RNA Conformational Changes Research Articles

Synthesis and Enzymatic Incorporation of a Dual-App Nucleotide Probe That Reports Antibiotics-Induced Conformational Change in the Bacterial Ribosomal Decoding Site RNA.

Natural nucleosides are nonfluorescent and do not have intrinsic labels that can be readily utilized for analyzing nucleic acid structure and recognition. In this regard, researchers typically use the so-called "one-label, one-technique" approach to study nucleic acids. However, we envisioned that a responsive dual-app nucleoside system that harnesses the power of two complementing biophysical techniques namely, fluorescence and 19F NMR, will allow the investigation of nucleic acid conformations more comprehensively than before. We recently introduced a nucleoside analogue by tagging trifluoromethyl-benzofuran at the C5 position of 2'-deoxyuridine, which serves as an excellent fluorescent and 19F NMR probe to study G-quadruplex and i-motif structures. Taking forward, here, we report the development of a ribonucleotide version of the dual-app probe to monitor antibiotics-induced conformational changes in RNA. The ribonucleotide analog is derived by conjugating trifluoromethyl-benzofuran at the C5 position of uridine (TFBF-UTP). The analog is efficiently incorporated by T7 RNA polymerase to produce functionalized RNA transcripts. Detailed photophysical and 19F NMR of the nucleoside and nucleotide incorporated into RNA oligonucleotides revealed that the analog is structurally minimally invasive and can be used for probing RNA conformations by fluorescence and 19F NMR techniques. Using the probe, we monitored and estimated aminoglycoside antibiotics binding to the bacterial ribosomal decoding site RNA (A-site, a very important RNA target). While 2-aminopurine, a famous fluorescent nucleic acid probe, fails to detect structurally similar aminoglycoside antibiotics binding to the A-site, our probe reports the binding of different aminoglycosides to the A-site. Taken together, our results demonstrate that TFBF-UTP is a very useful addition to the nucleic acid analysis toolbox and could be used to devise discovery platforms to identify new RNA binders of therapeutic potential.

TRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the ninth nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 13 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) that allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.

Principles of human pre-60S biogenesis.

During the early stages of human large ribosomal subunit (60S) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers of pre-60S particles by an unknown mechanism. Here, we report a series of cryo-electron microscopy structures of human nucleolar and nuclear pre-60S assembly intermediates at resolutions of 2.5 to 3.2 angstroms. These structures show how protein interaction hubs tether assembly factor complexes to nucleolar particles and how guanosine triphosphatases and adenosine triphosphatase couple irreversible nucleotide hydrolysis steps to the installation of functional centers. Nuclear stages highlight how a conserved RNA-processing complex, the rixosome, couples large-scale RNA conformational changes with pre-ribosomal RNA processing by the RNA degradation machinery. Our ensemble of human pre-60S particles provides a rich foundation with which to elucidate the molecular principles of ribosome formation.

Real-time label-free detection of dynamic aptamer–small molecule interactions using a nanopore nucleic acid conformational sensor

Nucleic acids can undergo conformational changes upon binding small molecules. These conformational changes can be exploited to develop new therapeutic strategies through control of gene expression or triggering of cellular responses and can also be used to develop sensors for small molecules such as neurotransmitters. Many analytical approaches can detect dynamic conformational change of nucleic acids, but they need labeling, are expensive, and have limited time resolution. The nanopore approach can provide a conformational snapshot for each nucleic acid molecule detected, but has not been reported to detect dynamic nucleic acid conformational change in response to small -molecule binding. Here we demonstrate a modular, label-free, nucleic acid-docked nanopore capable of revealing time-resolved, small molecule-induced, single nucleic acid molecule conformational transitions with millisecond resolution. By using the dopamine-, serotonin-, and theophylline-binding aptamers as testbeds, we found that these nucleic acids scaffolds can be noncovalently docked inside the MspA protein pore by a cluster of site-specific charged residues. This docking mechanism enables the ion current through the pore to characteristically vary as the aptamer undergoes conformational changes, resulting in a sequence of current fluctuations that report binding and release of single ligand molecules from the aptamer. This nanopore tool can quantify specific ligands such as neurotransmitters, elucidate nucleic acid-ligand interactions, and pinpoint the nucleic acid motifs for ligand binding, showing the potential for small molecule biosensing, drug discovery assayed via RNA and DNA conformational changes, and the design of artificial riboswitch effectors in synthetic biology.

Internal Normal Mode Analysis Applied to RNA Flexibility and Conformational Changes.

