Building a stable RNA U-turn with a protonated cytidine

The U-turn is a classical three-dimensional RNA folding motif first identified in the anticodon and T-loops of tRNAs. It also occurs frequently as a building block in other functional RNA structures in many different sequence and structural contexts. U-turns induce sharp changes in the direction of the RNA backbone and often conform to the 3-nt consensus sequence 5′-UNR-3′ (N = any nucleotide, R = purine). The canonical U-turn motif is stabilized by a hydrogen bond between the N3 imino group of the U residue and the 3′ phosphate group of the R residue as well as a hydrogen bond between the 2′-hydroxyl group of the uridine and the N7 nitrogen of the R residue. Here, we demonstrate that a protonated cytidine can functionally and structurally replace the uridine at the first position of the canonical U-turn motif in the apical loop of the neomycin riboswitch. Using NMR spectroscopy, we directly show that the N3 imino group of the protonated cytidine forms a hydrogen bond with the backbone phosphate 3′ from the third nucleotide of the U-turn analogously to the imino group of the uridine in the canonical motif. In addition, we compare the stability of the hydrogen bonds in the mutant U-turn motif to the wild type and describe the NMR signature of the C+-phosphate interaction. Our results have implications for the prediction of RNA structural motifs and suggest simple approaches for the experimental identification of hydrogen bonds between protonated C-imino groups and the phosphate backbone.     

Molecular Dynamics Study of Intrinsic Stability in Six RNA Terminal Loop Motifs

Abstract

Single stranded RNA molecules can assume a wide range of tertiary structures beyond the canonical A-form double helix. Certain sequences, termed motifs, are more common than a random distribution would suggest. The existence of such motifs can be rationalized in structural terms. In this study, we have investigated the intrinsic structural stability of RNA terminal loop motifs using multiple MD simulations in explicit water. Representative loops were chosen from the major tetraloop motifs, including also the U-turn motif. Not all loops retain their folded starting structure, but lowering the temperature to 277 K, or adding adjacent base pairs from the stem to which the motif is attached, helps stabilizing the folded loop structure.     

Molecular dynamics study of intrinsic stability in six RNA terminal loop motifs

Single stranded RNA molecules can assume a wide range of tertiary structures beyond the canonical A-form double helix. Certain sequences, termed motifs, are more common than a random distribution would suggest. The existence of such motifs can be rationalized in structural terms. In this study, we have investigated the intrinsic structural stability of RNA terminal loop motifs using multiple MD simulations in explicit water. Representative loops were chosen from the major tetraloop motifs, including also the U-turn motif. Not all loops retain their folded starting structure, but lowering the temperature to 277 K, or adding adjacent base pairs from the stem to which the motif is attached, helps stabilizing the folded loop structure.     

Probing the structural hierarchy and energy landscape of an RNA T-loop hairpin

The T-loop motif is an important recurrent RNA structural building block consisting of a U-turn sub-motif and a UA trans Watson–Crick/Hoogsteen base pair. In the presence of a hairpin stem, the UA non-canonical base pair becomes part of the UA-handle motif. To probe the hierarchical organization and energy landscape of the T-loop, we performed replica exchange molecular dynamics (REMD) simulations of the T-loop in isolation and as part of a hairpin. Our simulations reveal that the isolated T-loop adopts coil conformers stabilized by base stacking. The T-loop hairpin shows a highly rugged energy landscape featuring multiple local minima with a transition state for folding consisting of partially zipped states. The U-turn displays a high conformational flexibility both when the T-loop is in isolation and as part of a hairpin. On the other hand, the stability of the UA non-canonical base pair is enhanced in the presence of the UA-handle. This motif is apparently a key component for stabilizing the T-loop, while the U-turn is mostly involved in long-range interaction. Our results suggest that the stability and folding of small RNA motifs are highly dependent on local context.     

