Showing papers on "Nucleic acid structure published in 2015"

Structural imprints in vivo decode RNA regulatory mechanisms

Abstract: Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA. Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

Dynamic response of RNA editing to temperature in Drosophila

Abstract: Adenosine-to-inosine RNA editing is a highly conserved process that post-transcriptionally modifies mRNA, generating proteomic diversity, particularly within the nervous system of metazoans. Transcripts encoding proteins involved in neurotransmission predominate as targets of such modifications. Previous reports suggest that RNA editing is responsive to environmental inputs in the form of temperature alterations. However, the molecular determinants underlying temperature-dependent RNA editing responses are not well understood. Using the poikilotherm Drosophila, we show that acute temperature alterations within a normal physiological range result in substantial changes in RNA editing levels. Our examination of particular sites reveals diversity in the patterns with which editing responds to temperature, and these patterns are conserved across five species of Drosophilidae representing over 10 million years of divergence. In addition, we show that expression of the editing enzyme, ADAR (adenosine deaminase acting on RNA), is dramatically decreased at elevated temperatures, partially, but not fully, explaining some target responses to temperature. Interestingly, this reduction in editing enzyme levels at elevated temperature is only partially reversed by a return to lower temperatures. Lastly, we show that engineered structural variants of the most temperature-sensitive editing site, in a sodium channel transcript, perturb thermal responsiveness in RNA editing profile for a particular RNA structure. Our results suggest that the RNA editing process responds to temperature alterations via two distinct molecular mechanisms: through intrinsic thermo-sensitivity of the RNA structures that direct editing, and due to temperature sensitive expression or stability of the RNA editing enzyme. Environmental cues, in this case temperature, rapidly reprogram the Drosophila transcriptome through RNA editing, presumably resulting in altered proteomic ratios of edited and unedited proteins.

Crystal structure reveals specific recognition of a G-quadruplex RNA by a β-turn in the RGG motif of FMRP

Abstract: Fragile X Mental Retardation Protein (FMRP) is a regulatory RNA binding protein that plays a central role in the development of several human disorders including Fragile X Syndrome (FXS) and autism. FMRP uses an arginine-glycine-rich (RGG) motif for specific interactions with guanine (G)-quadruplexes, mRNA elements implicated in the disease-associated regulation of specific mRNAs. Here we report the 2.8-A crystal structure of the complex between the human FMRP RGG peptide bound to the in vitro selected G-rich RNA. In this model system, the RNA adopts an intramolecular K+-stabilized G-quadruplex structure composed of three G-quartets and a mixed tetrad connected to an RNA duplex. The RGG peptide specifically binds to the duplex–quadruplex junction, the mixed tetrad, and the duplex region of the RNA through shape complementarity, cation–π interactions, and multiple hydrogen bonds. Many of these interactions critically depend on a type I β-turn, a secondary structure element whose formation was not previously recognized in the RGG motif of FMRP. RNA mutagenesis and footprinting experiments indicate that interactions of the peptide with the duplex–quadruplex junction and the duplex of RNA are equally important for affinity and specificity of the RGG–RNA complex formation. These results suggest that specific binding of cellular RNAs by FMRP may involve hydrogen bonding with RNA duplexes and that RNA duplex recognition can be a characteristic RNA binding feature for RGG motifs in other proteins.

Detection of RNA-Protein Interactions in Living Cells with SHAPE.

Abstract: SHAPE-MaP is unique among RNA structure probing strategies in that it both measures flexibility at single-nucleotide resolution and quantifies the uncertainties in these measurements. We report a straightforward analytical framework that incorporates these uncertainties to allow detection of RNA structural differences between any two states, and we use it here to detect RNA-protein interactions in healthy mouse trophoblast stem cells. We validate this approach by analysis of three model cytoplasmic and nuclear ribonucleoprotein complexes, in 2 min in-cell probing experiments. In contrast, data produced by alternative in-cell SHAPE probing methods correlate poorly (r = 0.2) with those generated by SHAPE-MaP and do not yield accurate signals for RNA-protein interactions. We then examine RNA-protein and RNA-substrate interactions in the RNase MRP complex and, by comparing in-cell interaction sites with disease-associated mutations, characterize these noncoding mutations in terms of molecular phenotype. Together, these results reveal that SHAPE-MaP can define true interaction sites and infer RNA functions under native cellular conditions with limited preexisting knowledge of the proteins or RNAs involved.

