Changes In RNA Structure Research Articles

Tracking RNA structures as RNAs transit through the cell.

RNAs perform diverse cellular functions that are mediated at least in part by their structure. However, how RNA structure changes throughout the RNA lifecycle and how these changes affect RNA function remain incompletely understood. A detailed in vivo characterization of RNA structure in various cellular subcompartments now provides insights into how RNA structural changes influence translation, RNA decay, protein binding and RNA modification.

RNA structure maps across mammalian cellular compartments.

RNA structure is intimately connected to each step of gene expression. Recent advances have enabled transcriptome-wide maps of RNA secondary structure, termed RNA structuromes. However, previous whole-cell analyses lacked the resolution to unravel the landscape and also the regulatory mechanisms of RNA structural changes across subcellular compartments. Here we reveal the RNA structuromes in three compartments — chromatin, nucleoplasm and cytoplasm in human and mouse cells. The cytotopic structuromes substantially expand RNA structural information, and enable detailed investigation of the central role of RNA structure in linking transcription, translation, and RNA decay. We develop a resource to visualize the interplay of RNA-protein interactions, RNA modifications, and RNA structure, and predict both direct and indirect reader proteins of RNA modifications. We also validate a novel role of the RNA binding protein LIN28A as an N6-methyladenosine modification “anti-reader”. Our results highlight the dynamic nature of RNA structures and its functional significance in gene regulation.

Visualizing RNA Conformational Changes via Pattern Recognition of RNA by Small Molecules.

Conformational changes in RNA play vital roles in the regulation of many biological systems, yet these changes can be challenging to visualize. Previously, we demonstrated that Pattern Recognition of RNA by Small Molecules (PRRSM) can unbiasedly cluster defined RNA secondary structure motifs utilizing an aminoglycoside receptor library. In this work, we demonstrate the power of this method to visualize changes in folding at the secondary structure level within two distinct riboswitch structures. After labeling at three independent positions on each riboswitch, PRRSM accurately classified all apo and ligand-bound riboswitch structures, including changes in the size of a structural motif, and revealed modification sites that prevented folding and/or led to a mixture of states. These data underscore the utility and robustness of the PRRSM assay for rapid assessment of RNA structural changes and for gaining ready insight into nucleotide positions critical to RNA folding.

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2'-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2'-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2'-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Isothermal Titration Calorimetry Measurements of Riboswitch-Ligand Interactions.

One of the many ways by which bacteria control gene expression is through cis-acting regulatory mRNA elements called riboswitches. By specifically binding to small molecules or metabolites and pairing the binding event to an RNA structure change, riboswitches link a metabolic input to a transcriptional or translational output. For over a decade, isothermal titration calorimetry (ITC) has been used to investigate how riboswitches interact with small molecules. We present methods for assaying RNA-ligand interactions using ITC and analyzing resulting data to estimate thermodynamic parameters associated with binding.

Genome-wide RNA structurome reprogramming by acute heat shock globally regulates mRNA abundance

The heat shock response is crucial for organism survival in natural environments. RNA structure is known to influence numerous processes related to gene expression, but there have been few studies on the global RNA structurome as it prevails in vivo. Moreover, how heat shock rapidly affects RNA structure genome-wide in living systems remains unknown. We report here in vivo heat-regulated RNA structuromes. We applied Structure-seq chemical [dimethyl sulfate (DMS)] structure probing to rice (Oryza sativa L.) seedlings with and without 10 min of 42 °C heat shock and obtained structural data on >14,000 mRNAs. We show that RNA secondary structure broadly regulates gene expression in response to heat shock in this essential crop species. Our results indicate significant heat-induced elevation of DMS reactivity in the global transcriptome, revealing RNA unfolding over this biological temperature range. Our parallel Ribo-seq analysis provides no evidence for a correlation between RNA unfolding and heat-induced changes in translation, in contrast to the paradigm established in prokaryotes, wherein melting of RNA thermometers promotes translation. Instead, we find that heat-induced DMS reactivity increases correlate with significant decreases in transcript abundance, as quantified from an RNA-seq time course, indicating that mRNA unfolding promotes transcript degradation. The mechanistic basis for this outcome appears to be mRNA unfolding at both 5' and 3'-UTRs that facilitates access to the RNA degradation machinery. Our results thus reveal unexpected paradigms governing RNA structural changes and the eukaryotic RNA life cycle.