Nuclear magnetic resonance structure of the Varkud satellite ribozyme stem-loop V RNA and magnesium-ion binding from chemical-shift mapping

An important step in the substrate recognition of the Neurospora Varkud Satellite (VS) ribozyme is the formation of a magnesium-dependent loop/loop interaction between the terminal loops of stem-loops I and V. We have studied the structure of stem-loop V by nuclear magnetic resonance spectroscopy and shown that it adopts a U-turn conformation, a common motif found in RNA. Structural comparisons indicate that the U-turn of stem-loop V fulfills some but not all of the structural characteristics found in canonical U-turn structures. This U-turn conformation exposes the Watson-Crick faces of the bases within stem-loop V (G697, A698, and C699) and makes them accessible for interaction with stem-loop I. Using chemical-shift mapping, we show that magnesium ions interact with the loop of the isolated stem-loop V and induce a conformational change that may be important for interaction with stem-loop I. This study expands our understanding of the role of U-turn motifs in RNA structure and function and provides insights into the mechanism of substrate recognition in the VS ribozyme.     

Predicting U-turns in ribosomal RNA with comparative sequence analysis

The U-turn is a well-known RNA motif characterized by a sharp reversal of the RNA backbone following a single-stranded uridine base. In experimentally determined U-turn motifs, the nucleotides 3' to the turn are frequently involved in tertiary interactions, rendering this motif particularly attractive in RNA modeling and functional studies. The U-turn signature is composed of an UNR sequence pattern flanked by a Y:Y, Y:A (Y=pyrimidine) or G:A base juxtaposition. We have identified 33 potential UNR-type U-turns and 25 related GNRA-type U-turns in a large set of aligned 16 S and 23 S rRNA sequences. U-turn candidates occur in hairpin loops (34 times) as well as in internal and multi-stem loops (24 times). These are classified into ten families based on loop type, sequence pattern (UNR or GNRA) and the nature of the closing base juxtaposition. In 13 cases, the bases on the 3' side of the turn, or on the immediate 5' side, are involved in tertiary covariations, making these sites strong candidates for tertiary interactions.     

RNAMotifScan: automatic identification of RNA structural motifs using secondary structural alignment

Recent studies have shown that RNA structural motifs play essential roles in RNA folding and interaction with other molecules. Computational identification and analysis of RNA structural motifs remains a challenging task. Existing motif identification methods based on 3D structure may not properly compare motifs with high structural variations. Other structural motif identification methods consider only nested canonical base-pairing structures and cannot be used to identify complex RNA structural motifs that often consist of various non-canonical base pairs due to uncommon hydrogen bond interactions. In this article, we present a novel RNA structural alignment method for RNA structural motif identification, RNAMotifScan, which takes into consideration the isosteric (both canonical and non-canonical) base pairs and multi-pairings in RNA structural motifs. The utility and accuracy of RNAMotifScan is demonstrated by searching for kink-turn, C-loop, sarcin-ricin, reverse kink-turn and E-loop motifs against a 23S rRNA (PDBid: 1S72), which is well characterized for the occurrences of these motifs. Finally, we search these motifs against the RNA structures in the entire Protein Data Bank and the abundances of them are estimated. RNAMotifScan is freely available at our supplementary website (http://genome.ucf.edu/RNAMotifScan).     

Clustering RNA structural motifs in ribosomal RNAs using secondary structural alignment

RNA structural motifs are the building blocks of the complex RNA architecture. Identification of non-coding RNA structural motifs is a critical step towards understanding of their structures and functionalities. In this article, we present a clustering approach for de novo RNA structural motif identification. We applied our approach on a data set containing 5S, 16S and 23S rRNAs and rediscovered many known motifs including GNRA tetraloop, kink-turn, C-loop, sarcin-ricin, reverse kink-turn, hook-turn, E-loop and tandem-sheared motifs, with higher accuracy than the state-of-the-art clustering method. We also identified a number of potential novel instances of GNRA tetraloop, kink-turn, sarcin-ricin and tandem-sheared motifs. More importantly, several novel structural motif families have been revealed by our clustering analysis. We identified a highly asymmetric bulge loop motif that resembles the rope sling. We also found an internal loop motif that can significantly increase the twist of the helix. Finally, we discovered a subfamily of hexaloop motif, which has significantly different geometry comparing to the currently known hexaloop motif. Our discoveries presented in this article have largely increased current knowledge of RNA structural motifs.     

