Sequence Design for RNA-RNA Interactions.

The design of RNA sequences with desired structural properties presents a challenging computational problem with promising applications in biotechnology and biomedicine. Most regulatory RNAs function by forming RNA-RNA interactions, e.g., in order to regulate mRNA expression. It is therefore natural to consider problems where a sequence is designed to form a desired RNA-RNA interaction and switch between structures upon binding. This contribution demonstrates the use of the Infrared framework to design interacting sequences. Specifically, we consider the regulation of the rpoS mRNA by the sRNA DsrA and design artificial 5 ' UTRs that place a downstream protein coding gene under control of DsrA. The design process is explained step by step in a Jupyter notebook, accompanied by Python code. The text discusses setting up design constraints for sampling sequences in Infrared, computing quality measures, constructing a suitable cost function, as well as the optimization procedure. We show that not only thermodynamic but also kinetic folding features can be relevant. Kinetics of interaction formation can be estimated efficiently using the RRIkinDP tool, and the chapter explains how to include kinetic folding features from RRIkinDP directly in the cost function. The protocol implemented in our Jupyter notebook can easily be extended to consider additional requirements or adapted to novel design scenarios.

Phase separation and viral factories: unveiling the physical processes supporting RNA packaging in dsRNA viruses.

Understanding of the physicochemical properties and functions of biomolecular condensates has rapidly advanced over the past decade. More recently, many RNA viruses have been shown to form cytoplasmic replication factories, or viroplasms, via phase separation of their components, akin to numerous cellular membraneless organelles. Notably, diverse viruses from the Reoviridae family containing 10-12 segmented double-stranded RNA genomes induce the formation of viroplasms in infected cells. Little is known about the inner workings of these membraneless cytoplasmic inclusions and how they may support stoichiometric RNA assembly in viruses with segmented RNA genomes, raising questions about the roles of phase separation in coordinating viral genome packaging. Here, we discuss how the molecular composition of viroplasms determines their properties, highlighting the interplay between RNA structure, RNA remodelling, and condensate self-organisation. Advancements in RNA structural probing and theoretical modelling of condensates can reveal the mechanisms through which these ribonucleoprotein complexes support the selective enrichment and stoichiometric assembly of distinct viral RNAs.

Long Intergenic Non-Coding RNAs of Human Chromosome 18: Focus on Cancers.

Malignant neoplasms are characterized by high molecular heterogeneity due to multilevel deregulation of gene expression and cellular functions. It is known that non-coding RNAs, including long intergenic non-coding RNAs (lincRNAs), can play significant roles in cancer biology. The current review focuses on a systematical analysis of genomic, transcriptomic, epigenomic, interactomic, and literature data on 65 lincRNAs of human chromosome 18 in the context of pan-cancer studies. The entire group of lincRNAs can be conditionally divided into 4 subgroups depending on experimental evidence on direct or indirect involvement in cancers and the biological associations with cancers, which we found during the data-mining process: the most studied (5 lincRNAs), moderately or poorly studied (11 lincRNAs), and understudied (31 lincRNAs). For the remaining 18 lincRNAs, data for analysis were fragmentary or missing. Among the key findings were the following: Of the lincRNAs of human chromosome 18, 40% have tissue-specific expression patterns, 22% of lincRNAs are known to have gene fusions, 40% of lincRNAs are prone to gene amplifications and/or deletions in cancers at a frequency greater than 3%, and 23% of lincRNAs are differentially expressed across cancer types, whereas 7% have subtype-specific expression patterns. LincRNAs' interactomes consist of 'master' microRNAs and 47 proteins (including cancer-associated proteins and microRNAs) that can interact with 3 or more lincRNAs. Functional enrichment analysis of a set of highly co-expressed genes retrieved for 17 lincRNAs in different cancer types indicated the potential associations of these lincRNAs with cellular signaling pathways. Six lincRNAs encoded small open-reading frame (smORF) proteins with emerging roles in cancers, and microRNAs as well as proteins with known functions in molecular carcinogenesis can bind to coding regions of smORFs. We identified seven transcriptomic signatures with potential prognostic value, consisting of two to seven different lincRNAs only. Taken together, the literature, biomedical, and molecular biology data analyzed indicated that only five of all lincRNAs of human chromosome 18 are cancer-associated, while eleven other lincRNAs have the tendency to be associated with cancers.