We investigated the capability of internal normal modes to reproduce RNA flexibility and predict observed RNA conformational changes and, notably, those induced by the formation of RNA-protein and RNA-ligand complexes. Here, we extended our iNMA approach developed for proteins to study RNA molecules using a simplified representation of the RNA structure and its potential energy. Three data sets were also created to investigate different aspects. Despite all the approximations, our study shows that iNMA is a suitable method to take into account RNA flexibility and describe its conformational changes opening the route to its applicability in any integrative approach where these properties are crucial.

Structural basis and dynamics of Chikungunya alphavirus RNA capping by nsP1 capping pores

Alphaviruses are emerging positive-stranded RNA viruses which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The nonstructural protein 1 (nsP1) is responsible for viral RNA capping and gates the replication organelles by assembling into monotopic membrane-associated dodecameric pores. The capping pathway is unique to Alphaviruses; beginning with the N7 methylation of a guanosine triphosphate (GTP) molecule, followed by the covalent linkage of an m7GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA. Here, we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and S-adenosyl methionine (SAM), how the enzyme reaches a metastable postmethylation state with SAH and m7GTP in the active site, and the subsequent covalent transfer of m7GMP to nsP1 triggered by the presence of RNA and postdecapping reaction conformational changes inducing the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of reaction intermediates. Our data identify the molecular determinants allowing each pathway transition, providing an explanation for the need for the SAM methyl donor all along the pathway and clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together, our results set ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals.

Mechanics of divalent ion sensitivity of the manganese-sensing riboswitch measured by combined high-resolution optical tweezers and single-molecule fluorescence.

Riboswitches are structured non-coding RNA typically found in the 5′ untranslated regions of bacterial mRNAs. They modulate gene expression through the binding of specific ligands that induce changes in the riboswitch structure, most often at the level of transcription and translation. The Mn2+-sensing yybP-ykoY RNA motif is one of the most widespread riboswitches across bacteria, including human and plant pathogens. It contains two divalent metal ion binding sites: one is specific for Mn2+ whereas the other is nonspecific for a divalent metal ion, including Mg2+. Previous crystal structures have shown that Mn2+ binds in between the two distal helical legs of a four-way junction and induces a large “docking” conformational change. It is unclear how competition between Mn2+ and Mg2+ for the two binding sites and the associated RNA conformational changes give rise to Mn2+ detection function and sensitivity. Therefore, we explored the global structural dynamics of the L. lactis Mn2+-sensing riboswitch in the presence of competing divalent cations using high-resolution optical tweezers simultaneous with confocal Förster resonance energy transfer (FRET) spectroscopy. Initial non-equilibrium force ramp measurements revealed force-dependent docking distribution, suggesting a highly selective docked state at suboptimal Mn2+ ion concentration. Follow-up high-resolution equilibrium fixed-force measurements directly measured docking and undocking rate constants versus force to determine docking and undocking mechanics, including transition states and pathways, at varying Mn2+ vs Mg2+ concentrations. Simultaneous FRET measurements confirmed specific conformational transitions. Docking and undocking were symmetric processes with nearly equal force sensitivity and a transition state located nearly midway between the docked and undocked states. Mn2+ sensitivity appears to optimize at a concentration where further increasing Mn2+ competes with Mg2+ ions for the non-specific divalent metal ion binding site.

X-Ray Crystallography to Study Conformational Changes in a TPP Riboswitch.

Conformational rearrangements are key to the function of riboswitches. These regulatory mRNA regions specifically bind to cellular metabolites using evolutionarily conserved sensing domains and modulate gene expression via adjacent downstream expression platforms, which carry gene expression signals. The regulation is achieved through the ligand-dependent formation of two alternative and mutually exclusive conformations involving the same RNA region. While X-ray crystallography cannot visualize dynamics of such dramatic conformational rearrangements, this method is pivotal to understand RNA-ligand interaction that stabilize the sensing domain and drive folding of the expression platform. X-ray crystallography can reveal local changes in RNA necessary for discriminating cognate and noncognate ligands. This chapter describes preparation of thiamine pyrophosphate riboswitch RNAs and its crystallization with different ligands, resulting in structures with local conformational changes in RNA. These structures can help to derive information on the dynamics of the RNA essential for specific binding to small molecules, with potential for using this information for developing designer riboswitch-ligand systems.

