Eukaryotic Riboswitches Research Articles

Cell-Free Biosensors Based on Modular Eukaryotic Riboswitches That Function in One Pot at Ambient Temperature.

Artificial riboswitches responsive to user-defined analytes can be constructed by successfully inserting in vitro selected aptamers, which bind to the analytes, into untranslated regions of mRNA. Among them, eukaryotic riboswitches are more promising as biosensors than bacterial ones because they function well at ambient temperature. In addition, cell-free expression systems allow the broader use of these riboswitches as cell-free biosensors in an environmentally friendly manner without cellular limitations. The current best cell-free eukaryotic riboswitch regulates eukaryotic canonical translation initiation through self-cleavage mediated by an implanted analyte-responsive ribozyme (i.e., an aptazyme, an aptamer-ribozyme fusion). However, it has critical flaws as a sensor: due to the less-active ribozyme used, self-cleavage and translation reactions must be conducted separately and sequentially, and a different aptazyme has to be selected to change the analyte specificity, even if an aptamer for the next analyte is available. We here stepwise engineered novel types of cell-free eukaryotic riboswitches that harness highly active self-cleavage and thus require no reaction partitioning. Despite the single-step and one-pot reaction, these riboswitches showed higher analyte dose dependency and sensitivities than the current best cell-free eukaryotic riboswitch requiring multistep reactions. In addition, the analyte specificity can be changed in an extremely facile way, simply by aptamer substitution (and the subsequent simple fine-tuning for giant aptamers). Given that cell-free systems can be lyophilized for storage and transport, the present one-pot and thus easy-to-handle cell-free biosensors utilizing eukaryotic riboswitches are expected to be widely used for on-the-spot sensing of analytes at ambient temperature.

Rational design of eukaryotic riboswitches that up-regulate IRES-mediated translation initiation with high switching efficiency through a kinetic trapping mechanism in vitro.

In general, riboswitches functioning through a cotranscriptional kinetic trapping mechanism (kt-riboswitches) show higher switching efficiencies in response to practical concentrations of their ligand molecules than eq-riboswitches, which function by an equilibrium mechanism. However, the former have been much more difficult to design due to their more complex mechanism. We here successfully developed a rational strategy for constructing eukaryotic kt-riboswitches that ligand-dependently enhance translation initiation mediated by an internal ribosome entry site (IRES). This was achieved both by utilizing some predicted structural features of a highly efficient bacterial kt-riboswitch identified through screening and by completely decoupling an aptamer domain from the IRES. Three kt-riboswitches optimized through this strategy, each responding to a different ligand, exhibited three- to sevenfold higher induction ratios (up to ∼90) than previously optimized eq-riboswitches regulating the same IRES-mediated translation in wheat germ extract. Because the IRES used functions well in various eukaryotic expression systems, these types of kt-riboswitches are expected to serve as major eukaryotic gene regulators based on RNA. In addition, the present strategy could be applied to the rational construction of other types of kt-riboswitches, including those functioning in bacterial expression systems.

A computational approach for the identification of distant homologs of bacterial riboswitches based on inverse RNA folding.

Riboswitches are conserved structural ribonucleic acid (RNA) sensors that are mainly found to regulate a large number of genes/operons in bacteria. Presently, >50 bacterial riboswitch classes have been discovered, but only the thiamine pyrophosphate riboswitch class is detected in a few eukaryotes like fungi, plants and algae. One of the most important challenges in riboswitch research is to discover existing riboswitch classes in eukaryotes and to understand the evolution of bacterial riboswitches. However, traditional search methods for riboswitch detection have failed to detect eukaryotic riboswitches besides just one class and any distant structural homologs of riboswitches. We developed a novel approach based on inverse RNA folding that attempts to find sequences that match the shape of the target structure with minimal sequence conservation based on key nucleotides that interact directly with the ligand. Then, to support our matched candidates, we expanded the results into a covariance model representing similar sequences preserving the structure. Our method transforms a structure-based search into a sequence-based search that considers the conservation of secondary structure shape and ligand-binding residues. This method enables us to identify a potential structural candidate in fungi that could be the distant homolog of bacterial purine riboswitches. Further, phylogenomic analysis and evolutionary distribution of this structural candidate indicate that the most likely point of origin of this structural candidate in these organisms is associated with the loss of traditional purine riboswitches. The computational approach could be applicable to other domains and problems in RNA research.

Discovery of 17 conserved structural RNAs in fungi.

Many non-coding RNAs with known functions are structurally conserved: their intramolecular secondary and tertiary interactions are maintained across evolutionary time. Consequently, the presence of conserved structure in multiple sequence alignments can be used to identify candidate functional non-coding RNAs. Here, we present a bioinformatics method that couples iterative homology search with covariation analysis to assess whether a genomic region has evidence of conserved RNA structure. We used this method to examine all unannotated regions of five well-studied fungal genomes (Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Aspergillus fumigatus, and Schizosaccharomyces pombe). We identified 17 novel structurally conserved non-coding RNA candidates, which include four H/ACA box small nucleolar RNAs, four intergenic RNAs and nine RNA structures located within the introns and untranslated regions (UTRs) of mRNAs. For the two structures in the 3′ UTRs of the metabolic genes GLY1 and MET13, we performed experiments that provide evidence against them being eukaryotic riboswitches.

