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Over the past five decades, DNA restriction enzymes have revolutionized biotechnology. While these enzymes are widely used in DNA research and DNA engineering, the emerging field of RNA and mRNA therapeutics requires sequence-specific RNA endoribonucleases. Here, we describe EcoToxN1, a member of the type III toxin-antitoxin family of sequence-specific RNA endoribonucleases, and its use in RNA and mRNA analysis. This enzyme recognizes a specific pentamer in a single-stranded RNA and cleaves the RNA within this sequence. The enzyme is neither dependent on annealing of guide RNA or DNA oligos to the template nor does it require magnesium. Furthermore, it performs over a wide range of temperatures. With its unique functions and characteristics, EcoToxN1 can be classified as an RNA restriction enzyme. EcoToxN1 enables new workflows in RNA analysis and biomanufacturing, meeting the demand for faster, cheaper, and more robust analysis methods., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Conserved signaling cascades monitor protein-folding homeostasis to ensure proper cellular function. One of the evolutionary conserved key players is IRE1, which maintains endoplasmic reticulum (ER) homeostasis through the unfolded protein response (UPR). Upon accumulation of misfolded proteins in the ER, IRE1 forms clusters on the ER membrane to initiate UPR signaling. What regulates IRE1 cluster formation is not fully understood. Here, we show that the ER lumenal domain (LD) of human IRE1α forms biomolecular condensates in vitro. IRE1α LD condensates were stabilized both by binding to unfolded polypeptides as well as by tethering to model membranes, suggesting their role in assembling IRE1α into signaling-competent stable clusters. Molecular dynamics simulations indicated that weak multivalent interactions drive IRE1α LD clustering. Mutagenesis experiments identified disordered regions in IRE1α LD to control its clustering in vitro and in cells. Importantly, dysregulated clustering of IRE1α mutants led to defects in IRE1α signaling. Our results revealed that disordered regions in IRE1α LD control its clustering and suggest their role as a common strategy in regulating protein assembly on membranes., (© 2024. The Author(s).)

Researchers worldwide are looking for molecules that might disrupt the COVID-19 life cycle. Endoribonuclease, which is responsible for processing viral RNA to avoid detection by the host defense system, and helicase, which is responsible for unwinding the RNA helices for replication, are two key non-structural proteins. This study performs a hierarchical structure-based virtual screening approach for NSP15 and helicase to reach compounds with high binding probabilities. In this investigation, we incorporated a variety of filtering strategies for predicting compound interactions. First, we evaluated 756,275 chemicals from four databases using a deep learning method (NCI, Drug Bank, Maybridge, and COCONUT). Following that, two docking techniques (extra precision and induced fit) were utilized to evaluate the compounds' binding affinity, followed by molecular dynamic simulation supported by the MM-GBSA free binding energy calculation. Remarkably, two compounds (90616 and CNP0111740) exhibited high binding affinity values of -66.03 and -12.34 kcal/mol for helicase and NSP15, respectively. The VERO-E6 cell line was employed to test their in vitro therapeutic impact. The CC 50 for CNP0111740 and 90616 were determined to be 102.767 μg/ml and 379.526 μg/ml, while the IC 50 values were 140.176 μg/ml and 5.147 μg/ml, respectively. As a result, the selectivity index for CNP0111740 and 90616 is 0.73 and 73.73, respectively. Finally, these compounds were found to be novel, effective inhibitors for the virus; however, further in vivo validation is needed.Communicated by Ramaswamy H. Sarma.

Tuberculosis is a worldwide plague caused by the pathogen Mycobacterium tuberculosis ( M. tb ). Toxin-antitoxin (TA) systems are genetic elements abundantly present in prokaryotic organisms and regulate important cellular processes. MazEF is a TA system implicated in the formation of "persisters cells" of M. tb , which contain more than 10 such members. However, the exact function and inhibition mode of each MazF are not fully understood. Here we report crystal structures of MazF-mt3 in its apo form and in complex with the C-terminal half of MazE-mt3. Structural analysis suggested that two long but disordered β1-β2 loops would interfere with the binding of the cognate MazE-mt3 antitoxin. Similar loops are also present in the MazF-mt1 and -mt9 but are sustainably shortened in other M. tb MazF members, and these TA pairs behave distinctly in terms of their binding modes and their RNase activities. Systematic crystallographic and biochemical studies further revealed that the biochemical activities of M. tb toxins were combined results between the interferences from the characteristic loops and the electrostatic interactions between the cognate TA pairs. This study provides structural insight into the binding mode and the inhibition mechanism of the MazE/F TA pairs, which facilitate the structure-based peptide designs.

Various approaches have been developed to target RNA and modulate its function with modes of action including binding and cleavage. Herein, we explored how small molecule binding is correlated with cleavage induced by heterobifunctional ribonuclease targeting chimeras (RiboTACs), where RNase L is recruited to cleave the bound RNA target, in a transcriptome-wide, unbiased fashion. Only a fraction of bound targets was cleaved by RNase L, induced by RiboTAC binding. Global analysis suggested that (i) cleaved targets generally form a region of stable structure that encompasses the small molecule binding site; (ii) cleaved targets have preferred RNase L cleavage sites nearby small molecule binding sites; (iii) RiboTACs facilitate a cellular interaction between cleaved targets and RNase L; and (iv) the expression level of the target influences the extent of cleavage observed. In one example, we converted a binder of LGALS1 (galectin-1) mRNA into a RiboTAC. In MDA-MB-231 cells, the binder had no effect on galectin-1 protein levels, while the RiboTAC cleaved LGALS1 mRNA, reduced galectin-1 protein abundance, and affected galectin-1-associated oncogenic cellular phenotypes. Using LGALS1 , we further assessed additional factors including the length of the linker that tethers the two components of the RiboTAC, cellular uptake, and the RNase L-recruiting module on RiboTAC potency. Collectively, these studies may facilitate triangulation of factors to enable the design of RiboTACs.

The complex multistep activation cascade of Ire1 involves changes in the Ire1 conformation and oligomeric state. Ire1 activation enhances ER folding capacity, in part by overexpressing the ER Hsp70 molecular chaperone BiP; in turn, BiP provides tight negative control of Ire1 activation. This study demonstrates that BiP regulates Ire1 activation through a direct interaction with Ire1 oligomers. Particularly, we demonstrated that the binding of Ire1 luminal domain (LD) to unfolded protein substrates not only trigger conformational changes in Ire1-LD that favour the formation of Ire1-LD oligomers but also exposes BiP binding motifs, enabling the molecular chaperone BiP to directly bind to Ire1-LD in an ATP-dependent manner. These transient interactions between BiP and two short motifs in the disordered region of Ire1-LD are reminiscent of interactions between clathrin and another Hsp70, cytoplasmic Hsc70. BiP binding to substrate-bound Ire1-LD oligomers enables unfolded protein substrates and BiP to synergistically and dynamically control Ire1-LD oligomerisation, helping to return Ire1 to its deactivated state when an ER stress response is no longer required., (© 2024 Dawes et al.)

