RNA Structure Probing Research Articles

The interplay between molecular flexibility and RNA chemical probing reactivities analyzed at the nucleotide level via an extensive molecular dynamics study.

Determination of the tridimensional structure of ribonucleic acid molecules is fundamental for understanding their function in the cell. A common method to investigate RNA structures of large molecules is the use of chemical probes such as SHAPE (2'-hydroxyl acylation analyzed by primer extension) reagents, DMS (dimethyl sulfate) and CMCT (1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfate), the reaction of which is dependent on the local structural properties of each nucleotide. In order to understand the interplay between local flexibility, sugar pucker, canonical pairing and chemical reactivity of the probes, we performed all-atom molecular dynamics simulations on a set of RNA molecules for which both tridimensional structure and chemical probing data are available and we analyzed the correlations between geometrical parameters and the chemical reactivity. Our study confirms that SHAPE reactivity is guided by the local flexibility of the different chemical moieties but suggests that a combination of multiple parameters is needed to better understand the implications of the reactivity at the molecular level. This is also the case for DMS and CMCT for which the reactivity appears to be more complex than commonly accepted.

Development of a ribonuclease containing a G4-specific binding motif for programmable RNA cleavage

We developed a ribonuclease for site-specific targeting and cleavage of single-stranded RNA. The engineered RNase protein was constructed by incorporating two independent functional domains, an RNase HI domain that could cleave the RNA strand in a DNA-RNA hybrid, and a domain of the RHAU protein that could selectively recognize a parallel DNA G-quadruplex (G4). The newly designed RNase first recruits a DNA guide oligonucleotide containing both a parallel G4 motif and a template sequence complementary to the target RNA. This RNase:DNA complex targets and efficiently cleaves the single-stranded RNA in a site-specific manner. A major cleavage site occurs at the RNA region that is complementary to the DNA template sequence. The newly designed RNase can serve as a simple tool for RNA manipulation and probing RNA structure.

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.

Probing RNA structure and interaction dynamics at the single molecule level

RNA structures and their dynamic fluctuations lie at the heart of understanding key biological process such as transcription, splicing, translation and RNA decay. While conventional bulk assays have proven to identify and characterize key pathway intermediates, the generally dynamic nature of RNA structures renders the information obtained from time and ensemble averaging techniques necessarily lacking in critical details. Here we detail Single-Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS), a method that readily monitors structural fluctuations of single RNA molecules through the repetitive interaction of fluorescent probes with an unlabeled, surface-immobilized RNA target of virtually any length and in any biological context. In addition, we demonstrate the broad applicability of SiM-KARTS by kinetically fingerprinting the binding of cognate tRNA ligand to single immobilized T-box riboswitch molecules. SiM-KARTS represents a valuable tool for probing biologically relevant structure and interaction features of potentially many diverse RNA metabolic pathways.

Structure‐Function Analysis of the 3′ CITE from the Blackcurrant Reversion Nepovirus Genomic RNA 2 (BRV2)

Canonical eukaryotic translation initiation is enhanced by interactions between the 5′ 7‐methylguanosine cap (m7G) and the 3′ poly(A) tail, which exist on opposite ends of the mRNA molecule in the untranslated regions (UTRs). Viruses, however, hijack translation components using non‐canonical methods; typically differing in the structure of their 5′ and/or 3′ UTRs. Blackcurrant reversion nepovirus (BRV), a virus that infects the blackcurrant plant, has a genome consisting of two positive‐sense RNAs (RNA1 and RNA2). Neither RNA has a 5′ m7G cap, yet they both have a poly(A) tail. Structures within the 3′ UTRs of RNA1 and RNA2 called cap‐independent translation enhancers (3′ CITEs), are hypothesized to interact with a region of their 5′ UTRs. These distinct structural interactions facilitate the recruitment of essential initiation factors needed for translation, but this exact mechanism is not well understood. It is thought that the 5′ and 3′ UTRs possess conserved and complementary sequences which facilitate interaction. This study seeks to elucidate the specific secondary structure of the RNA2 3′ CITE and to correlate such to its role in translation initiation. Using in silico modeling of folded RNA2 and in vitro RNA structural probing, we determined a preliminary secondary structure for the 3′ CITE. We also identified structural changes when the putative 5′ ‐ 3′ UTR complementarity is disrupted. Using a luciferase reporter gene flanked by the RNA2 3′ CITE and wild‐type BRV2 5′ UTR, we show how the putative 5′ ‐ 3′ UTR complementarity is required for the quantity of and the rate at which luciferase protein is made. We conclude that the 3′ CITE is important for promoting translation initiation and the putative 5′ ‐ 3′ UTR complementarity between the ends of the viral RNA is necessary but not sufficient for protein production. The structure‐function relationship of the BRV RNA2 3′ CITE will lead to a clearer understanding of how BRV can be controlled to prevent Blackcurrant Reversion Disease (BRD) and shed light on vital processes that govern this unique class of nepoviruses.Support or Funding InformationColorado‐Wyoming Alliance for Minority Participation in STEMMSU Denver College of Letters, Arts and Sciences Dean's Seed GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Electronic Relaxation Dynamics of UV-Photoexcited 2-Aminopurine-Thymine Base Pairs in Watson-Crick and Hoogsteen Conformations.