Polymorphisms in KSHV-encoded microRNA sequences affect levels of mature viral microRNA in Kaposi Sarcoma lesions.

BackgroundWe previously reported Kaposi sarcoma-associated herpesvirus (KSHV) microRNA sequence variants in clinical samples correlated with increased risk of multicentric Castleman's disease (MCD). We then demonstrated that microRNAs with variant sequence have different maturation and mature microRNA expression in vitro. Here, we illustrate the association between microRNA sequence and changes in mature microRNA levels within Kaposi sarcoma (KS) lesions.MethodsKSHV microRNA sequences were determined from 20 KS lesions and 4 control skin biopsies from individuals evaluated for KS. Levels of mature KSHV microRNAs were measured with 21 custom small RNA qRT-PCR assays using RNA RNU6B as endogenous control.ResultsThe levels of 13 KSHV-encoded microRNAs were elevated in KS lesions compared to control biopsies. MicroRNA 9-5p was strongly down regulated in South African vs. US biopsies. Low levels of K12-9-5p were associated with single nucleotide polymorphisms (SNPs) in miR-K12-9-5p, 4-5p, 5-3p, 7-3p and pri-miR-K12-3. One SNP in pri-miR-K12-3 resulted in down regulation of miR-K12-6-3p, 8-3p, 10-3p, 12-5p and the upregulation of 5-5p, illustrating sequence variants outside pre-microRNAs were also associated with changes in mature microRNA levels.ConclusionsThe levels of mature KSHV-encoded microRNAs in KS lesions correlate with sequence variation reflecting changes in secondary and tertiary RNA structure.

Abstract SY43-02: RNA modifications and cell identity: The importance of forgetting the past to embrace the future

Abstract Impaired gene regulation lies at the heart of many disorders, including cancer. Gene expression is controlled through multiple mechanisms that are coordinated to ensure the proper and timely expression of each gene. From transcription to translation, every step of mRNA processing is controlled by interaction between the mRNA transcript and regulatory factors, such as RNA binding proteins or small noncoding RNAs. We now understand that these regulatory interactions can be modulated by RNA modifications. Presence of a modified nucleotide can create, or abrogate, sites for regulatory interactions by at least two distinct mechanisms: by inducing changes in RNA structure or through direct recognition by the RNA binding protein of the modified nucleotide. The RNA modification N6-methyladenosine (m6A) is present in several classes of RNAs, including mRNAs and lncRNAs, where it is the most abundant internal RNA modification. In mRNAs and lncRNAs, the m6A modification is present in a well-defined RNA motif, RRACH (R = A or G, H = A, C or U), and is enriched at specific transcript landmarks such as the last transcribed exon, near the STOP codon. The METTL3/METTL14 heterodimer is at the core of the large protein complex that catalyzes addition of m6A to mRNAs. To date, two enzymes capable of removing m6A have been identified: fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5). Our studies on the role of m6A in ESCs revealed that thousands of messenger and long noncoding RNAs are m6A-modified, including transcripts encoding core pluripotency transcription factors such as Nanog, Sox2, and Myc. Our observations suggest that m6A marks unstable transcripts, including transcripts that need to be turned over upon differentiation. Loss of Mettl3 did not affect self-renewal, but resulted in impaired ESC exit from self-renewal towards differentiation to several lineages. Mettl3 KO cells did not differentiate into cardiomyocites or neurons in vitro, and subcutaneous injection of Mettl3 KO cells formed tumors consistent in morphology with teratomas that tended to be larger than tumors derived from wild-type cells. Furthermore, teratomas derived from Mettl3 KO cells were predominantly composed of poorly differentiated cells with very high mitotic indices and numerous apoptotic bodies, whereas wild-type cells differentiated predominantly into neuroectoderm. The role of m6A in facilitating transition between cell states is not limited to embryonic stem cells. The m6A interacting protein YTHDC2 is required for a clean transition from mitosis to meiosis in male and female germ cells. Ythdc2-/- male germ cells attempt to enter meiotic prophase but appear to have a mixed identity, maintaining expression of mitotic Cyclin A2 and failing to properly express many meiotic markers. Instead of executing meiotic prophase, the cells attempt an abnormal mitotic-like division, then quickly die. YTHDC2 is required during the early stages of meiotic entry in mice to both downregulate the previous mitotic program and facilitate proper expression of meiotic and differentiation genes. We propose that m6A makes the transition between cell states possible by facilitating a reset mechanism between stages, as occurs in ESCs and during gametogenesis. In contrast to epigenetic mechanisms that provide cellular memory of gene expression states, m6A enforces the transience of genetic information, helping cells to forget the past and thereby embrace the future. Citation Format: Pedro J. Batista. RNA modifications and cell identity: The importance of forgetting the past to embrace the future [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY43-02.

