Showing papers on "Nucleic acid secondary structure published in 2013"

Accurate SHAPE-directed RNA secondary structure modeling, including pseudoknots

Abstract: A pseudoknot forms in an RNA when nucleotides in a loop pair with a region outside the helices that close the loop. Pseudoknots occur relatively rarely in RNA but are highly overrepresented in functionally critical motifs in large catalytic RNAs, in riboswitches, and in regulatory elements of viruses. Pseudoknots are usually excluded from RNA structure prediction algorithms. When included, these pairings are difficult to model accurately, especially in large RNAs, because allowing this structure dramatically increases the number of possible incorrect folds and because it is difficult to search the fold space for an optimal structure. We have developed a concise secondary structure modeling approach that combines SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) experimental chemical probing information and a simple, but robust, energy model for the entropic cost of single pseudoknot formation. Structures are predicted with iterative refinement, using a dynamic programming algorithm. This melded experimental and thermodynamic energy function predicted the secondary structures and the pseudoknots for a set of 21 challenging RNAs of known structure ranging in size from 34 to 530 nt. On average, 93% of known base pairs were predicted, and all pseudoknots in well-folded RNAs were identified.

Nucleic Acids With Metal‐Mediated Base Pairs and Their Applications

Abstract: Metal-mediated base pairs represent an elegant way for the site-specific functionalisation of nucleic acids. In this type of base pair, the hydrogen bonds between complementary nucleobases are replaced formally by coordinative bonds to one or more transition-metal ions. This Review presents an overview of metal-mediated base pairs reported so far. It gives an insight into the characterisation of metal-mediated base pairs and into the structures of the resulting metal-modified nucleic acids. In addition, a summary of applications exploiting the formation of metal-mediated base pairs is presented. The applications include, for example, metal-ion sensors of various types, a protein sensor, and DNA-based molecular machines. Initial reports on the potential use of metal-mediated base pairs in the context of charge transfer through nucleic acid double helices are summarised as well.

Analysis of RNA base modification and structural rearrangement by single-molecule real-time detection of reverse transcription

Abstract: Zero-mode waveguides (ZMWs) are photonic nanostructures that create highly confined optical observation volumes, thereby allowing single-molecule-resolved biophysical studies at relatively high concentrations of fluorescent molecules. This principle has been successfully applied in single-molecule, real-time (SMRT®) DNA sequencing for the detection of DNA sequences and DNA base modifications. In contrast, RNA sequencing methods cannot provide sequence and RNA base modifications concurrently as they rely on complementary DNA (cDNA) synthesis by reverse transcription followed by sequencing of cDNA. Thus, information on RNA modifications is lost during the process of cDNA synthesis. Here we describe an application of SMRT technology to follow the activity of reverse transcriptase enzymes synthesizing cDNA on thousands of single RNA templates simultaneously in real time with single nucleotide turnover resolution using arrays of ZMWs. This method thereby obtains information from the RNA template directly. The analysis of the kinetics of the reverse transcriptase can be used to identify RNA base modifications, shown by example for N6-methyladenine (m6A) in oligonucleotides and in a specific mRNA extracted from total cellular mRNA. Furthermore, the real-time reverse transcriptase dynamics informs about RNA secondary structure and its rearrangements, as demonstrated on a ribosomal RNA and an mRNA template. Our results highlight the feasibility of studying RNA modifications and RNA structural rearrangements in ZMWs in real time. In addition, they suggest that technology can be developed for direct RNA sequencing provided that the reverse transcriptase is optimized to resolve homonucleotide stretches in RNA.

The RNAsnp web server: predicting SNP effects on local RNA secondary structure

Abstract: The function of many non-coding RNA genes and cis-regulatory elements of messenger RNA largely depends on the structure, which is in turn determined by their sequence. Single nucleotide polymorphisms (SNPs) and other mutations may disrupt the RNA structure, interfere with the molecular function and hence cause a phenotypic effect. RNAsnp is an efficient method to predict the effect of SNPs on local RNA secondary structure based on the RNA folding algorithms implemented in the Vienna RNA package. The SNP effects are quantified in terms of empirical P-values, which, for computational efficiency, are derived from extensive pre-computed tables of distributions of substitution effects as a function of gene length and GC content. Here, we present a web service that not only provides an interface for RNAsnp but also features a graphical output representation. In addition, the web server is connected to a local mirror of the UCSC genome browser database that enables the users to select the genomic sequences for analysis and visualize the results directly in the UCSC genome browser. The RNAsnp web server is freely available at: http://rth.dk/resources/rnasnp/.

