Base-pairing Interactions Research Articles

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.

Development and Scale-Up of Oligo-Dt Monolithic Chromatographic Column for Mrna Capture Through Understanding of Base-Pairing Interactions

Development and Scale-Up of Oligo-Dt Monolithic Chromatographic Column for Mrna Capture Through Understanding of Base-Pairing Interactions

Guide RNA acrobatics: positioning consecutive uridines for pseudouridylation by H/ACA pseudouridylation loops with dual guide capacity.

Site-specific pseudouridylation of human ribosomal and spliceosomal RNAs is directed by H/ACA guide RNAs composed of two hairpins carrying internal pseudouridylation guide loops. The distal "antisense" sequences of the pseudouridylation loop base-pair with the target RNA to position two unpaired target nucleotides 5'-UN-3', including the 5' substrate U, under the base of the distal stem topping the guide loop. Therefore, each pseudouridylation loop is expected to direct synthesis of a single pseudouridine (Ψ) in the target sequence. However, in this study, genetic depletion and restoration and RNA mutational analyses demonstrate that at least four human H/ACA RNAs (SNORA53, SNORA57, SCARNA8, and SCARNA1) carry pseudouridylation loops supporting efficient and specific synthesis of two consecutive pseudouridines (ΨΨ or ΨNΨ) in the 28S (Ψ3747/Ψ3749), 18S (Ψ1045/Ψ1046), and U2 (Ψ43/Ψ44 and Ψ89/Ψ91) RNAs, respectively. In order to position two substrate Us for pseudouridylation, the dual guide loops form alternative base-pairing interactions with their target RNAs. This remarkable structural flexibility of dual pseudouridylation loops provides an unexpected versatility for RNA-directed pseudouridylation without compromising its efficiency and accuracy. Besides supporting synthesis of at least 6% of human ribosomal and spliceosomal Ψs, evidence indicates that dual pseudouridylation loops also participate in pseudouridylation of yeast and archaeal rRNAs.

RNAglib: a python package for RNA 2.5 D graphs.

RNA 3D architectures are stabilized by sophisticated networks of (non-canonical) base pair interactions, which can be conveniently encoded as multi-relational graphs and efficiently exploited by graph theoretical approaches and recent progresses in machine learning techniques. RNAglib is a library that eases the use of this representation, by providing clean data, methods to load it in machine learning pipelines and graph-based deep learning models suited for this representation. RNAglib also offers other utilities to model RNA with 2.5 D graphs, such as drawing tools, comparison functions or baseline performances on RNA applications. The method is distributed as a pip package, RNAglib. Data are available in a repository and can be accessed on rnaglib's web page. The source code, data and documentation are available at https://rnaglib.cs.mcgill.ca. Supplementary data are available at Bioinformatics online.

How can the cisplatin analogs with different amine act on DNA during cancer treatment theoretically?

Cisplatin is a widely used anti-cancer drug which inhibits the replication and polymerization of DNA molecule while showing some side effects and drug resistance. For this reason, to enhance its therapeutic index, researchers have synthesized several thousand analogs and tested their properties. In this project, several cisplatin analogs were designed to theoretically study the biological activity and lipophilicity effects on amine changes. The amines of the cisplatin molecule were substituted with aliphatic amines in different analogs. Computational methods such as molecular dynamics simulation, molecular docking, and molecular mechanics Poisson-Boltzmann surface area analysis were performed to investigate the binding of six cisplatin derivatives with DNA. The binding affinity and potential interactions of these drugs with double-strand DNA were analyzed. The stability effect of these drugs was investigated via root-mean-square deviation and root-mean-square fluctuation analysis, which showed that some analogs can break base-pair interaction at the end of DNA and reduced the stability of DNA. Also, the results revealed that the hydrogen bond is one of the most important factors in the binding of cisplatin's adduct to DNA. Molecular mechanics Poisson-Boltzmann surface area analysis indicated that electrostatic and van der Waals interactions are the most important deriving forces to the binding of cisplatin's drug to DNA. Finally, data revealed that cisplatin and the cis-dichloro-dimethylamine-platin tendency for binding to DNA are greater than that of other analogs.

The "guiding" principles of noncoding RNA function.