The first line of defense against invading pathogens usually relies on innate immune systems. In this context, the recognition of exogenous RNA structures is primordial to fight, notably, against RNA viruses. One of the most efficient immune response pathways is based on the sensing of RNA double helical motifs by the oligoadenylate synthase (OAS) proteins, which in turn triggers the activity of RNase L and, thus, cleaves cellular and viral RNA. In this contribution, by using long-range molecular dynamics simulations, complemented with enhanced sampling techniques, we elucidate the structural features leading to the activation of OAS by interaction with a model double-strand RNA oligomer mimicking a viral RNA. We characterize the allosteric regulation induced by the nucleic acid leading to the population of the active form of the protein. Furthermore, we also identify the free energy profile connected to the active vs. inactive conformational transitions in the presence and absence of RNA. Finally, the role of two RNA mutations, identified as able to downregulate OAS activation, in shaping the protein/nucleic acid interface and the conformational landscape of OAS is also analyzed.

RNA helicases-central enzymes in RNA metabolism-often feature intrinsically disordered regions (IDRs) that enable phase separation and complex molecular interactions. In the bacterial pathogen Pseudomonas aeruginosa, the non-redundant RhlE1 and RhlE2 RNA helicases share a conserved REC catalytic core but differ in C-terminal IDRs. Here, we show how the IDR diversity defines RhlE RNA helicase specificity of function. Both IDRs facilitate RNA binding and phase separation, localizing proteins in cytoplasmic clusters. However, RhlE2 IDR is more efficient in enhancing REC core RNA unwinding, exhibits a greater tendency for phase separation, and interacts with the RNase E endonuclease, a crucial player in mRNA degradation. Swapping IDRs results in chimeric proteins that are biochemically active but functionally distinct as compared to their native counterparts. The RECRhlE1-IDRRhlE2 chimera improves cold growth of a rhlE1 mutant, gains interaction with RNase E and affects a subset of both RhlE1 and RhlE2 RNA targets. The RECRhlE2-IDRRhlE1 chimera instead hampers bacterial growth at low temperatures in the absence of RhlE1, with its detrimental effect linked to aberrant RNA droplets. By showing that IDRs modulate both protein core activities and subcellular localization, our study defines the impact of IDR diversity on the functional differentiation of RNA helicases., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The ability of obstacles in cellular transcripts to protect downstream but not upstream sites en masse from attack by RNase E has prompted the hypothesis that this mRNA-degrading endonuclease may scan 5'-monophosphorylated RNA linearly for cleavage sites, starting at the 5' end. However, despite its proposed regulatory importance, the migration of RNase E on RNA has never been directly observed. We have now used single-molecule FRET to monitor the dynamics of this homotetrameric enzyme on RNA. Our findings reveal that RNase E slides along unpaired regions of RNA without consuming a molecular source of energy such as ATP and that its forward progress can be impeded when it encounters a large structural discontinuity. This movement, which is bidirectional, occurs in discrete steps of variable length and requires an RNA ligand much longer than needed to occupy a single RNase E subunit. These results indicate that RNase E scans for cleavage sites by one-dimensional diffusion and suggest a possible molecular mechanism., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In recent years, several noncanonical RNA caps derived from cofactors and metabolites have been identified. Purine-containing RNA caps have been extensively studied, with multiple decapping enzymes identified and efficient capture and sequencing protocols developed for nicotinamide adenine dinucleotide (NAD)-RNA, which allowed for a stepwise elucidation of capping functions. Despite being identified as an abundant noncanonical RNA-cap, UDP-sugar-capped RNA remains poorly understood, which is partly due to its complex in vitro preparation. Here, we describe a scalable synthesis of sugar-capped uridine-guanosine dinucleotides from readily available protected building blocks and their enzymatic conversion into several cell wall precursor-capped dinucleotides. We employed these capped dinucleotides in T7 RNA polymerase-catalyzed in vitro transcription reactions to efficiently generate RNAs capped with uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), its N-azidoacetyl derivative UDP-GlcNAz, and various cell wall precursors. We furthermore identified four enzymes capable of processing UDP-GlcNAc-capped RNA in vitro: MurA, MurB and MurC from Escherichia coli can sequentially modify the sugar-cap structure and were used to introduce a bioorthogonal, clickable moiety, and the human Nudix hydrolase Nudt5 was shown to efficiently decap UDP-GlcNAc-RNA. Our findings underscore the importance of efficient synthetic methods for capped model RNAs. Additionally, we provide useful enzymatic tools that could be utilized in the development and application of UDP-GlcNAc capture and sequencing protocols. Such protocols are essential for deepening our understanding of the widespread yet enigmatic GlcNAc modification of RNA and its physiological significance., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Oligoadenylate synthetase 3 (OAS3) and ribonuclease L (RNase L) are components of a pathway that combats viral infection in mammals. Upon detection of viral double-stranded RNA (dsRNA), OAS3 synthesizes 2'-5'-oligo(A), which activates the RNase domain of RNase L by promoting the homodimerization and oligomerization of RNase L monomers. Activated RNase L rapidly degrades all cellular mRNAs, shutting off several cellular processes. We sought to understand the molecular mechanisms underlying the rapid activation of RNase L in response to viral infection. Through superresolution microscopy and live-cell imaging, we showed that OAS3 and RNase L concentrated into higher-order cytoplasmic complexes known as dsRNA-induced foci (dRIF) in response to dsRNA or infection with dengue virus, Zika virus, or West Nile virus. The concentration of OAS3 and RNase L at dRIF corresponded with the activation of RNase L-mediated RNA decay. We showed that dimerized/oligomerized RNase L concentrated in a liquid-like shell surrounding a core OAS3-dRIF structure and dynamically exchanged with the cytosol. These data establish that the condensation of dsRNA, OAS3, and RNase L into dRIF is a molecular switch that promotes the rapid activation of RNase L upon detection of dsRNA in mammalian cells.