The fluorescent analogue 2-aminopurine (2AP) of the canonical nucleobase adenine (6-aminopurine) base-pairs with thymine (T) without disrupting the helical structure of DNA. It therefore finds frequent use in molecular biology for probing DNA and RNA structures and conformational dynamics. However, detailed understanding of the processes responsible for fluorescence quenching remains largely elusive on a fundamental level. Although attempts have been made to ascribe decreased excited-state lifetimes to intrastrand charge-transfer and stacking interactions, possible influences from dynamic interstrand H-bonding have been widely ignored. Here, we investigate the electronic relaxation of UV-excited 2AP·T in Watson-Crick (WC) and Hoogsteen (HS) conformations. Although the WC conformation features slowed-down, monomer-like electronic relaxation in τ ∼ 1.6 ns toward ground-state recovery and triplet formation, the dynamics associated with 2AP·T in the HS motif exhibit faster deactivation in τ ∼ 70 ps. As recent research has revealed abundant transient interstrand H-bonding in the Hoogsteen motif for duplex DNA, the established model for dynamic fluorescence quenching may need to be revised in the light of our results. The underlying supramolecular photophysical mechanisms are discussed in terms of a proposed excited-state double-proton transfer as an efficient deactivation channel for recovery of the HS species in the electronic ground state.

Illuminating the molecular choreography of multi-segmented RNA genomes

Genome segmentation offers certain evolutionary benefits to a number of pathogenic RNA viruses, including rotaviruses and influenza viruses. However, as the number of RNA segments per virion increases, the task of a non-random selection of a full set of distinct genomic RNAs poses a formidable challenge to maintaining the integrity of segmented genomes. Recently we have identified sequence-specific inter-segment interactions between rotavirus (+)ssRNA genome segment precursors. We have shown that binding of the rotavirus-encoded non-structural protein NSP2 to viral ssRNAs results in the remodeling of RNA, which is conducive to formation of inter-segment contacts. These protein-RNA interactions result in the stabilisation of extended intermolecular RNA-RNA contacts, potentially underpinning transient inter-segment interactions prior to genome encapsidation and replication. Using this approach, we have identified a number of RNA-RNA interaction sites in the rotavirus genome, which are likely to be involved in genome segment assortment process. Having established the role of NSP2 in promoting inter-segment RNA-RNA contacts, we have developed multiplexed imaging tools for directvisualization of the RNA assortment process in rotavirus-infected cells by employing single-molecule RNA FISH. To unravel the mechanisms, by which NSP2 controls the formation of inter-molecular RNA helices, we have applied RNA structure probing methods that allowed us to monitor conformational rearrangements, which are prerequisite for theformation of the RNA assortment complex. Our findings open up unique avenues for understanding the challenges for further improvement of the recently developed fully plasmid-based reverse genetics systems for rotaviruses.

A Metabolic Engineering Approach to Incorporate Modified Pyrimidine Nucleosides into Cellular RNA.

