RNA Structural Motifs Research Articles

Genome-Wide Identification of Stable RNA Secondary Structures Across Multiple Organisms Using Chemical Probing Data: Insights into Short Structural Motifs and RNA-Targeting Therapeutics.

Small molecules targeting specific RNA binding sites, including stable and transient RNA structures, are emerging as effective pharmacological approaches for modulating gene expression. However, little is understood about how stable RNA secondary structures are shared across organisms, an important factor in controlling drug selectivity. In this study, I provide an analytical pipeline named RNA Secondary Structure Finder (R2S-Finder) to discover short, stable RNA structural motifs for humans, Escherichia coli ( E. coli ), SARS-CoV-2, and Zika virus by leveraging existing in vivo and in vitro genome-wide chemical RNA-probing datasets. I found several common features across organisms. For example, apart from the well-documented tetraloops, AU-rich tetraloops are widely present in different organisms. I also found that the 5' untranslated region (UTR) contains a higher proportion of stable structures than the coding sequences in humans, SARS-CoV-2, and Zika virus. In general, stable structures predicted from in vitro (protein-free) and in vivo datasets are consistent in humans, E. coli , and SARS-CoV-2, indicating that most stable structure formation were driven by RNA folding alone, while a larger variation was found between in vitro and in vivo data with certain RNA types, such as human long intergenic non-coding RNAs (lincRNAs). Finally, I predicted stable three- and four-way RNA junctions that exist both in vivo and in vitro conditions, which can potentially serve as drug targets. All results of stable sequences, stem-loops, internal loops, bulges, and three- and four-way junctions have been collated in the R2S-Finder database ( https://github.com/JingxinWangLab/R2S-Finder ), which is coded in hyperlinked HTML pages and tabulated in CSV files.

RNAMotifProfile: a graph-based approach to build RNA structural motif profiles.

RNA structural motifs are the recurrent segments in RNA three-dimensional structures that play a crucial role in the functional diversity of RNAs. Understanding the similarities and variations within these recurrent motif groups is essential for gaining insights into RNA structure and function. While recurrent structural motifs are generally assumed to be composed of the same isosteric base interactions, this consistent pattern is not observed across all examples of these motifs. Existing methods for analyzing and comparing RNA structural motifs may overlook variations in base interactions and associated nucleotides. RNAMotifProfile is a novel profile-to-profile alignment algorithm that generates a comprehensive profile from a group of structural motifs, incorporating all base interactions and associated nucleotides at each position. By structurally aligning input motif instances using a guide-tree-based approach, RNAMotifProfile captures the similarities and variations within recurrent motif groups. Additionally, RNAMotifProfile can function as a motif search tool, enabling the identification of instances of a specific motif family by searching with the corresponding profile. The ability to generate accurate and comprehensive profiles for RNA structural motif families, and to search for these motifs, facilitates a deeper understanding of RNA structure-function relationships and potential applications in RNA engineering and therapeutic design.

Development of an RNA Nanostructure for Effective Botrytis cinerea Control through Spray-Induced Gene Silencing without an Extra Nanocarrier.

Spray-induced gene silencing represents an eco-friendly approach for crop protection through the use of double-stranded RNA (dsRNA) to activate the RNA interference (RNAi) pathway, thereby silencing crucial genes in pathogens. The major challenges associated with dsRNA are its limited stability and poor cellular uptake, necessitating repeated applications for effective crop protection. In this study, RNA nanoparticles (NPs) were proposed as effectors in plants and pathogens by inducing the RNAi pathway and silencing gene expression. RNA structural motifs, such as hairpin-loop, kissing-loop, and tetra-U motifs, were used to link multiple siRNAs into a long, single-stranded RNA (lssRNA). The lssRNA, synthesized in Escherichia coli, self-assembled into stable RNA nanostructures via local base pairing. Comparative analyses between dsRNA and RNA NPs revealed that the latter displayed superior efficacy in inhibiting spore germination and mycelial growth of Botrytis cinerea. Moreover, RNA NPs had a more robust protective effect on plants against B. cinerea than did dsRNA. In addition, RNA squares are processed into expected siRNA in plants, thereby inhibiting the expression of the target gene. These findings suggest the potential of RNA NPs for use in plant disease control by providing a more efficient and specific alternative to dsRNA without requiring nanocarriers.

Partial RNA design.

