Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Development of a ribonuclease containing a G4-specific binding motif for programmable RNA cleavage.

ABSTRACT: We developed a ribonuclease for site-specific targeting and cleavage of single-stranded RNA. The engineered RNase protein was constructed by incorporating two independent functional domains, an RNase HI domain that could cleave the RNA strand in a DNA-RNA hybrid, and a domain of the RHAU protein that could selectively recognize a parallel DNA G-quadruplex (G4). The newly designed RNase first recruits a DNA guide oligonucleotide containing both a parallel G4 motif and a template sequence complementary to the target RNA. This RNase:DNA complex targets and efficiently cleaves the single-stranded RNA in a site-specific manner. A major cleavage site occurs at the RNA region that is complementary to the DNA template sequence. The newly designed RNase can serve as a simple tool for RNA manipulation and probing RNA structure.

SUBMITTER: Dang DT

PROVIDER: S-EPMC6520340 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Development of a ribonuclease containing a G4-specific binding motif for programmable RNA cleavage.

Dang Dung Thanh DT Phan Anh Tuân AT

Scientific reports 20190515 1

We developed a ribonuclease for site-specific targeting and cleavage of single-stranded RNA. The engineered RNase protein was constructed by incorporating two independent functional domains, an RNase HI domain that could cleave the RNA strand in a DNA-RNA hybrid, and a domain of the RHAU protein that could selectively recognize a parallel DNA G-quadruplex (G4). The newly designed RNase first recruits a DNA guide oligonucleotide containing both a parallel G4 motif and a template sequence compleme ...[more]

PMID: 31092834

Similar Datasets

Human endonuclease V is a ribonuclease specific for inosine-containing RNA.

Project description:Deamination of DNA bases can create missense mutations predisposing humans to cancer and also interfere with other basic molecular genetic processes; this deamination generates deoxyinosine from deoxyadenosine. In Escherichia coli, the highly conserved endonuclease V is involved in alternative excision repair that removes deoxyinosine from DNA. However, its exact activities and roles in humans are unknown. Here we characterize the FLJ35220 protein, the human homologue of E. coli endonuclease V, hEndoV as a ribonuclease specific for inosine-containing RNA. hEndoV preferentially binds to RNA and efficiently hydrolyses the second phosphodiester bond located 3' to the inosine in unpaired inosine-containing ssRNA regions in dsRNA. It localizes to the cytoplasm in cells. The ribonuclease activity is promoted by Tudor staphylococcal nuclease and detected on inosine-containing dsRNA created by the action of adenosine deaminases acting on RNA. These results demonstrate that hEndoV controls the fate of inosine-containing RNA in humans.

| S-EPMC3741642 | biostudies-literature

Ribonuclease III mechanisms of double-stranded RNA cleavage.

Project description:Double-stranded(ds) RNA has diverse roles in gene expression and regulation, host defense, and genome surveillance in bacterial and eukaryotic cells. A central aspect of dsRNA function is its selective recognition and cleavage by members of the ribonuclease III (RNase III) family of divalent-metal-ion-dependent phosphodiesterases. The processing of dsRNA by RNase III family members is an essential step in the maturation and decay of coding and noncoding RNAs, including miRNAs and siRNAs. RNase III, as first purified from Escherichia coli, has served as a biochemically well-characterized prototype, and other bacterial orthologs provided the first structural information. RNase III family members share a unique fold (RNase III domain) that can dimerize to form a structure that binds dsRNA and cleaves phosphodiesters on each strand, providing the characteristic 2 nt, 3'-overhang product ends. Ongoing studies are uncovering the functions of additional domains, including, inter alia, the dsRNA-binding and PAZ domains that cooperate with the RNase III domain to select target sites, regulate activity, confer processivity, and support the recognition of structurally diverse substrates. RNase III enzymes function in multicomponent assemblies that are regulated by diverse inputs, and at least one RNase III-related polypeptide can function as a noncatalytic, dsRNA-binding protein. This review summarizes the current knowledge of the mechanisms of catalysis and target site selection of RNase III family members, and also addresses less well understood aspects of these enzymes and their interactions with dsRNA.

| S-EPMC3867540 | biostudies-literature

Programmable RNA targeting by bacterial Argonaute nucleases with unconventional guide binding and cleavage specificity.