MRNA structure determines specificity of a polyQ-driven phase separation.

RNA promotes liquid-liquid phase separation (LLPS) to build membraneless compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is unknown. Here, we report that secondary structure allows messenger RNAs (mRNAs) to self-associate and determines whether an mRNA is recruited to or excluded from liquid compartments. The polyQ-protein Whi3 induces conformational changes in RNA structure and generates distinct molecular fluctuations depending on the RNA sequence. These data support a model in which structure-based, RNA-RNA interactions promote assembly of distinct droplets and protein-driven, conformational dynamics of the RNA maintain this identity. Thus, the shape of RNA can promote the formation and coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.

Small Molecule Differentiation of RNA Structures Using Pattern Recognition

The critical importance of RNA elements in gene regulation and the potential of targeting RNA with small molecules are increasingly appreciated, yet many questions remain unanswered in small molecule:RNA recognition. These questions include which RNA structures can best be differentiated by small molecules and how small molecules can be used as tools to probe RNA structure and function. We developed a method termed Pattern Recognition of RNA with Small Molecules (PRRSM) that evaluates the ability of small molecules to classify RNA structures under a variety of conditions and in turn uses these small molecule “receptors” to identify site‐specific RNA structural changes. Insights gained from the assay, the current scope of the method, and future directions will be discussed.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation

BackgroundNSC260594, a quinolinium derivative from the NCI diversity set II compound library, was previously identified in a target-based assay as an inhibitor of the interaction between the HIV-1 (ψ) stem-loop 3 (SL3) RNA and Gag. This compound was shown to exhibit potent antiviral activity. Here, the effects of this compound on individual stages of the viral lifecycle were examined by qRT-PCR, ELISA and Western blot, to see if its actions were specific to the viral packaging stage. The structural effects of NSC260594 binding to the HIV-1 gRNA were also examined by SHAPE and dimerization assays.ResultsTreatment of cells with NSC260594 did not reduce the number of integration events of incoming virus, and treatment of virus producing cells did not affect the level of intracellular Gag protein or viral particle release as determined by immunoblot. However, NSC260594 reduced the incorporation of gRNA into virions by up to 82%, without affecting levels of gRNA inside the cell. This reduction in packaging correlated closely with the reduction in infectivity of the released viral particles. To establish the structural effects of NSC260594 on the HIV-1 gRNA, we performed SHAPE analyses to pinpoint RNA structural changes. NSC260594 had a stabilizing effect on the wild type RNA that was not confined to SL3, but that was propagated across the structure. A packaging mutant lacking SL3 did not show this effect.ConclusionsNSC260594 acts as a specific inhibitor of HIV-1 RNA packaging. No other viral functions are affected. Its action involves preventing the interaction of Gag with SL3 by stabilizing this small RNA stem-loop which then leads to stabilization of the global packaging signal region (psi or ψ). This confirms data, previously only shown in analyses of isolated SL3 oligonucleotides, that SL3 is structurally labile in the presence of Gag and that this is critical for the complete psi region to be able to adopt different conformations. Since replication is otherwise unaffected by NSC260594 the flexibility of SL3 appears to be a unique requirement for genome encapsidation and identifies this process as a highly specific drug target. This study is proof of principle that development of a new class of antiretroviral drugs that specifically target viral packaging by binding to the viral genomic RNA is achievable.

Characterizing the Structure-Function Relationship of a Naturally Occurring RNA Thermometer.