The incorporation of modified nucleotides into RNA is a powerful strategy to probe RNA structure and function. While a wide variety of modified nucleotides can be incorporated into RNA in vitro using chemical or enzymatic synthesis, strategies for the metabolic incorporation of artificial nucleotides into cellular RNA are limited, largely due to the incompatibility of modified nucleobases and nucleosides with nucleotide salvage pathways. In this work, we develop a metabolic engineering strategy to facilitate the labeling of cellular RNA with noncanonical pyrimidine nucleosides. First, we use structure-based protein engineering to alter the substrate specificity of uridine-cytidine kinase 2 (UCK2), a key enzyme in the pyrimidine nucleotide salvage pathway. Next, we show that expression of mutant UCK2 in HeLa and U2OS cells is sufficient to enable the incorporation of 5-azidomethyl uridine (5-AmU) into cellular RNA and promotes RNA labeling by other C5-modified pyrimidines. Finally, we apply UCK2-mediated RNA labeling with 5-AmU to study RNA trafficking and turnover during normal and stress conditions and find diminished RNA localization in the cytosol during arsenite stress. Taken together, our study provides a general strategy for the incorporation of modified pyrimidine nucleosides into cellular RNA and expands the chemical toolkit of modified bases for studying dynamic RNA behavior in living cells.

In Vivo Genome-Wide RNA Structure Probing with Structure-seq.

In vivo genome-wide RNA structure probing provides a global view of RNA structure as it occurs in the cell and can assist in elucidating important functional aspects of RNA structure. Structure-seq2 provides high-quality data on transcriptome-wide RNA structure in vivo but contains numerous steps that require technical precision. In this chapter we present the steps needed to produce high-quality structural data with a focus on controls and troubleshooting. Structure-seq2 can be applied to numerous organisms including plants, humans, and bacteria and is amenable to a wide variety of RNA-modifying chemicals including DMS, glyoxal, and SHAPE reagents. Notably, the data generated by the method highlighted here can be readily analyzed using our StructureFold2 computational pipeline.

Genome-wide probing RNA structure with the modified DMS-MaPseq in Arabidopsis.

Transcripts have intrinsic propensity to form stable secondary structure that is fundamental to regulate RNA transcription, splicing, translation, RNA localization and turnover. Numerous methods that integrate chemical reactions with next-generation sequencing (NGS) have been applied to study in vivo RNA structure, providing new insights into RNA biology. Dimethyl sulfate (DMS) probing coupled with mutational profiling through NGS (DMS-MaPseq) is a newly developed method for revealing genome-wide or target-specific RNA structure. Herein, we present our experimental protocol of a modified DMS-MaPseq method for plant materials. The DMS treatment condition was optimized, and library preparation procedures were simplified. We also provided custom scripts for bioinformatic analysis of genome-wide DMS-MaPseq data. Bioinformatic results showed that our method could generate high-quality and reproducible data. Further, we assessed sequencing depth and coverage for genome-wide RNA structure profiling in Arabidopsis, and provided two examples of in vivo structure of mobile RNAs. We hope that our modified DMS-MaPseq method will serve as a powerful tool for analyzing in vivo RNA structurome in plants.

The evolution of RNA structural probing methods: From gels to next-generation sequencing.

RNA molecules are important players in all domains of life and the study of the relationship between their multiple flexible states and the associated biological roles has increased in recent years. For several decades, chemical and enzymatic structural probing experiments have been used to determine RNA structure. During this time, there has been a steady improvement in probing reagents and experimental methods, and today the structural biologist community has a large range of tools at its disposal to probe the secondary structure of RNAs in vitro and in cells. Early experiments used radioactive labeling and polyacrylamide gel electrophoresis as read-out methods. This was superseded by capillary electrophoresis, and more recently by next-generation sequencing. Today, powerful structural probing methods can characterize RNA structure on a genome-wide scale. In this review, we will provide an overview of RNA structural probing methodologies from a historical and technical perspective. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Methods > RNA Analyses in vitro and In Silico RNA Methods > RNA Analyses in Cells.

Assaying RNA structure with LASER-Seq.

Chemical probing methods are crucial to our understanding of the structure and function of RNA molecules. The majority of chemical methods used to probe RNA structure report on Watson–Crick pairing, but tertiary structure parameters such as solvent accessibility can provide an additional layer of structural information, particularly in RNA-protein complexes. Herein we report the development of Light Activated Structural Examination of RNA by high-throughput sequencing, or LASER-Seq, for measuring RNA structure in cells with deep sequencing. LASER relies on a light-generated nicotinoyl nitrenium ion to form covalent adducts with the C8 position of adenosine and guanosine. Reactivity is governed by the accessibility of C8 to the light-generated probe. We compare structure probing by RT-stop and mutational profiling (MaP), demonstrating that LASER can be integrated with both platforms for RNA structure analyses. We find that LASER reactivity correlates with solvent accessibility across the entire ribosome, and that LASER can be used to rapidly survey for ligand binding sites in an unbiased fashion. LASER has a particular advantage in this last application, as it readily modifies paired nucleotides, enabling the identification of binding sites and conformational changes in highly structured RNA.

