Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Small RNAs generated in vitro by budding-yeast Dicer

ABSTRACT: 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.

ORGANISM(S): Vanderwaltozyma polyspora

PROVIDER: GSE29168 | GEO | 2011/05/11

SECONDARY ACCESSION(S): PRJNA140091

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

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. High-throughput sequencing of small RNAs generated by in vitro processing of long dsRNA using Kluyveromyces polysporus Dicer

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

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

An Extended dsRBD with a Novel Zinc-Binding Motif Mediates Nuclear Retention of Fission Yeast Dicer

Project description:Dicer proteins function in RNA interference (RNAi) pathways by generating small RNAs (sRNAs). Here we report the solution structure of the C-terminal domain of Schizosaccharomyces pombe Dicer (Dcr1). The structure reveals an unusual double-stranded RNA binding domain (dsRBD) fold embedding a novel zinc-binding motif that is conserved among dicers in yeast. Although the C-terminal domain of Dcr1 still binds nucleic acids, this property is dispensable for proper functioning of Dcr1. In contrast, disruption of zinc coordination renders Dcr1 mainly cytoplasmic and leads to remarkable changes in gene expression and loss of heterochromatin assembly. In summary, our results reveal novel insights into the mechanism of nuclear retention of Dcr1 and raise the possibility that this new class of dsRBDs might generally function in nucleo-cytoplasmic trafficking and not substrate binding. The C-terminal domain of Dcr1 constitutes a novel regulatory module that might represent a potential target for therapeutic intervention with fungal diseases.

2011-09-23 | GSE30586 | GEO

An Extended dsRBD with a Novel Zinc-Binding Motif Mediates Nuclear Retention of Fission Yeast Dicer

2011-09-23 | GSE30549 | GEO

An Extended dsRBD with a Novel Zinc-Binding Motif Mediates Nuclear Retention of Fission Yeast Dicer

2011-09-22 | E-GEOD-30586 | biostudies-arrayexpress

An Extended dsRBD with a Novel Zinc-Binding Motif Mediates Nuclear Retention of Fission Yeast Dicer

2011-09-22 | E-GEOD-30549 | biostudies-arrayexpress

Bacterial RNase III cleaves long dsRNA at preferred sites

Project description: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.

2019-05-26 | GSE120052 | GEO

Function of three homologs of RNase III in Synechococcus sp. PCC 7002

Project description:RNase III is a ribonucleases that recognizes and cleaves double-stranded RNA. RNase III has been known be involved in rRNA processing, but has many additional roles controlling both expression and RNA turnover of specific messages. Many organisms have just one RNase III while some have both a full length RNase III and a mini-III that lacks the double-stranded RNA binding domain. The cyanobacteria Synechococcus sp. PCC 7002 has three homologs of RNase III that are unessential even when deleted in combination. We were interested what coding regions these RNase III enzymes were influencing and if they had redundant or distinct specificities. To address these questions we collected samples for RNA-sequencing from WT, the single, double, and triple RNase III mutants in triplicate. Approximately 20% of genes were differentially expressed in various mutants with some operons and regulons showing complex changes in expression levels between mutants. We describe the role of two RNase III’s in 23S rRNA maturation, and show how the third is involved in copy number regulation of one of the six plasmids (pAQ3). Purified enzymes were capable of cleaving some E. coli RNase III target sequences, highlighting the remarkably conserved substrate specificity between organisms yet complex regulation of gene expression.

2018-01-16 | GSE99279 | 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

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

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