RNA-RNA Interactions Research Articles

Hfq-licensed RNA–RNA interactome in Pseudomonas aeruginosa reveals a keystone sRNA

The RNA chaperone Hfq plays important regulatory roles in many bacteria by facilitating the base pairing between small RNAs (sRNAs) and their cognate mRNA targets. In the gram-negative opportunistic pathogen Pseudomonas aeruginosa, over a hundred putative sRNAs have been identified but for most, their regulatory targets remained unknown. Using RIL-seq with Hfq in P. aeruginosa, we identified the mRNA targets for dozens of previously known and unknown sRNAs. Strikingly, hundreds of the RNA-RNA interactions we discovered involved PhrS. This sRNA was thought to mediate its effects by pairing with a single target mRNA and regulating the abundance of the transcription regulator MvfR required for the synthesis of the quorum sensing signal PQS. We present evidence that PhrS controls many transcripts by pairing with them directly and employs a two-tiered mechanism for governing PQS synthesis that involves control of an additional transcription regulator called AntR. Our findings in P. aeruginosa expand the repertoire of targets for previously known sRNAs, reveal potential regulatory targets for previously unknown sRNAs, and suggest that PhrS may be a keystone sRNA with the ability to pair with an unusually large number of transcripts in this organism.

Triplex-Forming Peptide Nucleic Acid Controls Dynamic Conformations of RNA Bulges.

RNA folding is driven by the formation of double-helical segments interspaced by loops of unpaired nucleotides. Among the latter, bulges formed by one or several unpaired nucleotides are one of the most common structural motifs that play an important role in stabilizing RNA-RNA, RNA-protein, and RNA-small molecule interactions. Single-nucleotide bulges can fold in alternative structures where the unpaired nucleobase is either looped-out (flexible) in a solvent or stacked-in (intercalated) between the base pairs. In the present study, we discovered that triplex-forming peptide nucleic acids (PNAs) had unusually high affinity for single-purine-nucleotide bulges in double-helical RNA. Depending on the PNA's sequence, the triplex formation shifted the equilibrium between looped-out and stacked-in conformations. The ability to control the dynamic equilibria of RNA's structure will be an important tool for studying structure-function relationships in RNA biology and may have potential in novel therapeutic approaches targeting disease-related RNAs.

Dengue Virus Capsid Protein Facilitates Genome Compaction and Packaging.

Dengue virus (DENV) is a single-stranded (+)-sense RNA virus that infects humans and mosquitoes, posing a significant health risk in tropical and subtropical regions. Mature virions are composed of an icosahedral shell of envelope (E) and membrane (M) proteins circumscribing a lipid bilayer, which in turn contains a complex of the approximately 11 kb genomic RNA with capsid (C) proteins. Whereas the structure of the envelope is clearly defined, the structure of the packaged genome in complex with C proteins remains elusive. Here, we investigated the interactions of C proteins with viral RNA, in solution and inside mature virions, via footprinting and cross-linking experiments. We demonstrated that C protein interaction with DENV genomes saturates at an RNA:C protein ratio below 1:250. Moreover, we also showed that the length of the RNA genome interaction sites varies, in a multimodal distribution, consistent with the C protein binding to each RNA site mostly in singlets or pairs (and, in some instances, higher numbers). We showed that interaction sites are preferentially sites with low base pairing, as previously measured by 2'-acetylation analyzed by primer extension (SHAPE) reactivity indicating structuredness. We found a clear association pattern emerged: RNA-C protein binding sites are strongly associated with long-range RNA-RNA interaction sites, particularly inside virions. This, in turn, explains the need for C protein in viral genome packaging: the protein has a chief role in coordinating these key interactions, promoting proper packaging of viral RNA. Such sites are, thus, highly consequential for viral assembly, and, as such, may be targeted in future drug development strategies against these and related viruses.