Parallel screening and cheminformatics modeling of flavonoid activated aptasensors

A parallel screening of 27 different flavonoids and chalcones was conducted using 6 artificial naringenin-activated riboswitches (M1, M2, M3, O, L and H). A quantitative structure-property relationship approach was applied to understand the physicochemical properties of the flavonoid structures resulting in specificity differences relied on the fluorescence intensity of a green fluorescent protein reporter. Robust models of riboswitches M1, M2 and O that had good predictive power were constructed with descriptors selected for their high correlation. Increased electronegativity and hydrophilicity of the flavonoids structures were identified as two properties that increased binding affinity to RNA riboswitches. Hydroxyl groups at the C-3′ and C-4’ positions of the flavonoid molecule were strictly required for ligand-activation with riboswitches M1 and M2. Riboswitches O and L preferred multi-hydroxylated flavones as ligands. Substitutions on the A ring of the flavonoid molecule were not important in the molecular recognition process. O-glycosylated derivatives were not recognized by any of the riboswitches, presumably due to steric hindrances. Despite the challenges of detecting RNA conformational change after ligand binding, the resulting models elucidate important physicochemical features in the ligands for conformational structural studies of artificial aptamer complexes and for design of ligands having higher binding specificity.

Using DMS-MaPseq to uncover the roles of DEAD-box proteins in ribosome assembly.

DMS (dimethylsulfate) is a time-tested chemical probe for nucleic acid secondary structure that has recently re-emerged as a powerful tool to study RNA structure and structural changes, by coupling it to high throughput sequencing techniques. This variant, termed DMS-MaPseq, allows for mapping of all RNAs in a cell at the same time. However, if an RNA adopts different structures, for example during the assembly of an RNA-protein complex, or as part of its functional cycle, then DMS-MaPseq cannot differentiate between these structures, and an ensemble average will be produced. This is especially challenging for long-lived RNAs, such as ribosomes, whose steady-state abundance far exceeds that of any assembly intermediates, rendering those inaccessible to DMS-MaPseq on total RNAs. These challenges can be overcome by purification of assembly intermediates stalled at specific assembly steps (or steps in the functional cycle), via a combination of affinity tags and mutants stalled at defined steps, and subsequent DMS probing of these intermediates. Interpretation of the differences in DMS accessibility is facilitated by additional structural information, e.g. from cryo-EM experiments, available for many functional RNAs. While this approach is generally useful for studying RNA folding or conformational changes within RNA-protein complexes, it can be particularly valuable for studying the role(s) of DEAD-box proteins, as these tend to lead to larger conformational rearrangements, often resulting from the release of an RNA-binding protein from a bound RNA. Here we provide an adaptation of the DMS-MaPseq protocol to study RNA conformational transitions during ribosome assembly, which addresses the challenges arising from the presence of many assembly intermediates, all at concentrations far below that of mature ribosomes. While this protocol was developed for the yeast S. cerevisiae, we anticipate that it should be readily transferable to other model organisms for which affinity purification has been established.

A loosened gating mechanism of RIG-I leads to autoimmune disorders.

DDX58 encodes RIG-I, a cytosolic RNA sensor that ensures immune surveillance of nonself RNAs. Individuals with RIG-IE510V and RIG-IQ517H mutations have increased susceptibility to Singleton-Merten syndrome (SMS) defects, resulting in tissue-specific (mild) and classic (severe) phenotypes. The coupling between RNA recognition and conformational changes is central to RIG-I RNA proofreading, but the molecular determinants leading to dissociated disease phenotypes remain unknown. Herein, we employed hydrogen/deuterium exchange mass spectrometry (HDX-MS) and single molecule magnetic tweezers (MT) to precisely examine how subtle conformational changes in the helicase insertion domain (HEL2i) promote impaired ATPase and erroneous RNA proofreading activities. We showed that the mutations cause a loosened latch-gate engagement in apo RIG-I, which in turn gradually dampens its self RNA (Cap2 moiety:m7G cap and N1-2-2′-O-methylation RNA) proofreading ability, leading to increased immunopathy. These results reveal HEL2i as a unique checkpoint directing two specialized functions, i.e. stabilizing the CARD2-HEL2i interface and gating the helicase from incoming self RNAs; thus, these findings add new insights into the role of HEL2i in the control of antiviral innate immunity and autoimmunity diseases.

Incorporation of a FRET Pair into a Riboswitch RNA to Measure Mg2+ Concentration and RNA Conformational Change in Cell.