RNA:DNA hybrids in the human genome have distinctive nucleotide characteristics, chromatin composition, and transcriptional relationships

Abstract: RNA:DNA hybrids represent a non-canonical nucleic acid structure that has been associated with a range of human diseases and potential transcriptional regulatory functions. Mapping of RNA:DNA hybrids in human cells reveals them to have a number of characteristics that give insights into their functions. We find RNA:DNA hybrids to occupy millions of base pairs in the human genome. A directional sequencing approach shows the RNA component of the RNA:DNA hybrid to be purine-rich, indicating a thermodynamic contribution to their in vivo stability. The RNA:DNA hybrids are enriched at loci with decreased DNA methylation and increased DNase hypersensitivity, and within larger domains with characteristics of heterochromatin formation, indicating potential transcriptional regulatory properties. Mass spectrometry studies of chromatin at RNA:DNA hybrids shows the presence of the ILF2 and ILF3 transcription factors, supporting a model of certain transcription factors binding preferentially to the RNA:DNA conformation. Overall, there is little to indicate a dependence for RNA:DNA hybrids forming co-transcriptionally, with results from the ribosomal DNA repeat unit instead supporting the intriguing model of RNA generating these structures in trans. The results of the study indicate heterogeneous functions of these genomic elements and new insights into their formation and stability in vivo.

Functionally conserved architecture of hepatitis C virus RNA genomes

Abstract: Hepatitis C virus (HCV) infects over 170 million people worldwide and is a leading cause of liver disease and cancer. The virus has a 9,650-nt, single-stranded, messenger-sense RNA genome that is infectious as an independent entity. The RNA genome has evolved in response to complex selection pressures, including the need to maintain structures that facilitate replication and to avoid clearance by cell-intrinsic immune processes. Here we used high-throughput, single-nucleotide resolution information to generate and functionally test data-driven structural models for three diverse HCV RNA genomes. We identified, de novo, multiple regions of conserved RNA structure, including all previously characterized cis-acting regulatory elements and also multiple novel structures required for optimal viral fitness. Well-defined RNA structures in the central regions of HCV genomes appear to facilitate persistent infection by masking the genome from RNase L and double-stranded RNA-induced innate immune sensors. This work shows how structure-first comparative analysis of entire genomes of a pathogenic RNA virus enables comprehensive and concise identification of regulatory elements and emphasizes the extensive interrelationships among RNA genome structure, viral biology, and innate immune responses.

New Perspectives on DNA and RNA Triplexes As Effectors of Biological Activity.

Abstract: Since the first description of the canonical B-form DNA double helix, it has been suggested that alternative DNA, DNA–RNA, and RNA structures exist and act as functional genomic elements. Indeed, over the past few years it has become clear that, in addition to serving as a repository for genetic information, genomic DNA elicits biological responses by adopting conformations that differ from the canonical right-handed double helix, and by interacting with RNA molecules to form complex secondary structures. This review focuses on recent advances on three-stranded (triplex) nucleic acids, with an emphasis on DNA–RNA and RNA–RNA interactions. Emerging work reveals that triplex interactions between noncoding RNAs and duplex DNA serve as platforms for delivering site-specific epigenetic marks critical for the regulation of gene expression. Additionally, an increasing body of genetic and structural studies demonstrates that triplex RNA–RNA interactions are essential for performing catalytic and regulatory functions in cellular nucleoprotein complexes, including spliceosomes and telomerases, and for enabling protein recoding during programmed ribosomal frameshifting. Thus, evidence is mounting that DNA and RNA triplex interactions are implemented to perform a range of diverse biological activities in the cell, some of which will be discussed in this review.