E-RNA: a collection of web-servers for the prediction and visualisation of RNA secondary structure and their functional features.

e-RNA is a collection of web-servers for the prediction and visualisation of RNA secondary structures and their functional features, including in particular RNA-RNA interactions. In this updated version, we have added novel tools for RNA secondary structure prediction and have significantly updated the visualisation functionality. The new method CoBold can identify transient RNA structure features and their potential functional effects on a known RNA structure during co-transcriptional structure formation. New tool ShapeSorter can predict evolutionarily conserved RNA secondary structure features while simultaneously taking experimental SHAPE probing evidence into account. The web-server R-Chie which visualises RNA secondary structure information in terms of arc diagrams, can now be used to also visualise and intuitively compare RNA-RNA, RNA-DNA and DNA-DNA interactions alongside multiple sequence alignments and quantitative information. The prediction generated by any method in e-RNA can be readily visualised on the web-server. For completed tasks, users can download their results and readily visualise them later on with R-Chie without having to re-run the predictions. e-RNA can be found at http://www.e-rna.org.

ADAR1 Biology Can Hinder Effective Antiviral RNA Interference.

Viruses constantly evolve and adapt to the antiviral defenses of their hosts. The biology of viral circumvention of these selective pressures can often be attributed to the acquisition of novel antagonistic gene products or by rapid genome change that prevents host recognition. To study viral evasion of RNA interference (RNAi)-based defenses, we established a robust antiviral system in mammalian cells using recombinant Sendai virus designed to be targeted by endogenous host microRNAs (miRNAs) with perfect complementarity. Using this system, we previously demonstrated the intrinsic ability of positive-strand RNA viruses to escape this selective pressure via homologous recombination, which was not observed in negative-strand RNA viruses. Here, we show that given extensive time, escape of miRNA-targeted Sendai virus was enabled by host adenosine deaminase acting on RNA 1 (ADAR1). Independent of the viral transcript targeted, ADAR1 editing resulted in disruption of the miRNA-silencing motif, suggesting an intolerance for extensive RNA-RNA interactions necessary for antiviral RNAi. This was further supported in Nicotiana benthamiana, where exogenous expression of ADAR1 interfered with endogenous RNAi. Together, these results suggest that ADAR1 diminishes the effectiveness of RNAi and may explain why it is absent in species that utilize this antiviral defense system. IMPORTANCE All life at the cellular level has the capacity to induce an antiviral response. Here, we examine the result of imposing the antiviral response of one branch of life onto another and find evidence for conflict. To determine the consequences of eliciting an RNAi-like defense in mammals, we applied this pressure to a recombinant Sendai virus in cell culture. We find that ADAR1, a host gene involved in regulation of the mammalian response to virus, prevented RNAi-mediated silencing and subsequently allowed for viral replication. In addition, the expression of ADAR1 in Nicotiana benthamiana, which lacks ADARs and has an endogenous RNAi system, suppresses gene silencing. These data indicate that ADAR1 is disruptive to RNAi biology and provide insight into the evolutionary relationship between ADARs and antiviral defenses in eukaryotic life.

Investigating RNA-RNA interactions through computational and biophysical analysis.

Numerous viruses utilize essential long-range RNA-RNA genome interactions, specifically flaviviruses. Using Japanese encephalitis virus (JEV) as a model system, we computationally predicted and then biophysically validated and characterized its long-range RNA-RNA genomic interaction. Using multiple RNA computation assessment programs, we determine the primary RNA-RNA interacting site among JEV isolates and numerous related viruses. Following in vitro transcription of RNA, we provide, for the first time, characterization of an RNA-RNA interaction using size-exclusion chromatography coupled with multi-angle light scatteringand analytical ultracentrifugation. Next, we demonstrate that the 5' and 3' terminal regions of JEV interact with nM affinity using microscale thermophoresis, and this affinity is significantly reduced when the conserved cyclization sequence is not present. Furthermore, we perform computational kinetic analyses validating the cyclization sequence as the primary driver of this RNA-RNA interaction. Finally, we examined the 3D structure of the interaction using small-angle X-ray scattering, revealing a flexible yet stable interaction. This pathway can be adapted and utilized to study various viral and human long-non-coding RNA-RNA interactions and determine their binding affinities, a critical pharmacological property of designing potential therapeutics.

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

Cis-Acting Element at the 5' Noncoding Region of Tacaribe Virus S RNA Modulates Genome Replication.

