Zasha Weinberg

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 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.

CMfinder---a covariance model based RNA motif finding algorithm

MOTIVATION The recent discoveries of large numbers of non-coding RNAs and computational advances in genome-scale RNA search create a need for tools for automatic, high quality identification and characterization of conserved RNA motifs that can be readily used for database search. Previous tools fall short of this goal. RESULTS CMfinder is a new tool to predict RNA motifs in unaligned sequences. It is an expectation maximization algorithm using covariance models for motif description, featuring novel integration of multiple techniques for effective search of motif space, and a Bayesian framework that blends mutual information-based and folding energy-based approaches to predict structure in a principled way. Extensive tests show that our method works well on datasets with either low or high sequence similarity, is robust to inclusion of lengthy extraneous flanking sequence and/or completely unrelated sequences, and is reasonably fast and scalable. In testing on 19 known ncRNA families, including some difficult cases with poor sequence conservation and large indels, our method demonstrates excellent average per-base-pair accuracy--79% compared with at most 60% for alternative methods. More importantly, the resulting probabilistic model can be directly used for homology search, allowing iterative refinement of structural models based on additional homologs. We have used this approach to obtain highly accurate covariance models of known RNA motifs based on small numbers of related sequences, which identified homologs in deeply-diverged species.

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.

A widespread riboswitch candidate that controls bacterial genes involved in molybdenum cofactor and tungsten cofactor metabolism

We have identified a highly conserved RNA motif located upstream of genes encoding molybdate transporters, molybdenum cofactor (Moco) biosynthesis enzymes, and proteins that utilize Moco as a coenzyme. Bioinformatics searches have identified 176 representatives in γ‐Proteobacteria, δ‐Proteobacteria, Clostridia, Actinobacteria, Deinococcus‐Thermus species and DNAs from environmental samples. Using genetic assays, we demonstrate that a Moco RNA in Escherichia coli associated with the Moco biosynthetic operon controls gene expression in response to Moco production. In addition, we provide evidence indicating that this conserved RNA discriminates against closely related analogues of Moco. These results, together with extensive phylogenetic conservation and typical gene control structures near some examples, indicate that representatives of this structured RNA represent a novel class of riboswitches that sense Moco. Furthermore, we identify variants of this RNA that are likely to be triggered by the related tungsten cofactor (Tuco), which carries tungsten in place of molybdenum as the metal constituent.

A widespread self-cleaving ribozyme class is revealed by bioinformatics

Ribozymes are noncoding RNAs that promote chemical transformations with rate enhancements approaching those of protein enzymes. Although ribozymes are likely to have been abundant during the RNA world era, only ten classes are known to exist among contemporary organisms. We report the discovery and analysis of an additional self-cleaving ribozyme class, called twister, which is present in many species of bacteria and eukarya. Nearly 2700 twister ribozymes were identified that conform to a secondary structure consensus that is small yet complex, with three stems conjoined by internal and terminal loops. Two pseudoknots provide tertiary structure contacts that are critical for catalytic activity. The twister ribozyme motif provides another example of a natural RNA catalyst and calls attention to the potentially varied biological roles of this and other classes of widely distributed self-cleaving RNAs.

Sequence-based heuristics for faster annotation of non-coding RNA families

MOTIVATION Non-coding RNAs (ncRNAs) are functional RNA molecules that do not code for proteins. Covariance Models (CMs) are a useful statistical tool to find new members of an ncRNA gene family in a large genome database, using both sequence and, importantly, RNA secondary structure information. Unfortunately, CM searches are extremely slow. Previously, we created rigorous filters, which provably sacrifice none of a CMs accuracy, while making searches significantly faster for virtually all ncRNA families. However, these rigorous filters make searches slower than heuristics could be. RESULTS In this paper we introduce profile HMM-based heuristic filters. We show that their accuracy is usually superior to heuristics based on BLAST. Moreover, we compared our heuristics with those used in tRNAscan-SE, whose heuristics incorporate a significant amount of work specific to tRNAs, where our heuristics are generic to any ncRNA. Performance was roughly comparable, so we expect that our heuristics provide a high-quality solution that--unlike family-specific solutions--can scale to hundreds of ncRNA families. AVAILABILITY The source code is available under GNU Public License at the supplementary web site.

R2R--software to speed the depiction of aesthetic consensus RNA secondary structures.

BackgroundWith continuing identification of novel structured noncoding RNAs, there is an increasing need to create schematic diagrams showing the consensus features of these molecules. RNA structural diagrams are typically made either with general-purpose drawing programs like Adobe Illustrator, or with automated or interactive programs specific to RNA. Unfortunately, the use of applications like Illustrator is extremely time consuming, while existing RNA-specific programs produce figures that are useful, but usually not of the same aesthetic quality as those produced at great cost in Illustrator. Additionally, most existing RNA-specific applications are designed for drawing single RNA molecules, not consensus diagrams.ResultsWe created R2R, a computer program that facilitates the generation of aesthetic and readable drawings of RNA consensus diagrams in a fraction of the time required with general-purpose drawing programs. Since the inference of a consensus RNA structure typically requires a multiple-sequence alignment, the R2R user annotates the alignment with commands directing the layout and annotation of the RNA. R2R creates SVG or PDF output that can be imported into Adobe Illustrator, Inkscape or CorelDRAW. R2R can be used to create consensus sequence and secondary structure models for novel RNA structures or to revise models when new representatives for known RNA classes become available. Although R2R does not currently have a graphical user interface, it has proven useful in our efforts to create 100 schematic models of distinct noncoding RNA classes.ConclusionsR2R makes it possible to obtain high-quality drawings of the consensus sequence and structural models of many diverse RNA structures with a more practical amount of effort. R2R software is available at http://breaker.research.yale.edu/R2R and as an Additional file.

The aptamer core of SAM-IV riboswitches mimics the ligand-binding site of SAM-I riboswitches

A novel family of riboswitches, called SAM-IV, is the fourth distinct set of mRNA elements to be reported that regulate gene expression via direct sensing of S-adenosylmethionine (SAM or AdoMet). SAM-IV riboswitches share conserved nucleotide positions with the previously described SAM-I riboswitches, despite rearranged structures and nucleotide positions with family-specific nucleotide identities. Sequence analysis and molecular recognition experiments suggest that SAM-I and SAM-IV riboswitches share similar ligand binding sites, but have different scaffolds. Our findings support the view that RNA has considerable structural versatility and reveal that riboswitches exploit this potential to expand the scope of RNA in genetic regulation.