A guide RNA sequence design platform for the CRISPR/Cas9 system for model organism genomes.

Abstract: Cas9/CRISPR has been reported to efficiently induce targeted gene disruption and homologous recombination in both prokaryotic and eukaryotic cells. Thus, we developed a Guide RNA Sequence Design Platform for the Cas9/CRISPR silencing system for model organisms. The platform is easy to use for gRNA design with input query sequences. It finds potential targets by PAM and ranks them according to factors including uniqueness, SNP, RNA secondary structure, and AT content. The platform allows users to upload and share their experimental results. In addition, most guide RNA sequences from published papers have been put into our database.

SeqFold: Genome-scale reconstruction of RNA secondary structure integrating high-throughput sequencing data

Abstract: We present an integrative approach, SeqFold, that combines high-throughput RNA structure profiling data with computational prediction for genome-scale reconstruction of RNA secondary structures. SeqFold transforms experimental RNA structure information into a structure preference profile (SPP) and uses it to select stable RNA structure candidates representing the structure ensemble. Under a high-dimensional classification framework, SeqFold efficiently matches a given SPP to the most likely cluster of structures sampled from the Boltzmann-weighted ensemble. SeqFold is able to incorporate diverse types of RNA structure profiling data, including parallel analysis of RNA structure (PARS), selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), fragmentation sequencing (FragSeq) data generated by deep sequencing, and conventional SHAPE data. Using the known structures of a wide range of mRNAs and noncoding RNAs as benchmarks, we demonstrate that SeqFold outperforms or matches existing approaches in accuracy and is more robust to noise in experimental data. Application of SeqFold to reconstruct the secondary structures of the yeast transcriptome reveals the diverse impact of RNA secondary structure on gene regulation, including translation efficiency, transcription initiation, and protein-RNA interactions. SeqFold can be easily adapted to incorporate any new types of high-throughput RNA structure profiling data and is widely applicable to analyze RNA structures in any transcriptome.

Comparison of SIV and HIV-1 Genomic RNA Structures Reveals Impact of Sequence Evolution on Conserved and Non-Conserved Structural Motifs

Abstract: RNA secondary structure plays a central role in the replication and metabolism of all RNA viruses, including retroviruses like HIV-1. However, structures with known function represent only a fraction of the secondary structure reported for HIV-1NL4-3. One tool to assess the importance of RNA structures is to examine their conservation over evolutionary time. To this end, we used SHAPE to model the secondary structure of a second primate lentiviral genome, SIVmac239, which shares only 50% sequence identity at the nucleotide level with HIV-1NL4-3. Only about half of the paired nucleotides are paired in both genomic RNAs and, across the genome, just 71 base pairs form with the same pairing partner in both genomes. On average the RNA secondary structure is thus evolving at a much faster rate than the sequence. Structure at the Gag-Pro-Pol frameshift site is maintained but in a significantly altered form, while the impact of selection for maintaining a protein binding interaction can be seen in the conservation of pairing partners in the small RRE stems where Rev binds. Structures that are conserved between SIVmac239 and HIV-1NL4-3 also occur at the 5′ polyadenylation sequence, in the plus strand primer sites, PPT and cPPT, and in the stem-loop structure that includes the first splice acceptor site. The two genomes are adenosine-rich and cytidine-poor. The structured regions are enriched in guanosines, while unpaired regions are enriched in adenosines, and functionaly important structures have stronger base pairing than nonconserved structures. We conclude that much of the secondary structure is the result of fortuitous pairing in a metastable state that reforms during sequence evolution. However, secondary structure elements with important function are stabilized by higher guanosine content that allows regions of structure to persist as sequence evolution proceeds, and, within the confines of selective pressure, allows structures to evolve.

RNAiFOLD: a constraint programming algorithm for RNA inverse folding and molecular design.