Genome-wide profiling of in vivo RNA structure at single-nucleotide resolution using structure-seq

Abstract: Structure-seq is a high-throughput and quantitative method that provides genome-wide information on RNA structure at single-nucleotide resolution. Structure-seq can be performed both in vivo and in vitro to study RNA structure-function relationships, RNA regulation of gene expression and RNA processing. Structure-seq can be carried out by an experienced molecular biologist with a basic understanding of bioinformatics. Structure-seq begins with chemical RNA structure probing under single-hit kinetics conditions. Certain chemical modifications, e.g., methylation of the Watson-Crick face of unpaired adenine and cytosine residues by dimethyl sulfate, result in a stop in reverse transcription. Modified RNA is then subjected to reverse transcription using random hexamer primers, which minimizes 3' end bias; reverse transcription proceeds until it is blocked by a chemically modified residue. Resultant cDNAs are amplified by adapter-based PCR and subjected to high-throughput sequencing, subsequently allowing retrieval of the structural information on a genome-wide scale. In contrast to classical methods that provide information only on individual transcripts, a single structure-seq experiment provides information on tens of thousands of RNA structures in ∼1 month. Although the procedure described here is for Arabidopsis thaliana seedlings in vivo, structure-seq is widely applicable, thereby opening new avenues to explore RNA structure-function relationships in living organisms.

Synthesis and applications of RNAs with position-selective labelling and mosaic composition

Abstract: Knowledge of the structure and dynamics of RNA molecules is critical to understanding their many biological functions. Furthermore, synthetic RNAs have applications as therapeutics and molecular sensors. Both research and technological applications of RNA would be dramatically enhanced by methods that enable incorporation of modified or labelled nucleotides into specifically designated positions or regions of RNA. However, the synthesis of tens of milligrams of such RNAs using existing methods has been impossible. Here we develop a hybrid solid-liquid phase transcription method and automated robotic platform for the synthesis of RNAs with position-selective labelling. We demonstrate its use by successfully preparing various isotope- or fluorescently labelled versions of the 71-nucleotide aptamer domain of an adenine riboswitch for nuclear magnetic resonance spectroscopy or single-molecule Forster resonance energy transfer, respectively. Those RNAs include molecules that were selectively isotope-labelled in specific loops, linkers, a helix, several discrete positions, or a single internal position, as well as RNA molecules that were fluorescently labelled in and near kissing loops. These selectively labelled RNAs have the same fold as those transcribed using conventional methods, but they greatly simplify the interpretation of NMR spectra. The single-position isotope- and fluorescently labelled RNA samples reveal multiple conformational states of the adenine riboswitch. Lastly, we describe a robotic platform and the operation that automates this technology. Our selective labelling method may be useful for studying RNA structure and dynamics and for making RNA sensors for a variety of applications including cell-biological studies, substance detection, and disease diagnostics.

Targeted Detection of G-Quadruplexes in Cellular RNAs

Abstract: The G-quadruplex (G4) is a non-canonical nucleic acid structure which regulates important cellular processes. RNA G4s have recently been shown to exist in human cells and be biologically significant. Described herein is a new approach to detect and map RNA G4s in cellular transcripts. This method exploits the specific control of RNA G4–cation and RNA G4–ligand interactions during reverse transcription, by using a selective reverse transcriptase to monitor RNA G4-mediated reverse transcriptase stalling (RTS) events. Importantly, a ligation-amplification strategy is coupled with RTS, and enables detection and mapping of G4s in important, low-abundance cellular RNAs. Strong evidence is provided for G4 formation in full-length cellular human telomerase RNA, offering important insights into its cellular function.

New hypotheses derived from the structure of a flaviviral Xrn1-resistant RNA: Conservation, folding, and host adaptation

Abstract: Arthropod-borne flaviviruses (FVs) are a growing world-wide health threat whose incidence and range are increasing. The pathogenicity and cytopathicity of these single-stranded RNA viruses are influenced by viral subgenomic non-protein-coding RNAs (sfRNAs) that the viruses produce to high levels during infection. To generate sfRNAs the virus co-opts the action of the abundant cellular exonuclease Xrn1, which is part of the cell's normal RNA turnover machinery. This exploitation of the cellular machinery is enabled by discrete, highly structured, Xrn1-resistant RNA elements (xrRNAs) in the 3'UTR that interact with Xrn1 to halt processive 5' to 3' decay of the viral genomic RNA. We recently solved the crystal structure of a functional xrRNA, revealing a novel fold that provides a mechanistic model for Xrn1 resistance. Continued analysis and interpretation of the structure reveals that the tertiary contacts that knit the xrRNA fold together are shared by a wide variety of arthropod-borne FVs, conferring robust Xrn1 resistance in all tested. However, there is some variability in the structures that correlates with unexplained patterns in the viral 3' UTRs. Finally, examination of these structures and their behavior in the context of viral infection leads to a new hypothesis linking RNA tertiary structure, overall 3' UTR architecture, sfRNA production, and host adaptation.