RNA design is a key technique to achieve new functionality in fields like synthetic biology or biotechnology. Computational tools could help to find such RNA sequences but they are often limited in their formulation of the search space. In this work, we propose partial RNA design, a novel RNA design paradigm that addresses the limitations of current RNA design formulations. Partial RNA design describes the problem of designing RNAs from arbitrary RNA sequences and structure motifs with multiple design goals. By separating the design space from the objectives, our formulation enables the design of RNAs with variable lengths and desired properties, while still allowing precise control over sequence and structure constraints at individual positions. Based on this formulation, we introduce a new algorithm, libLEARNA, capable of efficiently solving different constraint RNA design tasks. A comprehensive analysis of various problems, including a realistic riboswitch design task, reveals the outstanding performance of libLEARNA and its robustness. libLEARNA is open-source and publicly available at: https://github.com/automl/learna_tools.

Viroid Replication, Movement, and the Host Factors Involved.

Viroids represent distinctive infectious agents composed solely of short, single-stranded, circular RNA molecules. In contrast to viruses, viroids do not encode for proteins and lack a protective coat protein. Despite their apparent simplicity, viroids have the capacity to induce diseases in plants. Currently, extensive research is being conducted on the replication cycle of viroids within both the Pospiviroidae and Avsunviroidae families, shedding light on the intricacies of the associated host factors. Utilizing the potato spindle tuber viroid as a model, investigations into the RNA structural motifs involved in viroid trafficking between different cell types have been thorough. Nevertheless, our understanding of the host factors responsible for the intra- and inter-cellular movement of viroids remains highly incomplete. This review consolidates our current knowledge of viroid replication and movement within both families, emphasizing the structural basis required and the identified host factors involved. Additionally, we explore potential host factors that may mediate the intra- and inter-cellular movement of viroids, addressing gaps in our understanding. Moreover, the potential application of viroids and the emergence of novel viroid-like cellular parasites are also discussed.

A comprehensive survey of long-range tertiary interactions and motifs in non-coding RNA structures.

Understanding the 3D structure of RNA is key to understanding RNA function. RNA 3D structure is modular and can be seen as a composition of building blocks of various sizes called tertiary motifs. Currently, long-range motifs formed between distant loops and helical regions are largely less studied than the local motifs determined by the RNA secondary structure. We surveyed long-range tertiary interactions and motifs in a non-redundant set of non-coding RNA 3D structures. A new dataset of annotated LOng-RAnge RNA 3D modules (LORA) was built using an approach that does not rely on the automatic annotations of non-canonical interactions. An original algorithm, ARTEM, was developed for annotation-, sequence- and topology-independent superposition of two arbitrary RNA 3D modules. The proposed methods allowed us to identify and describe the most common long-range RNA tertiary motifs. Along with the prevalent canonical A-minor interactions, a large numberof previously undescribed staple interactions were observed. The most frequent long-range motifs were found to belong to three main motif families: planar staples, tilted staples, and helical packing motifs.

Systematic detection of tertiary structural modules in large RNAs and RNP interfaces by Tb-seq

Compact RNA structural motifs control many aspects of gene expression, but we lack methods for finding these structures in the vast expanse of multi-kilobase RNAs. To adopt specific 3-D shapes, many RNA modules must compress their RNA backbones together, bringing negatively charged phosphates into close proximity. This is often accomplished by recruiting multivalent cations (usually Mg2+), which stabilize these sites and neutralize regions of local negative charge. Coordinated lanthanide ions, such as terbium (III) (Tb3+), can also be recruited to these sites, where they induce efficient RNA cleavage, thereby revealing compact RNA 3-D modules. Until now, Tb3+ cleavage sites were monitored via low-throughput biochemical methods only applicable to small RNAs. Here we present Tb-seq, a high-throughput sequencing method for detecting compact tertiary structures in large RNAs. Tb-seq detects sharp backbone turns found in RNA tertiary structures and RNP interfaces, providing a way to scan transcriptomes for stable structural modules and potential riboregulatory motifs.

Molecular Dynamics Simulations with Grand-Canonical Reweighting Suggest Cooperativity Effects in RNA Structure Probing Experiments.

Chemical probing experiments such as SHAPE are routinely used to probe RNA molecules. In this work, we use atomistic molecular dynamics simulations to test the hypothesis that binding of RNA with SHAPE reagents is affected by cooperative effects leading to an observed reactivity that is dependent on the reagent concentration. We develop a general technique that enables the calculation of the affinity for arbitrary molecules as a function of their concentration in the grand-canonical ensemble. Our simulations of an RNA structural motif suggest that, at the concentration typically used in SHAPE experiments, cooperative binding would lead to a measurable concentration-dependent reactivity. We also provide a qualitative validation of this statement by analyzing a new set of experiments collected at different reagent concentrations.