The human genome is pervasively transcribed and yet only a small fraction of these RNAs (less than 2%) are known to code for proteins. The vast majority of the RNAs are classified as noncoding RNAs (ncRNAs) and are further subgrouped as small (shorter than 200 bases) and long noncoding RNAs. The ncRNAs have been identified in all three domains of life and regulate diverse cellular processes through transcriptional and posttranscriptional gene regulation. Most of these RNAs work in conjunction with proteins forming a wide array of base pairing interactions. The determinants of these base pairing interactions are now becoming more evident and show striking similarities among the diverse group of ncRNAs. Here we present a mechanistic overview of pairing between RNA-RNA or RNA-DNA that dictates the function of ncRNAs; we provide examples to illustrate that ncRNAs work through shared evolutionary mechanisms that encompasses a guide-target interaction, involving not only classical Watson-Crick but also noncanonical Wobble and Hoogsteen base pairing. We also highlight the similarities in target selection, proofreading, and the ruler mechanism of ncRNA-protein complexes that confers target specificity and target site selection. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA-Based Catalysis > RNA-Mediated Cleavage RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.

A new RNA-DNA interaction required for integration of group II intron retrotransposons into DNA targets.

Mobile group II introns are site-specific retrotransposable elements abundant in bacterial and organellar genomes. They are composed of a large and highly structured ribozyme and an intron-encoded reverse transcriptase that binds tightly to its intron to yield a ribonucleoprotein (RNP) particle. During the first stage of the mobility pathway, the intron RNA catalyses its own insertion directly into the DNA target site. Recognition of the proper target rests primarily on multiple base-pairing interactions between the intron RNA and the target DNA, while the protein makes contacts with only a few target positions by yet-unidentified mechanisms. Using a combination of comparative sequence analyses and in vivo mobility assays we demonstrate the existence of a new base-pairing interaction named EBS2a–IBS2a between the intron RNA and its DNA target site. This pairing adopts a Watson–Crick geometry and is essential for intron mobility, most probably by driving unwinding of the DNA duplex. Importantly, formation of EBS2a–IBS2a also requires the reverse transcriptase enzyme which stabilizes the pairing in a non-sequence-specific manner. In addition to bringing to light a new structural device that allows subgroup IIB1 and IIB2 introns to invade their targets with high efficiency and specificity our work has important implications for the biotechnological applications of group II introns in bacterial gene targeting.

Vernal: a tool for mining fuzzy network motifs in RNA.

RNA 3D motifs are recurrent substructures, modeled as networks of base pair interactions, which are crucial for understanding structure-function relationships. The task of automatically identifying such motifs is computationally hard, and remains a key challenge in the field of RNA structural biology and network analysis. State-of-the-art methods solve special cases of the motif problem by constraining the structural variability in occurrences of a motif, and narrowing the substructure search space. Here, we relax these constraints by posing the motif finding problem as a graph representation learning and clustering task. This framing takes advantage of the continuous nature of graph representations to model the flexibility and variability of RNA motifs in an efficient manner. We propose a set of node similarity functions, clustering methods and motif construction algorithms to recover flexible RNA motifs. Our tool, Vernal can be easily customized by users to desired levels of motif flexibility, abundance and size. We show that Vernal is able to retrieve and expand known classes of motifs, as well as to propose novel motifs. The source code, data and a webserver are available at vernal.cs.mcgill.ca. We also provide a flexible interface and a user-friendly webserver to browse and download our results. Supplementary data are available at Bioinformatics online.

Reconfigurable Two-Dimensional DNA Lattices: Static and Dynamic Angle Control.

Branched DNA motifs serve as the basic construction elements for all synthetic DNA nanostructures. However, precise control of branching orientation remains a key challenge to further heighten the overall structural order. In this study, we use two strategies to control the branching orientation. The first one is based on immobile Holliday junctions which employ specific nucleotide sequences at the branch points which dictate their orientation. The second strategy is to use angle-enforcing struts to fix the branching orientation with flexible spacers at the branch points. We have also demonstrated that the branching orientation control can be achieved dynamically, either by canonical Watson-Crick base pairing or non-canonical nucleobase interactions (e.g., i-motif and G-quadruplex). With precise angle control and feedback from the chemical environment, these results will enable novel DNA nanomechanical sensing devices, and precisely-ordered three-dimensional architectures.

RNA multimerization as an organizing force for liquid-liquid phase separation.