A large number of bacteria have been found to govern virulence and heat shock responses using temperature-sensing RNAs known as RNA thermometers. A prime example is the agsA thermometer known to regulate the production of the AgsA heat shock protein in Salmonella enterica using a "fourU" structural motif. Using the SHAPE-Seq RNA structure-probing method in vivo and in vitro, we found that the regulator functions by a subtle shift in equilibrium RNA structure populations that leads to a partial melting of the helix containing the ribosome binding site. We also demonstrate that binding of the ribosome to the agsA mRNA causes changes to the thermometer structure that appear to facilitate thermometer helix unwinding. These results demonstrate how subtle RNA structural changes can govern gene expression and illuminate the function of an important bacterial regulatory motif.

Long-Range Interactions in Riboswitch Control of Gene Expression.

Riboswitches are widespread RNA motifs that regulate gene expression in response to fluctuating metabolite concentrations. Known primarily from bacteria, riboswitches couple specific ligand binding and changes in RNA structure to mRNA expression in cis. Crystal structures of the ligand binding domains of most of the phylogenetically widespread classes of riboswitches, each specific to a particular metabolite or ion, are now available. Thus, the bound states-one end point-have been thoroughly characterized, but the unbound states have been more elusive. Consequently, it is less clear how the unbound, sensing riboswitch refolds into the ligand binding-induced output state. The ligand recognition mechanisms of riboswitches are diverse, but we find that they share a common structural strategy in positioning their binding sites at the point of the RNA three-dimensional fold where the residues farthest from one another in sequence meet. We review how riboswitch folds adhere to this fundamental strategy and propose future research directions for understanding and harnessing their ability to specifically control gene expression.

A DEAD-Box Helicase Mediates an RNA Structural Transition in the HIV-1 Rev Response Element

Nuclear export of partially spliced or unspliced HIV-1 RNA transcripts requires binding of the viral protein regulator of expression of virion (Rev) to the Rev response element (RRE) and subsequent oligomerization in a cooperative manner. Cellular DEAD-box helicase DEAD-box protein 1 (DDX1) plays a role in HIV replication, interacting with and affecting Rev-containing HIV transcripts in vivo, interacting directly with the RRE and Rev in vitro, and promoting Rev oligomerization in vitro. Binding of DDX1 results in enhancement of Rev oligomerization on the RRE that is correlated with an RNA structural change within the RRE that persists even after dissociation of DDX1. Furthermore, this structural transition is likely located within the three-way junction of stem II of the RRE that is responsible for initial Rev binding. This discovery of the stem II structural transition leads to a model wherein DDX1 can act as an RNA chaperone, folding stem IIB into a proper Rev binding conformation.

Classification of RNA structure change by ‘gazing’ at experimental data

MotivationMutations (or Single Nucleotide Variants) in folded RiboNucleic Acid structures that cause local or global conformational change are riboSNitches. Predicting riboSNitches is challenging, as it requires making two, albeit related, structure predictions. The data most often used to experimentally validate riboSNitch predictions is Selective 2′ Hydroxyl Acylation by Primer Extension, or SHAPE. Experimentally establishing a riboSNitch requires the quantitative comparison of two SHAPE traces: wild-type (WT) and mutant. Historically, SHAPE data was collected on electropherograms and change in structure was evaluated by ‘gel gazing.’ SHAPE data is now routinely collected with next generation sequencing and/or capillary sequencers. We aim to establish a classifier capable of simulating human ‘gazing’ by identifying features of the SHAPE profile that human experts agree ‘looks’ like a riboSNitch.ResultsWe find strong quantitative agreement between experts when RNA scientists ‘gaze’ at SHAPE data and identify riboSNitches. We identify dynamic time warping and seven other features predictive of the human consensus. The classSNitch classifier reported here accurately reproduces human consensus for 167 mutant/WT comparisons with an Area Under the Curve (AUC) above 0.8. When we analyze 2019 mutant traces for 17 different RNAs, we find that features of the WT SHAPE reactivity allow us to improve thermodynamic structure predictions of riboSNitches. This is significant, as accurate RNA structural analysis and prediction is likely to become an important aspect of precision medicine.Availability and ImplementationThe classSNitch R package is freely available at http://classsnitch.r-forge.r-project.org.Supplementary information Supplementary data are available at Bioinformatics online.