A potential long-range RNA-RNA interaction in the HIV-1 RNA

It is well-established that viral and cellular mRNAs alike harbour functional long-range intra-molecular RNA-RNA interactions. Despite the biological importance of such interactions, their identification and characterization remain challenging. Here we present a computational method for the identification of certain kinds of long-range intra-molecular RNA-RNA interactions involving the loop nucleotides of a hairpin loop. Using the computational method, we analysed 4272 HIV-1 genomic mRNAs. A potential long-range intra-molecular RNA-RNA interaction within the HIV-1 genomic RNA was identified. The long-range interaction is mediated by a kissing loop structure between two stem-loops of the previously reported SHAPE-based secondary structure of the entire HIV-1 genome. Structural modelling studies were carried out to show that the kissing loop structure not only is sterically feasible, but also contains a conserved RNA structural motif often found in compact RNA pseudoknots. The computational method should be generally applicable to the identification of potential long-range intra-molecular RNA-RNA interactions in any viral or cellular mRNA sequence. Communicated by Ramaswamy H. Sarma

Structural and Functional RNA Motifs of SARS-CoV-2 and Influenza A Virus as a Target of Viral Inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic, whereas the influenza A virus (IAV) causes seasonal epidemics and occasional pandemics. Both viruses lead to widespread infection and death. SARS-CoV-2 and the influenza virus are RNA viruses. The SARS-CoV-2 genome is an approximately 30 kb, positive sense, 5' capped single-stranded RNA molecule. The influenza A virus genome possesses eight single-stranded negative-sense segments. The RNA secondary structure in the untranslated and coding regions is crucial in the viral replication cycle. The secondary structure within the RNA of SARS-CoV-2 and the influenza virus has been intensively studied. Because the whole of the SARS-CoV-2 and influenza virus replication cycles are dependent on RNA with no DNA intermediate, the RNA is a natural and promising target for the development of inhibitors. There are a lot of RNA-targeting strategies for regulating pathogenic RNA, such as small interfering RNA for RNA interference, antisense oligonucleotides, catalytic nucleic acids, and small molecules. In this review, we summarized the knowledge about the inhibition of SARS-CoV-2 and influenza A virus propagation by targeting their RNA secondary structure.

Computer-Aided Design and Production of RNA Origami as Protein Scaffolds and Biosensors.

RNA nanotechnology is able to take advantage of the modularity of RNA to build a wide variety of structures and functional devices from a common set of structural modules. The RNA origami architecture harnesses the property of RNA to fold as it is being enzymatically synthesized by the RNA polymerase and enables the design of single-stranded devices that integrate multiple structural and functional RNA motifs. Here, we provide detailed procedures on how to design and characterize RNA origami structures. The process is illustrated by two examples: one that forms lattices and another example that acts as biosensors.

Comprehensive in virio structure probing analysis of the influenza A virus identifies functional RNA structures involved in viral genome replication

The influenza A virus genome is segmented into eight viral RNAs (vRNA). Secondary structures of vRNA are known to be involved in the viral proliferation process. Comprehensive vRNA structures in vitro, in virio, and in cellulo have been analyzed. However, the resolution of the structure map can be improved by comparative analysis and statistical modeling. Construction of a more high-resolution and reliable RNA structure map can identify uncharacterized functional structure motifs on vRNA in virion. Here, we establish the global map of the vRNA secondary structure in virion using the combination of dimethyl sulfate (DMS)-seq and selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE)-seq with a robust statistical analysis. Our high-resolution analysis identified a stem-loop structure at nucleotide positions 39 – 60 of segment 6 and further validated the structure at nucleotide positions 87 – 130 of segment 5 that was previously predicted to form a pseudoknot structure in silico. Notably, when the cells were infected with recombinant viruses which possess the mutations to disrupt the structure, the replication and packaging of the viral genome were drastically decreased. Our results provide comprehensive and high-resolution information on the influenza A virus genome structures in virion and evidence that the functional RNA structure motifs on the influenza A virus genome are associated with appropriate replication and packaging of the viral genome.

Characterizing the Conformational Free-Energy Landscape of RNA Stem-Loops Using Single-Molecule Field-Effect Transistors.