RNase P and RNase mitochondrial RNA processing (MRP) are ribonucleoproteins (RNPs) that consist of a catalytic RNA and a varying number of protein cofactors. RNase P is responsible for precursor tRNA maturation in all three domains of life, while RNase MRP, exclusive to eukaryotes, primarily functions in rRNA biogenesis. While eukaryotic RNase P is associated with more protein cofactors and has an RNA subunit with fewer auxiliary structural elements compared to its bacterial cousin, the double-anchor precursor tRNA recognition mechanism has remarkably been preserved during evolution. RNase MRP shares evolutionary and structural similarities with RNase P, preserving the catalytic core within the RNA moiety inherited from their common ancestor. By incorporating new protein cofactors and RNA elements, RNase MRP has established itself as a distinct RNP capable of processing ssRNA substrates. The structural information on RNase P and MRP helps build an evolutionary trajectory, depicting how emerging protein cofactors harmonize with the evolution of RNA to shape different functions for RNase P and MRP. Here, we outline the structural and functional relationship between RNase P and MRP to illustrate the coevolution of RNA and protein cofactors, a key driver for the extant, diverse RNP world., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacteria harbor diverse mechanisms to defend themselves against their viral predators, bacteriophages. In response, phages can evolve counter-defense systems, most of which are poorly understood. In T4-like phages, the gene tifA prevents bacterial defense by the type III toxin-antitoxin (TA) system toxIN, but the mechanism by which TifA inhibits ToxIN remains unclear. Here, we show that TifA directly binds both the endoribonuclease ToxN and RNA, leading to the formation of a high molecular weight ribonucleoprotein complex in which ToxN is inhibited. The RNA binding activity of TifA is necessary for its interaction with and inhibition of ToxN. Thus, we propose that TifA inhibits ToxN during phage infection by trapping ToxN on cellular RNA, particularly the abundant 16S rRNA, thereby preventing cleavage of phage transcripts. Taken together, our results reveal a novel mechanism underlying inhibition of a phage-defensive RNase toxin by a small, phage-encoded protein., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Fungal ribotoxins are extracellular RNases that inactivate ribosomes by cleaving a single phosphodiester bond at the universally conserved sarcin-ricin loop of the large rRNA. However, to reach the ribosomes, they need to cross the plasma membrane. It is there where these toxins show their cellular specificity, being especially active against tumoral or virus-infected cells. Previous studies have shown that fungal ribotoxins interact with negatively charged membranes, typically containing phosphatidylserine or phosphatidylglycerol. This ability is rooted on their long, non-structured, positively charged loops, and its N-terminal β-hairpin. However, its effect on complex lipid mixtures, including sphingophospholipids or cholesterol, remains poorly studied. Here, wild-type α-sarcin was used to evaluate its interaction with a variety of membranes not assayed before, which resemble much more closely mammalian cell membranes. The results confirm that α-sarcin is particularly sensitive to charge density on the vesicle surface. Its ability to induce vesicle aggregation is strongly influenced by both the lipid headgroup and the degree of saturation of the fatty acid chains. Acyl chain length is indeed particularly important for lipid mixing. Finally, cholesterol plays an important role in diluting the concentration of available negative charges and modulates the ability of α-sarcin to cross the membrane., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Prokaryotic type III CRISPR-Cas antiviral systems employ cyclic oligoadenylate (cA n ) signaling to activate a diverse range of auxiliary proteins that reinforce the CRISPR-Cas defense. Here we characterize a class of cA n -dependent effector proteins named CRISPR-Cas-associated messenger RNA (mRNA) interferase 1 (Cami1) consisting of a CRISPR-associated Rossmann fold sensor domain fused to winged helix-turn-helix and a RelE-family mRNA interferase domain. Upon activation by cyclic tetra-adenylate (cA 4 ), Cami1 cleaves mRNA exposed at the ribosomal A-site thereby depleting mRNA and leading to cell growth arrest. The structures of apo-Cami1 and the ribosome-bound Cami1-cA 4 complex delineate the conformational changes that lead to Cami1 activation and the mechanism of Cami1 binding to a bacterial ribosome, revealing unexpected parallels with eukaryotic ribosome-inactivating proteins.

Transposable elements are genomic parasites that expand within and spread between genomes 1 . PIWI proteins control transposon activity, notably in the germline 2,3 . These proteins recognize their targets through small RNA co-factors named PIWI-interacting RNAs (piRNAs), making piRNA biogenesis a key specificity-determining step in this crucial genome immunity system. Although the processing of piRNA precursors is an essential step in this process, many of the molecular details remain unclear. Here, we identify an endoribonuclease, precursor of 21U RNA 5'-end cleavage holoenzyme (PUCH), that initiates piRNA processing in the nematode Caenorhabditis elegans. Genetic and biochemical studies show that PUCH, a trimer of Schlafen-like-domain proteins (SLFL proteins), executes 5'-end piRNA precursor cleavage. PUCH-mediated processing strictly requires a 7-methyl-G cap (m 7 G-cap) and a uracil at position three. We also demonstrate how PUCH interacts with PETISCO, a complex that binds to piRNA precursors 4 , and that this interaction enhances piRNA production in vivo. The identification of PUCH concludes the search for the 5'-end piRNA biogenesis factor in C. elegans and uncovers a type of RNA endonuclease formed by three SLFL proteins. Mammalian Schlafen (SLFN) genes have been associated with immunity 5 , exposing a molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control transposable elements., (© 2023. The Author(s).)

