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Angewandte Chemie (International Ed. in... Mar 2018We describe a selective and mild chemical approach for controlling RNA hybridization, folding, and enzyme interactions. Reaction of RNAs in aqueous buffer with an...
We describe a selective and mild chemical approach for controlling RNA hybridization, folding, and enzyme interactions. Reaction of RNAs in aqueous buffer with an azide-substituted acylating agent (100-200 mm) yields several 2'-OH acylations per RNA strand in as little as 10 min. This poly-acylated ("cloaked") RNA is strongly blocked from hybridization with complementary nucleic acids, from cleavage by RNA-processing enzymes, and from folding into active aptamer structures. Importantly, treatment with a water-soluble phosphine triggers a Staudinger reduction of the azide groups, resulting in spontaneous loss of acyl groups ("uncloaking"). This fully restores RNA folding and biochemical activity.
Topics: Acylation; Azides; Molecular Structure; Phosphines; RNA; RNA Folding
PubMed: 29370460
DOI: 10.1002/anie.201708696 -
RNA Biology 2014RNAs play pivotal roles in the cell, ranging from catalysis (e.g., RNase P), acting as adaptor molecule (tRNA) to regulation (e.g., riboswitches). Precise understanding... (Review)
Review
RNAs play pivotal roles in the cell, ranging from catalysis (e.g., RNase P), acting as adaptor molecule (tRNA) to regulation (e.g., riboswitches). Precise understanding of its three-dimensional structures has given unprecedented insight into the molecular basis for all of these processes. Nevertheless, structural studies on RNA are still limited by the very special nature of this polymer. The most common methods for the determination of 3D RNA structures are NMR and X-ray crystallography. Both methods have their own set of requirements and give different amounts of information about the target RNA. For structural studies, the major bottleneck is usually obtaining large amounts of highly pure and homogeneously folded RNA. Especially for X-ray crystallography it can be necessary to screen a large number of variants to obtain well-ordered single crystals. In this mini-review we give an overview about strategies for the design, in vitro production, and purification of RNA for structural studies.
Topics: Crystallography, X-Ray; Nuclear Magnetic Resonance, Biomolecular; Nucleic Acid Conformation; RNA; RNA Folding; RNA, Catalytic
PubMed: 24667346
DOI: 10.4161/rna.28076 -
RNA (New York, N.Y.) Jun 2019Nearest neighbor parameters for estimating the folding stability of RNA are commonly used in secondary structure prediction, for generating folding ensembles of...
Nearest neighbor parameters for estimating the folding stability of RNA are commonly used in secondary structure prediction, for generating folding ensembles of structures, and for analyzing RNA function. Previously, we demonstrated that we could quantify the uncertainties in each nearest neighbor parameter by perturbing the underlying optical melting data within experimental error and rederiving the parameters, which accounts for the substantial correlations that exist between the parameters. In this contribution, we describe a method to estimate uncertainty in the estimated folding stabilities of RNA structures, accounting for correlations in the nearest neighbor parameters. This method is incorporated in the RNA structure software package.
Topics: Algorithms; Base Pairing; Base Sequence; Humans; RNA; RNA Folding; Software; Thermodynamics; Uncertainty
PubMed: 30952689
DOI: 10.1261/rna.069203.118 -
Proceedings of the National Academy of... Oct 2021Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through...
Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.
Topics: Cryoelectron Microscopy; Genome, Viral; Models, Molecular; Molecular Chaperones; RNA Folding; RNA, Viral; RNA-Binding Proteins; Ribonucleoproteins; Rotavirus; Viral Genome Packaging; Viral Nonstructural Proteins
PubMed: 34615715
DOI: 10.1073/pnas.2100198118 -
Cell Nov 2019Ribosome assembly is an efficient but complex and heterogeneous process during which ribosomal proteins assemble on the nascent rRNA during transcription. Understanding...
Ribosome assembly is an efficient but complex and heterogeneous process during which ribosomal proteins assemble on the nascent rRNA during transcription. Understanding how the interplay between nascent RNA folding and protein binding determines the fate of transcripts remains a major challenge. Here, using single-molecule fluorescence microscopy, we follow assembly of the entire 3' domain of the bacterial small ribosomal subunit in real time. We find that co-transcriptional rRNA folding is complicated by the formation of long-range RNA interactions and that r-proteins self-chaperone the rRNA folding process prior to stable incorporation into a ribonucleoprotein (RNP) complex. Assembly is initiated by transient rather than stable protein binding, and the protein-RNA binding dynamics gradually decrease during assembly. This work questions the paradigm of strictly sequential and cooperative ribosome assembly and suggests that transient binding of RNA binding proteins to cellular RNAs could provide a general mechanism to shape nascent RNA folding during RNP assembly.