Native Circular RNA Pulldown Method to Simultaneously Profile RNA and Protein Interactions.

Circular RNAs (circRNAs) are a widespread, cell-, tissue-, and disease-specific class of largely non-coding RNA transcripts. These single-stranded, covalently-closed transcripts arise through non-canonical splicing of pre-mRNA, a process called back-splicing. Back-splicing results in circRNAs which are distinguishable from their cognate mRNA as they possess a unique sequence of nucleic acids called the backsplice junction (BSJ). CircRNAs have been shown to play key functional roles in various cellular contexts and achieve this through their interaction with other macromolecules, particularly other RNA molecules and proteins. To elucidate the molecular mechanisms underlying circRNA function, it is necessary to identify these interacting partners. Herein, we present an optimized strategy for the simultaneous purification of the circRNA interactome within eukaryotic cells, allowing the identification of both circRNA-RNA and circRNA-protein interactions.

Experimental Validation of RNA-RNA Interactions by Electrophoretic Mobility Shift Assay.

Regulatory RNAs in bacteria are known to act by base pairing with other RNAs. Interactions between two partner RNAs can be investigated by electrophoretic mobility shift assays. The regions predicted to be engaged in base pairing are analyzed by introducing mutations in one RNA that prevent RNA-RNA complex formation. Next, base pairing is restored by introducing complementary mutations in its partner RNA. Here, we describe the mutational strategy and experimental methods used to validate specific base pairing between two RNA species.

Intronic small nucleolar RNAs regulate host gene splicing through base pairing with their adjacent intronic sequences.

BACKGROUND: Small nucleolar RNAs (snoRNAs) are abundant noncoding RNAs best known for their involvement in ribosomal RNA maturation. In mammals, most expressed snoRNAs are embedded in introns of longer genes and produced through transcription and splicing of their host. Intronic snoRNAs were long viewed as inert passengers with little effect on host expression. However, a recent study reported a snoRNA influencing the splicing and ultimate output of its host gene. Overall, the general contribution of intronic snoRNAs to host expression remains unclear. RESULTS: Computational analysis of large-scale human RNA-RNA interaction datasets indicates that 30% of detected snoRNAs interact with their host transcripts. Many snoRNA-host duplexes are located near alternatively spliced exons and display high sequence conservation suggesting a possible role in splicing regulation. The study of the model SNORD2-EIF4A2 duplex indicates that the snoRNA interaction with the host intronic sequence conceals the branch point leading to decreased inclusion of the adjacent alternative exon. Extended SNORD2 sequence containing the interacting intronic region accumulates in sequencing datasets in a cell-type-specific manner. Antisense oligonucleotides and mutations that disrupt the formation of the snoRNA-intron structure promote the splicing of the alternative exon, shifting the EIF4A2 transcript ratio away from nonsense-mediated decay. CONCLUSIONS: Many snoRNAs form RNA duplexes near alternative exons of their host transcripts, placing them in optimal positions to control host output as shown for the SNORD2-EIF4A2 model system. Overall, our study supports a more widespread role for intronic snoRNAs in the regulation of their host transcript maturation.

Subcellular spatially resolved gene neighborhood networks in single cells.