Human ribonuclease (RNase) 1 and bovine RNase A are the proto-types of the secretory "pancreatic-type" (pt)-RNase super-family. RNase A can oligomerize through the 3D domain swapping (DS) mechanism upon acetic acid (HAc) lyophilisation, producing enzymatically active oligomeric conformers by swapping both N- and C-termini. Also some RNase 1 mutants were found to self-associate through 3D-DS, however forming only N-swapped dimers. Notably, enzymatically active dimers and larger oligomers of wt-RNase 1 were collected here, in higher amount than RNase A, from HAc lyophilisation. In particular, RNase 1 self-associates through the 3D-DS of its N-terminus and, at a higher extent, of the C-terminus. Since RNase 1 is four-residues longer than RNase A, we further analyzed its oligomerization tendency in a mutant lacking the last four residues. The C-terminus role has been investigated also in amphibian onconase (ONC®), a pt-RNase that can form only a N-swapped dimer, since its C-terminus, that is three-residues longer than RNase A, is locked by a disulfide bond. While ONC mutants designed to unlock or cut this constraint were almost unable to dimerize, the RNase 1 mutant self-associated at a higher extent than the wt, suggesting a specific role of the C-terminus in the oligomerization of different RNases. Overall, RNase 1 reaches here the highest ability, among pt-RNases, to extensively self-associate through 3D-DS, paving the way for new investigations on the structural and biological properties of its oligomers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Through its role in intron cleavage, tRNA splicing endonuclease (TSEN) plays a critical function in the maturation of intron-containing pre-tRNAs. The catalytic mechanism and core requirement for this process is conserved between archaea and eukaryotes, but for decades, it has been known that eukaryotic TSENs have evolved additional modes of RNA recognition, which have remained poorly understood. Recent research identified new roles for eukaryotic TSEN, including processing or degradation of additional RNA substrates, and determined the first structures of pre-tRNA-bound human TSEN complexes. These recent discoveries have changed our understanding of how the eukaryotic TSEN targets and recognizes substrates. Here, we review these recent discoveries, their implications, and the new questions raised by these findings., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Target occupancy is often insufficient to elicit biological activity, particularly for RNA, compounded by the longstanding challenges surrounding the molecular recognition of RNA structures by small molecules. Here we studied molecular recognition patterns between a natural-product-inspired small-molecule collection and three-dimensionally folded RNA structures. Mapping these interaction landscapes across the human transcriptome defined structure-activity relationships. Although RNA-binding compounds that bind to functional sites were expected to elicit a biological response, most identified interactions were predicted to be biologically inert as they bind elsewhere. We reasoned that, for such cases, an alternative strategy to modulate RNA biology is to cleave the target through a ribonuclease-targeting chimera, where an RNA-binding molecule is appended to a heterocycle that binds to and locally activates RNase L 1 . Overlay of the substrate specificity for RNase L with the binding landscape of small molecules revealed many favourable candidate binders that might be bioactive when converted into degraders. We provide a proof of concept, designing selective degraders for the precursor to the disease-associated microRNA-155 (pre-miR-155), JUN mRNA and MYC mRNA. Thus, small-molecule RNA-targeted degradation can be leveraged to convert strong, yet inactive, binding interactions into potent and specific modulators of RNA function., (© 2023. The Author(s).)

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped positive-stranded RNA virus which causes serious economic losses to pig industry worldwide. Type I IFN induces expression of interferon-stimulated genes 15 (ISG15) to inhibit virus replication. To survive in the host, PRRSV has evolved to antagonize the antiviral response of ISGylation. Previous studies have reported that nonstructural protein 2 of PRRSV inhibits the ISGylation and antiviral function of ISG15 depending on its ovarian tumor (OTU) domain/papain-like protease domain (PLP2). However, whether there are other PRRSV proteins inhibiting ISGylation of cellular proteins is less well understood. In this study, we first found that PRRSV Nsp11 decreased ISGylation of cellular proteins. Meanwhile, the expression level of ISG15 was significantly inhibited by Nsp11. Further mechanistic studies demonstrated that the transcription of ISG15 was reduced by endoribonuclease activity of Nsp11. Finally, we found that the Nsp11-induced degradation of ISG15 was partially relied on autophagy-lysosome system. Taken together, PRRSV Nsp11 antagonizes the antiviral response of ISG15 by its endoribonuclease activity to promote PRRSV replication. Our results reveal a novel mechanism that PRRSV inhibits ISGylation of cellular proteins and impairs host innate immune response., Competing Interests: Conflicts of Interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Inositol-requiring enzyme 1α (IRE1α) is the most conserved endoplasmic reticulum (ER) stress sensor with two catalytic domains, kinase and RNase, in its cytosolic portion. IRE1α inhibitors have been used to improve existing clinical treatments against various cancers. In this study we identified toxoflavin (TXF) as a new-type potent small molecule IRE1α inhibitor. We used luciferase reporter systems to screen compounds that inhibited the IRE1α-XBP1s signaling pathway. As a result, TXF was found to be the most potent IRE1α RNase inhibitor with an IC 50 value of 0.226 μM. Its inhibitory potencies on IRE1α kinase and RNase were confirmed in a series of cellular and in vitro biochemical assays. Kinetic analysis showed that TXF caused time- and reducing reagent-dependent irreversible inhibition on IRE1α, implying that ROS might participate in the inhibition process. ROS scavengers decreased the inhibition of IRE1α by TXF, confirming that ROS mediated the inhibition process. Mass spectrometry analysis revealed that the thiol groups of four conserved cysteine residues (CYS-605, CYS-630, CYS-715 and CYS-951) in IRE1α were oxidized to sulfonic groups by ROS. In molecular docking experiments we affirmed the binding of TXF with IRE1α, and predicted its binding site, suggesting that the structure of TXF itself participates in the inhibition of IRE1α. Interestingly, CYS-951 was just near the docked site. In addition, the RNase IC 50 and ROS production in vitro induced by TXF and its derivatives were negative correlated (r = -0.872). In conclusion, this study discovers a new type of IRE1α inhibitor that targets a predicted new alternative site located in the junction between RNase domain and kinase domain, and oxidizes conserved cysteine residues of IRE1α active sites to inhibit IRE1α. TXF could be used as a small molecule tool to study IRE1α's role in ER stress., (© 2022. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

The quick widespread of the coronavirus and speedy upsurge in the tally of cases demand the fast development of effective drugs. The uridine-directed endoribonuclease activity of nonstructural protein 15 (Nsp15) of the coronavirus is responsible for the invasion of the host immune system. Therefore, developing potential inhibitors against Nsp15 is a promising strategy. In this concern, the in silico approach can play a significant role, as it is fast and cost-effective in comparison to the trial and error approaches of experimental investigations. In this study, six turmeric derivatives (curcuminoids) were chosen for in silico analysis. The molecular interactions, pharmacokinetics, and drug-likeness of all the curcuminoids were measured. Further, the stability of Nsp15-curcuminoids complexes was appraised by employing molecular dynamics (MD) simulations and MM-PBSA approaches. All the molecules were affirmed to have strong interactions and pharmacokinetic profile. The MD simulations data stated that the Nsp15-curcuminoids complexes were stable during simulations. All the curcuminoids showed stable and high binding affinity, and these curcuminoids could be admitted as potential modulators for Nsp15 inhibition., (© 2022 John Wiley & Sons Ltd.)