Abstract: Synthetic biology is a rapidly emerging discipline with long-term ramifications that range from single-molecule detection within cells to the creation of synthetic genomes and novel life forms. Truly phenomenal results have been obtained by pioneering groups--for instance, the combinatorial synthesis of genetic networks, genome synthesis using BioBricks, and hybridization chain reaction (HCR), in which stable DNA monomers assemble only upon exposure to a target DNA fragment, biomolecular self-assembly pathways, etc. Such work strongly suggests that nanotechnology and synthetic biology together seem poised to constitute the most transformative development of the 21st century. In this paper, we present a Constraint Programming (CP) approach to solve the RNA inverse folding problem. Given a target RNA secondary structure, we determine an RNA sequence which folds into the target structure; i.e. whose minimum free energy structure is the target structure. Our approach represents a step forward in RNA design--we produce the first complete RNA inverse folding approach which allows for the specification of a wide range of design constraints. We also introduce a Large Neighborhood Search approach which allows us to tackle larger instances at the cost of losing completeness, while retaining the advantages of meeting design constraints (motif, GC-content, etc.). Results demonstrate that our software, RNAiFold, performs as well or better than all state-of-the-art approaches; nevertheless, our approach is unique in terms of completeness, flexibility, and the support of various design constraints. The algorithms presented in this paper are publicly available via the interactive webserver http://bioinformatics.bc.edu/clotelab/RNAiFold; additionally, the source code can be downloaded from that site.

CoFold: an RNA secondary structure prediction method that takes co-transcriptional folding into account

Abstract: Existing state-of-the-art methods that take a single RNA sequence and predict the corresponding RNA secondary structure are thermodynamic methods. These aim to predict the most stable RNA structure. There exists by now ample experimental and theoretical evidence that the process of structure formation matters and that sequences in vivo fold while they are being transcribed. None of the thermodynamic methods, however, consider the process of structure formation. Here, we present a conceptually new method for predicting RNA secondary structure, called CoFold, that takes effects of co-transcriptional folding explicitly into account. Our method significantly improves the state-of-art in terms of prediction accuracy, especially for long sequences of >1000 nt in length.

Sensitive measurement of single-nucleotide polymorphism-induced changes of RNA conformation: application to disease studies

Abstract: Single-nucleotide polymorphisms (SNPs) are often linked to critical phenotypes such as diseases or responses to vaccines, medications and environmental factors. However, the specific molecular mechanisms by which a causal SNP acts is usually not obvious. Changes in RNA secondary structure emerge as a possible explanation necessitating the development of methods to measure the impact of single-nucleotide variation on RNA structure. Despite the recognition of the importance of considering the changes in Boltzmann ensemble of RNA conformers in this context, a formal method to perform directly such comparison was lacking. Here, we solved this problem and designed an efficient method to compute the relative entropy between the Boltzmann ensembles of the native and a mutant structure. On the basis of this theoretical progress, we developed a software tool, remuRNA, and investigated examples of its application. Comparing the impact of common SNPs naturally occurring in populations with the impact of random point mutations, we found that structural changes introduced by common SNPs are smaller than those introduced by random point mutations. This suggests a natural selection against mutations that significantly change RNA structure and demonstrates, surprisingly, that randomly inserted point mutations provide inadequate estimation of random mutations effects. Subsequently, we applied remuRNA to determine which of the disease-associated non-coding SNPs are potentially related to RNA structural changes.

Mean-field interactions between nucleic-acid-base dipoles can drive the formation of a double helix.

Abstract: A proposed coarse-grained model of nucleic acids demonstrates that average interactions between base dipoles, together with chain connectivity and excluded-volume interactions, are sufficient to form double-helical structures of DNA and RNA molecules. Additionally, local interactions determine helix handedness and direction of strand packing. This result, and earlier research on reduced protein models, suggests that mean-field multipole-multipole interactions are the principal factors responsible for the formation of regular structure of biomolecules.

Functional Analysis of the Murine Coronavirus Genomic RNA Packaging Signal

Abstract: Coronaviruses selectively package genomic RNA into assembled virions, despite the great molar excess of subgenomic RNA species that is present in infected cells. The genomic packaging signal (PS) for the coronavirus mouse hepatitis virus (MHV) was originally identified as an element that conferred packaging capability to defective interfering RNAs. The MHV PS is an RNA structure that maps to the region of the replicase gene encoding the nonstructural protein 15 subunit of the viral replicase-transcriptase complex. To begin to understand the role and mechanism of action of the MHV PS in its native genomic locus, we constructed viral mutants in which this cis-acting element was altered, deleted, or transposed. Our results demonstrated that the PS is pivotal in the selection of viral genomic RNA for incorporation into virions. Mutants in which PS RNA secondary structure was disrupted or entirely ablated packaged large quantities of subgenomic RNAs, in addition to genomic RNA. Moreover, the PS retained its function when displaced to an ectopic site in the genome. Surprisingly, the PS was not essential for MHV viability, nor did its elimination have a severe effect on viral growth. However, the PS was found to provide a distinct selective advantage to MHV. Viruses containing the PS readily outcompeted their otherwise isogenic counterparts lacking the PS.