A New Method to Predict Ion Effects in RNA Folding.

The strong interaction between metal ions in solution and highly charged RNA molecules is critical for RNA structure formation and stabilization. Metal ions binding to RNA can induce RNA structural changes that are important for RNA cellular functions. Therefore, quantitative modeling of the ion effects is essential for RNA structure prediction and RNA-based molecular design. Recently, inspired by the increasing experimental evidence that supports the importance of ion correlation and fluctuation in ion-RNA interactions, we developed a new computational model, Monte Carlo Tightly Bound Ion (MCTBI) model. The validity of the model is shown by the improved accuracy in the predictions for ion binding properties and ion-dependent free energies for RNA structures. In this chapter, using homodimeric tetraloop-receptor docking as an illustrativeexample, we showcase the MCTBI method for the computational prediction of the ion effects in RNA folding.

Classification of RNA structure change by “gazing” at experimental data

Classification of RNA structure change by “gazing” at experimental data

The X-ray Structures of Six Octameric RNA Duplexes in the Presence of Different Di- and Trivalent Cations.

Due to the polyanionic nature of RNA, the principles of charge neutralization and electrostatic condensation require that cations help to overcome the repulsive forces in order for RNA to adopt a three-dimensional structure. A precise structural knowledge of RNA-metal ion interactions is crucial to understand the mechanism of metal ions in the catalytic or regulatory activity of RNA. We solved the crystal structure of an octameric RNA duplex in the presence of the di- and trivalent metal ions Ca2+, Mn2+, Co2+, Cu2+, Sr2+, and Tb3+. The detailed investigation reveals a unique innersphere interaction to uracil and extends the knowledge of the influence of metal ions for conformational changes in RNA structure. Furthermore, we could demonstrate that an accurate localization of the metal ions in the X-ray structures require the consideration of several crystallographic and geometrical parameters as well as the anomalous difference map.

Understanding nucleic acid structural changes by comparing wide-angle x-ray scattering (WAXS) experiments to molecular dynamics simulations.

Wide-angle x-ray scattering (WAXS) is emerging as a powerful tool for increasing the resolution of solution structure measurements of biomolecules. Compared to its better known complement, small angle x-ray scattering (SAXS), WAXS targets higher scattering angles and can enhance structural studies of molecules by accessing finer details of solution structures. Although the extension from SAXS to WAXS is easy to implement experimentally, the computational tools required to fully harness the power of WAXS are still under development. Currently, WAXS is employed to study structural changes and ligand binding in proteins; however, the methods are not as fully developed for nucleic acids. Here, we show how WAXS can qualitatively characterize nucleic acid structures as well as the small but significant structural changes driven by the addition of multivalent ions. We show the potential of WAXS to test all-atom molecular dynamics (MD) simulations and to provide insight into understanding how the trivalent ion cobalt(III) hexammine (CoHex) affects the structure of RNA and DNA helices. We find that MD simulations capture the RNA structural change that occurs due to addition of CoHex.

Interplay between PCBP2 and miRNA modulates ARHGDIA expression and function in glioma migration and invasion.

RNA-RNA and protein-RNA interactions are essential for post-transcriptional regulationin normal development and may be deregulated in cancer initiation and progression. The RNA-binding protein PCBP2, an oncogenic protein in human malignant gliomas, is an essential regulator of mRNA and miRNA biogenesis, stability and activity. Here, we identified Rho GDP dissociation inhibitor α (ARHGDIA) as a target mRNA that binds to PCBP2, and we uncovered the role of ARHGDIA as a putative metastasis suppressor through analyses of in vitro and in vivo models of EMT and metastasis. Furthermore, we demonstrated that ARHGDIA is a potential target of miR-151-5p and miR-16 in gliomas. The interaction between PCBP2 and the 3′UTR of the ARHGDIA mRNA may induce a local change in RNA structure that favors subsequent binding of miR-151-5p and miR-16, thus leading to the suppression of ARHGDIA expression. PCBP2 may facilitate miR-151-5p and miR-16 promotion of glioma cell migration and invasion through mitigating the function of ARHGDIA.