RNA structure determination by solid-state NMR spectroscopy

Abstract: Knowledge of the RNA three-dimensional structure, either in isolation or as part of RNP complexes, is fundamental to understand the mechanism of numerous cellular processes. Because of its flexibility, RNA represents a challenge for crystallization, while the large size of cellular complexes brings solution-state NMR to its limits. Here, we demonstrate an alternative approach on the basis of solid-state NMR spectroscopy. We develop a suite of experiments and RNA labeling schemes and demonstrate for the first time that ssNMR can yield a RNA structure at high-resolution. This methodology allows structural analysis of segmentally labelled RNA stretches in high-molecular weight cellular machines—independent of their ability to crystallize—and opens the way to mechanistic studies of currently difficult-to-access RNA-protein assemblies.

A stable RNA G-quadruplex within the 5'-UTR of Arabidopsis thaliana ATR mRNA inhibits translation.

Abstract: Guanine quadruplex structures (GQSs) play important roles in the regulation of gene expression and cellular processes. Recent studies provide strong evidence for the formation and function of DNA and RNA GQSs in human cells. However, whether GQSs form and are functional in plants remains essentially unexplored. On the basis of circular dichroism (CD)-detected titration, UV-detected melting, in-line probing (ILP) and reporter gene assay studies, we report the first example of a plant RNA GQS that inhibits translation. This GQS is located within the 5′-UTR of the ATAXIA TELANGIECTASIA-MUTATED AND RAD3-RELATED ( ATR ) mRNA of Arabidopsis thaliana (mouse-ear cress). We show that this GQS is highly stable and is thermodynamically favoured over a competing hairpin structure in the 5′-UTR at physiological K+ and Mg2+ concentrations. Results from ILP reveal the secondary structure of the RNA and support formation of the GQS in vitro in the context of the complete 5′-UTR. Transient reporter gene assays performed in living plants reveal that the GQS inhibits translation but not transcription, implicating this GQS as a translational repressor in vivo . Our results provide the first complete demonstration of the formation and function of a regulatory RNA GQS in plants and open new avenues to explore potential functional roles of GQS in the plant kingdom. Abbreviations: ATR, ATAXIA TELANGIECTASIA-MUTATED AND RAD3-RELATED; CIP, calf intestinal phosphatase; DMS, dimethyl sulfate; GQS, guanine quadruplex structures; GUS, β-glucuronidase; ILP, in-line probing; L, loop; LiCac, lithium cacodylate; mGQS, mutant GQS; 4-MU, 4-methylumbelliferone; MUG, 4-methylumbelliferyl-β-D-glucuronide; nt, nucleotide; PNK, polynucleotide kinase; qRT-PCR, quantitative reverse transcription–PCR; RACE, rapid amplification of cDNA ends; SHAPE, selective 2′-hydroxyl acylation analysed by primer extension

A Large-Scale Assessment of Nucleic Acids Binding Site Prediction Programs

Abstract: Computational prediction of nucleic acid binding sites in proteins are necessary to disentangle functional mechanisms in most biological processes and to explore the binding mechanisms. Several strategies have been proposed, but the state-of-the-art approaches display a great diversity in i) the definition of nucleic acid binding sites; ii) the training and test datasets; iii) the algorithmic methods for the prediction strategies; iv) the performance measures and v) the distribution and availability of the prediction programs. Here we report a large-scale assessment of 19 web servers and 3 stand-alone programs on 41 datasets including more than 5000 proteins derived from 3D structures of protein-nucleic acid complexes. Well-defined binary assessment criteria (specificity, sensitivity, precision, accuracy…) are applied. We found that i) the tools have been greatly improved over the years; ii) some of the approaches suffer from theoretical defects and there is still room for sorting out the essential mechanisms of binding; iii) RNA binding and DNA binding appear to follow similar driving forces and iv) dataset bias may exist in some methods.