RNA structure and dynamics: a base pairing perspective.

Abstract: RNA is now known to possess various structural, regulatory and enzymatic functions for survival of cellular organisms. Functional RNA structures are generally created by three-dimensional organization of small structural motifs, formed by base pairing between self-complementary sequences from different parts of the RNA chain. In addition to the canonical Watson–Crick or wobble base pairs, several non-canonical base pairs are found to be crucial to the structural organization of RNA molecules. They appear within different structural motifs and are found to stabilize the molecule through long-range intra-molecular interactions between basic structural motifs like double helices and loops. These base pairs also impart functional variation to the minor groove of A-form RNA helices, thus forming anchoring site for metabolites and ligands. Non-canonical base pairs are formed by edge-to-edge hydrogen bonding interactions between the bases. A large number of theoretical studies have been done to detect and analyze these non-canonical base pairs within crystal or NMR derived structures of different functional RNA. Theoretical studies of these isolated base pairs using ab initio quantum chemical methods as well as molecular dynamics simulations of larger fragments have also established that many of these non-canonical base pairs are as stable as the canonical Watson–Crick base pairs. This review focuses on the various structural aspects of non-canonical base pairs in the organization of RNA molecules and the possible applications of these base pairs in predicting RNA structures with more accuracy.

Recognition of RNA duplexes by chemically modified triplex-forming oligonucleotides

Abstract: Triplex is emerging as an important RNA tertiary structure motif, in which consecutive non-canonical base pairs form between a duplex and a third strand. RNA duplex region is also often functionally important site for protein binding. Thus, triplex-forming oligonucleotides (TFOs) may be developed to regulate various biological functions involving RNA, such as viral ribosomal frameshifting and reverse transcription. How chemical modification in TFOs affects RNA triplex stability, however, is not well understood. Here, we incorporated locked nucleic acid, 2-thio U- and 2 0 -O methyl-modified residues in a series of all pyrimidine RNA TFOs, and we studied the binding to two RNA hairpin structures. The 12-base-triple major-groove pyrimidine–purine– pyrimidine triplex structures form between the duplex regions of RNA/DNA hairpins and the complementary RNA TFOs. Ultraviolet-absorbance-detected thermal melting studies reveal that the locked nucleic acid and 2-thio U modifications in TFOs strongly enhance triplex formation with both parental RNA and DNA duplex regions. In addition, we found that incorporation of 2 0 -O methyl-modified residues in a TFO destabilizes and stabilizes triplex formation with RNA and DNA duplex regions, respectively. The (de)stabilization of RNA triplex formation may be facilitated through modulation of van der Waals contact, base stacking, hydrogen bonding, backbone pre-organization, geometric compatibility and/or dehydration energy. Better understanding of the molecular determinants of RNA triplex structure stability lays the foundation for designing and discovering novel sequence-specific duplex-binding ligands as diagnostic and therapeutic agents targeting RNA.

APOBEC3G cytosine deamination hotspots are defined by both sequence context and single-stranded DNA secondary structure