Global sequencing efforts from the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, continue to provide insight into the evolution of the viral genome. Coronaviruses encode 16 nonstructural proteins, within the first two-thirds of their genome, that facilitate viral replication and transcription as well as evasion of the host immune response. However, many of these viral proteins remain understudied. Nsp15 is a uridine-specific endoribonuclease conserved across all coronaviruses. The nuclease activity of Nsp15 helps the virus evade triggering an innate immune response. Understanding how Nsp15 has changed over the course of the pandemic, and how mutations affect its RNA processing function, will provide insight into the evolution of an oligomerization-dependent endoribonuclease and inform drug design. In combination with previous structural data, bioinformatics analyses of 1.9 + million SARS-CoV-2 sequences revealed mutations across Nsp15's three structured domains (N-terminal, Middle, EndoU). Selected Nsp15 variants were characterized biochemically and compared to wild type Nsp15. We found that mutations to important catalytic residues decreased cleavage activity but increased the hexamer/monomer ratio of the recombinant protein. Many of the highly prevalent variants we analyzed led to decreased nuclease activity as well as an increase in the inactive, monomeric form. Overall, our work establishes how Nsp15 variants seen in patient samples affect nuclease activity and oligomerization, providing insight into the effect of these variants in vivo., Competing Interests: Conflicts of Interests The authors declare no conflicts of interest., (Published by Elsevier Ltd.)

With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, the development of analytical tools for characterization of long, complex RNAs has become essential. Tandem liquid chromatography-mass spectrometry (LC-MS/MS) permits direct assessment of the mRNA primary sequence and modifications thereof without conversion to cDNA or amplification. It relies upon digestion of mRNA with site-specific endoribonucleases to generate pools of short oligonucleotides that are then amenable to MS-based sequence analysis. Here, we showed that the uridine-specific human endoribonuclease hRNase 4 improves mRNA sequence coverage, in comparison with the benchmark enzyme RNase T1, by producing a larger population of uniquely mappable cleavage products. We deployed hRNase 4 to characterize mRNAs fully substituted with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U), as well as mRNAs selectively depleted of uridine-two key strategies to reduce synthetic mRNA immunogenicity. Lastly, we demonstrated that hRNase 4 enables direct assessment of the 5' cap incorporation into in vitro transcribed mRNA. Collectively, this study highlights the power of hRNase 4 to interrogate mRNA sequence, identity, and modifications by LC-MS/MS., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The RNA-binding protein tristetraprolin (TTP) is a potent activator of mRNA decay, specifically for transcripts bearing AU-rich elements (AREs) in their 3'-untranslated regions. TTP functions as a mediator for mRNA decay by interacting with the decay machinery and recruiting it to the target ARE-mRNA. In this study, we report a weak, but direct interaction between TTP and the human decapping enzyme DCP2, which impacts the stability of ARE transcripts. The TTP-DCP2 interaction is unusual as it involves intrinsically disordered regions (IDRs) of both binding partners. We show that the IDR of DCP2 has a propensity for oligomerization and liquid-liquid phase separation in vitro. Binding of TTP to DCP2 leads to its partitioning into phase-separated droplets formed by DCP2, suggesting that molecular crowding might facilitate the weak interaction between the two proteins and enable assembly of a decapping-competent mRNA-protein complex on TTP-bound transcripts in cells. Our studies underline the role of weak interactions in the cellular interaction network and their contribution towards cellular functionality., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The World Health Organization has classified the COVID-19 outbreak a pandemic which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) and declared it a global health emergency. Repurposing drugs with minimum side effects are one approach to quickly respond in attempt to prevent the spread of COVID-19. SARS-CoV-2 encodes several RNA processing enzymes that are unusual and unique for single-stranded RNA viruses, including Nsp15, a hexameric endoribonuclease that discriminatory cleaves immediately 3' of uridines. The structure of SARS-CoV-2 Nsp15 is reported to be homologous to that of the Nsp15 endoribonucleases of SARS-CoV and MERS-CoV, but it exhibits differences that may contribute to the greater virulence of SARS-CoV-2. This study aimed to identify drugs that targeted SARS-COV-2 Nsp15 using a molecular docking-based virtual screening of a library containing 10,000 approved and experimental drugs. The molecular docking results revealed 19 medications that demonstrated a good ability to inhibit Nsp15. Among all the candidated 19 drugs only five FDA approved drugs were used for further investigation by molecular dynamics simulation, the stability of Nsp15-ligand system was evaluated by calculating the RMSD, RMSF, radius of gyration and hydrogen bond profile. Furthermore, MM-PBSA method was employed to validate the binding affinity. According to the obtained results of MD, the complex of Olaparib was showed more stability and lower binding free energy than the control inhibitor during MD simulation time. Finally, we suggest that Olaparib is a potential drug for treating patients infected with SARS-CoV-2 and provide insight into the host immune response to viral RNA.Communicated by Ramaswamy H. Sarma.