Mutational interference mapping experiment (MIME) for studying RNA structure and function

Abstract: RNA regulates many biological processes; however, identifying functional RNA sequences and structures is complex and time-consuming. We introduce a method, mutational interference mapping experiment (MIME), to identify, at single-nucleotide resolution, the primary sequence and secondary structures of an RNA molecule that are crucial for its function. MIME is based on random mutagenesis of the RNA target followed by functional selection and next-generation sequencing. Our analytical approach allows the recovery of quantitative binding parameters and permits the identification of base-pairing partners directly from the sequencing data. We used this method to map the binding site of the human immunodeficiency virus-1 (HIV-1) Pr55(Gag) protein on the viral genomic RNA in vitro, and showed that, by analyzing permitted base-pairing patterns, we could model RNA structure motifs that are crucial for protein binding.

Challenge of mimicking the influences of the cellular environment on RNA structure by PEG-induced macromolecular crowding.

Abstract: There are large differences between the cellular environment and the conditions widely used to study RNA in vitro. SHAPE RNA structure probing in Escherichia coli cells has shown that the cellular environment stabilizes both long-range and local tertiary interactions in the adenine riboswitch aptamer domain. Synthetic crowding agents are widely used to understand the forces that stabilize RNA structure and in efforts to recapitulate the cellular environment under simplified experimental conditions. Here, we studied the structure and ligand binding ability of the adenine riboswitch in the presence of the macromolecular crowding agent, polyethylene glycol (PEG). Ethylene glycol and low-molecular mass PEGs destabilized RNA structure and caused the riboswitch to sample secondary structures different from those observed in simple buffered solutions or in cells. In the presence of larger PEGs, longer-range loop–loop interactions were more similar to those in cells than in buffer alone, consistent with prior wor...

Rich RNA Structure Landscapes Revealed by Mutate-and-Map Analysis

Abstract: Landscapes exhibiting multiple secondary structures arise in natural RNA molecules that modulate gene expression, protein synthesis, and viral. We report herein that high-throughput chemical experiments can isolate an RNA’s multiple alternative secondary structures as they are stabilized by systematic mutagenesis (mutate-and-map, M2) and that a computational algorithm, REEFFIT, enables unbiased reconstruction of these states’ structures and populations. In an in silico benchmark on non-coding RNAs with complex landscapes, M2-REEFFIT recovers 95% of RNA helices present with at least 25% population while maintaining a low false discovery rate (10%) and conservative error estimates. In experimental benchmarks, M2-REEFFIT recovers the structure landscapes of a 35-nt MedLoop hairpin, a 110-nt 16S rRNA four-way junction with an excited state, a 25-nt bistable hairpin, and a 112-nt three-state adenine riboswitch with its expression platform, molecules whose characterization previously required expert mutational analysis and specialized NMR or chemical mapping experiments. With this validation, M2-REEFFIT enabled tests of whether artificial RNA sequences might exhibit complex landscapes in the absence of explicit design. An artificial flavin mononucleotide riboswitch and a randomly generated RNA sequence are found to interconvert between three or more states, including structures for which there was no design, but that could be stabilized through mutations. These results highlight the likely pervasiveness of rich landscapes with multiple secondary structures in both natural and artificial RNAs and demonstrate an automated chemical/computational route for their empirical characterization.

Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy.

Abstract: The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.

StructureFold: genome-wide RNA secondary structure mapping and reconstruction in vivo.

Abstract: MOTIVATION RNAs fold into complex structures that are integral to the diverse mechanisms underlying RNA regulation of gene expression. Recent development of transcriptome-wide RNA structure profiling through the application of structure-probing enzymes or chemicals combined with high-throughput sequencing has opened a new field that greatly expands the amount of in vitro and in vivo RNA structural information available. The resultant datasets provide the opportunity to investigate RNA structural information on a global scale. However, the analysis of high-throughput RNA structure profiling data requires considerable computational effort and expertise. RESULTS We present a new platform, StructureFold, that provides an integrated computational solution designed specifically for large-scale RNA structure mapping and reconstruction across any transcriptome. StructureFold automates the processing and analysis of raw high-throughput RNA structure profiling data, allowing the seamless incorporation of wet-bench structural information from chemical probes and/or ribonucleases to restrain RNA secondary structure prediction via the RNAstructure and ViennaRNA package algorithms. StructureFold performs reads mapping and alignment, normalization and reactivity derivation, and RNA structure prediction in a single user-friendly web interface or via local installation. The variation in transcript abundance and length that prevails in living cells and consequently causes variation in the counts of structure-probing events between transcripts is accounted for. Accordingly, StructureFold is applicable to RNA structural profiling data obtained in vivo as well as to in vitro or in silico datasets. StructureFold is deployed via the Galaxy platform. AVAILABILITY AND IMPLEMENTATION StructureFold is freely available as a component of Galaxy available at: https://usegalaxy.org/. CONTACT yxt148@psu.edu or sma3@psu.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands

Abstract: Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as Gquadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescencebased technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5 to 3 DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures.