We have developed and used single-molecule field-effect transistors (smFETs) to characterize the conformational free-energy landscape of RNA stem-loops. Stem-loops are one of the most common RNA structural motifs and serve as building blocks for the formation of complex RNA structures. Given their prevalence and integral role in RNA folding, the kinetics of stem-loop (un)folding has been extensively characterized using both experimental and computational approaches. Interestingly, these studies have reported vastly disparate timescales of (un)folding, which has been interpreted as evidence that (un)folding of even simple stem-loops occurs on a highly rugged conformational energy landscape. Because smFETs do not rely on fluorophore reporters of conformation or mechanical (un)folding forces, they provide a unique approach that has allowed us to directly monitor tens of thousands of (un)folding events of individual stem-loops at a 200 μs time resolution. Our results show that under our experimental conditions, stem-loops (un)fold over a 1-200 ms timescale during which they transition between ensembles of unfolded and folded conformations, the latter of which is composed of at least two sub-populations. The 1-200 ms timescale of (un)folding we observe here indicates that smFETs report on complete (un)folding trajectories in which unfolded conformations of the RNA spend long periods of time wandering the free-energy landscape before sampling one of several misfolded conformations or the natively folded conformation. Our findings highlight the extremely rugged landscape on which even the simplest RNA structural elements fold and demonstrate that smFETs are a unique and powerful approach for characterizing the conformational free-energy of RNA.

Ornate, large, extremophilic (OLE) RNA forms a kink turn necessary for OapC protein recognition and RNA function

Ornate, large, extremophilic (OLE) RNAs represent a class of noncoding RNAs prevalent in Gram-positive, extremophilic/anaerobic bacterial species. OLE RNAs (∼600nt), whose precise biochemical functions remain mysterious, form an intricate secondary structure interspersed with regions of highly conserved nucleotides. In the alkali-halophilic bacterium Bacillus halodurans, OLE RNA is a component of a ribonucleoprotein (RNP) complex involving at least two proteins named OapA and OapB, but additional components may exist that could point to functional roles for the RNA. Disruption of the genes for either OLE RNA, OapA, or OapB result in the inability of cells to overcome cold, alcohol, or Mg2+ stresses. In the current study, we used invivo crosslinking followed by OLE RNA isolation to identify the protein YbxF as a potential additional partner in the OLE RNP complex. Notably, a mutation in the gene for this same protein was also reported to be present in a strain wherein the complex is nonfunctional. The B.halodurans YbxF (herein renamed OapC) is homologous to a bacterial protein earlier demonstrated to bind kink turn (k-turn) RNA structural motifs. Invitro RNA-protein binding assays reveal that OLE RNA forms a previously unrecognized k-turn that serves as the natural binding site for YbxF/OapC. Moreover, B.halodurans cells carrying OLE RNAs with disruptive mutations in the k-turn exhibit phenotypes identical to cells lacking functional OLE RNP complexes. These findings reveal that the YbxF/OapC protein of B.halodurans is important for the formation of a functional OLE RNP complex.

An atypical RNA quadruplex marks RNAs as vectors for gene silencing.

The addition of poly(UG) ('pUG') repeats to 3' termini of mRNAs drives gene silencing and transgenerational epigenetic inheritance in the metazoan Caenorhabditis elegans. pUG tails promote silencing by recruiting an RNA-dependent RNA polymerase (RdRP) that synthesizes small interfering RNAs. Here we show that active pUG tails require a minimum of 11.5 repeats and adopt a quadruplex (G4) structure we term the pUG fold. The pUG fold differs from known G4s in that it has a left-handed backbone similar to Z-RNA, no consecutive guanosines in its sequence, and three G quartets and one U quartet stacked non-sequentially. The compact pUG fold binds six potassium ions and brings the RNA ends into close proximity. The biological importance of the pUG fold is emphasized by our observations that porphyrin molecules bind to the pUG fold and inhibit both gene silencing and binding of RdRP. Moreover, specific 7-deaza substitutions that disrupt the pUG fold neither bind RdRP nor induce RNA silencing. These data define the pUG fold as a previously unrecognized RNA structural motif that drives gene silencing. The pUG fold can also form internally within larger RNA molecules. Approximately 20,000 pUG-fold sequences are found in noncoding regions of human RNAs, suggesting that the fold probably has biological roles beyond gene silencing.

Small-Molecule 3D Ligand for RNA Recognition: Tuning Selectivity through Scaffold Hopping.