Image-based spatial omics methods such as fluorescence in situ hybridization (FISH) generate molecular profiles of single cells at single-molecule resolution. Current spatial transcriptomics methods focus on the distribution of single genes. However, the spatial proximity of RNA transcripts can play an important role in cellular function. We demonstrate a spatially resolved gene neighborhood network (spaGNN) pipeline for the analysis of subcellular gene proximity relationships. In spaGNN, machine-learning-based clustering of subcellular spatial transcriptomics data yields subcellular density classes of multiplexed transcript features. The nearest-neighbor analysis produces heterogeneous gene proximity maps in distinct subcellular regions. We illustrate the cell-type-distinguishing capability of spaGNN using multiplexed error-robust FISH data of fibroblast and U2-OS cells and sequential FISH data of mesenchymal stem cells (MSCs), revealing tissue-source-specific MSC transcriptomics and spatial distribution characteristics. Overall, the spaGNN approach expands the spatial features that can be used for cell-type classification tasks.

Inter- and Intramolecular RNA-RNA Interactions Modulate the Regulation of Translation Mediated by the 3' UTR in West Nile Virus.

RNA viruses rely on genomic structural elements to accomplish the functions necessary to complete the viral cycle. These elements participate in a dynamic network of RNA-RNA interactions that determine the overall folding of the RNA genome and may be responsible for the fine regulation of viral replication and translation as well as the transition between them. The genomes of members of the genus Flavivirus are characterized by a complexly folded 3' UTR with a number of RNA structural elements that are conserved across isolates of each species. The present work provides evidence of intra- and intermolecular RNA-RNA interactions involving RNA structural elements in the 3' UTR of the West Nile virus genome. The intermolecular interactions can be visualized in vitro by the formation of molecular dimers involving the participation of at least the SLI and 3'DB elements. Certainly, the 3' UTR of dengue virus, which lacks the SLI element, forms molecular dimers in lower quantities via a single interaction site, probably 3'DB. The functional analysis of sequence or deletion mutants revealed an inverse relationship between 3' UTR dimerization and viral translation efficiency in cell cultures. A network of RNA-RNA interactions involving 3' UTR structural elements might therefore exist, helping to regulate viral translation.

MicroRNA Target Identification: Revisiting Accessibility and Seed Anchoring.

By pairing to messenger RNAs (mRNAs for short), microRNAs (miRNAs) regulate gene expression in animals and plants. Accurately identifying which mRNAs interact with a given miRNA and the precise location of the interaction sites is crucial to reaching a more complete view of the regulatory network of an organism. Only a few experimental approaches, however, allow the identification of both within a single experiment. Computational predictions of miRNA-mRNA interactions thus remain generally the first step used, despite their drawback of a high rate of false-positive predictions. The major computational approaches available rely on a diversity of features, among which anchoring the miRNA seed and measuring mRNA accessibility are the key ones, with the first being universally used, while the use of the second remains controversial. Revisiting the importance of each is the aim of this paper, which uses Cross-Linking, Ligation, And Sequencing of Hybrids (CLASH) datasets to achieve this goal. Contrary to what might be expected, the results are more ambiguous regarding the use of the seed match as a feature, while accessibility appears to be a feature worth considering, indicating that, at least under some conditions, it may favour anchoring by miRNAs.

Generation of Functional-RNA Arrays by In Vitro Transcription and In Situ RNA Capture for the Detection of RNA-RNA Interactions.

RNA performs a wide variety of vital cellular functions. These functions typically require interactions with other biological macromolecules, often as part of an intricate communication network. High-throughput techniques capable of analyzing RNA-based interactions are therefore essential. Functional-RNA arrays address this need, providing the capability of performing hundreds of miniature assays in parallel. Here we describe a method to generate functional-RNA arrays using in vitro transcription of a DNA template array and in situ RNA capture. We also suggest how functional-RNA arrays could be applied to investigating RNA-RNA interactions.