Project description:Argonaute proteins are programmable nucleases that have defense and regulatory functions in both eukaryotes and prokaryotes. All known prokaryotic Argonautes (pAgos) characterized so far act on DNA targets. Here, we describe a new class of pAgos that uniquely use DNA guides to process RNA targets. The biochemical and structural analysis of Pseudooceanicola lipolyticus pAgo (PliAgo) reveals an unusual organization of the guide binding pocket that does not rely on divalent cations and the canonical set of contacts for 5'-end interactions. Unconventional interactions of PliAgo with the 5'-phosphate of guide DNA define its new position within pAgo and shift the site of target RNA cleavage in comparison with known Argonautes. The specificity for RNA over DNA is defined by ribonucleotide residues at the cleavage site. The analysed pAgos sense mismatches and modifications in the RNA target. The results broaden our understanding of prokaryotic defense systems and extend the spectrum of programmable nucleases with potential use in RNA technology.

| S-EPMC9360449 | biostudies-literature

Programmable RNA recognition and cleavage by CRISPR/Cas9.

Project description:The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

| S-EPMC4268322 | biostudies-literature

Sequence-specific RNA cleavage by PNA conjugates of the metal-free artificial ribonuclease tris(2-aminobenzimidazole).

Project description:Tris(2-aminobenzimidazole) conjugates with antisense oligonucleotides are effective site-specific RNA cleavers. Their mechanism of action is independent of metal ions. Here we investigate conjugates with peptide nucleic acids (PNA). RNA degradation occurs with similar rates and substrate specificities as in experiments with DNA conjugates we performed earlier. Although aggregation phenomena are observed in some cases, proper substrate recognition is not compromised. While our previous synthesis of 2-aminobenzimidazoles required an HgO induced cyclization step, a mercury free variant is described herein.

| S-EPMC4419560 | biostudies-literature

RNA-binding strategies common to cold-shock domain- and RNA recognition motif-containing proteins.

Project description:Numerous RNA-binding proteins have modular structures, comprising one or several copies of a selective RNA-binding domain generally coupled to an auxiliary domain that binds RNA non-specifically. We have built and compared homology-based models of the cold-shock domain (CSD) of the Xenopus protein, FRGY2, and of the third RNA recognition motif (RRM) of the ubiquitous nucleolar protein, nucleolin. Our model of the CSD(FRG)-RNA complex constitutes the first prediction of the three-dimensional structure of a CSD-RNA complex and is consistent with the hypothesis of a convergent evolution of CSD and RRM towards a related single-stranded RNA-binding surface. Circular dichroism spectroscopy studies have revealed that these RNA-binding domains are capable of orchestrating similar types of RNA conformational change. Our results further show that the respective auxiliary domains, despite their lack of sequence homology, are functionally equivalent and indispensable for modulating the properties of the specific RNA-binding domains. A comparative analysis of FRGY2 and nucleolin C-terminal domains has revealed common structural features representing the signature of a particular type of auxiliary domain, which has co-evolved with the CSD and the RRM.

| S-EPMC55715 | biostudies-literature

Enhanced RNA cleavage within bulge-loops by an artificial ribonuclease.

Project description:Cleavage of phosphodiester bonds by small ribonuclease mimics within different bulge-loops of RNA was investigated. Bulge-loops of different size (1-7 nt) and sequence composition were formed in a 3' terminal fragment of influenza virus M2 RNA (96 nt) by hybridization of complementary oligodeoxynucleotides. Small bulges (up to 4 nt) were readily formed upon oligonucleotide hybridization, whereas hybridization of the RNA to the oligonucleotides designed to produce larger bulges resulted in formation of several alternative structures. A synthetic ribonuclease mimic displaying Pyr-Pu cleavage specificity cleaved CpA motifs located within bulges faster than similar motifs within the rest of the RNA. In the presence of 10 mM MgCl2, 75% of the cleavage products resulted from the attack of this motif. Thus, selective RNA cleavage at a single target phosphodiester bond was achieved by using bulge forming oligonucleotides and a small ribonuclease A mimic.

| S-EPMC549568 | biostudies-literature

Sequence-specific cleavage of the RNA strand in DNA-RNA hybrids by the fusion of ribonuclease H with a zinc finger.