RNA structural motif recognition based on least-squares distance

RNA structural motifs are recurrent structural elements occurring in RNA molecules. RNA structural motif recognition aims to find RNA substructures that are similar to a query motif, and it is important for RNA structure analysis and RNA function prediction. In view of this, we propose a new method known as RNA Structural Motif Recognition based on Least-Squares distance (LS-RSMR) to effectively recognize RNA structural motifs. A test set consisting of five types of RNA structural motifs occurring in Escherichia coli ribosomal RNA is compiled by us. Experiments are conducted for recognizing these five types of motifs. The experimental results fully reveal the superiority of the proposed LS-RSMR compared with four other state-of-the-art methods.     

RNAMotifScanX: a graph alignment approach for RNA structural motif identification

RNA structural motifs are recurrent three-dimensional (3D) components found in the RNA architecture. These RNA structural motifs play important structural or functional roles and usually exhibit highly conserved 3D geometries and base-interaction patterns. Analysis of the RNA 3D structures and elucidation of their molecular functions heavily rely on efficient and accurate identification of these motifs. However, efficient RNA structural motif search tools are lacking due to the high complexity of these motifs. In this work, we present RNAMotifScanX, a motif search tool based on a base-interaction graph alignment algorithm. This novel algorithm enables automatic identification of both partially and fully matched motif instances. RNAMotifScanX considers noncanonical base-pairing interactions, base-stacking interactions, and sequence conservation of the motifs, which leads to significantly improved sensitivity and specificity as compared with other state-of-the-art search tools. RNAMotifScanX also adopts a carefully designed branch-and-bound technique, which enables ultra-fast search of large kink-turn motifs against a 23S rRNA. The software package RNAMotifScanX is implemented using GNU C++, and is freely available from http://genome.ucf.edu/RNAMotifScanX.     

Antisense RNA regulation in prokaryotes: rapid RNA/RNA interaction facilitated by a general U-turn loop structure

Efficient gene control by antisense RNA requires rapid bi-molecular interaction with a cognate target RNA. A comparative analysis revealed that a YUNR motif (Y=pyrimidine, R=purine) is ubiquitous in RNA recognition loops in antisense RNA-regulated gene systems. The (Y)UNR sequence motif specifies two intraloop hydrogen bonds forming U-turn structures in many anticodon-loops and all T-loops of tRNAs, the hammerhead ribozyme and in other conserved RNA loops. This structure creates a sharp bend in the RNA phosphate-backbone and presents the following three to four bases in a solvent-exposed, stacked configuration providing a scaffold for rapid interaction with complementary RNA. Sok antisense RNA from plasmid R1 inhibits translation of the hok mRNA by preventing ribosome entry at the mok Shine & Dalgarno element. The 5' single-stranded region of Sok-RNA recognizes a loop in the hok mRNA. We show here, that the initial pairing between Sok antisense RNA and its target in hok mRNA occurs with an observed second-order rate-constant of 2 x 10(6) M(-1) s(-1). Mutations that eliminate the YUNR motif in the target loop of hok mRNA resulted in reduced antisense RNA pairing kinetics, whereas mutations maintaining the YUNR motif were silent. In addition, RNA phosphate-backbone accessibility probing by ethylnitrosourea was consistent with a U-turn structure formation promoted by the YUNR motif. Since the YUNR U-turn motif is present in the recognition units of many antisense/target pairs, the motif is likely to be a generally employed enhancer of RNA pairing rates. This suggestion is consistent with the re-interpretation of the mutational analyses of several antisense control systems including RNAI/RNAII of ColE1, CopA/CopT of R1 and RNA-IN/RNA-OUT of IS10.     

Automated classification of RNA 3D motifs and the RNA 3D Motif Atlas

The analysis of atomic-resolution RNA three-dimensional (3D) structures reveals that many internal and hairpin loops are modular, recurrent, and structured by conserved non-Watson–Crick base pairs. Structurally similar loops define RNA 3D motifs that are conserved in homologous RNA molecules, but can also occur at nonhomologous sites in diverse RNAs, and which often vary in sequence. To further our understanding of RNA motif structure and sequence variability and to provide a useful resource for structure modeling and prediction, we present a new method for automated classification of internal and hairpin loop RNA 3D motifs and a new online database called the RNA 3D Motif Atlas. To classify the motif instances, a representative set of internal and hairpin loops is automatically extracted from a nonredundant list of RNA-containing PDB files. Their structures are compared geometrically, all-against-all, using the FR3D program suite. The loops are clustered into motif groups, taking into account geometric similarity and structural annotations and making allowance for a variable number of bulged bases. The automated procedure that we have implemented identifies all hairpin and internal loop motifs previously described in the literature. All motif instances and motif groups are assigned unique and stable identifiers and are made available in the RNA 3D Motif Atlas (http://rna.bgsu.edu/motifs), which is automatically updated every four weeks. The RNA 3D Motif Atlas provides an interactive user interface for exploring motif diversity and tools for programmatic data access.     