In Vitro Selection of RNA Aptamers Binding to Nanosized DNA for Constructing Artificial Riboswitches.

We here designed an in vitro selection scheme for obtaining an aptamer with which to rationally construct an artificial riboswitch as its component part. In fact, a nanosized DNA-binding aptamer obtained through this scheme allowed us to easily and successfully create eukaryotic riboswitches that upregulate internal ribosome entry site-mediated translation in response to the ligand (nanosized DNA) in wheat germ extract, a eukaryotic cell-free expression system. The induction ratio of the best riboswitch ligand-dose-dependently increased to 21 at 300 μM ligand. This switching efficiency is much higher than that of the same type of riboswitch with a widely used theophylline-binding aptamer, which was in vitro selected without considering its utility for constructing riboswitches. The selection scheme described here would facilitate obtaining various ligand/aptamer pairs suitable for constructing artificial riboswitches, which could serve as elements of synthetic gene circuits in synthetic biology.

Artificial Protein-Responsive Riboswitches Upregulate Non-AUG Translation Initiation in Yeast.

Artificial control of gene expression is one of the core technologies for engineering biological systems. Riboswitches are cis-acting elements on mRNA that regulate gene expression in a ligand-dependent manner often seen in prokaryotes, but rarely in eukaryotes. Because of the poor variety of such elements available in eukaryotic systems, the number of artificially engineered eukaryotic riboswitches, especially of the upregulation type, is still limited. Here, we developed a design principle for upregulation-type riboswitches that utilize non-AUG initiation induced by ribosomal stalling in a ligand-dependent manner in Saccharomyces cerevisiae. Our design principle simply required the proper positioning of a near-cognate start codon relative to the RNA aptamer. Intriguingly, the CUG codon was the most preferable for non-AUG ON switches in terms of output level and switch performance. This work establishes novel choices for artificial genetic control in eukaryotes with versatile potential for industrial and biomedical applications as well as basic research.

Role of riboswitches in gene regulation and their potential for algal biotechnology.

Riboswitches are regulatory elements in messenger RNA to which specific ligands can bind directly in the absence of proteins. Ligand binding alters the mRNA secondary structure, thereby affecting expression of the encoded protein. Riboswitches are widespread in prokaryotes, with over 20 different effector ligands known, including amino acids, cofactors, and Mg(2+) ions, and gene expression is generally regulated by affecting translation or termination of transcription. In plants, fungi, and microalgae, riboswitches have been found, but only those that bind thiamine pyrophosphate. These eukaryotic riboswitches operate by causing alternative splicing of the transcript. Here, we review the current status of riboswitch research with specific emphasis on microalgae. We discuss new riboswitch discoveries and insights into the underlying mechanism of action, and how next generation sequencing technology provides the motivation and opportunity to improve our understanding of these rare but important regulatory elements. We also highlight the potential of microalgal riboswitches as a tool for synthetic biology and industrial biotechnology.

An Efficient Minimum Free Energy Structure-Based Search Method for Riboswitch Identification Based on Inverse RNA Folding.

Riboswitches are RNA genetic control elements that were originally discovered in bacteria and provide a unique mechanism of gene regulation. They work without the participation of proteins and are believed to represent ancient regulatory systems in the evolutionary timescale. One of the biggest challenges in riboswitch research is to find additional eukaryotic riboswitches since more than 20 riboswitch classes have been found in prokaryotes but only one class has been found in eukaryotes. Moreover, this single known class of eukaryotic riboswitch, namely the TPP riboswitch class, has been found in bacteria, archaea, fungi and plants but not in animals. The few examples of eukaryotic riboswitches were identified using sequence-based bioinformatics search methods such as a combination of BLAST and pattern matching techniques that incorporate base-pairing considerations. None of these approaches perform energy minimization structure predictions. There is a clear motivation to develop new bioinformatics methods, aside of the ongoing advances in covariance models, that will sample the sequence search space more flexibly using structural guidance while retaining the computational efficiency of sequence-based methods. We present a new energy minimization approach that transforms structure-based search into a sequence-based search, thereby enabling the utilization of well established sequence-based search utilities such as BLAST and FASTA. The transformation to sequence space is obtained by using an extended inverse RNA folding problem solver with sequence and structure constraints, available within RNAfbinv. Examples in applying the new method are presented for the purine and preQ1 riboswitches. The method is described in detail along with its findings in prokaryotes. Potential uses in finding novel eukaryotic riboswitches and optimizing pre-designed synthetic riboswitches based on ligand simulations are discussed. The method components are freely available for use.

Alternatives to vitamin B1 uptake revealed with discovery of riboswitches in multiple marine eukaryotic lineages.

