Cleavage Of Double-stranded RNA Research Articles

Understanding preferences for double-stranded RNA cleavage by SARS-Cov-2 enzyme Nsp15

Understanding preferences for double-stranded RNA cleavage by SARS-Cov-2 enzyme Nsp15

Structural Insights into the Advances and Mechanistic Understanding of Human Dicer.

The RNase III endoribonuclease Dicer was discovered to be associated with cleavage of double-stranded RNA in 2001. Since then, many advances in our understanding of Dicer function have revealed that the enzyme plays a major role not only in microRNA biology but also in multiple RNA interference-related pathways. Yet, there is still much to be learned regarding Dicer structure-function in relation to how Dicer and Dicer-like enzymes initiate their cleavage reaction and release the desired RNA product. This Perspective describes the latest advances in Dicer structural studies, expands on what we have learned from this data, and outlines key gaps in knowledge that remain to be addressed. More specifically, we focus on human Dicer and highlight the intermediate processing steps where there is a lack of structural data to understand how the enzyme traverses from pre-cleavage to cleavage-competent states. Understanding these details is necessary to model Dicer's function as well as develop more specific microRNA-targeted therapeutics for the treatment of human diseases.

StepRNA: Identification of Dicer cleavage signatures and passenger strand lengths in small RNA sequences.

The enzyme Dicer is a component of many small RNA (sRNA) pathways involved in RNA processing for post-transcriptional regulation, anti-viral response and control of transposable elements. Cleavage of double-stranded RNA by Dicer produces a signature overhanging sequence at the 3' end of the sRNA sequence relative to a complementary passenger strand in a RNA duplex. There is a need for reliable tools to computationally search for Dicer cleavage signatures to help characterise families of sRNAs. This is increasingly important due to the rising popularity of sRNA sequencing, especially in non-model organisms. Here, we present stepRNA, a fast, local tool that identifies (i) overhang signatures strongly indicative of Dicer cleavage in RNA sequences, and (ii) the length of the passenger strand in sRNAs duplexes. We demonstrate the use of stepRNA with simulated and biological datasets to detect Dicer cleavage signatures in experimentally validated examples. Compared to currently available tools, stepRNA is more accurate, requires only sRNA sequence data rather than a reference genome, and provides information about other important features such as passenger strand length. stepRNA is freely available at https://github.com/Vicky-Hunt-Lab/stepRNA and is easily installable.

Detection of SARS-CoV-2 Derived Small RNAs and Changes in Circulating Small RNAs Associated with COVID-19.

Cleavage of double-stranded RNA is described as an evolutionary conserved host defense mechanism against viral infection. Small RNAs are the product and triggers of post transcriptional gene silencing events. Up until now, the relevance of this mechanism for SARS-CoV-2-directed immune responses remains elusive. Herein, we used high throughput sequencing to profile the plasma of active and convalescent COVID-19 patients for the presence of small circulating RNAs. The existence of SARS-CoV-2 derived small RNAs in plasma samples of mild and severe COVID-19 cases is described. Clusters of high siRNA abundance were discovered, homologous to the nsp2 3′-end and nsp4 virus sequence. Four virus-derived small RNA sequences have the size of human miRNAs, and a target search revealed candidate genes associated with ageusia and long COVID symptoms. These virus-derived small RNAs were detectable also after recovery from the disease. The additional analysis of circulating human miRNAs revealed differentially abundant miRNAs, discriminating mild from severe cases. A total of 29 miRNAs were reduced or absent in severe cases. Several of these are associated with JAK-STAT response and cytokine storm.

Single-molecule analysis of processive double-stranded RNA cleavage by Drosophila Dicer-2

Drosophila Dicer-2 (Dcr-2) produces small interfering RNAs from long double-stranded RNAs (dsRNAs), playing an essential role in antiviral RNA interference. The dicing reaction by Dcr-2 is enhanced by Loquacious-PD (Loqs-PD), a dsRNA-binding protein that partners with Dcr-2. Previous biochemical analyses have proposed that Dcr-2 uses two distinct—processive or distributive—modes of cleavage by distinguishing the terminal structures of dsRNAs and that Loqs-PD alters the terminal dependence of Dcr-2. However, the direct evidence for this model is lacking, as the dynamic movement of Dcr-2 along dsRNAs has not been traced. Here, by utilizing single-molecule imaging, we show that the terminal structures of long dsRNAs and the presence or absence of Loqs-PD do not essentially change Dcr-2’s cleavage mode between processive and distributive, but rather simply affect the probability for Dcr-2 to undergo the cleavage reaction. Our results provide a refined model for how the dicing reaction by Dcr-2 is regulated.