RNA interactions are exceptionally strong and highly redundant. As such, nearly any two RNAs have the potential to interact with one another over relatively short stretches, especially at high RNA concentrations. This is especially true for pairs of RNAs that do not form strong self-structure. Such phenomena can drive liquid–liquid phase separation, either solely from RNA–RNA interactions in the presence of divalent or organic cations, or in concert with proteins. RNA interactions can drive multimerization of RNA strands via both base-pairing and tertiary interactions. In this article, we explore the tendency of RNA to form stable monomers, dimers, and higher order structures as a function of RNA length and sequence through a focus on the intrinsic thermodynamic, kinetic, and structural properties of RNA. The principles we discuss are independent of any specific type of biomolecular condensate, and thus widely applicable. We also speculate how external conditions experienced by living organisms can influence the formation of nonmembranous compartments, again focusing on the physical and structural properties of RNA. Plants, in particular, are subject to diverse abiotic stresses including extreme temperatures, drought, and salinity. These stresses and the cellular responses to them, including changes in the concentrations of small molecules such as polyamines, salts, and compatible solutes, have the potential to regulate condensate formation by melting or strengthening base-pairing. Reversible condensate formation, perhaps including regulation by circadian rhythms, could impact biological processes in plants, and other organisms.

Modeling Noncanonical RNA Base Pairs by a Coarse-Grained IsRNA2 Model.

Noncanonical base pairs contribute crucially to the three-dimensional architecture of large RNA molecules; however, how to accurately model them remains an open challenge in RNA 3D structure prediction. Here, we report a promising coarse-grained (CG) IsRNA2 model to predict noncanonical base pairs in large RNAs through molecular dynamics simulations. By introducing a five-bead per nucleotide CG representation to reserve the three interacting edges of nucleobases, IsRNA2 accurately models various base-pairing interactions, including both canonical and noncanonical base pairs. A benchmark test indicated that IsRNA2 achieves a comparable performance to the atomic model in de novo modeling of noncanonical RNA structures. In addition, IsRNA2 was able to refine the 3D structure predictions for large RNAs in RNA-puzzle challenges. Finally, the graphics processing unit acceleration was introduced to speed up the sampling efficiency in IsRNA2 for very large RNA molecules. Therefore, the CG IsRNA2 model reported here offers a reliable approach to predict the structures and dynamics of large RNAs.

SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (COVID-19), a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry (CLMS) identified a region that drives interactions between N proteins in condensates, and deletion of this region disrupts phase separation. We also identified small molecules that alter the size and shape of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.

A New Perspective on the Maillard Reaction and the Origin of Life

The Maillard reaction, a spontaneous 'one pot' reaction between amino acids and reducing sugars that occurs at low reactant concentrations and low temperatures, is a good candidate for having played a role in the origin of life on the Earth. In view of the probability that RNA and DNA were preceded by an evolutionary forerunner with a more straightforward prebiotic synthesis, it is a testament to the prescience of Oró and colleagues that, in 1975, they drew attention to the Maillard reaction, in particular evidence that melanoidin polymers (the end-product of the reaction) contain '…heterocyclic nitrogen compounds similar to the nitrogenous bases' (Nissenbaum in J Mol Evol 6:253-270, 1975). Indeed, reports of the Maillard reaction product, 2-Acetyl-6-(Hydroxymethyl)-5,6-Dihydro-4H-Pyridinone (AHDP), with a structure reminiscent of the pyrimidine nucleobase uracil, suggest the Maillard reaction might have played a key role in the synthesis of components of a proto-RNA polymer, with AHDP and two structurally related products predicted to be similar to uracil in the latter's ability to form non-standard base pair interactions. It is possible that the primary function of these interactions was to allow molecules such as AHDP to separate out of the prebiotic chemical clutter. If this were the case, catalysis, and coding-made possible by the polymerization of proto-nucleoside monomers into linear sequence strings-would have been evolving properties.

The RNA chaperone StpA enables fast RNA refolding by destabilization of mutually exclusive base pairs within competing secondary structure elements.

In bacteria RNA gene regulatory elements refold dependent on environmental clues between two or more long-lived conformational states each associated with a distinct regulatory state. The refolding kinetics are strongly temperature-dependent and especially at lower temperatures they reach timescales that are biologically not accessible. To overcome this problem, RNA chaperones have evolved. However, the precise molecular mechanism of how these proteins accelerate RNA refolding reactions remains enigmatic. Here we show how the RNA chaperone StpA of Escherichia coli leads to an acceleration of a bistable RNA’s refolding kinetics through the selective destabilization of key base pairing interactions. We find in laser assisted real-time NMR experiments on photocaged bistable RNAs that the RNA chaperone leads to a two-fold increase in refolding rates at low temperatures due to reduced stability of ground state conformations. Further, we can show that upon interaction with StpA, base pairing interactions in the bistable RNA are modulated to favor refolding through the dominant pseudoknotted transition pathway. Our results shed light on the molecular mechanism of the interaction between RNA chaperones and bistable RNAs and are the first step into a functional classification of chaperones dependent on their biophysical mode of operation.

