Shape similarity measures for the design of small RNA switches

Conformational switching in the secondary structure of RNAs has recently attracted considerable attention, fostered by the discovery of 'riboswitches' in living organisms. These are genetic control elements that were found in bacteria and offer a unique regulation mechanism based on switching between two highly stable states, separated by an energy barrier between them. In riboswitches, the energy barrier is crossed by direct metabolite binding, which facilitates regulation by allosteric means. However, other event triggers can cause switching to occur, such as single-point mutations and slight variations in temperature. Examples of switches with these event triggers have already been reported experimentally in the past. Here, the goal is to computationally design small RNA switches that rely on these triggers. Towards this end, our computer simulations utilize a variety of different similarity measures to assess the distances between an initial state and triggered states, based on the topology of the secondary structure itself. We describe these combined similarity measures that rely on both coarse-grained and fine-grained graph representations of the RNA secondary structure. As a result of our simulations, we provide some candidate sequences of approximately 30-50 nt, along with the exact triggers that drive the switching. The event triggers under consideration can be modelled by Zuker's mfold or the Vienna package. The proposed methodology that rely on shape measures can further be used to computationally generate more candidates by simulating various event triggers and calculating their effect on the shape.     

Riboswitch regulation mechanisms: RNA,metabolites and regulatory proteins

Riboswitches are RNA sensors that have been shown to modulate the expression of downstream genes by altering their structure upon metabolite binding. Riboswitches are unique among cellular regulators in that metabolite detection is strictly performed using RNA interactions with the sensed metabolite and in which no regulatory protein is needed to mediate the interaction. However, recent studies have shed light on riboswitch control mechanisms relying on protein regulators to harness metabolite binding for the mediation of gene expression, thereby increasing the range of cellular factors involved in riboswitch regulation. The interaction between riboswitches and proteins adds another level of evolutionary pressure as riboswitches must maintain key residues for metabolite detection, structural switching and protein binding sites. Here, we review regulatory mechanisms involving Escherichia coli riboswitches that have recently been shown to rely on regulatory proteins. We also discuss the implication of such protein-based riboswitch regulatory mechanisms for genetic regulation.     

STR2: a structure to string approach for locating G-box riboswitch shapes in pre-selected genes

Traditional sequence-based search methods such as BLAST and FASTA can be used to identify sequence similarities. Recently, there is a growing interest in performing RNA shape similarity searches inside selected genes to locate RNA structure motifs that are known to possess functionally important roles. For example, in the newly discovered RNA genetic control elements called "riboswitches", the box domain is known to be highly conserved among various bacterial species in both its nucleotide composition and shape. However, in non-bacterial species, shape conservation is likely to become more important than sequence conservation when searching for riboswitch patterns. For this purpose, we present an approach tailored for detecting RNA shape similarities. We extend the Structure to String (ST R2) method that was initially proposed to locate shape similarities in proteins to identify predicted secondary structures of RNAs. The ST R2 for RNAs is a translation of a secondary structure to a string of characters, after which known sequence-based search algorithms with an efficient implementation are being used. We validate that the ST R2 succeeds to locate G-box riboswitches in prokaryotes, as expected. Subsequently we show running examples when attempting to detect G-box riboswitch candidates in eukaryotes.     

Engineered riboswitches as novel tools in molecular biology

During the last years the great importance of RNA for regulating gene expression in all organisms has become obvious. Consequently, several recent approaches aim to utilize the outstanding chemical properties of RNA to develop artificial RNA regulators for conditional gene expression systems. A combination of rational design, in vitro selection and in vivo screening systems has been used to create a versatile set of RNA based molecular switches. These tools rely on diverse mechanisms and exhibit activity in several organisms. In this review, we summarize recent developments in the application of engineered riboswitches for gene regulation in vivo.     

依赖于5′端非编码区高级结构的真核生物mRNA翻译调控

翻译水平的调控是真核基因表达调控的重要环节.近年来的研究表明,许多真核基因的翻译依赖于RNA 5′端非编码区的结构元件.一些小结构元件,如铁离子反应元件,具有1个茎环结构,由铁离子介导控制转铁蛋白的翻译. 核糖开关通过结合特定代谢分子在2种结构状态下切换,调控可变剪接和翻译起始.另1个高度结构化的mRNA元件是内部核糖体进入位点,通过富集核糖体和起始因子促进基因的表达.本文综述了依赖于小结构元件、内部核糖体进入位点和核糖开关的真核基因翻译起始调控相应的研究成果和研究方法.对于研究的前景以及可能存在的挑战也作出阐述.     

Folding path of P5abc RNA involves direct coupling of secondary and tertiary structures

Folding mechanisms in which secondary structures are stabilized through the formation of tertiary interactions are well documented in protein folding but challenge the folding hierarchy normally assumed for RNA. However, it is increasingly clear that RNA could fold by a similar mechanism. P5abc, a small independently folding tertiary domain of the Tetrahymena thermophila group I ribozyme, is known to fold by a secondary structure rearrangement involving helix P5c. However, the extent of this rearrangement and the precise stage of folding that triggers it are unknown. We use experiments and simulations to show that the P5c helix switches to the native secondary structure late in the folding pathway and is directly coupled to the formation of tertiary interactions in the A-rich bulge. P5c mutations show that the switch in P5c is not rate-determining and suggest that non-native interactions in P5c aid folding rather than impede it. Our study illustrates that despite significant differences in the building blocks of proteins and RNA, there may be common ways in which they self-assemble.     