Identification of protein structural elements responsible for the diversity of sequence preferences among Mini-III RNases

Many known endoribonucleases select their substrates based on the presence of one or a few specific nucleotides at or near the cleavage site. In some cases, selectivity is also determined by the structural features of the substrate. We recently described the sequence-specific cleavage of double-stranded RNA by Mini-III RNase from Bacillus subtilis in vitro. Here, we characterized the sequence specificity of eight other members of the Mini-III RNase family from different bacterial species. High-throughput analysis of the cleavage products of Φ6 bacteriophage dsRNA indicated subtle differences in sequence preference between these RNases, which were confirmed and characterized by systematic analysis of the cleavage kinetics of a set of short dsRNA substrates. We also showed that the sequence specificities of Mini-III RNases are not reflected by different binding affinities for cognate and non-cognate sequences, suggesting that target selection occurs predominantly at the cleavage step. We were able to identify two structural elements, the α4 helix and α5b-α6 loop that were involved in target selection. Characterization of the sequence specificity of the eight Mini-III RNases may provide a basis for better understanding RNA substrate recognition by Mini-III RNases and adopting these enzymes and their engineered derivatives as tools for RNA research.

Arabidopsis RNASE THREE LIKE2 Modulates the Expression of Protein-Coding Genes via 24-Nucleotide Small Interfering RNA-Directed DNA Methylation.

RNaseIII enzymes catalyze the cleavage of double-stranded RNA (dsRNA) and have diverse functions in RNA maturation. Arabidopsis thaliana RNASE THREE LIKE2 (RTL2), which carries one RNaseIII and two dsRNA binding (DRB) domains, is a unique Arabidopsis RNaseIII enzyme resembling the budding yeast small interfering RNA (siRNA)-producing Dcr1 enzyme. Here, we show that RTL2 modulates the production of a subset of small RNAs and that this activity depends on both its RNaseIII and DRB domains. However, the mode of action of RTL2 differs from that of Dcr1. Whereas Dcr1 directly cleaves dsRNAs into 23-nucleotide siRNAs, RTL2 likely cleaves dsRNAs into longer molecules, which are subsequently processed into small RNAs by the DICER-LIKE enzymes. Depending on the dsRNA considered, RTL2-mediated maturation either improves (RTL2-dependent loci) or reduces (RTL2-sensitive loci) the production of small RNAs. Because the vast majority of RTL2-regulated loci correspond to transposons and intergenic regions producing 24-nucleotide siRNAs that guide DNA methylation, RTL2 depletion modifies DNA methylation in these regions. Nevertheless, 13% of RTL2-regulated loci correspond to protein-coding genes. We show that changes in 24-nucleotide siRNA levels also affect DNA methylation levels at such loci and inversely correlate with mRNA steady state levels, thus implicating RTL2 in the regulation of protein-coding gene expression.

Characterization of the psbH precursor RNAs reveals a precise endoribonuclease cleavage site in the psbT/psbH intergenic region that is dependent on psbN gene expression.

The plastid psbB operon harbours 5 genes, psbB, psbT, psbH, petB and petD. A sixth gene, the psbN gene, is located on the opposite DNA strand in the psbT/psbH intergenic region. Its transcription produces antisense RNA to a large part of the psbB pentacistronic mRNA. We have investigated whether transcription of the psbN gene, i.e. production of antisense RNA, influences psbT/psbH intergenic processing. Results reveal the existence of four different psbH precursor RNAs. Three of them result from processing and one is produced by transcription initiation. One of the processed RNAs is probably created by site-specific RNA cleavage. This RNA is absent in plants where the psbN gene is not transcribed suggesting that cleavage at this site is dependent on the formation of sense/antisense double-stranded RNA. In order to characterize the nuclease that might be responsible for double-stranded RNA cleavage, we analysed csp41a and csp41b knock-out mutants and the corresponding double mutant. Both CSP41 proteins are known to interact physically and CSP41a had been shown to cleave within 3'-untranslated region stem-loop structures, which contain double-stranded RNA, in vitro. We demonstrate that the psbH RNA, that is absent in plants where the psbN gene is not transcribed, is also strongly diminished in all csp41 plants. Altogether, results reveal a site-specific endoribonuclease cleavage event that seems to depend on antisense RNA and might implicate endoribonuclease activity of CSP41a.