Abstract: Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (i.e., APOBEC3G or A3G) is an evolutionarily conserved cytosine deaminase that potently restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons and other viruses. A3G has a nucleotide target site specificity for cytosine dinucleotides, though only certain cytosine dinucleotides are ‘hotspots’ for cytosine deamination, and others experience little or no editing by A3G. The factors that define these critical A3G hotspots are not fully understood. To investigate how A3G hotspots are defined, we used an in vitro fluorescence resonance energy transfer-based oligonucleotide assay to probe the site specificity of A3G. Our findings strongly suggest that the target single-stranded DNA (ssDNA) secondary structure as well as the bases directly 3′ and 5′ of the cytosine dinucleotide are critically important A3G recognition. For instance, A3G cannot readily deaminate a cytosine dinucleotide in ssDNA stem structures or in nucleotide base loops composed of three bases. Single-stranded nucleotide loops up to seven bases in length were poor targets for A3G activity unless cytosine residues flanked the cytosine dinucleotide. Furthermore, we observed that A3G favors adenines, cytosines and thymines flanking the cytosine dinucleotide target in unstructured regions of ssDNA. Low cytosine deaminase activity was detected when guanines flanked the cytosine dinucleotide. Taken together, our findings provide the first demonstration that A3G cytosine deamination hotspots are defined by both the sequence context of the cytosine dinucleotide target as well as the ssDNA secondary structure. This knowledge can be used to better trace the origins of mutations to A3G activity, and illuminate its impact on processes such as HIV-1 genetic variation.

A Simple Fluorescence Method for pKa Determination in RNA and DNA Reveals Highly Shifted pKa’s

Abstract: Charged nucleobases exist in RNA and DNA at neutral pH owing to pKa shifting. These bases can affect polymerase fidelity and participate in ribozyme general acid–base catalysis. Protonated RNA bases further influence miRNA processing and viral frameshifting. It is therefore important to have a simple and rapid method for determining the pKa of nucleobases in RNA and DNA. Here we describe the application of 2-aminopurine (2AP), a fluorescent isomer of adenine, to report on the pKa of a nearby ionizing base both in DNA secondary structure and RNA tertiary structure. We observe large, up to 5-fold quenching in fluorescence upon protonation of a nearby base. Using this method, we identify highly shifted pKa’s of 7.6 for adenine in a DNA oligonucleotide and 8.15 for cytidine in a tertiary structure element from beet western yellows virus (BWYV) RNA. These pKa values, which were corroborated by 31P NMR measurements and comparison to literature, are shifted over 4 units from their standard values. This fluoresce...

The origin of biased sequence depth in sequence-independent nucleic acid amplification and optimization for efficient massive parallel sequencing.

Abstract: Sequence Independent Single Primer Amplification is one of the most widely used random amplification approaches in virology for sequencing template preparation. This technique relies on oligonucleotides consisting of a 3′ random part used to prime complementary DNA synthesis and a 5′ defined tag sequence for subsequent amplification. Recently, this amplification method was combined with next generation sequencing to obtain viral sequences. However, these studies showed a biased distribution of the resulting sequence reads over the analyzed genomes. The aim of this study was to elucidate the mechanisms that lead to biased sequence depth when using random amplification. Avian paramyxovirus type 8 was used as a model RNA virus to investigate these mechanisms. We showed, based on in silico analysis of the sequence depth in relation to GC-content, predicted RNA secondary structure and sequence complementarity to the 3′ part of the tag sequence, that the tag sequence has the main contribution to the observed bias in sequence depth. We confirmed this finding experimentally using both fragmented and non-fragmented viral RNAs as well as primers differing in random oligomer length (6 or 12 nucleotides) and in the sequence of the amplification tag. The observed oligonucleotide annealing bias can be reduced by extending the random oligomer sequence and by in silico combining sequence data from SISPA experiments using different 5′ defined tag sequences. These findings contribute to the optimization of random nucleic acid amplification protocols that are currently required for downstream applications such as viral metagenomics and microarray analysis.

RNAsnp: Efficient Detection of Local RNA Secondary Structure Changes Induced by SNPs: HUMAN MUTATION

Abstract: The original article to which this Erratum refers was published in Human Mutation 34(4):546–556 (DOI 10.1002/humu.22273). In the article cited above, the symbols for Mik and ξ < i were inadvertently changed during the production process. The corrected text appears below. The Publisher sincerely regrets this error. At face value, this minimization is rather expensive because for each index pair u,v, the values of π i[u,v] need to be determined. The naı̈ve evaluation of Eq. (7) can be replaced by a recursive scheme (Supp. Fig. S1). Consider sequence positions k < i < l and denote by Mik and Nil, the probabilities that i has a pairing partner in the interval [k,i – 1] and the interval [i + 1,l], respectively. Clearly, these auxiliary variables satisfy Mik = Mi(k – 1) + Pki and Nil = Ni(l + 1) + Pil. Obviously, for all k < i < l, we have π i[k,l] = Mik + Nil, ξ < i [k, l] = Mik , and ξ > i [k, l] = Nil . Precomputing M and N thus allows the evaluation of distances and correlation coefficients in linear time for each sequence interval.

