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RNA Biology 2015Arthropod-borne flaviviruses (FVs) are a growing world-wide health threat whose incidence and range are increasing. The pathogenicity and cytopathicity of these... (Review)
Review
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.
Topics: 3' Untranslated Regions; Adaptation, Biological; Base Sequence; Evolution, Molecular; Exoribonucleases; Flavivirus; Genome, Viral; Host-Pathogen Interactions; Models, Biological; Molecular Sequence Data; Nucleic Acid Conformation; RNA Folding; RNA Stability; RNA, Viral; Sequence Alignment
PubMed: 26399159
DOI: 10.1080/15476286.2015.1094599 -
Proceedings of the National Academy of... Jun 2014The effects of "molecular crowding" on elementary biochemical processes due to high solute concentrations are poorly understood and yet clearly essential to the folding...
The effects of "molecular crowding" on elementary biochemical processes due to high solute concentrations are poorly understood and yet clearly essential to the folding of nucleic acids and proteins into correct, native structures. The present work presents, to our knowledge, first results on the single-molecule kinetics of solute molecular crowding, specifically focusing on GAAA tetraloop-receptor folding to isolate a single RNA tertiary interaction using time-correlated single-photon counting and confocal single-molecule FRET microscopy. The impact of crowding by high-molecular-weight polyethylene glycol on the RNA folding thermodynamics is dramatic, with up to ΔΔG° ∼ -2.5 kcal/mol changes in free energy and thus >60-fold increase in the folding equilibrium constant (Keq) for excluded volume fractions of 15%. Most importantly, time-correlated single-molecule methods permit crowding effects on the kinetics of RNA folding/unfolding to be explored for the first time (to our knowledge), which reveal that this large jump in Keq is dominated by a 35-fold increase in tetraloop-receptor folding rate, with only a modest decrease in the corresponding unfolding rate. This is further explored with temperature-dependent single-molecule RNA folding measurements, which identify that crowding effects are dominated by entropic rather than enthalpic contributions to the overall free energy change. Finally, a simple "hard-sphere" treatment of the solute excluded volume is invoked to model the observed kinetic trends, and which predict ΔΔG° ∼ -5 kcal/mol free-energy stabilization at excluded volume fractions of 30%.
Topics: Base Sequence; Carbocyanines; Entropy; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Kinetics; Microscopy, Confocal; Nucleic Acid Conformation; Protein Folding; Proteins; RNA; RNA Folding; Solutions; Thermodynamics
PubMed: 24850865
DOI: 10.1073/pnas.1316039111 -
Proceedings of the National Academy of... Jun 2023A synthetic biology approach toward constructing an RNA-based genome expands our understanding of living things and opens avenues for technological advancement. For the...
A synthetic biology approach toward constructing an RNA-based genome expands our understanding of living things and opens avenues for technological advancement. For the precise design of an artificial RNA replicon either from scratch or based on a natural RNA replicon, understanding structure-function relationships of RNA sequences is critical. However, our knowledge remains limited to a few particular structural elements intensively studied so far. Here, we conducted a series of site-directed mutagenesis studies of yeast narnaviruses ScNV20S and ScNV23S, perhaps the simplest natural autonomous RNA replicons, to identify RNA elements required for maintenance and replication. RNA structure disruption corresponding to various portions of the entire narnavirus genome suggests that pervasive RNA folding, in addition to the precise secondary structure of genome termini, is essential for maintenance of the RNA replicon in vivo. Computational RNA structure analyses suggest that this scenario likely applies to other "narna-like" viruses. This finding implies selective pressure on these simplest autonomous natural RNA replicons to fold into a unique structure that acquires both thermodynamic and biological stability. We propose the importance of pervasive RNA folding for the design of RNA replicons that could serve as a platform for in vivo continuous evolution as well as an interesting model to study the origin of life.
Topics: RNA, Viral; RNA Folding; Genome, Viral; RNA Viruses; Base Sequence; Replicon; Virus Replication
PubMed: 37339222
DOI: 10.1073/pnas.2304082120 -
International Journal of Molecular... Jul 2021Due to the high exposition to changing environmental conditions, bacteria have developed many mechanisms enabling immediate adjustments of gene expression. In many... (Review)
Review
Due to the high exposition to changing environmental conditions, bacteria have developed many mechanisms enabling immediate adjustments of gene expression. In many cases, the required speed and plasticity of the response are provided by RNA-dependent regulatory mechanisms. This is possible due to the very high dynamics and flexibility of an RNA structure, which provide the necessary sensitivity and specificity for efficient sensing and transduction of environmental signals. In this review, we will discuss the current knowledge about known bacterial regulatory mechanisms which rely on RNA structure. To better understand the structure-driven modulation of gene expression, we describe the basic theory on RNA structure folding and dynamics. Next, we present examples of multiple mechanisms employed by RNA regulators in the control of bacterial transcription and translation.