Topics: Models, Biological; Nucleic Acid Conformation; Protein Binding; RNA Folding; RNA Stability; RNA, Ribosomal; RNA-Binding Proteins; Transcription, Genetic
PubMed: 31761533
DOI: 10.1016/j.cell.2019.10.035 -
PloS One 2014The ever increasing discovery of non-coding RNAs leads to unprecedented demand for the accurate modeling of RNA folding, including the predictions of two-dimensional...
BACKGROUND
The ever increasing discovery of non-coding RNAs leads to unprecedented demand for the accurate modeling of RNA folding, including the predictions of two-dimensional (base pair) and three-dimensional all-atom structures and folding stabilities. Accurate modeling of RNA structure and stability has far-reaching impact on our understanding of RNA functions in human health and our ability to design RNA-based therapeutic strategies.
RESULTS
The Vfold server offers a web interface to predict (a) RNA two-dimensional structure from the nucleotide sequence, (b) three-dimensional structure from the two-dimensional structure and the sequence, and (c) folding thermodynamics (heat capacity melting curve) from the sequence. To predict the two-dimensional structure (base pairs), the server generates an ensemble of structures, including loop structures with the different intra-loop mismatches, and evaluates the free energies using the experimental parameters for the base stacks and the loop entropy parameters given by a coarse-grained RNA folding model (the Vfold model) for the loops. To predict the three-dimensional structure, the server assembles the motif scaffolds using structure templates extracted from the known PDB structures and refines the structure using all-atom energy minimization.
CONCLUSIONS
The Vfold-based web server provides a user friendly tool for the prediction of RNA structure and stability. The web server and the source codes are freely accessible for public use at "http://rna.physics.missouri.edu".
Topics: Databases, Protein; Humans; Internet; Nucleic Acid Conformation; RNA; RNA Folding; RNA Stability; Software; Thermodynamics
PubMed: 25215508
DOI: 10.1371/journal.pone.0107504 -
Proceedings of the National Academy of... Jan 2022Accurate knowledge of RNA hybridization is essential for understanding RNA structure and function. Here we mechanically unzip and rezip a 2-kbp RNA hairpin and derive...
Accurate knowledge of RNA hybridization is essential for understanding RNA structure and function. Here we mechanically unzip and rezip a 2-kbp RNA hairpin and derive the 10 nearest-neighbor base pair (NNBP) RNA free energies in sodium and magnesium with 0.1 kcal/mol precision using optical tweezers. Notably, force-distance curves (FDCs) exhibit strong irreversible effects with hysteresis and several intermediates, precluding the extraction of the NNBP energies with currently available methods. The combination of a suitable RNA synthesis with a tailored pulling protocol allowed us to obtain the fully reversible FDCs necessary to derive the NNBP energies. We demonstrate the equivalence of sodium and magnesium free-energy salt corrections at the level of individual NNBP. To characterize the irreversibility of the unzipping-rezipping process, we introduce a barrier energy landscape of the stem-loop structures forming along the complementary strands, which compete against the formation of the native hairpin. This landscape correlates with the hysteresis observed along the FDCs. RNA sequence analysis shows that base stacking and base pairing stabilize the stem-loops that kinetically trap the long-lived intermediates observed in the FDC. Stem-loops formation appears as a general mechanism to explain a wide range of behaviors observed in RNA folding.
Topics: Biomechanical Phenomena; Magnesium; Nucleic Acid Conformation; RNA; RNA Folding; Sodium; Thermodynamics
PubMed: 35022230
DOI: 10.1073/pnas.2025575119 -
Angewandte Chemie (International Ed. in... Mar 2020Spinach and Broccoli are fluorogenic RNA aptamers that bind DFHBI, a mimic of the chromophore in green fluorescent protein, and activate its fluorescence....