Selection of a novel class of RNA-RNA interaction motifs based on the ligase ribozyme with defined modular architecture

To develop molecular tools for the detection and control of RNA molecules whose functions rely on their 3D structures, we have devised a selection system to isolate novel RNA motifs that interact with a target RNA structure within a given structural context. In this system, a GAAA tetraloop and its specific receptor motif (11-ntR) from an artificial RNA ligase ribozyme with modular architecture (the DSL ribozyme) were replaced with a target structure and random sequence, respectively. Motifs recognizing the target structure can be identified by in vitro selection based on ribozyme activity. A model selection targeting GAAA-loop successfully identified motifs previously known as GAAA-loop receptors. In addition, a new selection targeting a C-loop motif also generated novel motifs that interact with this structure. Biochemical analysis of one of the C-loop receptor motifs revealed that it could also function as an independent structural unit.     

An in vitro-selected RNA receptor for the GAAC loop: modular receptor for non–GNRA-type tetraloop

Although artificial RNA motifs that can functionally replace the GNRA/receptor interaction, a class of RNA–RNA interacting motifs, were isolated from RNA libraries and used to generate designer RNA structures, receptors for non-GNRA tetraloops have not been found in nature or selected from RNA libraries. In this study, we report successful isolation of a receptor motif interacting with GAAC, a non-GNRA tetraloop, from randomized sequences embedded in a catalytic RNA. Biochemical characterization of the GAAC/receptor interacting motif within three structural contexts showed its binding affinity, selectivity and structural autonomy. The motif has binding affinity comparable with that of a GNRA/receptor, selectivity orthogonal to GNRA/receptors and structural autonomy even in a large RNA context. These features would be advantageous for usage of the motif as a building block for designer RNAs. The isolated motif can also be used as a query sequence to search for unidentified naturally occurring GANC receptor motifs.     

A computational pipeline for high- throughput discovery of cis-regulatory noncoding RNA in prokaryotes

Noncoding RNAs (ncRNAs) are important functional RNAs that do not code for proteins. We present a highly efficient computational pipeline for discovering cis-regulatory ncRNA motifs de novo. The pipeline differs from previous methods in that it is structure-oriented, does not require a multiple-sequence alignment as input, and is capable of detecting RNA motifs with low sequence conservation. We also integrate RNA motif prediction with RNA homolog search, which improves the quality of the RNA motifs significantly. Here, we report the results of applying this pipeline to Firmicute bacteria. Our top-ranking motifs include most known Firmicute elements found in the RNA family database (Rfam). Comparing our motif models with Rfam's hand-curated motif models, we achieve high accuracy in both membership prediction and base-pair–level secondary structure prediction (at least 75% average sensitivity and specificity on both tasks). Of the ncRNA candidates not in Rfam, we find compelling evidence that some of them are functional, and analyze several potential ribosomal protein leaders in depth.     

RNAMotifContrast: a method to discover and visualize RNA structural motif subfamilies

Understanding the 3D structural properties of RNAs will play a critical role in identifying their functional characteristics and designing new RNAs for RNA-based therapeutics and nanotechnology. While several existing computational methods can help in the analysis of RNA properties by recognizing structural motifs, they do not provide the means to compare and contrast those motifs extensively. We have developed a new method, RNAMotifContrast, which focuses on analyzing the similarities and variations of RNA structural motif characteristics. In this method, a graph is formed to represent the similarities among motifs, and a new traversal algorithm is applied to generate visualizations of their structural properties. Analyzing the structural features among motifs, we have recognized and generalized the concept of motif subfamilies. To asses its effectiveness, we have applied RNAMotifContrast on a dataset of known RNA structural motif families. From the results, we observed that the derived subfamilies possess unique structural variations while holding standard features of the families. Overall, the visualization approach of this method presents a new perspective to observe the relation among motifs more closely, and the discovered subfamilies provide opportunities to achieve valuable insights into RNA’s diverse roles.     

