswitches—are found in the messenger RNAs of many bacteria, plants, and fungi. They bind to small-molecule metabolites and, through switching between alternate RNA secondary structures, regulate the expression of the linked RNA. Lee et al. (p. 845) have identified a c-di-GMP (cyclic di-guanosyl-5′-monophosphate)–binding riboswitch in the bacterium Clostridium difficile that regulates the splicing of a group I self-splicing ribozyme. Binding of c-di-GMP to the riboswitch favors a conformation of the ribozyme that promotes splicing in the presence of guanosine triphosphate (as is typical for this class of ribozymes). Concomitantly, splicing promotes the formation of a ribosome binding site, thereby stimulating protein production from the downstream pathogenesis-related gene. This regulatory region may thus constitute a two-input gene-control system that reads the concentration of both GTP and c-di-GMP. Thus, not all group I self-splicing ribozymes represent selfish genetic elements. A metabolite-sensing riboswitch regulates the splicing of a “selfish” group I self-splicing ribozyme. Group I self-splicing ribozymes commonly function as components of selfish mobile genetic elements. We identified an allosteric group I ribozyme, wherein self-splicing is regulated by a distinct riboswitch class that senses the bacterial second messenger c-di-GMP. The tandem RNA sensory system resides in the 5′ untranslated region of the messenger RNA for a putative virulence gene in the pathogenic bacterium Clostridium difficile. c-di-GMP binding by the riboswitch induces folding changes at atypical splice site junctions to modulate alternative RNA processing. Our findings indicate that some self-splicing ribozymes are not selfish elements but are harnessed by cells as metabolite sensors and genetic regulators.

Immobilized RNA switches for the analysis of complex chemical and biological mixtures

A prototype biosensor array has been assembled from engineered RNA molecular switches that undergo ribozyme-mediated self-cleavage when triggered by specific effectors. Each type of switch is prepared with a 5′-thiotriphosphate moiety that permits immobilization on gold to form individually addressable pixels. The ribozymes comprising each pixel become active only when presented with their corresponding effector, such that each type of switch serves as a specific analyte sensor. An addressed array created with seven different RNA switches was used to report the status of targets in complex mixtures containing metal ion, enzyme cofactor, metabolite, and drug analytes. The RNA switch array also was used to determine the phenotypes of Escherichia coli strains for adenylate cyclase function by detecting naturally produced 3′,5′- cyclic adenosine monophosphate (cAMP) in bacterial culture media.

Widespread Genetic Switches and Toxicity Resistance Proteins for Fluoride

Fluoride Riboswitch Riboswitches are found in prokaryote and eukaryote messenger RNAs (mRNAs), where they regulate expression of the linked mRNA through ligand binding and conformational change. Baker et al. (p. 233, published online 22 December) analyzed the binding properties of the “crcB motif” found in the noncoding RNA at the 5′ end of a diverse collection of prokaryotic genes. A crcB motif from Pseudomonas syringae was capable of selectively sensing the very small and highly charged fluoride ion. Some of the crcB and eriC genes associated with the fluoride riboswitch showed evidence of being fluoride transporters. The bacterium Methylobacterium extorquens DM4, which can use halogenated hydrocarbons as an energy source, was found to encode at least 10 fluoride riboswitches in its genome. A fluoride-sensing riboswitch regulates the expression of putative fluoride channels in prokaryotes. Most riboswitches are metabolite-binding RNA structures located in bacterial messenger RNAs where they control gene expression. We have discovered a riboswitch class in many bacterial and archaeal species whose members are selectively triggered by fluoride but reject other small anions, including chloride. These fluoride riboswitches activate expression of genes that encode putative fluoride transporters, enzymes that are known to be inhibited by fluoride, and additional proteins of unknown function. Our findings indicate that most organisms are naturally exposed to toxic levels of fluoride and that many species use fluoride-sensing RNAs to control the expression of proteins that alleviate the deleterious effects of this anion.

Riboswitches in eubacteria sense the second messenger c-di-AMP.

Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered bacterial second messenger implicated in the control of cell wall metabolism, osmotic stress responses, and sporulation. However, the mechanisms by which c-di-AMP triggers these physiological responses have remained largely unknown. Intriguingly, a candidate riboswitch class called ydaO associates with numerous genes involved in these same processes. Although a representative ydaO motif RNA recently was reported to weakly bind ATP, we report that numerous members of this noncoding RNA class selectively respond to c-di-AMP with sub-nanomolar affinity. Our findings resolve the mystery regarding the primary ligand for this extremely common riboswitch class and expose a major portion of the super-regulon of genes that are controlled by the widespread bacterial second messenger c-di-AMP.

Control of bacterial exoelectrogenesis by c-AMP-GMP

Significance The cyclic dinucleotides c-di-GMP and c-di-AMP are responsible for controlling broad changes in cell phenotypes during the life cycles of many bacterial species. To date, riboswitches that sense c-di-GMP and c-di-AMP have been discovered. The genomic locations of riboswitches reveal numerous genes that are controlled by these signaling compounds and thereby reveal the biological processes that are regulated. Herein, we report that a subset of conserved noncoding RNA domains previously annotated as c-di-GMP-I riboswitches in fact bind the newly discovered second messenger c-AMP-GMP. These riboswitches control many genes involved in the utilization of extracellular iron(III) oxide as an electron sink by various Geobacter species. Our findings reveal additional roles for this recently discovered signaling molecule. Major changes in bacterial physiology including biofilm and spore formation involve signaling by the cyclic dinucleotides c-di-GMP and c-di-AMP. Recently, another second messenger dinucleotide, c-AMP-GMP, was found to control chemotaxis and colonization by Vibrio cholerae. We have identified a superregulon of genes controlled by c-AMP-GMP in numerous Deltaproteobacteria, including Geobacter species that use extracellular insoluble metal oxides as terminal electron acceptors. This exoelectrogenic process has been studied for its possible utility in energy production and bioremediation. Many genes involved in adhesion, pilin formation, and others that are important for exoelectrogenesis are controlled by members of a variant riboswitch class that selectively bind c-AMP-GMP. These RNAs constitute, to our knowledge, the first known specific receptors for c-AMP-GMP and reveal that this molecule is used by many bacteria to control specialized physiological processes.

Challenges of ligand identification for riboswitch candidates.

Expanding DNA sequence databases and improving methods for comparative analysis are being exploited to identify numerous noncoding RNA elements including riboswitches. Ligands for many riboswitch classes usually can be inferred based on the genomic contexts of representative RNAs, and complex formation or genetic regulation subsequently demonstrated experimentally. However, there are several candidate riboswitches for which ligands have not been identified. In this report, we discuss three of the most compelling riboswitch candidates: the ykkC/ykkD, yybP/ykoY and pfl RNAs. Each of these RNAs is numerous, phylogenetically widespread, and carries features that are hallmarks of metabolite-binding riboswitches, such as a well-conserved aptamer-like structure and apparent interactions with gene regulation elements such as ribosome binding sites or intrinsic transcription termination stems. These RNAs likely represent only a small sampling of the challenging motifs that researchers will encounter as new noncoding RNAs are identified.

Identification of ligand analogues that control c-di-GMP riboswitches.

Riboswitches for the bacterial second messenger c-di-GMP control the expression of genes involved in numerous cellular processes such as virulence, competence, biofilm formation, and flagella synthesis. Therefore, the two known c-di-GMP riboswitch classes represent promising targets for developing novel modulators of bacterial physiology. Here, we examine the binding characteristics of circular and linear c-di-GMP analogues for representatives of both class I and II c-di-GMP riboswitches derived from the pathogenic bacterium Vibrio choleae (class I) and Clostridium difficile (class II). Some compounds exhibit values for apparent dissociation constant (K(D)) below 1 μM and associate with riboswitch RNAs during transcription with a speed that is sufficient to influence riboswitch function. These findings are consistent with the published structural models for these riboswitches and suggest that large modifications at various positions on the ligand can be made to create novel compounds that target c-di-GMP riboswitches. Moreover, we demonstrate the potential of an engineered allosteric ribozyme for the rapid screening of chemical libraries for compounds that bind c-di-GMP riboswitches.