Targeting RNAs with small molecules is considered the next frontier for drug discovery. In this context, the development of compounds capable of binding RNA structural motifs of low complexity with high affinity and selectivity would greatly expand the number of targets of potential therapeutic value. In this study, we demonstrate that tuning the three-dimensional shape of promiscuous nucleic acid binders is a valuable strategy for the design of new selective RNA ligands. Indeed, starting from a known cyanine, the simple replacement of a phenyl ring with a [2.2]paracyclophane moiety led to a new compound able to discriminate between nucleic acids showing different structural characteristics with a marked affinity and selectivity for an octahairpin loop RNA sequence. This shape modification also affected the in cellulo behavior of the cyanine. These results suggest that scaffold hopping is a valuable strategy to improve the selectivity of RNA/small-molecule interactions and highlight the need to explore a new chemical space for the design of selective RNA ligands.

Analysis of key genes in Mycobacterium ulcerans reveals conserved RNA structural motifs and regions with apparent pressure to remain unstructured.

Buruli Ulcer is a neglected tropical disease that results in disfiguring and dangerous lesions in affected persons across a wide geographic area, including much of West Africa. The causative agent of Buruli Ulcer is Mycobacterium ulcerans, a relative of the bacterium that causes tuberculosis and leprosy. Few therapeutic options exist for the treatment of this disease beyond antibiotics in the early stages, which are frequently ineffective, and surgical removal in the later stage. In this study we analyze six genes in Mycobacterium ulcerans that have high potential of therapeutic targeting. We focus our analysis on a combined in silico and comparative sequence study of potential RNA secondary structure across these genes. The result of this work was the comprehensive local RNA structural landscape across each of these significant genes. This revealed multiple sites of ordered and evolved RNA structure interspersed between sequences that either have no bias for structure or, indeed, appear to be ordered to be unstructured and (potentially) accessible. In addition to providing data that could be of interest to basic biology, our results provide guides for efforts aimed at targeting this pathogen at the RNA level. We explore this latter possibility through the in silico analysis of antisense oligonucleotides that could potentially be used to target pathogen RNA.

Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS

Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS

PRIESSTESS: interpretable, high-performing models of the sequence and structure preferences of RNA-binding proteins.

Modelling both primary sequence and secondary structure preferences for RNA binding proteins (RBPs) remains an ongoing challenge. Current models use varied RNA structure representations and can be difficult to interpret and evaluate. To address these issues, we present a universal RNA motif-finding/scanning strategy, termed PRIESSTESS (Predictive RBP-RNA InterpretablE Sequence-Structure moTif regrESSion), that can be applied to diverse RNA binding datasets. PRIESSTESS identifies dozens of enriched RNA sequence and/or structure motifs that are subsequently reduced to a set of core motifs by logistic regression with LASSO regularization. Importantly, these core motifs are easily visualized and interpreted, and provide a measure of RBP secondary structure specificity. We used PRIESSTESS to interrogate new HTR-SELEX data for 23 RBPs with diverse RNA binding modes and captured known primary sequence and secondary structure preferences for each. Moreover, when applying PRIESSTESS to 144 RBPs across 202 RNA binding datasets, 75% showed an RNA secondary structure preference but only 10% had a preference besides unpaired bases, suggesting that most RBPs simply recognize the accessibility of primary sequences.

Designing and executing prime editing experiments in mammalian cells.

Prime editing (PE) is a precision gene editing technology that enables the programmable installation of substitutions, insertions and deletions in cells and animals without requiring double-strand DNA breaks (DSBs). The mechanism of PE makes it less dependent on cellular replication and endogenous DNA repair than homology-directed repair-based approaches, and its ability to precisely install edits without creating DSBs minimizes indels and other undesired outcomes. The capabilities of PE have also expanded since its original publication. Enhanced PE systems, PE4 and PE5, manipulate DNA repair pathways to increase PE efficiency and reduce indels. Other advances that improve PE efficiency include engineered pegRNAs (epegRNAs), which include a structured RNA motif to stabilize and protect pegRNA 3' ends, and the PEmax architecture, which improves editor expression and nuclear localization. New applications such as twin PE (twinPE) can precisely insert or delete hundreds of base pairs of DNA and can be used in tandem with recombinases to achieve gene-sized (>5 kb) insertions and inversions. Achieving optimal PE requires careful experimental design, and the large number of parameters that influence PE outcomes can be daunting. This protocol describes current best practices for conducting PE and twinPE experiments and describes the design and optimization of pegRNAs. We also offer guidelines for how to select the proper PE system (PE1 to PE5 and twinPE) for a given application. Finally, we provide detailed instructions on how to perform PE in mammalian cells. Compared with other procedures for editing human cells, PE offers greater precision and versatility, and can be completed within 2-4 weeks.