Identification and initial characterization of a new pair of sibling sRNAs of Neisseria gonorrhoeae involved in type IV pilus biogenesis

Non-coding regulatory RNAs mediate post-transcriptional gene expression control by a variety of mechanisms relying mostly on base-pairing interactions with a target mRNA. Though a plethora of putative non-coding regulatory RNAs have been identified by global transcriptome analysis, knowledge about riboregulation in the pathogenic Neisseriae is still limited. Here we report the initial characterization of a pair of sRNAs of N. gonorrhoeae, TfpR1 and TfpR2, which exhibit a similar secondary structure and identical single-stranded seed regions, and therefore might be considered as sibling sRNAs. By combination of in silico target prediction and sRNA pulse expression followed by differential RNA sequencing we identified target genes of TfpR1 which are involved in type IV pilus biogenesis and DNA damage repair. We provide evidence that members of the TfpR1 regulon can also be targeted by the sibling TfpR2.

Kinetic modeling reveals additional regulation at co-transcriptional level by post-transcriptional sRNA regulators.

SUMMARYSmall RNAs (sRNAs) are important gene regulators in bacteria. Many sRNAs act post-transcriptionally by affecting translation and degradation of the target mRNAs upon base-pairing interactions. Here we present a general approach combining imaging and mathematical modeling to determine kinetic parameters at different levels of sRNA-mediated gene regulation that contribute to overall regulation efficacy. Our data reveal that certain sRNAs previously characterized as post-transcriptional regulators can regulate some targets co-transcriptionally, leading to a revised model that sRNA-mediated regulation can occur early in an mRNA’s lifetime, as soon as the sRNA binding site is transcribed. This co-transcriptional regulation is likely mediated by Rho-dependent termination when transcription-coupled translation is reduced upon sRNA binding. Our data also reveal several important kinetic steps that contribute to the differential regulation of mRNA targets by an sRNA. Particularly, binding of sRNA to the target mRNA may dictate the regulation hierarchy observed within an sRNA regulon.

How stop codon pseudouridylation induces nonsense suppression

Nonsense mutation can be an inherited disorder or caused by genetic mutation under certain conditions such as stress or exposure. Nonsense suppression, can have a broad potential as a therapeutic approach to nonsense mutation. Pseudouridine is proposed to result in nonsense suppression if induced in one of the three nonsense codons, Amber, Ochre, or Opal. Understanding how pseudouridine in stop codon induces nonsense suppression is vital for preventing nonsense mutation. Using in-silico analysis with DFT, we have simulated different near and non-cognate codon-anticodon interactions with pseudouridylated and normal nonsense codons. The structural analysis show that pseudouridylation of the nonsense codons in the first position leads to the mRNA-tRNA helix major groove conformation and formation of unusual Hoogsteen base pair in the purine bases at the second or third position. This leads to stabilized base-pair interaction and π…π stacking for the pseudouridylated nonsense codon compared its normal nonsense codon counterpart.

Long non-coding RNA LINC01224 promotes progression and cisplatin resistance in non-small lung cancer by sponging miR-2467

Copious evidence reveals that long non-coding RNAs (lncRNAs) exert great regulatory functions in various human cancers. LINC01224 is a novel lncRNA, identified as a cancer regulator of HCC. However, the underlying mechanisms and clinical significance of LINC01224 in other types of cancers need further researches to explore. In this study, we aimed to elucidate the biological role of LINC01224 in NSCLC progression. Presently, LINNC01224 expression was elevated and miR-2467 expression was down-regulated in NSCLC, compared with standard control. Then we described the reciprocal correlation between LINC01224 and miR 2467. Afterward, the dual-luciferase reporter assay, RIP assay and RNA pull-down assay validated the base-pair interaction between LINC01224 and miR-2467. Moreover, our findings demonstrated that the silence of LINC01224 inhibited cell proliferation and invasion in NSCLC and enhanced cisplatin (CDDP) sensitivity in vitro. Besides, rescue assays verified that miR-2467 inhibitor could reverse the effects on cell biological activities and CDDP resistance caused by knockdown of LINC01224. Finally, in vivo experiments implicated that knockdown of LINC01224 could inhibit NSCLC tumor growth. To sum up, LINC01224 can promote tumor progression and CDDP resistance in NSCLC via sponging miR-2467, suggesting a promising therapeutic target for better diagnosis and prognosis of NSCLC patients.

