Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Bacterial RNase III cleaves long dsRNA at preferred sites

ABSTRACT: Members of the ribonuclease (RNase) III family regulate gene expression by processing double-stranded (ds) RNA. The founding member of the family, Escherichia coli (Ec) RNase III, is the most comprehensively studied and its E38A mutant (EcE38A) is an economical reagent for the preparation of small interfering (si) RNA cocktails. Previously, it was shown that EcRNase III recognizes dsRNA with little specificity and that EcE38A mainly produces 23-nucleotide (nt) siRNAs. To characterize substrate specificity and product size, we performed in vitro cleavage of dsRNAs by bacterial RNase IIIs and delineated the cleavage products by next generation sequencing. Surprisingly, we found that RNase III cleaves dsRNA at preferred sites and most siRNAs produced by EcE38A are 22 nt long. We eliminated the sequence specificity of EcE38A through the introduction of additional mutations, thereby creating a reagent that is ideally suited for producing heterogeneous siRNA cocktails to be used in gene silencing studies.

ORGANISM(S): Escherichia coli Aquifex aeolicus

PROVIDER: GSE120052 | GEO | 2019/05/26

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Impact of two RNaseIII-like protein on the small RNA repertoire

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

2015-10-01 | GSE71782 | GEO

Bacterial RNase III cleaves long dsRNA at preferred sites

Project description:Bacterial RNase III cleaves long dsRNA at preferred sites

| PRJNA491502 | ENA

Determinants of selectivity in the dicing mechanism

Project description:The RNase III enzyme, DICER, is instrumental in the production of small RNAs, including miRNAs and siRNAs, by cleaving their precursors, such as pre-miRNAs, shRNAs, and long duplex RNAs. Utilizing High-throughput (HT) cleavage assays, our study delves into the cleavage activity of DICER. We challenge the widely accepted 2-nt loop counting rule in the previous study, revealing a divergent mechanism, the bipartite base pairing rule. This rule directs DICER's cleavage sites via the RNase III domain. Moreover, we demystify the recognition mechanism of the previously identified YCR motif. Building on this understanding, a secondary YCR motif that also influences DICER's cleavage sites has been discovered. We also address a long-debated issue concerning DICER's cleavage sites on long stem RNAs, such as pre-siRNAs or long shRNAs/pre-miRNAs. Our study shows that the dsRBD plays a crucial role in determining the cleavage sites of DICER in long-stem RNAs. In sum, our research provides a comprehensive understanding of several fundamental DICER mechanisms, challenging the long-standing model of the loop counting rule. This newfound knowledge reshapes our understanding of DICER's mechanisms, providing a robust foundation for future studies investigating the vast number of DICER mutations linked to various diseases.

2024-06-01 | GSE252434 | GEO

Determinants of selectivity in the dicing mechanism III

2024-07-13 | GSE272135 | GEO

Determinants of selectivity in the dicing mechanism

2024-06-01 | GSE252433 | GEO

Small RNAs generated in vitro by budding-yeast Dicer

Project description:The Dicer ribonuclease III (RNase III) enzymes process long double-stranded RNA (dsRNA) into the small interfering RNAs (siRNAs) that direct RNA interference. Here, we describe the structure and activity of a catalytically active fragment of Kluyveromyces polysporus Dcr1, which represents the noncanonical Dicers found in budding yeast. The crystal structure reveals a homodimer that resembles bacterial RNase III but includes a novel N-terminal domain and newly identified catalytic residues conserved throughout eukaryotic RNase III enzymes. Biochemical analyses show that Dcr1 dimers bind cooperatively along the dsRNA substrate and cleave at precise intervals based on the distance between consecutive active sites. Thus, unlike canonical Dicers, which successively remove siRNA duplexes from the dsRNA termini, Dcr1 initiates processing in the interior and works outward. The distinct mechanism of budding-yeast Dicers establishes a novel paradigm for natural protein-based molecular rulers and imparts substrate preferences with ramifications for biological function.

2011-05-11 | GSE29168 | GEO

Cotranscriptional RNA cleavage by Drosha homolog Pac1 triggers transcription termination in fission yeast. [RNA-Seq]

Project description:RNAse III is an evolutionarily conserved family of endoribonuclease that cleaves dsRNA structures. Here we studied RNAse III homolog Pac1 In fission yeast to shed light on underappreciated roles of Drosha. We found that Pac1 co-transcriptional cleavage of nascent hairpin RNA structures trigger transcriptional termination by creating an entry point for “torpedo” exonucleases – that is, exonuclease that degrade the nascent RNA until they bump into the transcribing polymerases. As such termination pathway decouple termination from pre-mRNA polyadenylation, the nascent transcript are destabilized upon Pac1 cleavage, therefore expending the paradigm of post-transcriptional regulation of gene expression.

2021-02-19 | GSE167041 | GEO

Cotranscriptional RNA cleavage by Drosha homolog Pac1 triggers transcription termination in fission yeast. [ChIP-Seq]

2021-02-19 | GSE167040 | GEO

Small RNAs generated in vitro by budding-yeast Dicer

2011-05-11 | E-GEOD-29168 | biostudies-arrayexpress

High-pressure sprayed siRNAs influence the efficiency but not the profile of transitive silencing

Project description:In plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)-inducing molecules produced by the endonucleolytic cleavage of double stranded RNAs (dsRNAs). In order to ensure robust RNAi, siRNAs are amplified through a positive feedback mechanism called transitivity. Transitivity relies on RNA-DIRECTED-RNA POLYMERASE 6 (RDR6)-mediated dsRNA synthesis using siRNA-targeted RNA. The newly synthesized dsRNA is subsequently cleaved into secondary siRNAs by DICER-LIKE (DCL) endonucleases. Just like primary siRNAs, secondary siRNAs are also loaded into ARGONAUTE proteins (AGOs) to form an RNA-induced silencing complex (RISC) reinforcing the cleavage of the target RNA. Although the molecular players underlying transitivity are well established, the mode of action of transitivity remains elusive. In this study, we investigated the influence of primary target sites on transgene silencing and transitivity using the GFP-expressing Nicotiana benthamiana 16C line, high pressure spraying protocol (HPSP), and synthetic 22-nucleotide (nt) long siRNAs. We found that the 22-nt siRNA targeting the 3’ of the GFP transgene was less efficient in inducing silencing when compared to the siRNAs targeting the 5’ and middle region of the GFP. Moreover, sRNA sequencing of locally silenced leaves showed that the amount but not the profile of secondary RNAs is shaped by the occupancy of the primary siRNA triggers on the target RNA. Our findings suggest that RDR6-mediated dsRNA synthesis is not primed by primary siRNAs and that dsRNA synthesis appears to be generally initiated at the 3’ end of the target RNA.

2021-12-01 | GSE189777 | GEO

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