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Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs.

ABSTRACT: Ribonuclease L (RNase L) is a metal-ion-independent endoribonuclease associated with antiviral and antibacterial defense, cancer and lifespan. Despite the biological significance of RNase L, the RNAs cleaved by this enzyme are poorly defined. In this study, we used deep sequencing methods to reveal the frequency and location of RNase L cleavage sites within host and viral RNAs. To make cDNA libraries, we exploited the 2', 3'-cyclic phosphate at the end of RNA fragments produced by RNase L and other metal-ion-independent endoribonucleases. We optimized and validated 2', 3'-cyclic phosphate cDNA synthesis and Illumina sequencing methods using viral RNAs cleaved with purified RNase L, viral RNAs cleaved with purified RNase A and RNA from uninfected and poliovirus-infected HeLa cells. Using these methods, we identified (i) discrete regions of hepatitis C virus and poliovirus RNA genomes that were profoundly susceptible to RNase L and other single-strand specific endoribonucleases, (ii) RNase L-dependent and RNase L-independent cleavage sites within ribosomal RNAs (rRNAs) and (iii) 2', 3'-cyclic phosphates at the ends of 5S rRNA and U6 snRNA. Monitoring the frequency and location of metal-ion-independent endoribonuclease cleavage sites within host and viral RNAs reveals, in part, how these enzymes contribute to health and disease.

SUBMITTER: Cooper DA

PROVIDER: S-EPMC4005677 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Ribonuclease L and Metal-Ion-Independent Endoribonuclease Cleavage Sites in Host and Viral RNAs

Project description:We used 2', 3'-cyclic phosphate cDNA synthesis and Illumina sequencing to identify and quantify metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs. To make cDNA libraries, we exploited the 2’, 3’-cyclic phosphate at the end of RNA fragments produced by RNase L and other metal-ion-independent endoribonucleases. Using Arabidopsis thaliana tRNA ligase, RNA fragments with 2’, 3’-cyclic phosphates were covalently attached to defined RNA linkers containing an 8-base long unique molecular identifier (UMI) sequence. Libraries prepared in this manner contain cDNA derived exclusively from RNA fragments with 2', 3'-cyclic phosphates. The UMI sequence allows for detailed quantitation. We optimized and validated 2’, 3’-cyclic phosphate cDNA synthesis and Illumina sequencing methods using viral RNAs cleaved with purified RNase L, viral RNAs cleaved with purified RNase A, and RNA from uninfected and poliovirus-infected HeLa cells.

2014-02-07 | E-GEOD-52489 | biostudies-arrayexpress

Ribonuclease L and Metal-Ion-Independent Endoribonuclease Cleavage Sites in Host and Viral RNAs

Project description:We used 2', 3'-cyclic phosphate cDNA synthesis and Illumina sequencing to identify and quantify metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs.

2014-02-07 | GSE52489 | GEO

Ribonuclease L and Metal-Ion-Independent Endoribonuclease Cleavage Sites in Host and Viral RNAs

Project description:Ribonuclease L and Metal-Ion-Independent Endoribonuclease Cleavage Sites in Host and Viral RNAs

| PRJNA229098 | ENA

Site-Specific Cleavage of RNAs Derived from the PIM1 3'-UTR by a Metal-Free Artificial Ribonuclease.

Project description:Oligonucleotide conjugates of tris(2-aminobenzimidazole) have been reported previously to cleave complementary RNA strands with high levels of sequence and site specificity. The RNA substrates used in these studies were oligonucleotides not longer than 29-mers. Here we show that ~150⁻400-mer model transcripts derived from the 3'-untranslated region of the PIM1 mRNA reacted with rates and specificities comparable to those of short oligonucleotide substrates. The replacement of DNA by DNA/LNA mixmers further increased the cleavage rate. Tris(2-aminobenzimidazoles) were designed to interact with phosphates and phosphate esters. A cell, however, contains large amounts of phosphorylated species that may cause competitive inhibition of RNA cleavage. It is thus important to note that no loss in reaction rates was observed in phosphate buffer. This opens the way to in-cell applications for this type of artificial nuclease. Furthermore, we disclose a new synthetic method giving access to tris(2-aminobenzimidazoles) in multigram amounts.

| S-EPMC6412833 | biostudies-literature

Homodimerisation-independent cleavage of dsRNA by a pestiviral nicking endoribonuclease.

Project description:The glycoprotein Erns plays a central role in the biology of the pestivirus bovine viral diarrhea virus (BVDV). This soluble endonuclease mediates the escape from an interferon (IFN) response in the infected fetus, thereby permitting the establishment of persistent infection. Viral single-stranded (ss) and double-stranded (ds) RNA act as potent IFN inducing signals and we previously showed that Erns efficiently cleaves these substrates, thereby inhibiting an IFN response that is crucial for successful fetal infection. Considering that a large variety of RNases and DNases require dimerisation to cleave double-stranded substrates, the activity of Erns against dsRNA was postulated to depend on homodimer formation mediated by disulfide bonds involving residue Cys171. Here, we show that monomeric Erns is equally able to cleave dsRNA and to inhibit dsRNA-induced IFN synthesis as the wild-type form. Furthermore, both forms were able to degrade RNA within a DNA/RNA- as well as within a methylated RNA/RNA-hybrid, with the DNA and the methylated RNA strand being resistant to degradation. These results support our model that Erns acts as 'nicking endoribonuclease' degrading ssRNA within double-stranded substrates. This efficiently prevents the activation of IFN and helps to maintain a state of innate immunotolerance in persistently infected animals.