Carbodiimide reagents for the chemical probing of RNA structure in cells.

Deciphering the conformations of RNAs in their cellular environment allows identification of RNA elements with potentially functional roles within biological contexts. Insight into the conformation of RNA in cells has been achieved using chemical probes that were developed to react specifically with flexible RNA nucleotides, or the Watson–Crick face of single-stranded nucleotides. The most widely used probes are either selective SHAPE (2′-hydroxyl acylation and primer extension) reagents that probe nucleotide flexibility, or dimethyl sulfate (DMS), which probes the base-pairing at adenine and cytosine but is unable to interrogate guanine or uracil. The constitutively charged carbodiimide N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) is widely used for probing G and U nucleotides, but has not been established for probing RNA in cells. Here, we report the use of a smaller and conditionally charged reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), as a chemical probe of RNA conformation, and the first reagent validated for structure probing of unpaired G and U nucleotides in intact cells. We showed that EDC demonstrates similar reactivity to CMC when probing transcripts in vitro. We found that EDC specifically reacted with accessible nucleotides in the 7SK noncoding RNA in intact cells. We probed structured regions within the Xist lncRNA with EDC and integrated these data with DMS probing data. Together, EDC and DMS allowed us to refine predicted structure models for the 3′ extension of repeat C within Xist. These results highlight how complementing DMS probing experiments with EDC allows the analysis of Watson–Crick base-pairing at all four nucleotides of RNAs in their cellular context.

In vivo RNA structural probing of uracil and guanine base-pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

Many biological functions performed by RNAs arise from their in vivo structures. The structure of the same RNA can differ in vitro and in vivo owing in part to the influence of molecules ranging from protons to secondary metabolites to proteins. Chemical reagents that modify the Watson–Crick (WC) face of unprotected RNA bases report on the absence of base-pairing and so are of value to determining structures adopted by RNAs. Reagents have thus been sought that can report on the native RNA structures that prevail in living cells. Dimethyl sulfate (DMS) and glyoxal penetrate cell membranes and inform on RNA secondary structure in vivo through modification of adenine (A), cytosine (C), and guanine (G) bases. Uracil (U) bases, however, have thus far eluded characterization in vivo. Herein, we show that the water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is capable of modifying the WC face of U and G in vivo, favoring the former nucleobase by a factor of ∼1.5, and doing so in the eukaryote rice, as well as in the Gram-negative bacterium Escherichia coli. While both EDC and glyoxal target Gs, EDC reacts with Gs in their typical neutral state, while glyoxal requires Gs to populate the rare anionic state. EDC may thus be more generally useful; however, comparison of the reactivity of EDC and glyoxal may allow the identification of Gs with perturbed pKas in vivo and genome-wide. Overall, use of EDC with DMS allows in vivo probing of the base-pairing status of all four RNA bases.

Technique Development for Probing RNA Structure In Vivo and Genome-Wide.

How organisms perceive and respond to their surroundings is one of the great questions in biology. It is clear that RNA plays key roles in sensing. Cellular and environmental cues that RNA responds to include temperature, ions, metabolites, and biopolymers. Recent advances in next-generation sequencing and in vivo chemical probing have provided unprecedented insights into RNA folding in vivo and genome-wide. Patterns of chemical reactivity have implicated control of gene expression by RNA and aided prediction of RNA structure. Central to these advances has been development of molecular biological and chemical techniques. Key advances are improvements in the quality, cost, and throughput of library preparation; availability of a wider array of chemicals for probing RNA structure in vivo; and robustness and user friendliness of data analysis. Insights from probing transcriptomes and future directions are provided.

RNA Structure-A Neglected Puppet Master for the Evolution of Virus and Host Immunity.