Interaction of Ribonuclease III with the Regulatory Macrodomain Protein YmdB Analyzed by Docking Calculations and SPR Experiments

Ribonuclease III (RNase III) is a conserved bacterial endonuclease that cleaves double-stranded(ds) RNA structures and is essential in diverse RNA maturation and decay pathways [1,2]. RNase III is subject to multiple levels of regulation, allowing fine-tuning of its catalytic activity depending on the cellular physiological state. The regulatory macrodomain protein YmdB interacts with RNase III, and an increase in YmdB levels correlates with a decrease in RNase III activity in vivo [3]. However, the molecular details of the YmdB-RNase III interaction are not yet known. Here, docking calculations and computationally-driven mutagenesis were combined with surface plasmon resonance (SPR) experiments to identify energetically important determinants of the Escherichia coli YmdB-RNase III interaction. The computational results reveal two alternative YmdB binding sites in RNase III: one located in the N-terminal nuclease domain (RIIID) (also indicated by co-immunoprecipitation and chemical cross-linking [3]), and a novel site in the C-terminal dsRNA-binding domain (dsRBD). The binding site in the RIIID is composed of a cluster of negatively charged residues that interact with a conserved arginine in YmdB, and the importance of this interaction is confirmed by SPR analysis of the YmdB Arg to Ala mutation. These results suggest a mechanism of RNase III regulation in which YmdB can bind separate sites in a concentration-dependent manner, leading to inhibition of catalytic activity.[1] Nicholson A.W. 2014. Ribonuclease III mechanisms of double-stranded RNA cleavage. WIREs RNA 5:31-48.[2] Court D.L., Gan J., Liang Y.-H., Shaw G.X., Tropea J.E., Costantino N., Waugh D.S., Ji X. 2013. RNase III: genetics and function; structure and mechanism. Annu. Rev. Genet. 47:405-431.[3] Kim K.-S., Manasherob R., Cohen S.N. 2008. YmdB: a stress-responsive ribonuclease-binding regulator of E. coli RNase III activity. Genes Dev. 22:3497-3508.

Vector-initiated transitive RNA interference in the filamentous fungus Aspergillus oryzae

RNA interference (RNAi), modulates gene expression via cleavage of double-stranded RNA (dsRNA) by Dicer, producing 21–25 nucleotide silence-inducing RNAs (siRNAs). In association with Argonaute containing complexes, these siRNAs target sequence-specific degradation of the homologous single-stranded messenger RNA. In the majority of eukaryotes, degradation occurs within the boundaries of the dsRNA target. In Arabidopsis thaliana and Caenorhabditis elegans, gene silencing can also take place transitively, impacting transcripts from coding sequences that are adjacent to the intended target gene. Here we demonstrate effective transitive RNAi in the ascomycete Aspergillus oryzae. Fragments of 174bp and 499bp derived from the A. oryzae wA gene involved in spore color development, were inserted immediately upstream of an inverted repeat derived from the Escherichia coli gene encoding for Hygromycin Phosphotransferase B (hph), which provided a double-stranded hph RNAi trigger. Introduction of this construct into A. oryzae host cells produced transformants with spores that were lighter in color than those of wild type. Real-time RT-qPCR analysis demonstrated a direct correspondence of steady-state wA mRNA level to spore color. An A. oryzae strain deficient in RNA-dependent RNA Polymerase (RdRP) produced exclusively wild type colored spores when transformed with a wA transitive RNAi construct. Conversely, increased expression of RdRP enhanced the incidence of wA gene silencing via transitive RNAi.