Topics: Bacteria; Bacterial Proteins; Gene Expression Regulation, Bacterial; Nucleic Acid Conformation; RNA Folding; RNA, Bacterial; Transcription, Genetic
PubMed: 34360611
DOI: 10.3390/ijms22157845 -
Journal of Nanobiotechnology May 2024Approximately 80 percent of the total RNA in cells is ribosomal RNA (rRNA), making it an abundant and inexpensive natural source of long, single-stranded nucleic acid,...
Approximately 80 percent of the total RNA in cells is ribosomal RNA (rRNA), making it an abundant and inexpensive natural source of long, single-stranded nucleic acid, which could be used as raw material for the fabrication of molecular origami. In this study, we demonstrate efficient and robust construction of 2D and 3D origami nanostructures utilizing cellular rRNA as a scaffold and DNA oligonucleotide staples. We present calibrated protocols for the robust folding of contiguous shapes from one or two rRNA subunits that are efficient to allow folding using crude extracts of total RNA. We also show that RNA maintains stability within the folded structure. Lastly, we present a novel and comprehensive analysis and insights into the stability of RNA:DNA origami nanostructures and demonstrate their enhanced stability when coated with polylysine-polyethylene glycol in different temperatures, low Mg concentrations, human serum, and in the presence of nucleases (DNase I or RNase H). Thus, laying the foundation for their potential implementation in emerging biomedical applications, where folding rRNA into stable structures outside and inside cells would be desired.
Topics: RNA, Ribosomal; Nucleic Acid Conformation; Nanostructures; Humans; RNA Folding; DNA; Polylysine; Polyethylene Glycols
PubMed: 38698435
DOI: 10.1186/s12951-024-02489-2 -
The Journal of Physical Chemistry. B Aug 2018On the basis of a helix-based transition rate model, we developed a new method for sampling cotranscriptional RNA conformational ensemble and the prediction of...
On the basis of a helix-based transition rate model, we developed a new method for sampling cotranscriptional RNA conformational ensemble and the prediction of cotranscriptional folding kinetics. Applications to E. coli. SRP RNA and pbuE riboswitch indicate that the model may provide reliable predictions for the cotranscriptional folding pathways and population kinetics. For E. coli. SRP RNA, the predicted population kinetics and the folding pathway are consistent with the SHAPE profiles in the recent cotranscriptional SHAPE-seq experiments. For the pbuE riboswitch, the model predicts the transcriptional termination efficiency as a function of the force. The theoretical results show (a) a force-induced transition from the aptamer (antiterminator) to the terminator structure and (b) the different folding pathways for the riboswitch with and without the ligand (adenine). More specifically, without adenine, the aptamer structure emerges as a short-lived kinetic transient state instead of a thermodynamically stable intermediate state. Furthermore, from the predicted extension-time curves, the model identifies a series of conformational switches in the pulling process, where the predicted relative residence times for the different structures are in accordance with the experimental data. The model may provide a new tool for quantitative predictions of cotranscriptional folding kinetics, and results can offer useful insights into cotranscriptional folding-related RNA functions such as regulation of gene expression with riboswitches.
Topics: Aptamers, Nucleotide; Escherichia coli; Kinetics; Models, Molecular; Nucleic Acid Conformation; RNA Folding; RNA Stability; Riboswitch; Thermodynamics
PubMed: 29985608
DOI: 10.1021/acs.jpcb.8b04249 -
Nucleic Acids Research Jan 2024The kinetics of folding is crucial for the function of many regulatory RNAs including RNA G-quadruplexes (rG4s). Here, we characterize the folding pathways of a...
The kinetics of folding is crucial for the function of many regulatory RNAs including RNA G-quadruplexes (rG4s). Here, we characterize the folding pathways of a G-quadruplex from the telomeric repeat-containing RNA by combining all-atom molecular dynamics and coarse-grained simulations with circular dichroism experiments. The quadruplex fold is stabilized by cations and thus, the ion atmosphere forming a double layer surrounding the highly charged quadruplex guides the folding process. To capture the ionic double layer in implicit solvent coarse-grained simulations correctly, we develop a matching procedure based on all-atom simulations in explicit water. The procedure yields quantitative agreement between simulations and experiments as judged by the populations of folded and unfolded states at different salt concentrations and temperatures. Subsequently, we show that coarse-grained simulations with a resolution of three interaction sites per nucleotide are well suited to resolve the folding pathways and their intermediate states. The results reveal that the folding progresses from unpaired chain via hairpin, triplex and double-hairpin constellations to the final folded structure. The two- and three-strand intermediates are stabilized by transient Hoogsteen interactions. Each pathway passes through two on-pathway intermediates. We hypothesize that conformational entropy is a hallmark of rG4 folding. Conformational entropy leads to the observed branched multi-pathway folding process for TERRA25. We corroborate this hypothesis by presenting the free energy landscapes and folding pathways of four rG4 systems with varying loop length.