Poxviruses encode decapping enzymes that remove the protective 5' cap from both host and viral mRNAs to commit transcripts for decay by the cellular exonuclease Xrn1. Decapping by these enzymes is critical for poxvirus pathogenicity by means of simultaneously suppressing host protein synthesis and limiting the accumulation of viral double-stranded RNA (dsRNA), a trigger for antiviral responses. Here we present a high-resolution structural view of the vaccinia virus decapping enzyme D9. This Nudix enzyme contains a domain organization different from other decapping enzymes in which a three-helix bundle is inserted into the catalytic Nudix domain. The 5' mRNA cap is positioned in a bipartite active site at the interface of the two domains. Specificity for the methylated guanosine cap is achieved by stacking between conserved aromatic residues in a manner similar to that observed in canonical cap-binding proteins VP39, eIF4E, and CBP20, and distinct from eukaryotic decapping enzyme Dcp2., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Aminothiophene is a scaffold that is widely present in drugs and biologically active small molecules as chemical probes. In this study, 43 compounds sharing a 2-aminothiophenone-3-carboxylate (ATPC) scaffold, known to activate the ribonuclease L (RNase L), were synthesized and selected ATPCs showed enhancement of thermal stability of RNase L upon binding. Screening of antiproliferation activities against human cancer cell lines revealed that ATPCs represented by compounds 4l and 50 showed potent single-digit micromolar antiproliferation activity against human cancer cell lines. Compounds 4l and 50 exhibited time- and dose-dependent proliferation inhibition, induced cellular apoptosis measured by cleaved PARP and via flow cytometry, inhibited cell migration, and inhibited cell colony formation. Combining the results reported in this work, ATPCs were evaluated as potential anticancer agents mediated by RNase L-binding and apoptosis induction. The work contributes to the study on the polypharmacological properties of aminothiophene-containing small molecules., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The Bacillus subtilis genome is predicted to encode numerous ribonucleases, including four 3' exoribonucleases that have been characterized to some extent. A strain containing gene knockouts of all four known 3' exoribonucleases is viable, suggesting that one or more additional RNases remain to be discovered. A protein extract from the quadruple RNase mutant strain was fractionated and RNase activity was followed, resulting in the identification of an enzyme activity catalyzed by the YloC protein. YloC is an endoribonuclease and is a member of the highly conserved "YicC family" of proteins that is widespread in bacteria. YloC is a metal-dependent enzyme that catalyzes the cleavage of single-stranded RNA, preferentially at U residues, and exists in an oligomeric form, most likely a hexamer. As such, YloC shares some characteristics with the SARS-CoV Nsp15 endoribonuclease. While the in vivo function of YloC in B. subtilis is yet to be determined, YloC was found to act similarly to YicC in an Escherichia coli in vivo assay that assesses decay of the small RNA, RyhB. Thus, YloC may play a role in small RNA regulation., (© 2022 Ingle et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Combinations of ribonucleases (RNases) are commonly used to digest RNA into oligoribonucleotide fragments prior to liquid chromatography-mass spectrometry (LC-MS) analysis. The distribution of the RNase target sequences or nucleobase sites within an RNA molecule is critical for achieving a high mapping coverage. Cusativin and MC1 are nucleotide-specific endoribonucleases encoded in the cucumber and bitter melon genomes, respectively. Their high specificity for cytidine (Cusativin) and uridine (MC1) make them ideal molecular biology tools for RNA modification mapping. However, heterogenous recombinant expression of either enzyme has been challenging because of their high toxicity to expression hosts and the requirement of posttranslational modifications. Here, we present two highly efficient and time-saving protocols that overcome these hurdles and enhance the expression and purification of these RNases. We first purified MC1 and Cusativin from bacteria by expressing and shuttling both enzymes to the periplasm as MBP-fusion proteins in T7 Express lysY/I q E. coli strain at low temperature. The RNases were enriched using amylose affinity chromatography, followed by a subsequent purification via a C-terminal 6xHIS tag. This fast, two-step purification allows for the purification of highly active recombinant RNases significantly surpassing yields reported in previous studies. In addition, we expressed and purified a Cusativin-CBD fusion enzyme in P. pastoris using chitin magnetic beads. Both Cusativin variants exhibited a similar sequence preference, suggesting that neither posttranslational modifications nor the epitope-tags have a substantial effect on the sequence specificity of the enzyme., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The genus Pestivirus , family Flaviviridae , includes four historically accepted species, i.e., bovine viral diarrhea virus (BVDV)-1 and -2, classical swine fever virus (CSFV), and border disease virus (BDV). A large number of new pestivirus species were identified in recent years. A common feature of most members is the presence of two unique proteins, N pro and E rns , that pestiviruses evolved to regulate the host's innate immune response. In addition to its function as a structural envelope glycoprotein, E rns is also released in the extracellular space, where it is endocytosed by neighboring cells. As an endoribonuclease, E rns is able to cleave viral ss- and dsRNAs, thus preventing the stimulation of the host's interferon (IFN) response. Here, we characterize the basic features of soluble E rns of a large variety of classified and unassigned pestiviruses that have not yet been described. Its ability to form homodimers, its RNase activity, and the ability to inhibit dsRNA-induced IFN synthesis were investigated. Overall, we found large differences between the various E rns proteins that cannot be predicted solely based on their primary amino acid sequences, and that might be the consequence of different virus-host co-evolution histories. This provides valuable information to delineate the structure-function relationship of pestiviral endoribonucleases.

Pillar[5]arenes containing sulfonate fragments have been shown to form supramolecular complexes with therapeutic proteins to facilitate targeted transport with an increased duration of action and enhanced bioavailability. Regioselective synthesis was used to obtain a water-soluble pillar[5]arene containing the fluorescent label FITC and nine sulfoethoxy fragments. The pillar[5]arene formed complexes with the therapeutic proteins binase, bleomycin, and lysozyme in a 1:2 ratio as demonstrated by UV-vis and fluorescence spectroscopy. The formation of stable spherical nanosized macrocycle/binase complexes with an average particle size of 200 nm was established by dynamic light scattering and transmission electron microscopy. Flow cytometry demonstrated the ability of macrocycle/binase complexes to penetrate into tumor cells where they exhibited significant cytotoxicity towards A549 cells at 10 -5 -10 -6 M while maintaining the enzymatic activity of binase., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The divide-and-conquer strategy is commonly used for protein structure determination, but its applications to high-resolution structure determination of RNAs have been limited. Here, we introduce an integrative approach based on the divide-and-conquer strategy that was undertaken to determine the solution structure of an RNA model system, the Neurospora VS ribozyme. NMR and SAXS studies were conducted on a minimal trans VS ribozyme as well as several isolated subdomains. A multi-step procedure was used for structure determination that first involved pairing refined NMR structures with SAXS data to obtain structural subensembles of the various subdomains. These subdomain structures were then assembled to build a large set of structural models of the ribozyme, which was subsequently filtered using SAXS data. The resulting NMR-SAXS structural ensemble shares several similarities with the reported crystal structures of the VS ribozyme. However, a local structural difference is observed that affects the global fold by shifting the relative orientation of the two three-way junctions. Thus, this finding highlights a global conformational change associated with substrate binding in the VS ribozyme that is likely critical for its enzymatic activity. Structural studies of other large RNAs should benefit from similar integrative approaches that allow conformational sampling of assembled fragments., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Integrator and protein phosphatase 2A (PP2A) form a complex that dephosphorylates paused RNA polymerase II (Pol II), cleaves the nascent RNA, and terminates transcription. We report the structure of the pretermination complex containing the human Integrator-PP2A complex bound to paused Pol II. Integrator binds Pol II and the pausing factors DSIF and NELF to exclude binding of the elongation factors SPT6 and PAF1 complex. Integrator also binds the C-terminal domain of Pol II and positions PP2A to counteract Pol II phosphorylation and elongation. The Integrator endonuclease docks to the RNA exit site and opens to cleave nascent RNA about 20 nucleotides from the Pol II active site. Integrator does not bind the DNA clamps formed by Pol II and DSIF, enabling release of DNA and transcription termination.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 relies on sRNAs to coordinate expression of metabolic and virulence factors to colonize the host. Here, we focus on the sRNA, named MavR (metabolism and virulence regulator), that is conserved among pathogenic Enterobacteriaceae. MavR is constitutively expressed under in vitro conditions that promote EHEC virulence gene expression. Using MS2-affinity purification coupled with RNA sequencing, the eutR transcript was identified as a putative target of MavR. EutR is a transcription factor that promotes expression of genes required for ethanolamine metabolism as well as virulence factors important for host colonization. MavR binds to the eutR coding sequence to protect the eutR transcript from RNase E-mediated degradation. Ultimately, MavR promotes EutR expression and in turn ethanolamine utilization and ethanolamine-dependent growth. RNAseq analyses revealed that MavR also affected expression of genes important for other metabolic pathways, motility, oxidative stress and attaching and effacing lesion formation, which contribute to EHEC colonization of the gastrointestinal tract. In support of the idea that MavR-dependent gene expression affects fitness during infection, deletion of mavR resulted in significant (∼10- to 100-fold) attenuation in colonization of the mammalian intestine. Altogether, these studies reveal an important, extensive, and robust phenotype for a bacterial sRNA in host-pathogen interactions., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The conserved endoribonuclease RNase E dominates the dynamic landscape of RNA metabolism and underpins control mediated by small regulatory RNAs in diverse bacterial species. We explored the enzyme's hydrolytic mechanism, allosteric activation, and interplay with partner proteins in the multicomponent RNA degradosome assembly of Escherichia coli. RNase E cleaves single-stranded RNA with preference to attack the phosphate located at the 5' nucleotide preceding uracil, and we corroborate key interactions that select that base. Unexpectedly, RNase E activity is impeded strongly when the recognized uracil is isomerized to 5-ribosyluracil (pseudouridine), from which we infer the detailed geometry of the hydrolytic attack process. Kinetics analyses support models for recognition of secondary structure in substrates by RNase E and for allosteric autoregulation. The catalytic power of the enzyme is boosted when it is assembled into the multienzyme RNA degradosome, most likely as a consequence of substrate capture and presentation. Our results rationalize the origins of substrate preferences of RNase E and illuminate its catalytic mechanism, supporting the roles of allosteric domain closure and cooperation with other components of the RNA degradosome complex., (© 2021 Islam et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