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Calcium inhibition of ribonuclease H1 two-metal ion catalysis.

Project description:Most phosphate-processing enzymes require Mg(2+) as a cofactor to catalyze nucleotide cleavage and transfer reactions. Ca(2+) ions inhibit many of these enzymatic activities, despite Ca(2+) and Mg(2+) having comparable binding affinities and overall biological abundances. Here we study the molecular details of the calcium inhibition mechanism for phosphodiester cleavage, an essential reaction in the metabolism of nucleic acids and nucleotides, by comparing Ca(2+)- and Mg(2+) catalyzed reactions. We study the functional roles of the specific metal ion sites A and B in enabling the catalytic cleavage of an RNA/DNA hybrid substrate by B. halodurans ribonuclease (RNase) H1 using hybrid quantum-mechanics/molecular mechanics (QM/MM) free energy calculations. We find that Ca(2+) substitution of either of the two active-site Mg(2+) ions substantially increases the height of the reaction barrier and thereby abolishes the catalytic activity. Remarkably, Ca(2+) at the A site is inactive also in Mg(2+)-optimized active-site structures along the reaction path, whereas Mg(2+) substitution recovers activity in Ca(2+)-optimized structures. Geometric changes resulting from Ca(2+) substitution at metal ion site A may thus be a secondary factor in the loss of catalytic activity. By contrast, at metal ion site B geometry plays a more important role, with only a partial recovery of activity after Mg(2+) substitution in Ca(2+)-optimized structures. Ca(2+)-substitution also leads to a change in mechanism, with deprotonation of the water nucleophile requiring a closer approach to the scissile phosphate, which in turn increases the barrier. As a result, Ca(2+) is less efficient in activating the water. As a likely cause for the different reactivities of Mg(2+) and Ca(2+) ions in site A, we identify differences in charge transfer to the ions and the associated decrease in the pKa of the oxygen nucleophile attacking the phosphate group.

| S-EPMC3985467 | biostudies-literature

Coronavirus endoribonuclease targets viral polyuridine sequences to evade activating host sensors.

Project description:Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5'-polyuridines from negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wild-type-infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections.

| S-EPMC7149396 | biostudies-literature

Phosphodiester cleavage in ribonuclease H occurs via an associative two-metal-aided catalytic mechanism.

Project description:Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase (NT) superfamily and hydrolyzes the phosphodiester linkages that form the backbone of the RNA strand in RNA x DNA hybrids. This enzyme is implicated in replication initiation and DNA topology restoration and represents a very promising target for anti-HIV drug design. Structural information has been provided by high-resolution crystal structures of the complex RNase H/RNA x DNA from Bacillus halodurans (Bh), which reveals that two metal ions are required for formation of a catalytic active complex. Here, we use classical force field-based and quantum mechanics/molecular mechanics calculations for modeling the nucleotidyl transfer reaction in RNase H, clarifying the role of the metal ions and the nature of the nucleophile (water versus hydroxide ion). During the catalysis, the two metal ions act cooperatively, facilitating nucleophile formation and stabilizing both transition state and leaving group. Importantly, the two Mg(2+) metals also support the formation of a meta-stable phosphorane intermediate along the reaction, which resembles the phosphorane intermediate structure obtained only in the debated beta-phosphoglucomutase crystal (Lahiri, S. D.; et al. Science 2003, 299 (5615), 2067-2071). The nucleophile formation (i.e., water deprotonation) can be achieved in situ, after migration of one proton from the water to the scissile phosphate in the transition state. This proton transfer is actually mediated by solvation water molecules. Due to the highly conserved nature of the enzymatic bimetal motif, these results might also be relevant for structurally similar enzymes belonging to the NT superfamily.

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Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX.

Project description:Many viruses express factors that reduce host gene expression through widespread degradation of cellular mRNA. An example of this class of proteins is the mRNA-targeting endoribonuclease SOX from the gamma-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV). Previous studies indicated that cleavage of messenger RNAs (mRNA) by SOX occurs at specific locations defined by the sequence of the target RNA, which is at odds with the down-regulation of a large portion of cellular transcripts. In this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify SOX cleavage sites across the mRNA transcriptome. These data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. This degenerate element is well represented in both human and KSHV mRNA, and its presence correlates with RNA destabilization by SOX. This represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables RNA cleavage at specific locations without restricting the range of targets. Furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity.

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Sequence-independent characterization of viruses based on the pattern of viral small RNAs produced by the host.

Project description:Virus surveillance in vector insects is potentially of great benefit to public health. Large-scale sequencing of small and long RNAs has previously been used to detect viruses, but without any formal comparison of different strategies. Furthermore, the identification of viral sequences largely depends on similarity searches against reference databases. Here, we developed a sequence-independent strategy based on virus-derived small RNAs produced by the host response, such as the RNA interference pathway. In insects, we compared sequences of small and long RNAs, demonstrating that viral sequences are enriched in the small RNA fraction. We also noted that the small RNA size profile is a unique signature for each virus and can be used to identify novel viral sequences without known relatives in reference databases. Using this strategy, we characterized six novel viruses in the viromes of laboratory fruit flies and wild populations of two insect vectors: mosquitoes and sandflies. We also show that the small RNA profile could be used to infer viral tropism for ovaries among other aspects of virus biology. Additionally, our results suggest that virus detection utilizing small RNAs can also be applied to vertebrates, although not as efficiently as to plants and insects.

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