Topics: Entropy; G-Quadruplexes; Molecular Dynamics Simulation; Nucleic Acid Conformation; RNA; RNA Folding
PubMed: 37986217
DOI: 10.1093/nar/gkad1065 -
Nucleic Acids Research Feb 2014Riboswitches are part of noncoding regions of messenger RNA (mRNA) that act as RNA sensors regulating gene expression of the downstream gene. Typically, one out of two...
Riboswitches are part of noncoding regions of messenger RNA (mRNA) that act as RNA sensors regulating gene expression of the downstream gene. Typically, one out of two distinct conformations is formed depending on ligand binding when the transcript leaves RNA polymerase (RNAP). Elongation of the RNA chain by RNAP, folding and binding all occurs simultaneously and interdependently on the seconds' timescale. To investigate the effect of transcript elongation velocity on folding for the S-adenosylmethionine (SAM)-I and adenine riboswitches we employ two complementary coarse-grained in silico techniques. Native structure-based molecular dynamics simulations provide a 3D, atomically resolved model of folding with homogenous energetics. Energetically more detailed kinetic Monte Carlo simulations give access to longer timescale by describing folding on the secondary structure level and feature the incorporation of competing aptamer conformations and a ligand-binding model. Depending on the extrusion scenarios, we observe and quantify different pathways in structure formation with robust agreements between the two techniques. In these scenarios, free-folding riboswitches exhibit different folding characteristics compared with transcription-rate limited folding. The critical transcription rate distinguishing these cases is higher than physiologically relevant rates. This result suggests that in vivo folding of the analyzed SAM-I and adenine riboswitches is transcription-rate limited.
Topics: Molecular Dynamics Simulation; Monte Carlo Method; RNA Folding; Riboswitch; Transcription, Genetic
PubMed: 24275497
DOI: 10.1093/nar/gkt1213 -
Nature Chemistry Jun 2021RNA origami is a framework for the modular design of nanoscaffolds that can be folded from a single strand of RNA and used to organize molecular components with...
RNA origami is a framework for the modular design of nanoscaffolds that can be folded from a single strand of RNA and used to organize molecular components with nanoscale precision. The design of genetically expressible RNA origami, which must fold cotranscriptionally, requires modelling and design tools that simultaneously consider thermodynamics, the folding pathway, sequence constraints and pseudoknot optimization. Here, we describe RNA Origami Automated Design software (ROAD), which builds origami models from a library of structural modules, identifies potential folding barriers and designs optimized sequences. Using ROAD, we extend the scale and functional diversity of RNA scaffolds, creating 32 designs of up to 2,360 nucleotides, five that scaffold two proteins, and seven that scaffold two small molecules at precise distances. Micrographic and chromatographic comparisons of optimized and non-optimized structures validate that our principles for strand routing and sequence design substantially improve yield. By providing efficient design of RNA origami, ROAD may simplify the construction of custom RNA scaffolds for nanomedicine and synthetic biology.
Topics: Base Sequence; Microscopy, Electron, Transmission; Nanostructures; Nanotechnology; Protein Biosynthesis; RNA; RNA Folding; Small Molecule Libraries; Software; Synthetic Biology
PubMed: 33972754
DOI: 10.1038/s41557-021-00679-1 -
RNA Biology Jan 2013RNA folding is an essential aspect underlying RNA-mediated cellular processes. Many RNAs, including large, multi-domain ribozymes, are capable of folding to the native,... (Review)
Review
RNA folding is an essential aspect underlying RNA-mediated cellular processes. Many RNAs, including large, multi-domain ribozymes, are capable of folding to the native, functional state without assistance of a protein cofactor in vitro. In the cell, trans-acting factors, such as proteins, are however known to modulate the structure and thus the fate of an RNA. DEAD-box proteins, including Mss116p, were recently found to assist folding of group I and group II introns in vitro and in vivo. The underlying mechanism(s) have been studied extensively to explore the contribution of ATP hydrolysis and duplex unwinding in helicase-stimulated intron splicing. Here we summarize the ongoing efforts to understand the novel role of DEAD-box proteins in RNA folding.
Topics: DEAD-box RNA Helicases; Exons; Introns; Mitochondria; Mutation; Protein Binding; Protein Interaction Domains and Motifs; RNA; RNA Folding; RNA Splicing; RNA Stability; Yeasts
PubMed: 23064153
DOI: 10.4161/rna.22492