Tacaribe virus (TCRV) is the prototype of New World mammarenaviruses, a group that includes several members that cause hemorrhagic fevers in humans. The TCRV genome comprises two RNA segments, named S (small) and L (large). Both genomic segments contain noncoding regions (NCRs) at their 5' and 3' ends. While the 5'- and 3'-terminal 19-nucleotide sequences are known to be essential for promoter function, the role of their neighboring internal noncoding region (iNCR) sequences remains poorly understood. To analyze the relevance of the 5' and 3' iNCRs in TCRV S RNA synthesis, mutant S-like minigenomes and miniantigenomes were generated. Using a minireplicon assay, Northern blotting, and reverse transcription-quantitative PCR, we demonstrated that the genomic 5' iNCR is specifically engaged in minigenome replication yet is not directly involved in minigenome transcription, and we showed that the S genome 3' iNCR is barely engaged in this process. Analysis of partial deletions and point mutations, as well as total or partial substitution of the 5' iNCR sequence, led us to conclude that the integrity of the whole genomic 5' iNCR is essential and that a local predicted secondary structure or RNA-RNA interactions between the 5' and 3' iNCRs are not strictly required for viral S RNA synthesis. Furthermore, we employed a TCRV reverse genetic approach to ask whether manipulation of the S genomic 5' iNCR sequence may be suitable for viral attenuation. We found that mutagenesis of the 5' promoter-proximal subregion slightly impacted recombinant TCRV virulence in vivo. IMPORTANCE The Mammarenavirus genus of the Arenaviridae family includes several members that cause severe hemorrhagic fevers associated with high morbidity and mortality rates, for which no FDA-approved vaccines and limited therapeutic resources are available. We provide evidence demonstrating the specific involvement of the TCRV S 5' noncoding sequence adjacent to the viral promoter in replication. In addition, we examined the relevance of this region in the context of an in vivo infection. Our findings provide insight into the mechanism through which this 5' viral RNA noncoding region assists the L polymerase for efficient viral S RNA synthesis. Also, these findings expand our understanding of the effect of genetic manipulation of New World mammarenavirus sequences aimed at the rational design of attenuated recombinant virus vaccine platforms.

Non-canonical interactions tune condensation and kinetically arrested material states of short RNA homopolymers.

Phase separation related processes have been frequently linked to biological compartmentalization and organization, and associated with cellular responses to stress, development, and disease. As a result, understanding biomolecular condensates and the parameters that modulate material and chemical properties has been an area of interest. Although much initial work focused on the role of proteins in phase separation, increasingly RNA-RNA interactions are being recognized for their potential to tune the condensate properties, influencing material properties, morphology, and dynamic organization. While many of these studies have focused on Watson-Crick base pairing as the primary networking interaction, previous work has also suggested a potential for non-canonical RNA-RNA interactions in long homopolymers to promote phase separation and modulate overall material properties. Here, we examine the phase separation of short RNA homopolymers in the presence of divalent cations and disordered peptides. Our studies reveal that particular short homopolymers form internally kinetically arrested droplets with limited monomer mixing despite continued fusion. We also probe the combined cooperative effects of various ions in modulating phase behavior and investigate the potential for homopolymer tracts in slightly more complex sequences to tune the material and phase separation propensities of different oligomers. Collectively, our results suggest a potential for tracts predominantly interacting through non-canonical interactions to tune the phase behavior and mesoscale properties of simplified condensates, as well as offer insight into the fundamental biophysics of RNA-based condensate systems and short homopolymer tracts in more complex biological systems.

Pseudoknots in RNA Structure Prediction.

RNA molecules play active roles in the cell and are important for numerous applications in biotechnology and medicine. The function of an RNA molecule stems from its structure. RNA structure determination is time consuming, challenging, and expensive using experimental methods. Thus, much research has been directed at RNA structure prediction through computational means. Many of these methods focus primarily on the secondary structure of the molecule, ignoring the possibility of pseudoknotted structures. However, pseudoknots are known to play functional roles in many RNA molecules or in their method of interaction with other molecules. Improving the accuracy and efficiency of computational methods that predict pseudoknots is an ongoing challenge for single RNA molecules, RNA-RNA interactions, and RNA-protein interactions. To improve the accuracy of prediction, many methods focus on specific applications while restricting the length and the class of the pseudoknotted structures they can identify. In recent years, computational methods for structure prediction have begun to catch up with the impressive developments seen in biotechnology. Here, we provide a non-comprehensive overview of available pseudoknot prediction methods and their best-use cases. © 2023 Wiley Periodicals LLC.

Uncovering the biophysical mechanisms of riboregulatory interactions involving the hepatitis C virus 3'X RNA using single-molecule FRET.