The central dogma of molecular biology describes the flow of genetic information from DNA to protein via an RNA intermediate. For many years, RNA has been considered simply as a messenger relaying information between DNA and proteins. Recent advances in next generation sequencing technology, bioinformatics, and non-coding RNA biology have highlighted the many important roles of RNA in virtually every biological process. Our understanding of RNA biology has been further enriched by a number of significant advances in probing RNA structures. It is now appreciated that many cellular and viral biological processes are highly dependent on specific RNA structures and/or sequences, and such reliance will undoubtedly impact on the evolution of both hosts and viruses. As a contribution to this special issue on host immunity and virus evolution, it is timely to consider how RNA sequences and structures could directly influence the co-evolution between hosts and viruses. In this manuscript, we begin by stating some of the basic principles of RNA structures, followed by describing some of the critical RNA structures in both viruses and hosts. More importantly, we highlight a number of available new tools to predict and to evaluate novel RNA structures, pointing out some of the limitations readers should be aware of in their own analyses.

Coordinate regulation of alternative pre-mRNA splicing events by the human RNA chaperone proteins hnRNPA1 and DDX5.

Alternative premessenger RNA (pre-mRNA) splicing is a post-transcriptional mechanism for controlling gene expression. Splicing patterns are determined by both RNA-binding proteins and nuclear pre-mRNA structure. Here, we analyzed pre-mRNA splicing patterns, RNA-binding sites, and RNA structures near these binding sites coordinately controlled by two splicing factors: the heterogeneous nuclear ribonucleoprotein hnRNPA1 and the RNA helicase DDX5. We identified thousands of alternative pre-mRNA splicing events controlled by these factors by RNA sequencing (RNA-seq) following RNAi. Enhanced cross-linking and immunoprecipitation (eCLIP) on nuclear extracts was used to identify protein-RNA-binding sites for both proteins in the nuclear transcriptome. We found a significant overlap between hnRNPA1 and DDX5 splicing targets and that they share many closely linked binding sites as determined by eCLIP analysis. In vivo SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical RNA structure probing data were used to model RNA structures near several exons controlled and bound by both proteins. Both sequence motifs and in vivo UV cross-linking sites for hnRNPA1 and DDX5 were used to map binding sites in their RNA targets, and often these sites flanked regions of higher chemical reactivity, suggesting an organized nature of nuclear pre-mRNPs. This work provides a first glimpse into the possible RNA structures surrounding pre-mRNA splicing factor-binding sites.

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

ABSTRACTPackaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5ʹ untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.

RNA Framework: an all-in-one toolkit for the analysis of RNA structures and post-transcriptional modifications.

RNA is emerging as a key regulator of a plethora of biological processes. While its study has remained elusive for decades, the recent advent of high-throughput sequencing technologies provided the unique opportunity to develop novel techniques for the study of RNA structure and post-transcriptional modifications. Nonetheless, most of the required downstream bioinformatics analyses steps are not easily reproducible, thus making the application of these techniques a prerogative of few laboratories. Here we introduce RNA Framework, an all-in-one toolkit for the analysis of most NGS-based RNA structure probing and post-transcriptional modification mapping experiments. To prove the extreme versatility of RNA Framework, we applied it to both an in-house generated DMS-MaPseq dataset, and to a series of literature available experiments. Notably, when starting from publicly available datasets, our software easily allows replicating authors' findings. Collectively, RNA Framework provides the most complete and versatile toolkit to date for a rapid and streamlined analysis of the RNA epistructurome. RNA Framework is available for download at: http://www.rnaframework.com.

New Era of Studying RNA Secondary Structure and Its Influence on Gene Regulation in Plants.

The dynamic structure of RNA plays a central role in post-transcriptional regulation of gene expression such as RNA maturation, degradation, and translation. With the rise of next-generation sequencing, the study of RNA structure has been transformed from in vitro low-throughput RNA structure probing methods to in vivo high-throughput RNA structure profiling. The development of these methods enables incremental studies on the function of RNA structure to be performed, revealing new insights of novel regulatory mechanisms of RNA structure in plants. Genome-wide scale RNA structure profiling allows us to investigate general RNA structural features over 10s of 1000s of mRNAs and to compare RNA structuromes between plant species. Here, we provide a comprehensive and up-to-date overview of: (i) RNA structure probing methods; (ii) the biological functions of RNA structure; (iii) genome-wide RNA structural features corresponding to their regulatory mechanisms; and (iv) RNA structurome evolution in plants.