Proteins and Enzymes from Marine Resources

Sea products are valuable resources of natural substances such as lipids, polysaccharides, enzymes, vitamins, and proteins. In this issue, a variety of fish proteases (trypsin, alcalase, etc.) have been characterized and sometimes purified. Many of these enzymes display potentially interesting new biochemical properties for industrial applications. Their potential adequacy for biotechnological applications such as chitin extraction was demonstrated. Biochemical characteristics of crude alkaline protease extracts from the viscera of goby (Zosterisessor ophiocephalus), thornback ray (Raja clavata), and scorpionfish (Scorpaena scrofa) were studied. At least four caseinolytic proteases bands were observed in zymogram of each enzyme preparation. These proteolytic preparations were successfully used in the deproteinization of shrimp wastes for chitin extraction. Furthermore, a trypsin was purified from the pyloric ceca of Pacific cod (Gadus macrocephalus) using chromatographic methods. The cod trypsin was successfully applied to catalyze dipeptide synthesis using series of “inverse substrates.” A Mackerel trypsin was also purified from defatted viscera by supercritical carbon dioxide, and its biochemical properties were studied. Recent biotechnological progresses require the research of new bioactive peptides from natural resources that can be used as an alternative to chemical products in various applications (food, cosmetics, drugs, etc.). Sea products proteins digestion using bacterial proteases generated protein hydrolysates displaying interesting biochemical properties and antioxidant activity. Antioxidative activities and biochemical properties of protein hydrolysates with different hydrolysis degrees, prepared from cuttlefish (Sepia officinalis) using an Alcalase and Bacillus licheniformis NH1 proteases were determined. The antioxidant activities of cuttlefish protein hydrolysates increased with increasing the hydrolysis degree. Antioxidative activity was concentration dependent. Furthermore, both Alcalase and NH1 protein hydrolysates were able to retard lipid peroxidation and β-carotene-linoleic acid oxidation. In addition, cuttlefish protein hydrolysates have a high percentage of essential amino acids and could be used as supplement to poorly balanced dietary proteins. Furthermore, carbohydrate-binding lectins were also extracted from many marine resources. This issue highlights the potential of sea products as valuable resources of bioactive peptides and proteins such as hydrolytic enzymes, lectins, and antioxidants. These resources might be of great interest for industrial biotechnological processes using safe and natural materials such as peptides or enzymes. Dicer is an RNase III enzyme with two catalytic subunits, which catalyzes the cleavage of double-stranded RNA to small interfering RNAs and micro-RNAs, which are mainly involved in invasive nucleic acid defense and endogenous genes regulation. In addition, Dicer is thought to be involved in defense mechanism against foreign nucleic acids such as viruses. A paper focused on the recent progress of Dicer-related research and discussed potential RNA interference pathways in aquatic species. Dicer is abundantly expressed in embryos, indicating the importance of the protein in early embryonic development. Nabil Miled Moncef Nasri Hideki Kishimura Faouzi Ben Rebah

Dicer Functions in Aquatic Species

Dicer is an RNase III enzyme with two catalytic subunits, which catalyzes the cleavage of double-stranded RNA to small interfering RNAs and micro-RNAs, which are mainly involved in invasive nucleic acid defense and endogenous genes regulation. Dicer is abundantly expressed in embryos, indicating the importance of the protein in early embryonic development. In addition, Dicer is thought to be involved in defense mechanism against foreign nucleic acids such as viruses. This paper will mainly focus on the recent progress of Dicer-related research and discuss potential RNA interference pathways in aquatic species.

Correction: Functional Role of Glutamine 28 and Arginine 39 in Double Stranded RNA Cleavage by Human Pancreatic Ribonuclease

Correction: Functional Role of Glutamine 28 and Arginine 39 in Double Stranded RNA Cleavage by Human Pancreatic Ribonuclease

Functional Role of Glutamine 28 and Arginine 39 in Double Stranded RNA Cleavage by Human Pancreatic Ribonuclease

Human pancreatic ribonuclease (HPR), a member of RNase A superfamily, has a high activity on double stranded (ds) RNA. By virtue of this activity HPR appears to be involved in the host-defense against pathogenic viruses. To delineate the mechanism of dsRNA cleavage by HPR, we have investigated the role of glutamine 28 and arginine 39 of HPR in its activity on dsRNA. A non-basic residue glycine 38, earlier shown to be important for dsRNA cleavage by HPR was also included in the study in the context of glutamine 28 and arginine 39. Nine variants of HPR respectively containing Q28A, Q28L, R39A, G38D, Q28A/R39A, Q28L/R39A, Q28A/G38D, R39A/G38D and Q28A/G38D/R39A mutations were generated and functionally characterized. The far-UV CD-spectral analysis revealed all variants, except R39A, to have structures similar to that of HPR. The catalytic activity of all HPR variants on single stranded RNA substrate was similar to that of HPR, whereas on dsRNA, the catalytic efficiency of all single residue variants, except for the Q28L, was significantly reduced. The dsRNA cleavage activity of R39A/G38D and Q28A/G38D/R39A variants was most drastically reduced to 4% of that of HPR. The variants having reduced dsRNA cleavage activity also had reduction in their dsDNA melting activity and thermal stability. Our results indicate that in HPR both glutamine 28 and arginine 39 are important for the cleavage of dsRNA. Although these residues are not directly involved in catalysis, both arginine 39 and glutamine 28 appear to be facilitating a productive substrate-enzyme interaction during the dsRNA cleavage by HPR.