Principles for Understanding the Accuracy of SHAPE-Directed RNA Structure Modeling

Abstract: Accurate RNA structure modeling is an important, incompletely solved, challenge. Single-nucleotide resolution SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) yields an experimental measurement of local nucleotide flexibility that can be incorporated as pseudo-free energy change constraints to direct secondary structure predictions. Prior work from our laboratory has emphasized both the overall accuracy of this approach and the need for nuanced interpretation of modeled structures. Recent studies by Das and colleagues [Kladwang, W., et al. (2011) Biochemistry 50, 8049; Nat. Chem. 3, 954], focused on analyzing six small RNAs, yielded poorer RNA secondary structure predictions than expected on the basis of prior benchmarking efforts. To understand the features that led to these divergent results, we re-examined four RNAs yielding the poorest results in this recent work: tRNA(Phe), the adenine and cyclic-di-GMP riboswitches, and 5S rRNA. Most of the errors reported by Das and colleagues reflected nonstandard experiment and data processing choices, and selective scoring rules. For two RNAs, tRNA(Phe) and the adenine riboswitch, secondary structure predictions are nearly perfect if no experimental information is included but were rendered inaccurate by the SHAPE data of Das and colleagues. When best practices were used, single-sequence SHAPE-directed secondary structure modeling recovered ~93% of individual base pairs and >90% of helices in the four RNAs, essentially indistinguishable from the results of the mutate-and-map approach with the exception of a single helix in the 5S rRNA. The field of experimentally directed RNA secondary structure prediction is entering a phase focused on the most difficult prediction challenges. We outline five constructive principles for guiding this field forward.

Role of DNA secondary structures in fragile site breakage along human chromosome 10

Abstract: The formation of alternative DNA secondary structures can result in DNA breakage leading to cancer and other diseases. Chromosomal fragile sites, which are regions of the genome that exhibit chromosomal breakage under conditions of mild replication stress, are predicted to form stable DNA secondary structures. DNA breakage at fragile sites is associated with regions that are deleted, amplified or rearranged in cancer. Despite the correlation, unbiased examination of the ability to form secondary structures has not been evaluated in fragile sites. Here, using the Mfold program, we predict potential DNA secondary structure formation on the human chromosome 10 sequence, and utilize this analysis to compare fragile and non-fragile DNA. We found that aphidicolin (APH)-induced common fragile sites contain more sequence segments with potential high secondary structure-forming ability, and these segments clustered more densely than those in non-fragile DNA. Additionally, using a threshold of secondary structure-forming ability, we refined legitimate fragile sites within the cytogenetically defined boundaries, and identified potential fragile regions within non-fragile DNA. In vitro detection of alternative DNA structure formation and a DNA breakage cell assay were used to validate the computational predictions. Many of the regions identified by our analysis coincide with genes mutated in various diseases and regions of copy number alteration in cancer. This study supports the role of DNA secondary structures in common fragile site instability, provides a systematic method for their identification and suggests a mechanism by which DNA secondary structures can lead to human disease.

Long-Range Architecture in a Viral RNA Genome

Abstract: We have developed a model for the secondary structure of the 1058-nucleotide plus-strand RNA genome of the icosahedral satellite tobacco mosaic virus (STMV) using nucleotide-resolution SHAPE chemical probing of the viral RNA isolated from virions and within the virion, perturbation of interactions distant in the primary sequence, and atomic force microscopy. These data are consistent with long-range base pairing interactions and a three-domain genome architecture. The compact domains of the STMV RNA have dimensions of 10–45 nm. Each of the three domains corresponds to a specific functional component of the virus: The central domain corresponds to the coding sequence of the single (capsid) protein encoded by the virus, whereas the 5′ and 3′ untranslated domains span signals essential for translation and replication, respectively. This three-domain architecture is compatible with interactions between the capsid protein and short RNA helices previously visualized by crystallography. STMV is among the simples...