Riboswitches are natural biosensors that can regulate gene expression by sensing small molecules. Knowledge of the structural dynamics of riboswitches is crucial to elucidate their regulatory mechanism and develop RNA biosensors. In this work, we incorporated the fluorophore, Cy3, and its quencher, TQ3, into a full-length adenine riboswitch RNA and its isolated aptamer domain to monitor the dynamics of the RNAs in vitro and in cell. The adenine riboswitch was sensitive to Mg2+ concentrations and could be used as a biosensor to measure cellular Mg2+ concentrations. Additionally, the TQ3/Cy3-labeled adenine riboswitch yielded a Mg2+ concentration that was similar to that measured using a commercial assay kit. Furthermore, the fluorescence response to the adenine of the TQ3/Cy3-labeled riboswitch RNA was applied to determine the proportions of multiple RNA conformational changes in cells. The strategy developed in this work can be used to probe the dynamics of other RNAs in cells and may facilitate the developments of RNA biosensors, drugs and engineering.

An ultra low-input method for global RNA structure probing uncovers Regnase-1-mediated regulation in macrophages

To enable diverse functions and precise regulation, an RNA sequence often folds into complex yet distinct structures in different cellular states. Probing RNA in its native environment is essential to uncovering RNA structures of biological contexts. However, current methods generally require large amounts of input RNA and are challenging for physiologically relevant use. Here, we report smartSHAPE, a new RNA structure probing method that requires very low amounts of RNA input due to the largely reduced artefact of probing signals and increased efficiency of library construction. Using smartSHAPE, we showcased the profiling of the RNA structure landscape of mouse intestinal macrophages upon inflammation, and provided evidence that RNA conformational changes regulate immune responses. These results demonstrate that smartSHAPE can greatly expand the scope of RNA structure-based investigations in practical biological systems, and also provide a research paradigm for the study of post-transcriptional regulation.

Three-dimensional structure of a flavivirus dumbbell RNA reveals molecular details of an RNA regulator of replication.

Mosquito-borne flaviviruses (MBFVs) including dengue, West Nile, yellow fever, and Zika viruses have an RNA genome encoding one open reading frame flanked by 5′ and 3′ untranslated regions (UTRs). The 3′ UTRs of MBFVs contain regions of high sequence conservation in structured RNA elements known as dumbbells (DBs). DBs regulate translation and replication of the viral RNA genome, functions proposed to depend on the formation of an RNA pseudoknot. To understand how DB structure provides this function, we solved the x-ray crystal structure of the Donggang virus DB to 2.1Å resolution and used structural modeling to reveal the details of its three-dimensional fold. The structure confirmed the predicted pseudoknot and molecular modeling revealed how conserved sequences form a four-way junction that appears to stabilize the pseudoknot. Single-molecule FRET suggests that the DB pseudoknot is a stable element that can regulate the switch between translation and replication during the viral lifecycle by modulating long-range RNA conformational changes.

DiffBUM-HMM: a robust statistical modeling approach for detecting RNA flexibility changes in high-throughput structure probing data

Advancing RNA structural probing techniques with next-generation sequencing has generated demands for complementary computational tools to robustly extract RNA structural information amidst sampling noise and variability. We present diffBUM-HMM, a noise-aware model that enables accurate detection of RNA flexibility and conformational changes from high-throughput RNA structure-probing data. diffBUM-HMM is widely compatible, accounting for sampling variation and sequence coverage biases, and displays higher sensitivity than existing methods while robust against false positives. Our analyses of datasets generated with a variety of RNA probing chemistries demonstrate the value of diffBUM-HMM for quantitatively detecting RNA structural changes and RNA-binding protein binding sites.

Synchronous RNA conformational changes trigger ordered phase transitions in crystals

Time-resolved studies of biomacromolecular crystals have been limited to systems involving only minute conformational changes within the same lattice. Ligand-induced changes greater than several angstroms, however, are likely to result in solid-solid phase transitions, which require a detailed understanding of the mechanistic interplay between conformational and lattice transitions. Here we report the synchronous behavior of the adenine riboswitch aptamer RNA in crystal during ligand-triggered isothermal phase transitions. Direct visualization using polarized video microscopy and atomic force microscopy shows that the RNA molecules undergo cooperative rearrangements that maintain lattice order, whose cell parameters change distinctly as a function of time. The bulk lattice order throughout the transition is further supported by time-resolved diffraction data from crystals using an X-ray free electron laser. The synchronous molecular rearrangements in crystal provide the physical basis for studying large conformational changes using time-resolved crystallography and micro/nanocrystals.

Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity.

Retroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a “Ψ” packaging signal located in the gRNA 5′-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs efficiently. The simplicity of RSV Ψ makes it an informative model to examine the mechanism of retroviral gRNA packaging, which is incompletely understood. Little is known about the structure of dimerization initiation sites or specific Gag interaction sites of RSV gRNA. Using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), we probed the secondary structure of the entire RSV 5′-leader RNA for the first time. We identified a putative bipartite dimerization initiation signal (DIS), and mutation of both sites was required to significantly reduce dimerization in vitro. These mutations failed to reduce viral replication, suggesting that in vitro dimerization results do not strictly correlate with in vivo infectivity, possibly due to additional RNA interactions that maintain the dimers in cells. UV crosslinking-coupled SHAPE (XL-SHAPE) was next used to determine Gag-induced RNA conformational changes, revealing G218 as a critical Gag contact site. Overall, our results suggest that disruption of either of the DIS sequences does not reduce virus replication and reveal specific sites of Gag–RNA interactions.

Early detection of cancer: Focus on antibody coated metal and magnetic nanoparticle-based biosensors

Cancer is a highly fatal disease, and due to contemporary lifestyle and growing, environmental pollution chances of cancer are increasing, which is a global threat. The high mortality rate in cancer is caused primarily due to its late detection, mostly in a metastatic third or fourth stage, resulting in a poor after therapy prognosis. The conventional detection methods include identification of carcinogenic features of cells such as DNA or RNA mutation, conformational changes and overexpression of some proteins, and cell morphology, which are called biomarkers or analytes. Those processes are specialist-dependent, time-consuming, and expensive. Recently, biosensors are becoming popular as easy, quick, cheap, and highly sensitive detection tools. The biosensor technique depends on the availability of biomarkers in the sample. Thus, identifying new molecular markers for various types of cancers is a parallel issue, which is, fortunately, in rapid progress. A biosensor has a biomarker-specific layer of biorecognition elements on a transducer, which acts as an electrode. Upon binding of biomarkers with the biorecognition elements, a chemical signal gets generated. The transducer converts that signal into a measurable output for further analysis. Among several biorecognition elements, antibodies (Abs) are highly demanding, especially for the cancer diagnosis, for their unique three-dimensional structures and high specificity. However, the conventional biosensors produce inaccurate results at the low concentration of biomarkers in the patient's sample in an early stage of cancer. The use of nanoparticles shows remarkable improvement in the sensitivity of biosensors. This article has reviewed recent applications of nanoparticles in biosensor development and made a recommendation based on safety, cost and convenience.

Second-harmonic generation-based methods to detect and characterize ligand-induced RNA conformational changes.

Second-harmonic generation (SHG) is a biophysical tool that senses ligand-induced conformational changes in biomolecules. The Biodesy Delta™ has been developed as a high-throughput screening platform to monitor conformational changes in proteins and oligonucleotides by SHG to support drug discovery efforts. This work will outline (1) an overview of this technology, (2) detailed protocols for optimizing screening-ready SHG assays on RNA targets, (3) practical considerations for developing robust and informative SHG measurements, and (4) a case study that demonstrates the application of these recommendations on an RNA target. The previously published theophylline aptamer SHG assay [1] was further optimized to maximize the assay window between the positive control (theophylline) and the negative control (caffeine). Optimization of this assay provides practical considerations for building a robust SHG assay on an RNA target, including testing for specific tethering of the conjugate to the surface as well as testing tool compound response stability, reversibility, and concentration-dependence/affinity. A more robust, better-performing theophylline aptamer SHG assay was achieved that would be more appropriate for conducting a screen.

Specific interaction of zinc finger protein Com with RNA and the crystal structure of a self-complementary RNA duplex recognized by Com.

The bacteriophage Mu Com is a small zinc finger protein that binds to its cognate mom mRNA and activates its translation. The Mom protein, in turn, elicits a chemical modification (momification) of the bacteriophage genome, rendering the DNA resistant to cleavage by bacterial restriction endonucleases, and thereby protecting it from defense mechanisms of the host. We examined the basis of specificity in Com–RNA interactions by in vitro selection and probing of RNA structure. We demonstrated that Com recognizes a sequence motif within a hairpin-loop structure of its target RNA. Our data support the model of Com interaction with mom mRNA, in which Com binds to the short hairpin structure proximal to the so-called translation inhibition structure. We also observed that Com binds its target motif weakly if it is within an RNA duplex. These results suggest that the RNA structure, in addition to its sequence, is crucial for Com to recognize its target and that RNA conformational changes may constitute another level of Mom regulation. We determined a crystal structure of a Com binding site variant designed to form an RNA duplex preferentially. Our crystal model forms a 19-mer self-complementary double helix composed of the canonical and non-canonical base pairs. The helical parameters of crystalized RNA indicate why Com may bind it more weakly than a monomeric hairpin form.