Exploring structural determinants and the role of nucleolin in formation of the long-range interactions between untranslated regions of p53 mRNA.

p53 protein is a key regulator of cellular homeostasis by coordinating the framework of antiproliferative pathways as a response to various stress factors. Although the main mechanism of stress-dependent induction of p53 protein relies on post-translational modifications influencing its stability and activity, a growing amount of evidence suggests that complex regulation of p53 expression occurs also at the mRNA level. This study explores structural determinants of long-range RNA-RNA interactions in p53 mRNA, crucial for stress-dependent regulation of p53 protein translation. We demonstrate that the 8-nt bulge motif plays a key structural role in base-pairing of complementary sequences from the 5' and 3' untranslated regions of p53 mRNA. We also show that one of the p53 translation regulators, nucleolin, displays an RNA chaperone activity and facilitates the association of sequences involved in the formation of long-range interactions in p53 mRNA. Nucleolin promotes base-pairing of complementary sequences through the bulge motif, because mutations of this region reduce or inhibit pairing while compensatory mutations restore this interaction. Mutational analysis of nucleolin reveals that all four RNA recognition motifs are indispensable for optimal RNA chaperone activity of nucleolin. These observations help to decipher the unique mechanism of p53 protein translation regulation pointing to bulge motif and nucleolin as the critical factors during intramolecular RNA-RNA recognition in p53 mRNA.

Cotranscriptional Assembly and Native Purification of Large RNA-RNA Complexes for Structural Analyses.

Recent technological developments such as cryogenic electron microscopy (Cryo-EM) and X-ray free electron lasers (XFEL) have significantly expanded the available toolkit to visualize large, complex noncoding RNAs and their complexes. Consequently, the quality of the RNA sample, as measured by its chemical monodispersity and conformational homogeneity, has become the bottleneck that frequently precludes effective structural analyses. Here we describe a general RNA sample preparation protocol that combines cotranscriptional RNA folding and RNA-RNA complex assembly, followed by native purification of stoichiometric complexes. We illustrate and discuss the utility of this versatile method in overcoming RNA misfolding and enabling the structural and mechanistic elucidations of the T-box riboswitch-tRNA complexes.

Probing RNA Structures and Interactions Using Fluorescence Lifetime Analyses.

Structural analyses of large, complex noncoding RNAs continue to lag behind their rapid discovery and functional descriptions. Site-specifically incorporated, minimally invasive fluorescent probes such as 2-aminopurine (2AP) and pyrrolo-cytosine (PyC) have provided essential complementary information about local RNA structure, conformational dynamics, and interactions. Here I describe a protocol that benchmarks and correlates local RNA conformations with their respective fluorescence lifetimes, as a general technique that confers key advantages over fluorescence intensity-based methods. The observation that fluorescence lifetimes are more sensitive to local structures than sequence contexts suggests broad utility across diverse RNA and ribonucleoprotein systems.

Advances in endogenous RNA pull-down: A straightforward dextran sulfate-based method enhancing RNA recovery.

Detecting RNA/RNA interactions in the context of a given cellular system is crucial to gain insights into the molecular mechanisms that stand beneath each specific RNA molecule. When it comes to non-protein coding RNA (ncRNAs), and especially to long noncoding RNAs (lncRNAs), the reliability of the RNA purification is dramatically dependent on their abundance. Exogenous methods, in which lncRNAs are in vitro transcribed and incubated with protein extracts or overexpressed by cell transfection, have been extensively used to overcome the problem of abundance. However, although useful to study the contribution of single RNA sub-modules to RNA/protein interactions, these exogenous practices might fail in revealing biologically meaningful contacts occurring in vivo and risk to generate non-physiological artifacts. Therefore, endogenous methods must be preferred, especially for the initial identification of partners specifically interacting with elected RNAs. Here, we apply an endogenous RNA pull-down to lncMN2-203, a neuron-specific lncRNA contributing to the robustness of motor neurons specification, through the interaction with miRNA-466i-5p. We show that both the yield of lncMN2-203 recovery and the specificity of its interaction with the miRNA dramatically increase in the presence of Dextran Sulfate Sodium (DSS) salt. This new set-up may represent a powerful means for improving the study of RNA-RNA interactions of biological significance, especially for those lncRNAs whose role as microRNA (miRNA) sponges or regulators of mRNA stability was demonstrated.