A Dicer-1 gene from white shrimp Litopenaeus vannamei: Expression pattern in the processes of immune response and larval development

Dicer is a member of the RNAase III family which catalyzes the cleavage of double-stranded RNA to small interfering RNAs and micro RNAs, and then directs sequence-specific gene silencing. In this paper, the full-length cDNA of Dicer-1 was cloned from white shrimp Litopenaeus vannamei (designated as LvDcr1). It was of 7636 bp, including a poly A tail, a 5′ UTR of 136 bp, a 3′ UTR of 78 bp, and an open reading frame (ORF) of 7422 bp encoding a putative protein of 2473 amino acids. The predicted amino acid sequence comprised all recognized functional domains found in other Dicer-1 homologues and showed the highest (97.7%) similarity to the Dicer-1 from tiger shrimp Penaeus mondon. Quantitative real-time PCR was employed to investigate the tissue distribution of LvDcr1 mRNA, and its expression in shrimps under virus challenge and larvae at different developmental stages. The LvDcr1 mRNA could be detected in all examined tissues with the highest expression level in hemocyte, and was up-regulated in hemocytes and gills after virus injection. These results indicated that LvDcr1 was involved in antiviral defense in adult shrimp. During the developmental stages from fertilized egg to postlarva VII, LvDcr1 was constitutively expressed at all examined development stages, but the expression level varied significantly. The highest expression level was observed in fertilized eggs and followed a decrease from fertilized egg to nauplius I stage. Then, the higher levels of expression were detected at nauplius V and postlarva stages. LvDcr1 expression regularly increased at the upper phase of nauplius, zoea and mysis stages than their prophase. The different expression of LvDcr1 in the larval stages could provide clues for understanding the early innate immunity in the process of shrimp larval development.

Continuous Fluorescence-Based Method for Assessing Dicer Cleavage Efficiency Reveals 3′ Overhang Nucleotide Preference

The cleavage of double-stranded RNA (dsRNA) molecules by Dicer is a critical step in silencing genes by the RNA induced silencing complex (RISC). The development of Dicer substrates as nucleic acid-based therapeutics brings about a need to rapidly evaluate chemically modified RNA molecules for their ability to be processed by Dicer. This study outlines a quantitative fluorescence quencher-based assay for studying the ability of Dicer substrates to be processed by Dicer. By using a dsRNA probe labeled with Cy5-Iowa Black RQ, a panel of unlabeled test substrates can be rapidly assessed in heterologous competition assays without the need for electrophoresis or radiolabeling. This assay was piloted by evaluation of 196 unlabeled 27-mer Dicer substrates with various overhang structures in a purified Dicer enzyme assay system. Results indicate that Dicer has no preference for the sequence of RNA in the main double-stranded region of the substrate. However, a preference for Dicer substrate RNAs (D-siRNAs) containing purine/purine 3' overhang nucleotides was observed. These results demonstrate that the method is useful for studying the effects of modified nucleic acids in addition to rapidly accessing the influence of potential regulatory factors on Dicer processing.

Carbodiimide EDC Induces Cross-Links That Stabilize RNase A C-Dimer against Dissociation: EDC Adducts Can Affect Protein Net Charge, Conformation, and Activity