The last 98 nucleotides of the hepatitis C virus genome form what is called the 3ʹX RNA, which is a structured and highly conserved non-coding RNA element that has been implicated in the regulation of several different RNA-dependent viral processes like replication, translation, and particle assembly. Current research suggests that this riboregulatory behavior arises from the ability of 3ʹX to form multiple mutually exclusive interactions with highly conserved RNA elements across the HCV genome. Here, we use time-resolved single-molecule fluorescence spectroscopy in conjunction with analytical size exclusion HPLC to characterize the structural, energetic, and kinetic principles governing these RNA-RNA interactions. In the absence of any interaction partners, we see that 3ʹX adopts two distinct conformations. Furthermore, these two conformations have different preferences for various inter- and intragenomic RNA-RNA interactions, with the interactions themselves depending on various external factors (e.g., [Mg2+]). These findings have made it possible for us to predict which of these mutually exclusive interactions will be formed under specific conditions and ultimately how the formation and disruption of them regulates various RNA-dependent viral processes.

RNA structure controls composition and network dynamics of condensates.

Biomolecular condensates can form by phase separation and likely serve myriad functions in cells. Many studies have characterized protein condensates, and sometimes these include model RNAs that are often nonspecific and/or homopolymeric, unlike native RNAs found in biomolecular condensates. How complex RNA sequences and structures control condensation with proteins remains poorly understood. We studied the influence of mRNA structure in phase separation using the Whi3 protein and a native mRNA binding partner, CLN3. We first created an evolutionary algorithm that designs shuffled CLN3 RNA sequences which maximize or minimize predicted free energies of folding while preserving mass, known Whi3 protein binding sites, encoded amino acid sequence, and nucleotide composition. RNAs with minimized free energies of folding contain many stable duplexes, while RNAs with maximized energies have unstable structures and long single-stranded regions. Using mass photometry, we show that RNAs with stable structures are mostly monomeric, while RNAs with unstable structures can multimerize. When mixed with protein, these differential RNA-RNA interactions affect dense phase compositions. Specifically, unstable RNAs form condensates with high RNA and low protein concentrations, while the converse is true for RNAs with stable structures. Using microbead rheology, we observe that unstable RNAs form condensates with long viscoelastic relaxation times that increase with condensate age, indicating elastic properties at many timescales. By contrast, stably-structured RNAs form less elastic condensates with shorter relaxation times that increase to a lesser degree with time. We hypothesize that such aging is controlled by the exchange of intra- for inter-molecular base pairing amongst RNAs whose folding free energies determine the timescale of these exchanges. Thus, RNA structure can have multi-scale consequences for biomolecular condensates, with impacts on condensate composition and material properties. This work underscores how specific RNA sequences can encode physical properties of condensates.

The structure and dynamics of a fully packaged RNA virus.

The vast majority of known viruses that infect humans have their genetic program written in molecules of RNA. Such RNA viruses are thought to attain their final, mature form via self-assembly of individual capsid proteins directed by specific interactions with the RNA genome. Despite tremendous amount of experimental information about the average structure of the protein capsids, much less is known about the structure and dynamics of the RNA genomes. Here, we characterized the structure, dynamics and ion atmosphere in a bacteriophage MS2 virus using all-atom molecular dynamics simulations. Starting from available experimental information, we built a computational model of a mature MS2 virion containing a 3569 nucleotide RNA genome enclosed inside a mature protein capsid that in turn consists of a single maturation protein embedded within an icosahedral lattice of the protein capsid. Our two 2.5-microsecond replica simulations of the complete virion differing by the initial configurations of the RNA genome revealed the stabilizing effect of the RNA—protein interactions on the structure of the mature capsid and on the conformation of the RNA genome. The virus particle as a whole was observed to undergo a breathing-like motion, whereas parts of the RNA genome not bound specifically to the protein capsid explored a range of structural transformations while retaining their secondary structure. A set of simulations performed upon removal of the RNA genome characterized the effect of the RNA on the internal pressure in the virus and on its ion atmosphere. Taken together, our work introduces a mature viral particle as a system of high dynamic complexity where repulsive RNA-RNA interactions compete with specific RNA-protein binding, with the ion atmosphere serving as intermolecular glue holding the assembly together.

Restricting intermolecular interactions among complementary sequences is required for the specificity of homotypic mRNA assembly.