The lncRNA ALPHA specifically targets chikungunya virus to control infection.

Arthropod-borne viruses, including the alphavirus chikungunya virus (CHIKV), cause acute disease in millions of people and utilize potent mechanisms to antagonize and circumvent innate immune pathways including the type I interferon (IFN) pathway. In response, hosts have evolved antiviral counterdefense strategies that remain incompletely understood. Recent studies have found that long noncoding RNAs (lncRNAs) regulate classical innate immune pathways; how lncRNAs contribute to additional antiviral counterdefenses remains unclear. Using high-throughput genetic screening, we identified a cytoplasmic antiviral lncRNA that we named antiviral lncRNA prohibiting human alphaviruses (ALPHA), which is transcriptionally induced by alphaviruses and functions independently of IFN to inhibit the replication of CHIKV and its closest relative, O'nyong'nyong virus (ONNV), but not other viruses. Furthermore, we showed that ALPHA interacts with CHIKV genomic RNA and restrains viral RNA replication. Together, our findings reveal that ALPHA and potentially other lncRNAs can mediate non-canonical antiviral immune responses against specific viruses.

Impact of Diminished Expression of circRNA on Multiple Sclerosis Pathomechanisms.

Circular RNA (circRNA) molecules represent a novel and unique class of endogenous non-coding RNAs controlling the expression and function of microRNA (miRNA) and post-transcriptional regulation. Recent studies implicated circRNA in the pathomechanism of multiple sclerosis (MS). Hybridization microarray was used to define the circRNA profile in the peripheral blood mononuclear cells (PBMCs) from 20 untreated patients with relapsing-remitting MS (RRMS: 10 in relapse, 10 in remission) and 10 healthy controls (HCs). We analyzed close to 14,000 individual circRNAs per sample. The discovery set data were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with an independent cohort of 45 RRMS patients (18 in relapse, 27 in remission) and 27 HCs. Microarray analysis revealed 246 circRNAs differentially downregulated (P < 0.05) in RRMS patients versus HCs. We validated two circRNAs of the three showing the lowest levels of differential expression in the RRMS remission group versus the HC group: hsa_circRNA_101145 and hsa_circRNA_001896. Their expression was significantly decreased during remission in RRMS (P = 0.0000332, FC = 0.385 and P = 0.0455, FC = 0.591, respectively) and in patients showing a lower level of disability (hsa_circRNA_101145, P = 0.0695; hsa_circRNA_001896, P = 0.0008). Bioinformatic analysis revealed 10 miRNAs interacting with these circRNAs in a complementary manner and led to the discovery of three protein-coding mRNAs downregulated in patients with RRMS during remission. These transcripts have been previously implicated in oxidative stress, blood-brain barrier permeability, microglia function, and extracellular matrix molecules altering the microenvironment and inhibiting oligodendrocyte progenitor cells. circRNAs displayed a distinct profile in PBMCs from patients with RRMS, and our results may implicate circRNAs with low expression in important mechanistic pathways of RRMS.

Uncovering Novel Viral Innate Immune Evasion Strategies: What Has SARS-CoV-2 Taught Us?

The ongoing SARS-CoV-2 pandemic has tested the capabilities of public health and scientific community. Since the dawn of the twenty-first century, viruses have caused several outbreaks, with coronaviruses being responsible for 2: SARS-CoV in 2007 and MERS-CoV in 2013. As the border between wildlife and the urban population continue to shrink, it is highly likely that zoonotic viruses may emerge more frequently. Furthermore, it has been shown repeatedly that these viruses are able to efficiently evade the innate immune system through various strategies. The strong and abundant antiviral innate immunity evasion strategies shown by SARS-CoV-2 has laid out shortcomings in our approach to quickly identify and modulate these mechanisms. It is thus imperative that there be a systematic framework for the study of the immune evasion strategies of these viruses, to guide development of therapeutics and curtail transmission. In this review, we first provide a brief overview of general viral evasion strategies against the innate immune system. Then, we utilize SARS-CoV-2 as a case study to highlight the methods used to identify the mechanisms of innate immune evasion, and pinpoint the shortcomings in the current paradigm with its focus on overexpression and protein-protein interactions. Finally, we provide a recommendation for future work to unravel viral innate immune evasion strategies and suitable methods to aid in the study of virus-host interactions. The insights provided from this review may then be applied to other viruses with outbreak potential to remain ahead in the arms race against viral diseases.