RNase A self-associates under certain conditions to form a series of domain-swapped oligomers. These oligomers show high catalytic activity against double-stranded RNA and striking antitumor actions that are lacking in the monomer. However, the dissociation of these metastable oligomers limits their therapeutic potential. Here, a widely used conjugating agent, 1-ethyl-3-(3-dimethylaminoisopropyl) carbodiimide (EDC), has been used to induce the formation of amide bonds between carboxylate and amine groups of different subunits of the RNase A C-dimer. A cross-linked C-dimer which does not dissociate was isolated and was found have augmented enzymatic activity toward double-stranded RNA relative to the unmodified C-dimer. Characterization using chromatography, electrophoresis, mass spectrometry, and NMR spectroscopy revealed that the EDC-treated C-dimer retains its structure and contains one to three novel amide bonds. Moreover, both the EDC-treated C-dimer and EDC-treated RNase A monomer were found to carry an increased number of positive charges (about 6 ± 2 charges per subunit). These additional positive charges are presumably due to adduct formation with EDC, which neutralizes a negatively charged carboxylate group and couples it to a positively charged tertiary amine. The increased net positive charge endowed by EDC adducts likely contributes to the heightened cleavage of double-stranded RNA of the EDC-treated monomer and EDC-treated C-dimer. Further evidence for EDC adduct formation is provided by the reaction of EDC with a dipeptide Ac-Asp-Ala-NH(2) monitored by NMR spectroscopy and mass spectrometry. To determine if EDC adduct formation with proteins is common and how this affects protein net charge, conformation, and activity, four well-characterized proteins, ribonuclease Sa, hen lysozyme, carbonic anhydrase, and hemoglobin, were incubated with EDC and the products were characterized. EDC formed adducts with all these proteins, as judged by mass spectrometry and electrophoresis. Moreover, all suffered conformational changes ranging from slight structural modifications in the case of lysozyme, to denaturation for hemoglobin as measured by NMR spectroscopy and enzyme assays. We conclude that EDC adduct formation with proteins can affect their net charge, conformation, and enzymatic activity.

Expression of shRNA using intron splicing

RNA is considered a highly promising candidate for several applications such as gene knock-down, gene repair and gene therapy, where double-stranded RNA and RNA with catalytic activity are key players. A group I intron, a ribozyme catalyzing its own splicing reactions in the absence of any proteins, has generated interest for its potential utility in gene repair using trans-splicing. On the other hand, the induction of small interfering RNA, via double-stranded RNA cleavage in short hairpin RNA (shRNA) by the RNase* *enzyme DICER is a convenient and powerful mechanism for gene silencing. We constructed shRNA expression vectors directed against Firefly luciferase, in which the loop region of the shRNA was interrupted by an intron. The decreased levels of luciferase activity were measured in cultured cells as an index of the ribozyme splicing activity.

Homodimeric Structure and Double-stranded RNA Cleavage Activity of the C-terminal RNase III Domain of Human Dicer

Human Dicer contains two RNase III domains (RNase IIIa and RNase IIIb) that are responsible for the production of short interfering RNAs and microRNAs. These small RNAs induce gene silencing known as RNA interference. Here, we report the crystal structure of the C-terminal RNase III domain (RNase IIIb) of human Dicer at 2.0 Å resolution. The structure revealed that the RNase IIIb domain can form a tightly associated homodimer, which is similar to the dimers of the bacterial RNase III domains and the two RNase III domains of Giardia Dicer. Biochemical analysis showed that the RNase IIIb homodimer can cleave double-stranded RNAs (dsRNAs), and generate short dsRNAs with 2 nt 3′ overhang, which is characteristic of RNase III products. The RNase IIIb domain contained two magnesium ions per monomer around the active site. The distance between two Mg-1 ions is approximately 20.6 Å, almost identical with those observed in bacterial RNase III enzymes and Giardia Dicer, while the locations of two Mg-2 ions were not conserved at all. We presume that Mg-1 ions act as catalysts for dsRNA cleavage, while Mg-2 ions are involved in RNA binding.

The 5S rRNA maturase, ribonuclease M5, is a Toprim domain family member

The maturation of 5S ribosomal RNA in low G+C Gram-positive bacteria is catalyzed by a highly conserved, ∼190 residue, enzyme, called ribonuclease M5 (RNase M5). Sequence alignment had predicted that the N-terminal half of RNase M5 would consist of a Toprim domain, a protein fold found in type IA and type II topoisomerases, DnaG-like primases, OLD family nucleases and RecR proteins [L. Aravind, D. D. Leipe and E. V. Koonin (1998) Nucleic Acids Res., 26, 4205–4213]. Here, we present structural modelling data and a mutational analysis of RNase M5 that confirms this hypothesis. The N-terminal half of RNase M5 can be fitted to the Toprim domain of the DnaG catalytic core. Mutation of amino acid residues highly conserved among RNase M5 enzymes and members of the Toprim domain family showed that alteration of residues critical for topoisomerase and primase activity also had a dramatic effect on the cleavage of 5S rRNA precursor by RNase M5 both in vivo and in vitro. This suggests that the mechanisms of double-stranded RNA cleavage by RNase M5 and double-stranded DNA cleavage by members of the topoisomerase family are related.