Vitamin B1 (thiamine pyrophosphate, TPP) is essential to all life but scarce in ocean surface waters. In many bacteria and a few eukaryotic groups thiamine biosynthesis genes are controlled by metabolite-sensing mRNA-based gene regulators known as riboswitches. Using available genome sequences and transcriptomes generated from ecologically important marine phytoplankton, we identified 31 new eukaryotic riboswitches. These were found in alveolate, cryptophyte, haptophyte and rhizarian phytoplankton as well as taxa from two lineages previously known to have riboswitches (green algae and stramenopiles). The predicted secondary structures bear hallmarks of TPP-sensing riboswitches. Surprisingly, most of the identified riboswitches are affiliated with genes of unknown function, rather than characterized thiamine biosynthesis genes. Using qPCR and growth experiments involving two prasinophyte algae, we show that expression of these genes increases significantly under vitamin B1-deplete conditions relative to controls. Pathway analyses show that several algae harboring the uncharacterized genes lack one or more enzymes in the known TPP biosynthesis pathway. We demonstrate that one such alga, the major primary producer Emiliania huxleyi, grows on 4-amino-5-hydroxymethyl-2-methylpyrimidine (a thiamine precursor moiety) alone, although long thought dependent on exogenous sources of thiamine. Thus, overall, we have identified riboswitches in major eukaryotic lineages not known to undergo this form of gene regulation. In these phytoplankton groups, riboswitches are often affiliated with widespread thiamine-responsive genes with as yet uncertain roles in TPP pathways. Further, taxa with ‘incomplete' TPP biosynthesis pathways do not necessarily require exogenous vitamin B1, making vitamin control of phytoplankton blooms more complex than the current paradigm suggests.

Riboswitches: Discovery of Drugs That Target Bacterial Gene-Regulatory RNAs

Riboswitches are messenger RNA (mRNA) domains that regulate gene function in response to the intracellular concentration of a variety of metabolites and second messengers. They control essential genes in many pathogenic bacteria, thus representing an inviting new class of biomolecular target for the development of antibiotics and chemical-biological tools. In this Account, we briefly review the discovery of riboswitches in the first years of the 21st century and their ensuing characterization over the past decade. We then discuss the progress achieved so far in using riboswitches as a focus for drug discovery, considering both the value of past serendipity and the particular challenges that confront current researchers. Five mechanisms of gene regulation have been determined for riboswitches. Most bacterial riboswitches modulate either transcription termination or translation initiation in response to ligand binding. All known examples of eukaryotic riboswitches, and some bacterial riboswitches, control gene expression by alternative splicing. The glmS riboswitch, which is widespread in Gram-positive bacteria, is a catalytic RNA activated by ligand binding: its self-cleavage destabilizes the mRNA of which it is part. Finally, one example of a trans-acting riboswitch is known. Three-dimensional structures have been determined for representatives of 13 structurally distinct riboswitch classes, providing atomic-level insight into their mechanisms of ligand recognition. While cellular and viral RNAs have attracted widespread interest as potential drug targets, riboswitches show special promise due to the diversity of small-molecule recognition strategies that are on display in their ligand-binding pockets. Moreover, riboswitches have evolved to recognize small-molecule ligands, which is unique among known structured RNA domains. Structural and biochemical advances in the study of riboswitches provide an impetus for the development of methods for the discovery of novel riboswitch activators and inhibitors. Recent rational drug design efforts focused on select riboswitch classes have yielded a small number of candidate antibiotic compounds, including one active in a mouse model of Staphylococcus aureus infection. The development of high-throughput methods suitable for riboswitch-specific drug discovery is ongoing. A fragment-based screening approach employing equilibrium dialysis that may be generically useful has demonstrated early success. Riboswitch-mediated gene regulation is widely employed by bacteria; however, only the thiamine pyrophosphate-responsive riboswitch has thus far been found in eukaryotes. Thus, riboswitches are particularly attractive as targets for antibacterials. Indeed, antimicrobials with previously unknown mechanisms have been found to function by binding riboswitches and causing aberrant gene expression.

Structural Basis of Thiamine Pyrophosphate Analogues Binding to the Eukaryotic Riboswitch

The thiamine pyrophosphate (TPP)-sensing riboswitch is the only riboswitch found in eukaryotes. In plants, TPP regulates its own production by binding to the 3' untranslated region of the mRNA encoding ThiC, a critical enzyme in thiamine biosynthesis, which promotes the formation of an unstable splicing variant. In order to better understand the molecular basis of TPP-analogue binding to the eukaryotic TPP-responsive riboswitch, we have determined the crystal structures of the Arabidopsis thaliana TPP-riboswitch in complex with oxythiamine pyrophosphate (OTPP) and with the antimicrobial compound pyrithiamine pyrophosphate (PTPP). The OTPP-riboswitch complex reveals that the pyrimidine ring of OTPP is stabilized in its enol form in order to retain key interactions with guanosine 28 of the riboswitch previously observed in the TPP complex. The structure of PTPP in complex with the riboswitch shows that the base moiety of guanosine 60 undergoes a conformational change to cradle the pyridine ring of the PTPP. Structural information from these complexes has implications for the design of novel antimicrobials targeting TPP-sensing riboswitches.

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.