Emerging roles of RNA-RNA interactions in transcriptional regulation.

Pervasive transcription of the human genome generates a massive amount of noncoding RNAs (ncRNAs) that lack protein-coding potential but play crucial roles in development, differentiation, and tumorigenesis. To achieve these biological functions, ncRNAs must first fold into intricate structures via intramolecular RNA-RNA interactions (RRIs) and then interact with different RNA substrates via intermolecular RRIs. RRIs are usually facilitated, stabilized, or mediated by RNA-binding proteins. With this guiding principle, several protein-based high-throughput methods have been developed for unbiased mapping of defined or all RNA-binding protein-mediated RRIs in various species and cell lines. In addition, some chemical-based approaches are also powerful to detect RRIs globally based on the fact that RNA duplex can be cross-linked by psoralen or its derivative 4'-aminomethyltrioxsalen. These efforts have significantly expanded our understanding of RRIs in determining the specificity and variability of gene regulation. Here, we review the current knowledge of the regulatory roles of RRI, focusing on their emerging roles in transcriptional regulation and nuclear body formation. This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry.

CLASH Analyst: A Web Server to Identify In Vivo RNA-RNA Interactions from CLASH Data.

Non-coding RNAs, such as miRNAs and piRNAs, play critical roles in gene regulation through base-pairing interactions with their target molecules. The recent development of the crosslinking, ligation, and sequencing of hybrids (CLASH) method has allowed scientists to map transcriptome-wide RNA-RNA interactions by identifying chimeric reads consisting of fragments from regulatory RNAs and their targets. However, analyzing CLASH data requires scientists to use advanced bioinformatics, and currently available tools are limited for users with little bioinformatic experience. In addition, many published CLASH studies do not show the full scope of RNA-RNA interactions that were captured, highlighting the importance of reanalyzing published data. Here, we present CLASH Analyst, a web server that can analyze raw CLASH data within a fully customizable and easy-to-use interface. CLASH Analyst accepts raw CLASH data as input and identifies the RNA chimeras containing the regulatory and target RNAs according to the user's interest. Detailed annotation of the captured RNA-RNA interactions is then presented for the user to visualize within the server or download for further analysis. We demonstrate that CLASH Analyst can identify miRNA- and piRNA-targeting sites reported from published CLASH data and should be applicable to analyze other RNA-RNA interactions. CLASH Analyst is freely available for academic use.

Cooperativity and Interdependency between RNA Structure and RNA-RNA Interactions.

Complex RNA-RNA interactions are increasingly known to play key roles in numerous biological processes from gene expression control to ribonucleoprotein granule formation. By contrast, the nature of these interactions and characteristics of their interfaces, especially those that involve partially or wholly structured RNAs, remain elusive. Herein, we discuss different modalities of RNA-RNA interactions with an emphasis on those that depend on secondary, tertiary, or quaternary structure. We dissect recently structurally elucidated RNA-RNA complexes including RNA triplexes, riboswitches, ribozymes, and reverse transcription complexes. These analyses highlight a reciprocal relationship that intimately links RNA structure formation with RNA-RNA interactions. The interactions not only shape and sculpt RNA structures but also are enabled and modulated by the structures they create. Understanding this two-way relationship between RNA structure and interactions provides mechanistic insights into the expanding repertoire of noncoding RNA functions, and may inform the design of novel therapeutics that target RNA structures or interactions.