RNAMotif, an RNA secondary structure definition and search algorithm

RNA molecules fold into characteristic secondary and tertiary structures that account for their diverse functional activities. Many of these RNA structures are assembled from a collection of RNA structural motifs. These basic building blocks are used repeatedly, and in various combinations, to form different RNA types and define their unique structural and functional properties. Identification of recurring RNA structural motifs will therefore enhance our understanding of RNA structure and help associate elements of RNA structure with functional and regulatory elements. Our goal was to develop a computer program that can describe an RNA structural element of any complexity and then search any nucleotide sequence database, including the complete prokaryotic and eukaryotic genomes, for these structural elements. Here we describe in detail a new computational motif search algorithm, RNAMotif, and demonstrate its utility with some motif search examples. RNAMotif differs from other motif search tools in two important aspects: first, the structure definition language is more flexible and can specify any type of base–base interaction; second, RNAMotif provides a user controlled scoring section that can be used to add capabilities that patterns alone cannot provide.     

On the occurrence of the T-loop RNA folding motif in large RNA molecules

The T-loop RNA folding motif may be considered as a five-nucleotide motif composed of a U-turn flanked by a noncanonical base pair. It was recently proposed that the flanking noncanonical base pair is always a UA trans Watson-Crick/Hoogsteen base pair stacked on a Watson-Crick base pair on one side. Here we show that structural analysis of several large RNA molecules, including the recently solved crystal structure of the specificity domain of Bacillus subtilis RNase P, combined with sequence analysis, indicates a broader sequence consensus for the motif. Additionally, we show that the flanking base pair does not necessarily stack on a Watson-Crick base pair and the 3' terminus of the five-nucleotide motif is often followed by a sharp turn in the phosphate backbone rather than just a bulged base or bases.     

The genomic HDV ribozyme utilizes a previously unnoticed U-turn motif to accomplish fast site-specific catalysis

The genome of the human hepatitis delta virus (HDV) harbors a self-cleaving catalytic RNA motif, the genomic HDV ribozyme, whose crystal structure shows the dangling nucleotides 5′ of the cleavage site projecting away from the catalytic core. This 5′-sequence contains a clinically conserved U − 1 that we find to be essential for fast cleavage, as the order of activity follows U − 1 > C − 1 > A − 1 > G − 1, with a >25-fold activity loss from U − 1 to G − 1. Terbium(III) footprinting detects conformations for the P1.1 stem, the cleavage site wobble pair and the A-minor motif of the catalytic trefoil turn that depend on the identity of the N − 1 base. The most tightly folded catalytic core, resembling that of the reaction product, is found in the U − 1 wild-type precursor. Molecular dynamics simulations demonstrate that a U − 1 forms the most robust kink around the scissile phosphate, exposing it to the catalytic C75 in a previously unnoticed U-turn motif found also, for example, in the hammerhead ribozyme and tRNAs. Strikingly, we find that the common structural U-turn motif serves distinct functions in the HDV and hammerhead ribozymes.     

NMR structures of double loops of an RNA aptamer against mammalian initiation factor 4A

A high affinity RNA aptamer (APT58, 58 nt long) against mammalian initiation factor 4A (eIF4A) requires nearly its entire nucleotide sequence for efficient binding. Since splitting either APT58 or eIF4A into two domains diminishes the affinity for each other, it is suggested that multiple interactions or a global interaction between the two molecules accounts for the high affinity. To understand the structural basis of APT58's global recognition of eIF4A, we determined the solution structure of two essential nucleotide loops (AUCGCA and ACAUAGA) within the aptamer using NMR spectroscopy. The AUCGCA loop is stabilized by a U-turn motif and contains a non-canonical A:A base pair (the single hydrogen bond mismatch: Hoogsteen/Sugar-edge). On the other hand, the ACAUAGA loop is stabilized by an AUA tri-nucleotide loop motif and contains the other type of A:A base pair (single hydrogen bond mismatch: Watson–Crick/Watson–Crick). Considering the known structural and functional properties of APT58, we propose that the AUCGCA loop is directly involved in the interaction with eIF4A, while the flexibility of the ACAUAGA loop is important to support this interaction. The Watson–Crick edges of C7 and C9 in the AUCGCA loop may directly interact with eIF4A.