All species transcribe ribosomal RNA in an immature form that requires several enzymes for processing into mature rRNA. The number and types of enzymes utilized for these processes vary greatly between different species. In low G + C Gram-positive bacteria including Bacillus subtilis and Geobacillus stearothermophilus , the endoribonuclease (RNase) M5 performs the final step in 5S rRNA maturation, by removing the 3'- and 5'-extensions from precursor (pre) 5S rRNA. This cleavage activity requires initial complex formation between the pre-rRNA and a ribosomal protein, uL18, making the full M5 substrate a ribonucleoprotein particle (RNP). M5 contains a catalytic N-terminal Toprim domain and an RNA-binding C-terminal domain, respectively, shown to assist in processing and binding of the RNP. Here, we present structural data that show how two Mg 2+ ions are accommodated in the active site pocket of the catalytic Toprim domain and investigate the importance of these ions for catalysis. We further perform solution studies that support the previously proposed 3'-before-5' order of removal of the pre-5S rRNA extensions and map the corresponding M5 structural rearrangements during catalysis.

The ribonucleoprotein RNase MRP is responsible for the processing of ribosomal RNA precursors. It is found in virtually all eukaryotes that have been examined. In the Euglenozoa, including the genera Euglena, Diplonema and kinetoplastids, MRP RNA and protein subunits have so far escaped detection using bioinformatic methods. However, we now demonstrate that the RNA component is widespread among the Euglenozoa and that these RNAs have secondary structures that conform to the structure of all other phylogenetic groups. In Euglena, we identified the same set of P/MRP protein subunits as in many other protists. However, we failed to identify any of these proteins in the kinetoplastids. This finding poses interesting questions regarding the structure and function of RNase MRP in these species.

The signaling network of the unfolded protein response (UPR) adjusts the protein-folding capacity of the endoplasmic reticulum (ER) according to need. The most conserved UPR sensor, IRE1α, spans the ER membrane and activates through oligomerization. IRE1α oligomers accumulate in dynamic foci. We determined the in situ structure of IRE1α foci by cryogenic correlated light and electron microscopy combined with electron cryo-tomography and complementary immuno–electron microscopy in mammalian cell lines. IRE1α foci localized to a network of narrow anastomosing ER tubes (diameter, ~28 nm) with complex branching. The lumen of the tubes contained protein filaments, which were likely composed of arrays of IRE1α lumenal domain dimers that were arranged in two intertwined, left-handed helices. This specialized ER subdomain may play a role in modulating IRE1α signaling.

The SARS-CoV-2 replication-transcription complex is an assembly of nonstructural viral proteins that collectively act to reproduce the viral genome and generate mRNA transcripts. While the structures of the individual proteins involved are known, how they assemble into a functioning superstructure is not. Applying molecular modeling tools, including protein-protein docking, to the available structures of nsp7-nsp16 and the nucleocapsid, we have constructed an atomistic model of how these proteins associate. Our principal finding is that the complex is hexameric, centered on nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10) 2 , which then recruit six nsp12/nsp7/(nsp8) 2 polymerase subunits to the complex. To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed. Polymerase subunits that coordinate dimers of nsp13 are capable of binding the nucleocapsid, which positions the 5'-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analysis of the viral RNA path through the complex indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14. The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping. The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping, and the role of the endonuclease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15-34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes., (© 2021. The Author(s).)

Type III CRISPR-Cas immunity is widespread in prokaryotes and is generally mediated by multisubunit effector complexes. These complexes recognize complementary viral transcripts and can activate ancillary immune proteins. Here, we describe a type III-E effector from Candidatus “Scalindua brodae” ( Sb -gRAMP), which is natively encoded by a single gene with several type III domains fused together. This effector uses CRISPR RNA to guide target RNA recognition and cleaves single-stranded RNA at two defined positions six nucleotides apart. Sb -gRAMP physically combines with the caspase-like TPR-CHAT peptidase to form the CRISPR-guided caspase (Craspase) complex, suggesting a potential mechanism of target RNA–induced protease activity to gain viral immunity.