Role of the unstructured N-terminal domain of the hAPE1 (human apurinic/apyrimidinic endonuclease 1) in the modulation of its interaction with nucleic acids and NPM1 (nucleophosmin)

Abstract: The hAPE1 (human apurinic/apyrimidinic endonuclease 1) is an essential enzyme, being the main abasic endonuclease in higher eukaryotes. However, there is strong evidence to show that hAPE1 can directly bind specific gene promoters, thus modulating their transcriptional activity, even in the absence of specific DNA damage. Recent findings, moreover, suggest a role for hAPE1 in RNA processing, which is modulated by the interaction with NPM1 (nucleophosmin). Independent domains account for many activities of hAPE1; however, whereas the endonuclease and the redox-active portions of the protein are well characterized, a better understanding of the role of the unstructured N-terminal region is needed. In the present study, we characterized the requirements for the interaction of hAPE1 with NPM1 and undamaged nucleic acids. We show that DNA/RNA secondary structure has an impact on hAPE1 binding in the absence of damage. Biochemical studies, using the isolated N-terminal region of the protein, reveal that the hAPE1 N-terminal domain represents an evolutionary gain of function, since its composition affects the protein9s stability and ability to interact with both nucleic acids and NPM1. Although required, however, this region is not sufficient itself to stably interact with DNA or NPM1.

Secondary Structure of a Conserved Domain in the Intron of Influenza A NS1 mRNA

Abstract: Influenza A virus is a segmented single-stranded (−)RNA virus that causes substantial annual morbidity and mortality. The transcriptome of influenza A is predicted to have extensive RNA secondary structure. The smallest genome segment, segment 8, encodes two proteins, NS1 and NEP, via alternative splicing. A conserved RNA domain in the intron of segment 8 may be important for regulating production of NS1. Two different multi-branch loop structures have been proposed for this region. A combination of in vitro chemical mapping and isoenergetic microarray techniques demonstrate that the consensus sequence for this region folds into a hairpin conformation. These results provide an alternative folding for this region and a foundation for designing experiments to probe its functional role in the influenza life cycle.

Influence of genome-scale RNA structure disruption on the replication of murine norovirus—similar replication kinetics in cell culture but attenuation of viral fitness in vivo

Abstract: Mechanisms by which certain RNA viruses, such as hepatitis C virus, establish persistent infections and cause chronic disease are of fundamental importance in viral pathogenesis. Mammalian positive-stranded RNA viruses establishing persistence typically possess genome-scale ordered RNA secondary structure (GORS) in their genomes. Murine norovirus (MNV) persists in immunocompetent mice and provides an experimental model to functionally characterize GORS. Substitution mutants were constructed with coding sequences in NS3/4- and NS6/7-coding regions replaced with sequences with identical coding and (di-)nucleotide composition but disrupted RNA secondary structure (F1, F2, F1/F2 mutants). Mutants replicated with similar kinetics to wild-type (WT) MNV3 in RAW264.7 cells and primary macrophages, exhibited similar (highly restricted) induction and susceptibility to interferon-coupled cellular responses and equal replication fitness by serial passaging of co-cultures. In vivo, both WT and F1/F2 mutant viruses persistently infected mice, although F1, F2 and F1/F2 mutant viruses were rapidly eliminated 1–7 days post-inoculation in competition experiments with WT. F1/F2 mutants recovered from tissues at 9 months showed higher synonymous substitution rates than WT and nucleotide substitutions that potentially restored of RNA secondary structure. GORS plays no role in basic replication of MNV but potentially contributes to viral fitness and persistence in vivo.

Visualizing RNA base-pairing probabilities with RNAbow diagrams.

Abstract: There are many effective ways to represent a minimum free energy RNA secondary structure that make it easy to locate its helices and loops. It is a greater challenge to visualize the thermal average probabilities of all folds in a partition function sum; dot plot representations are often puzzling. Therefore, we introduce the RNAbows visualization tool for RNA base pair probabilities. RNAbows represent base pair probabilities with line thickness and shading, yielding intuitive diagrams. RNAbows aid in disentangling incompatible structures, allow comparisons between clusters of folds, highlight differences between wild-type and mutant folds, and are also rather beautiful.

Efficient Detection of Hydrogen Bonds in Dynamic Regions of RNA by Sensitivity‐Optimized NMR Pulse Sequences

Abstract: Improved Sensitivity: Efficient NMR experiments are presented for determining the secondary structure in large and dynamic RNAs using J-couplings across hydrogen bonds. The experiments provide up to eight-fold improved sensitivity and thus enable detection of base pairs in dynamic regions even in large RNAs.