Project description:Ribonucleases (RNases) are valuable tools applied in the analysis of RNA sequence, structure and function. Their substrate specificity is limited to recognition of single bases or distinct secondary structures in the substrate. Currently, there are no RNases available for purely sequence-dependent fragmentation of RNA. Here, we report the development of a new enzyme that cleaves the RNA strand in DNA-RNA hybrids 5 nt from a nonanucleotide recognition sequence. The enzyme was constructed by fusing two functionally independent domains, a RNase HI, that hydrolyzes RNA in DNA-RNA hybrids in processive and sequence-independent manner, and a zinc finger that recognizes a sequence in DNA-RNA hybrids. The optimization of the fusion enzyme's specificity was guided by a structural model of the protein-substrate complex and involved a number of steps, including site-directed mutagenesis of the RNase moiety and optimization of the interdomain linker length. Methods for engineering zinc finger domains with new sequence specificities are readily available, making it feasible to acquire a library of RNases that recognize and cleave a variety of sequences, much like the commercially available assortment of restriction enzymes. Potentially, zinc finger-RNase HI fusions may, in addition to in vitro applications, be used in vivo for targeted RNA degradation.

| S-EPMC3526281 | biostudies-literature

Identification of RNA binding motif proteins essential for cardiovascular development.

Project description:BACKGROUND: We recently identified Rbm24 as a novel gene expressed during mouse cardiac development. Due to its tightly restricted and persistent expression from formation of the cardiac crescent onwards and later in forming vasculature we posited it to be a key player in cardiogenesis with additional roles in vasculogenesis and angiogenesis. RESULTS: To determine the role of this gene in cardiac development, we have identified its zebrafish orthologs (rbm24a and rbm24b), and functionally evaluated them during zebrafish embryogenesis. Consistent with our underlying hypothesis, reduction in expression of either ortholog through injection of morpholino antisense oligonucleotides results in cardiogenic defects including cardiac looping and reduced circulation, leading to increasing pericardial edema over time. Additionally, morphant embryos for either ortholog display incompletely overlapping defects in the forming vasculature of the dorsal aorta (DA), posterior caudal vein (PCV) and caudal vein (CV) which are the first blood vessels to form in the embryo. Vasculogenesis and early angiogenesis in the trunk were similarly compromised in rbm24 morphant embryos at 48 hours post fertilization (hpf). Subsequent vascular maintenance was impaired in both rbm24 morphants with substantial vessel degradation noted at 72 hpf. CONCLUSION: Taken collectively, our functional data support the hypothesis that rbm24a and rbm24b are key developmental cardiac genes with unequal roles in cardiovascular formation.

| S-EPMC3277282 | biostudies-literature

Specific Targeting and Imaging of RNA G-Quadruplex (rG4) Structure Using Non-G4-Containing l-RNA Aptamer and Fluorogenic l-Aptamer.

Project description:RNA G-quadruplex structures (rG4s) play important roles in the regulation of biological processes. So far, all the l-RNA aptamers developed to target rG4 of interest contain G4 motif itself, raising the question of whether non-G4-containing l-RNA aptamer can be developed to target rG4. Furthermore, it is unclear whether an l-Aptamer-based tool can be generated for G4 detection in vitro and imaging in cells. Herein, a new strategy is designed using a low GC content template library to develop a novel non-G4-containing l-RNA aptamer with strong binding affinity and improved binding specificity to rG4 of interest. The first non-G4-containing l-Aptamer, l-Apt.1-1, is identified with nanomolar binding affinity to amyloid precursor protein (APP) D-rG4. l-Apt.1-1 is applied to control APP gene expression in cells via targeting APP D-rG4 structure. Moreover, the first l-RNA-based fluorogenic bi-functional aptamer (FLAP) system is developed, and l-Apt.1-1_Pepper is engineered for in vitro detection and cellular imaging of APP D-rG4. This work provides an original approach for developing non-G4-containing l-RNA aptamer for rG4 targeting, and the novel l-Apt.1-1 developed for APP gene regulation, as well as the l-Apt.1-1_Pepper generated for imaging of APP rG4 structure can be further used in other applications in vitro and in cells.

| S-EPMC11926469 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data