Thermodynamic insights into 2-thiouridine-enhanced RNA hybridization

Abstract: Nucleobase modifications dramatically alter nucleic acid structure and thermodynamics. 2-thiouridine (s(2)U) is a modified nucleobase found in tRNAs and known to stabilize U:A base pairs and destabilize U:G wobble pairs. The recently reported crystal structures of s(2)U-containing RNA duplexes do not entirely explain the mechanisms responsible for the stabilizing effect of s(2)U or whether this effect is entropic or enthalpic in origin. We present here thermodynamic evaluations of duplex formation using ITC and UV thermal denaturation with RNA duplexes containing internal s(2)U:A and s(2)U:U pairs and their native counterparts. These results indicate that s(2)U stabilizes both duplexes. The stabilizing effect is entropic in origin and likely results from the s(2)U-induced preorganization of the single-stranded RNA prior to hybridization. The same preorganizing effect is likely responsible for structurally resolving the s(2)U:U pair-containing duplex into a single conformation with a well-defined H-bond geometry. We also evaluate the effect of s(2)U on single strand conformation using UV- and CD-monitored thermal denaturation and on nucleoside conformation using (1)H NMR spectroscopy, MD and umbrella sampling. These results provide insights into the effects that nucleobase modification has on RNA structure and thermodynamics and inform efforts toward improving both ribozyme-catalyzed and nonenzymatic RNA copying.

RNA:DNA hybrids in the human genome have distinctive nucleotide characteristics, chromatin composition, and transcriptional relationships

Abstract: Background: RNA:DNA hybrids represent a non-canonical nucleic acid structure that has been associated with a range of human diseases and potential transcriptional regulatory functions. Mapping of RNA:DNA hybrids in human cells reveals them to have a number of characteristics that give insights into their functions. Results: We find RNA:DNA hybrids to occupy millions of base pairs in the human genome. A directional sequencing approach shows the RNA component of the RNA:DNA hybrid to be purine-rich, indicating a thermodynamic contribution to their in vivo stability. The RNA:DNA hybrids are enriched at loci with decreased DNA methylation and increased DNase hypersensitivity, and within larger domains with characteristics of heterochromatin formation, indicating potential transcriptional regulatory properties. Mass spectrometry studies of chromatin at RNA:DNA hybrids shows the presence of the ILF2 and ILF3 transcription factors, supporting a model of certain transcription factors binding preferentially to the RNA:DNA conformation. Conclusions: Overall, there is little to indicate a dependence for RNA:DNA hybrids forming co-transcriptionally, with results from the ribosomal DNA repeat unit instead supporting the intriguing model of RNA generating these structures in trans. The results of the study indicate heterogeneous functions of these genomic elements and new insights into their formation and stability in vivo.

Model-Free RNA Sequence and Structure Alignment Informed by SHAPE Probing Reveals a Conserved Alternate Secondary Structure for 16S rRNA

Abstract: Discovery and characterization of functional RNA structures remains challenging due to deficiencies in de novo secondary structure modeling. Here we describe a dynamic programming approach for model-free sequence comparison that incorporates high-throughput chemical probing data. Based on SHAPE probing data alone, ribosomal RNAs (rRNAs) from three diverse organisms – the eubacteria E. coli and C. difficile and the archeon H. volcanii – could be aligned with accuracies comparable to alignments based on actual sequence identity. When both base sequence identity and chemical probing reactivities were considered together, accuracies improved further. Derived sequence alignments and chemical probing data from protein-free RNAs were then used as pseudo-free energy constraints to model consensus secondary structures for the 16S and 23S rRNAs. There are critical differences between these experimentally-informed models and currently accepted models, including in the functionally important neck and decoding regions of the 16S rRNA. We infer that the 16S rRNA has evolved to undergo large-scale changes in base pairing as part of ribosome function. As high-quality RNA probing data become widely available, structurally-informed sequence alignment will become broadly useful for de novo motif and function discovery.