Role of pH in the stability of cytosine-cytosine mismatch and canonical AT and GC base pairs mediated with silver ion: a DFT study

Metallo-nucleic acids have been investigated for their applications in the field of nanodevices and genetic expansion. The cytosine-Ag+-cytosine mismatch base pair interactions and their stability in nucleic acids have attracted the attention of chemists. We report a systematic study of canonical, mismatch, Ag+ mediated system with CC, AT, and GC base pairs computationally. The stability of such mismatch base pairs is dependent on the pH range ~ 5 to 9 and the duplexes beyond this range are unstable. The DFT calculations performed with the model DNA duplexes comprising of such mismatch pairs reveal the stability trend while varying the pH conditions. The stability of canonical Watson–Crick ATGC base pairs was compared with the CCAT and CCGC mismatch base pairs and the calculated results at B3LYP-D3/6-31G(d) level of theory in the aqueous phase suggest that the base stacking and hydrogen bonding are well maintained in the former case, however, the larger perturbations in the geometry are observed with the mispair and relatively unstable. The calculated binding energy B3LYP-D3/6-31G(d) level of theory of ATGC is energetically more stable (~ 20 kcal/mol) than the mismatch base pairs. The Ag+ mediated mismatch base pairs i.e., C_CAT and C_CGC examined at the same level of theory suggest that the CC mismatch base pairs complexed with proper alignment to the Ag+ ion and the AT and GC bases maintained the hydrogen bonding interactions. The relative binding energies have been calculated with 6–311 + G(d,p) basis set using B3LYP-D3 and wb97xd DFT functionals in the study. The mismatched base pair duplex systems i.e., C_CAT and C_CGC are structurally similar to the canonical Watson–Crick base pairs and energetically stable by ~ 40 and ~ 50 kcal/mol compared to the canonical ATGC base pairs. The experimental report on the thermal transition profile in 5’-(A)10C(A)10–3’ and 5’ (T)10C(T)10–3’ duplexes showed remarkable stability and corroborate the calculated results (Ono et al. in Chem Commun 4825−4827, 2008). The stability of Ag+ mediated mismatch bases at the higher pH 9 was also examined and the nucleobases such as guanine and thymine would be deprotonated under this condition. The calculated results suggest that the CCA_T and CCG_C duplexes are largely distorted with the complexation of Ag+ with the AT and GC base pairs and would in turn denature the duplex. The AIM analysis performed at B3LYP-D3/6-31G(d) level of theory for all the studied Ag+ mediated complexes reveals that the Ag+ interaction with the corresponding nucleobases was electrostatic in nature. The role of pH in governing the stability of C–Ag+-C complex formation in mismatch base nucleic acids is crucial for their application of genetic expansion and nucleic acid-based nanodevices.Graphical abstract

Diversity and Versatility in Small RNA-Mediated Regulation in Bacterial Pathogens.

Bacterial gene expression is under the control of a large set of molecules acting at multiple levels. In addition to the transcription factors (TFs) already known to be involved in global regulation of gene expression, small regulatory RNAs (sRNAs) are emerging as major players in gene regulatory networks, where they allow environmental adaptation and fitness. Developments in high-throughput screening have enabled their detection in the entire bacterial kingdom. These sRNAs influence a plethora of biological processes, including but not limited to outer membrane synthesis, metabolism, TF regulation, transcription termination, virulence, and antibiotic resistance and persistence. Almost always noncoding, they regulate target genes at the post-transcriptional level, usually through base-pair interactions with mRNAs, alone or with the help of dedicated chaperones. There is growing evidence that sRNA-mediated mechanisms of actions are far more diverse than initially thought, and that they go beyond the so-called cis- and trans-encoded classifications. These molecules can be derived and processed from 5' untranslated regions (UTRs), coding or non-coding sequences, and even from 3' UTRs. They usually act within the bacterial cytoplasm, but recent studies showed sRNAs in extracellular vesicles, where they influence host cell interactions. In this review, we highlight the various functions of sRNAs in bacterial pathogens, and focus on the increasing examples of widely diverse regulatory mechanisms that might compel us to reconsider what constitute the sRNA.