We reported that the Drosophila mRNAs often organize into homotypic assemblies that are composed of mRNAs with the same primary sequences. While the formation of these assemblies involves intermolecular interactions among proteins and mRNAs, the role of these interactions in generating the specificity of homotypic mRNA assembly is unknown. Here, we demonstrate that intermolecular RNA-RNA interactions are sufficient to sort a mixture of distinct Drosophila RNAs into homotypic assemblies in vitro in the absence of proteins. To determine whether intermolecular interactions among complementary sequences generate the specificity of homotypic assembly, we computationally identified these sequences transcriptome-wide. Strikingly, we observed that the complementary sequences are common and frequently shared among distinct mRNAs with different primary sequences. These results predict that the formation of heterotypic assemblies, which are composed of distinct mRNAs, should be favored, contrary to our in vivo measurements. To resolve this contradiction, we employed DMS-MaPseq to determine the structuredness of mRNAs within assemblies in vivo. We observed that most of the complementary sequences are structurally embedded and thus inaccessible to intermolecular interactions. We also demonstrate that higher RNA concentration in assemblies does not extensively unwind the RNA structure in vitro. Finally, we show that in vitro a single, structurally exposed complementary sequence is sufficient to disrupt the specificity of homotypic assembly, promoting instead heterotypic assembly. Our data demonstrate that restricting the intermolecular interactions among complementary sequences is critical for the specificity of mRNA homotypic assembly. Thus, we propose a model where a combination of intermolecular base stackings, electrostatic forces, and single nucleotide base pairings modulated by RNA structure, generate the specificity of homotypic assembly. This work is supported by the R35GM142737 NIGMS grant awarded to TT.

Viral-host RNA-RNA interactions in SARS-CoV-2: Study of miR-34a-5p binding interactions within the genome 3'-untranslated region.

SARS-CoV-2, the pathogen responsible for the COVID-19 pandemic, has contributed to 615 million cases and over 6.5 million deaths worldwide. COVID-19 has a higher death rate (1.1%) than other respiratory viruses, and while the implementation of vaccines has shown to be effective in mitigating disease spread, a lack of therapeutic options still exists. Further, the mechanisms behind COVID-19 disease severity are not well understood. Sequence analysis of SARS-CoV-2 has revealed that host miRNA miR-34a-5p can interact with the genome 3’-UTR, providing a potential mechanism of viral hijacking. MiR-34a-5p has been shown to indirectly regulate interleukin-6 (IL-6), a key cytokine in cytokine release syndrome (CRS). Viral hijacking of miR-34a-5p could contribute to CRS through IL-6 dysregulation and increase severity of COVID-19 cases. This study demonstrates that the viral genome 3’-UTR binds miR-34a-5p, and that these interactions are mediated by viral genome and miRNA secondary structures. Further, this study aims to elucidate disruption of the proposed interactions through intervention with competitive, complementary nucleic acid analogs. Native gel electrophoresis, 1H NMR spectroscopy, and fluorescence spectroscopy were used to characterize binding interactions of miR-34a-5p to the 3’-UTR at its predicted binding site, and in the context of the entire genome 3’-UTR. The molecular mechanisms detailed by this study have the potential to drive the development of antiviral treatments for severe SARS-CoV-2 infections with CRS and may provide a systematic approach to be replicated for the study of other RNA viruses with binding affinity for host cellular miRNAs.

Cancer-associated fibroblast-secreted IGFBP7 promotes gastric cancer by enhancing tumor associated macrophage infiltration via FGF2/FGFR1/PI3K/AKT axis