Spinach and Broccoli are fluorogenic RNA aptamers that bind DFHBI, a mimic of the chromophore in green fluorescent protein, and activate its fluorescence. Spinach/Broccoli-DFHBI complexes exhibit high fluorescence in vitro, but they exhibit lower fluorescence in mammalian cells. Here, computational screening was used to identify BI, a DFHBI derivative that binds Broccoli with higher affinity and leads to markedly higher fluorescence in cells compared to previous ligands. BI prevents thermal unfolding of Broccoli at 37 °C, leading to more folded Broccoli and thus more fluorescent Broccoli-BI complexes in cells. Broccoli-BI complexes are more photostable owing to impaired photoisomerization and rapid unbinding of photoisomerized cis-BI. These properties enable single mRNA containing 24 Broccoli aptamers to be imaged in live mammalian cells treated with BI. Small molecule ligands can thus promote RNA folding in cells, and thus allow single mRNA imaging with fluorogenic aptamers.
Topics: Aptamers, Nucleotide; Benzyl Compounds; Brassica; Fluorescent Dyes; Gene Expression Regulation; HEK293 Cells; Humans; Imidazolines; Isomerism; Optical Imaging; Photochemical Processes; RNA Folding; RNA, Messenger; Single Molecule Imaging; Transition Temperature
PubMed: 31850609
DOI: 10.1002/anie.201914576 -
Structure (London, England : 1993) Jan 2019RNA-protein complexes underlie numerous cellular processes including translation, splicing, and posttranscriptional regulation of gene expression. The structures of...
RNA-protein complexes underlie numerous cellular processes including translation, splicing, and posttranscriptional regulation of gene expression. The structures of these complexes are crucial to their functions but often elude high-resolution structure determination. Computational methods are needed that can integrate low-resolution data for RNA-protein complexes while modeling de novo the large conformational changes of RNA components upon complex formation. To address this challenge, we describe RNP-denovo, a Rosetta method to simultaneously fold-and-dock RNA to a protein surface. On a benchmark set of diverse RNA-protein complexes not solvable with prior strategies, RNP-denovo consistently sampled native-like structures with better than nucleotide resolution. We revisited three past blind modeling challenges involving the spliceosome, telomerase, and a methyltransferase-ribosomal RNA complex in which previous methods gave poor results. When coupled with the same sparse FRET, crosslinking, and functional data used previously, RNP-denovo gave models with significantly improved accuracy. These results open a route to modeling global folds of RNA-protein complexes from low-resolution data.
Topics: Binding Sites; Molecular Docking Simulation; Protein Binding; Protein Domains; Protein Folding; Proteins; RNA; RNA Folding; Software
PubMed: 30416038
DOI: 10.1016/j.str.2018.10.001 -
Journal of Molecular Biology Oct 2016Structured RNAs fold through multiple pathways, but we have little understanding of the molecular features that dictate folding pathways and determine rates along a...
Structured RNAs fold through multiple pathways, but we have little understanding of the molecular features that dictate folding pathways and determine rates along a given pathway. Here, we asked whether folding of a complex RNA can be understood from its structural modules. In a two-piece version of the Tetrahymena group I ribozyme, the separated P5abc subdomain folds to local native secondary and tertiary structure in a linked transition and assembles with the ribozyme core via three tertiary contacts: a kissing loop (P14), a metal core-receptor interaction, and a tetraloop-receptor interaction, the first two of which are expected to depend on native P5abc structure from the local transition. Native gel, NMR, and chemical footprinting experiments showed that mutations that destabilize the native P5abc structure slowed assembly up to 100-fold, indicating that P5abc folds first and then assembles with the core by conformational selection. However, rate decreases beyond 100-fold were not observed because an alternative pathway becomes dominant, with nonnative P5abc binding the core and then undergoing an induced-fit rearrangement. P14 is formed in the rate-limiting step along the conformational selection pathway but after the rate-limiting step along the induced-fit pathway. Strikingly, the assembly rate along the conformational selection pathway resembles that of an isolated kissing loop similar to P14, and the rate along the induced-fit pathway resembles that of an isolated tetraloop-receptor interaction. Our results indicate substantial modularity in RNA folding and assembly and suggest that these processes can be understood in terms of underlying structural modules.
Topics: DNA Mutational Analysis; Electrophoresis, Polyacrylamide Gel; Magnetic Resonance Spectroscopy; Nucleic Acid Conformation; RNA Folding; RNA, Catalytic; Tetrahymena
PubMed: 27452365
DOI: 10.1016/j.jmb.2016.07.013