Computational analysis of riboswitch-based regulation

Advances in computational analysis of riboswitches in the last decade have contributed greatly to our understanding of riboswitch regulatory roles and mechanisms. Riboswitches were originally discovered as part of the sequence analysis of the 5′-untranslated region of mRNAs in the hope of finding novel gene regulatory sites, and the existence of structural RNAs appeared to be a spurious phenomenon. As more riboswitches were discovered, they illustrated the diversity and adaptability of these RNA regulatory sequences. The fact that a chemically monotonous molecule like RNA can discern a wide range of substrates and exert a variety of regulatory mechanisms was subsequently demonstrated in diverse genomes and has hastened the development of sophisticated algorithms for their analysis and prediction. In this review, we focus on some of the computational tools for riboswitch detection and secondary structure prediction. The study of this simple yet efficient form of gene regulation promises to provide a more complete picture of a world that RNA once dominated and allows rational design of artificial riboswitches. This article is part of a Special Issue entitled: Riboswitches.     

Secondary structures and functional requirements for thiM riboswitches from Desulfovibrio vulgaris, Erwinia carotovora and Rhodobacter spheroides

Abstract Bacterial thiM riboswitches contain aptamer domains that bind the metabolite thiamine pyrophosphate (TPP). Binding of TPP to the aptamer domain induces structural rearrangements that are relayed to the expression domain, thereby interfering with gene expression. Here, we report identification of three putative thiM riboswitches from different bacteria and analysis of their secondary structures. Chemical probing revealed that the riboswitches share similar secondary structures in their aptamer domains that can communicate with the highly variant expression domains in a mechanism likely involving sequestration of the Shine-Dalgarno sequence. Remarkably, the aptamer domain of the thiM gene of Desulfovibrio vulgaris binds TPP with similar affinity and selectivity as that of Escherichia coli, although nucleotides previously shown to form direct contacts to the metabolite are mutated. We also designed small RNA hairpins for each riboswitch that bind the RNA only in the absence of the metabolite. Our study shows that aptamer domains in riboswitches with high similarity in their secondary structures can communicate with a broad variety of non-related expression domains by similar mechanisms.     

Riboswitch Detection Using Profile Hidden Markov Models

Background  

Riboswitches are a type of noncoding RNA that regulate gene expression by switching from one structural conformation to another on ligand binding. The various classes of riboswitches discovered so far are differentiated by the ligand, which on binding induces a conformational switch. Every class of riboswitch is characterized by an aptamer domain, which provides the site for ligand binding, and an expression platform that undergoes conformational change on ligand binding. The sequence and structure of the aptamer domain is highly conserved in riboswitches belonging to the same class. We propose a method for fast and accurate identification of riboswitches using profile Hidden Markov Models (pHMM). Our method exploits the high degree of sequence conservation that characterizes the aptamer domain.     

Mutational analysis in RNAs: comparing programs for RNA deleterious mutation prediction

Programs for RNA mutational analysis that are structure-based and rely on secondary structure prediction have been developed and expanded in the past several years. They can be used for a variety of purposes, such as in suggesting point mutations that will alter RNA virus replication or translation initiation, investigating the effect of deleterious and compensatory mutations in allosteric ribozymes and riboswitches, computing an optimal path of mutations to get from one ribozyme fold to another, or analyzing regulatory RNA sequences by their mutational profile. This review describes three different freeware programs (RNAMute, RDMAS and RNAmutants) that have been developed for such purposes. RNAMute and RDMAS in principle perform energy minimization prediction by available software such as RNAfold from the Vienna RNA package or Zuker's Mfold, while RNAmutants provides an efficient method using essential ingredients from energy minimization prediction. Both RNAMute in its extended version that uses RNAsubopt from the Vienna RNA package and the RNAmutants software are able to predict multiple-point mutations using developed methodologies, while RDMAS is currently restricted to single-point mutations. The strength of RNAMute in its extended version is the ability to predict a small number of point mutations in an accurate manner. RNAmutants is well fit for large scale simulations involving the calculation of all k-mutants, where k can be a large integer number, of a given RNA sequence.     

Searching genomes for noncoding RNA using FastR

The discovery of novel noncoding RNAs has been among the most exciting recent developments in biology. It has been hypothesized that there is, in fact, an abundance of functional noncoding RNAs (ncRNAs) with various catalytic and regulatory functions. However, the inherent signal for ncRNA is weaker than the signal for protein coding genes, making these harder to identify. We consider the following problem: Given an RNA sequence with a known secondary structure, efficiently detect all structural homologs in a genomic database by computing the sequence and structure similarity to the query. Our approach, based on structural filters that eliminate a large portion of the database while retaining the true homologs, allows us to search a typical bacterial genome in minutes on a standard PC. The results are two orders of magnitude better than the currently available software for the problem. We applied FastR to the discovery of novel riboswitches, which are a class of RNA domains found in the untranslated regions. They are of interest because they regulate metabolite synthesis by directly binding metabolites. We searched all available eubacterial and archaeal genomes for riboswitches from purine, lysine, thiamin, and riboflavin subfamilies. Our results point to a number of novel candidates for each of these subfamilies and include genomes that were not known to contain riboswitches.