We previously reported that IGFBP7 plays a role in maintaining mRNA stability of oncogenic lncRNA UBE2CP3 by RNA-RNA interaction in gastric cancer (GC). Clinical cohort studies had implied an oncogenic role of IGFBP7 in GC. However, the molecular mechanism of IGFBP7 in GC progression remains unknown. In this study, clinical analysis based on two independent cohorts showed that IGFBP7 was positively associated with poor prognosis and macrophage infiltration in GC. Loss-of-function studies confirmed the oncogenic properties of IGFBP7 in regulating GC cell proliferation and invasion. Mechanismly, IGFBP7 was highly expressed in cancer-associated fibroblasts (CAF) and mesenchymal cells, and was induced by epithelial-to-mesenchymal transition (EMT) signaling, since its expression was increased by TGF-beta treatment and reduced by overexpression of OVOL2 in GC. RNA sequencing, qRT-PCR, ELISA assay showed that IGFBP7 positively regulated FGF2 expression and secretion in GC. Transcriptome analysis revealed that FGFR1 was downregulated in M1 polarization but upregulated in M2 polarization. Exogenous recombinant IGFBP7 treatment in macrophages and GC cells further identified that IGFBP7 promotes tumor associated macrophage (TAM) polarization via FGF2/FGFR1/PI3K/AKT axis. Our finding here represented the first evidence that IGFBP7 promotes GC by enhancing TAM/M2 macrophage polarization through FGF2/FGFR1/PI3K/AKT axis.

High-throughput biochemistry in RNA sequence space: predicting structure and function.

RNAs are central to fundamental biological processes in all known organisms. The set of possible intramolecular interactions of RNA nucleotides defines the range of alternative structural conformations of a specific RNA that can coexist, and these structures enable functional catalytic properties of RNAs and/or their productive intermolecular interactions with other RNAs or proteins. However, the immense combinatorial space of potential RNA sequences has precluded predictive mapping between RNA sequence and molecular structure and function. Recent advances in high-throughput approaches in vitro have enabled quantitative thermodynamic and kinetic measurements of RNA-RNA and RNA-protein interactions, across hundreds of thousands of sequence variations. In this Review, we explore these techniques, how they can be used to understand RNA function and how they might form the foundations of an accurate model to predict the structure and function of an RNA directly from its nucleotide sequence. The experimental techniques and modelling frameworks discussed here are also highly relevant for the sampling of sequence-structure-function space of DNAs and proteins.

Profiling the in vivo RNA interactome associated with the endoribonuclease RNase III in Staphylococcus aureus.

Regulatory small RNA (sRNA) have been extensively studied in model Gram-negative bacteria, but the functional characterisation of these post-transcriptional gene regulators in Gram-positives remains a major challenge. Our previous work in enterohaemorrhagic E. coli utilised the proximity-dependant ligation technique termed CLASH (UV-crosslinking, ligation, and sequencing of hybrids) for direct high-throughput sequencing of the regulatory sRNA-RNA interactions within the cell. Recently, we adapted the CLASH technique and demonstrated that UV-crosslinking and RNA proximity-dependant ligation can be applied to Staphylococcus aureus, which uncovered the first RNA-RNA interaction network in a Gram-positive bacterium. In this chapter, we describe modifications to the CLASH technique that were developed to capture the RNA interactome associated with the double-stranded endoribonuclease RNase III in two clinical isolates of S. aureus. To briefly summarise our CLASH methodology, regulatory RNA-RNA interactions were first UV-crosslinked in vivo to the RNase III protein and protein-RNA complexes were affinity-purified using the His6-TEV-FLAG tags. Linkers were ligated to RNase III-bound RNA during library preparation and duplexed RNA-RNA species were ligated together to form a single contiguous RNA 'hybrid'. The RNase III-RNA binding sites and RNA-RNA interactions occurring on RNase III (RNA hybrids) were then identified by paired-end sequencing technology. RNase III-CLASH represents a step towards a systems-level understanding of regulatory RNA in Gram-positive bacteria.

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.

Into the Microscale: Low-input sequencing technologies and applications in medicine

<p>Sequencing technology has undergone major breakthroughs over time and has become indispensable in biological and medical research. Advances in protocols and analysis algorithms allow the analysis of small sample inputs, enabling the characterization of complex networks that govern biological processes in physiology and pathology, driving the development of precision medicine and targeted therapy. In this review, we comprehensively summarize low-input sequencing technologies that include methods for profiling the genome, epigenome, transcriptome, translatome, RNA modification, RNA-RNA interaction, RNA-protein interaction, and multi-omics. The key steps and innovations in different protocols are highlighted, and their advantages, limitations, and scope of application are described. With a focus on the impact of low-input sequencing technologies in biomedical fields, including embryonic development, prenatal and preimplantation genetic testing, oncology and immunology, and pandemic research, we discuss their potential to improve embryo implantation rates, prevent birth defects, develop prospective therapies, and predict prognosis. Lastly, we discuss current limitations and future prospects, providing new insights for medical research.</p>