The Unfolded Protein response is an adaptive pathway triggered upon alteration of endoplasmic reticulum (ER) homeostasis. It is transduced by three major ER stress sensors, among which the Inositol Requiring Enzyme 1 (IRE1) is the most evolutionarily conserved. IRE1 is an ER-resident type I transmembrane protein exhibiting an ER luminal domain that senses the protein folding status and a catalytic kinase and RNase cytosolic domain. In recent years, IRE1 has emerged as a relevant therapeutic target in various diseases including degenerative, inflammatory and metabolic pathologies and cancer. As such several drugs altering IRE1 activity were developed that target either catalytic activity and showed some efficacy in preclinical pathological mouse models. In this review, we describe the different drugs identified to target IRE1 activity as well as their mode of action from a structural perspective, thereby identifying common and different modes of action. Based on this information we discuss on how new IRE1-targeting drugs could be developed that outperform the currently available molecules., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies., (© 2021. The Author(s).)

In Leptospira interrogans serovar Copenhageni, the CRISPR-Cas I-B locus possesses a CRISPR array between the two independent cas-operons. Using the reverse transcription-PCR and the in vitro endoribonuclease assay with Cas6 of Leptospira (LinCas6), we account that the CRISPR is transcriptionally active and is conventionally processed. The LinCas6 specifically excises at one site within the synthetic cognate repeat RNA or the repeats of precursor-CRISPR RNA (pre-crRNA) in the sense direction. In contrast, the antisense repeat RNA is cleaved at multiple sites. LinCas6 functions as a single turnover endoribonuclease on its repeat RNA substrate, where substitution of one of predicted active site residues (His38) resulted in reduced activity. This study highlights the comprehensive understanding of the Leptospira CRISPR array transcription and its processing by LinCas6 that is central to RNA-mediated CRISPR-Cas I-B adaptive immunity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Immensely aggravated situation of COVID-19 has pushed the scientific community towards developing novel therapeutics to fight the pandemic. Small molecules can possibly prevent the spreading infection by targeting specific vital components of the viral genome. Non-structural protein 15 (Nsp15) has emerged as a promising target for such inhibitor molecules. In this investigation, we docked bioactive molecules of tea onto the active site of Nsp15. Based on their docking scores, top three molecules (Barrigenol, Kaempferol, and Myricetin) were selected and their conformational behavior was analyzed via molecular dynamics simulations and MMPBSA calculations. The results indicated that the protein had well adapted the ligands in the binding pocket thereby forming stable complexes. These molecules displayed low binding energy during MMPBSA calculations, substantiating their strong association with Nsp15. The inhibitory potential of these molecules could further be examined by in-vivo and in-vitro investigations to validate their use as inhibitors against Nsp15 of SARS-CoV2., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Since its initial characterization, Escherichia coli RNase I has been described as a single-strand specific RNA endonuclease that cleaves its substrate in a largely sequence independent manner. Here, we describe a strong calcium (Ca2+)-dependent activity of RNase I on double-stranded RNA (dsRNA), and a Ca2+-dependent novel hybridase activity, digesting the RNA strand in a DNA:RNA hybrid. Surprisingly, Ca2+ does not affect the activity of RNase I on single stranded RNA (ssRNA), suggesting a specific role for Ca2+ in the modulation of RNase I activity. Mutation of a previously overlooked Ca2+ binding site on RNase I resulted in a gain-of-function enzyme that is highly active on dsRNA and could no longer be stimulated by the metal. In summary, our data imply that native RNase I contains a bound Ca2+, allowing it to target both single- and double-stranded RNAs, thus having a broader substrate specificity than originally proposed for this traditional enzyme. In addition, the finding that the dsRNase activity, and not the ssRNase activity, is associated with the Ca2+-dependency of RNase I may be useful as a tool in applied molecular biology., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Coronavirus infectious disease 2019 (COVID-19), a viral infection caused by a novel coronavirus (nCoV), continues to emerge as a serious threat to public health. This pandemic caused by SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) has infected globally with 1,550,000 plus deaths to date, representing a high risk to public health. No effective drug or vaccine is available to curb down this deadly virus. The expedition for searching for a potential drug or vaccine against COVID-19 is of massive potential and favour to the community. This study is focused on finding an effective natural compound that can be processed further into a potential inhibitor to check the activity of SARS-CoV-2 with minimal side effects targeting NSP15 protein, which belongs to the EndoU enzyme family. The natural screening suggested two efficient compounds (PubChem ID: 95372568 and 1776037) with dihydroxyphenyl region of the compound, found to be important in the interaction with the viral protein showing promising activity which may act as a potent lead inhibitory molecule against the virus. In combination with virtual screening, modelling, drug likeliness, molecular docking, and 500 ns cumulative molecular dynamics simulations (100 ns for each complex) along with the decomposition analysis to calculate and confirm the stability and fold, we propose 95372568 and 1776037 as novel compounds of natural origin capable of getting developed into potent lead molecules against SARS-CoV-2 target protein NSP15.

Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis ( M. tb ). It is regarded as a major health threat all over the world, mainly because of its high mortality and drug-resistant nature. Toxin-antitoxin (TA) systems are modules ubiquitously found in prokaryotic organisms, and the well-studied MazEF systems (MazE means "what is it?" in Hebrew) are implicated in the formation of "persister cells" in the M. tb pathogen. Here, we report cocrystal structures of M. tb MazF-mt1 and -mt9, two important MazF members responsible for specific mRNA and tRNA cleavages, respectively, in complexes with truncated forms of their cognate antitoxin peptides. These peptides bind to the toxins with comparable affinities to their full-length antitoxins, which would reduce the RNA-cleavage capacities of the toxins in vitro. After structural analysis of the binding modes, we systemically tested the influence of the substitutions of individual residues in the truncated MazE-mt9 peptide on its affinity. This study provides structural insight into the binding modes and the inhibition mechanisms between the MazE/F-mt TA pairs. More importantly, it contributes to the future design of peptide-based antimicrobial agents against TB and potentially relieves the drug-resistance problems by targeting novel M. tb proteins.

The SARS-CoV-2, a positive-sense single-stranded RNA Coronavirus, is a global threat to human health. Thus, understanding its life cycle mechanistically would be important to facilitate the design of antiviral drugs. A key aspect of viral progression is the synthesis of viral proteins by the ribosome of the human host. In Coronaviruses, this process is regulated by the viral 5' and 3' untranslated regions (UTRs), but the precise regulatory mechanism has not yet been well understood. In particular, the 5'-UTR of the viral genome is most likely involved in translation initiation of viral proteins. Here, we performed inline probing and RNase V1 probing to establish a model of the secondary structure of SARS-CoV-2 5'-UTR. We found that the 5'-UTR contains stable structures including a very stable four-way junction close to the AUG start codon. Sequence alignment analysis of SARS-CoV-2 variants 5'-UTRs revealed a highly conserved structure with few co-variations that confirmed our secondary structure model based on probing experiments.