Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Dicer's helicase domain discriminates dsRNA termini to promote an altered reaction mode.

ABSTRACT: The role of Dicer's helicase domain is enigmatic, but in vivo it is required for processing certain endogenous siRNA, but not miRNA. By using Caenorhabditis elegans extracts or purified Drosophila Dicer-2 we compared activities of wild-type enzymes and those containing mutations in the helicase domain. We found the helicase domain was essential for cleaving dsRNA with blunt or 5'-overhanging termini, but not those with 3' overhangs, as found on miRNA precursors. Further, blunt termini, but not 3' overhangs, led to increased siRNAs from internal regions of dsRNA; this activity required ATP and a functional helicase domain. Our data suggest that blunt or 5'-overhanging termini engage Dicer's helicase domain to facilitate accumulation of siRNAs from internal regions of a dsRNA, an activity suited for processing long siRNA precursors of low abundance, but not necessary for the single cleavage required for miRNA processing.

SUBMITTER: Welker NC

PROVIDER: S-EPMC3061311 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Dicer's helicase domain discriminates dsRNA termini to promote an altered reaction mode.

Welker Noah C NC Maity Tuhin S TS Ye Xuecheng X Aruscavage P Joseph PJ Krauchuk Ammie A AA Liu Qinghua Q Bass Brenda L BL

Molecular cell 20110301 5

The role of Dicer's helicase domain is enigmatic, but in vivo it is required for processing certain endogenous siRNA, but not miRNA. By using Caenorhabditis elegans extracts or purified Drosophila Dicer-2 we compared activities of wild-type enzymes and those containing mutations in the helicase domain. We found the helicase domain was essential for cleaving dsRNA with blunt or 5'-overhanging termini, but not those with 3' overhangs, as found on miRNA precursors. Further, blunt termini, but not 3 ...[more]

PMID: 21362554

Similar Datasets

Drosophila dicer-2 cleavage is mediated by helicase- and dsRNA termini-dependent states that are modulated by Loquacious-PD.

Project description:In previous studies we observed that the helicase domain of Drosophila Dicer-2 (dmDcr-2) governs substrate recognition and cleavage efficiency, and that dsRNA termini are key to this discrimination. We now provide a mechanistic basis for these observations. We show that discrimination of termini occurs during initial binding. Without ATP, dmDcr-2 binds 3' overhanging, but not blunt, termini. By contrast, with ATP, dmDcr-2 binds both types of termini, with highest-affinity binding observed with blunt dsRNA. In the presence of ATP, binding, cleavage, and ATP hydrolysis are optimal with BLT termini compared to 3'ovr termini. Limited proteolysis experiments suggest the optimal reactivity of BLT dsRNA is mediated by a conformational change that is dependent on ATP and the helicase domain. We find that dmDcr-2's partner protein, Loquacious-PD, alters termini dependence, enabling dmDcr-2 to cleave substrates normally refractory to cleavage, such as dsRNA with blocked, structured, or frayed ends.

| S-EPMC4433149 | biostudies-literature

Dicer uses distinct modules for recognizing dsRNA termini.

Project description:Invertebrates rely on Dicer to cleave viral double-stranded RNA (dsRNA), and Drosophila Dicer-2 distinguishes dsRNA substrates by their termini. Blunt termini promote processive cleavage, while 3' overhanging termini are cleaved distributively. To understand this discrimination, we used cryo-electron microscopy to solve structures of Drosophila Dicer-2 alone and in complex with blunt dsRNA. Whereas the Platform-PAZ domains have been considered the only Dicer domains that bind dsRNA termini, unexpectedly, we found that the helicase domain is required for binding blunt, but not 3' overhanging, termini. We further showed that blunt dsRNA is locally unwound and threaded through the helicase domain in an adenosine triphosphate-dependent manner. Our studies reveal a previously unrecognized mechanism for optimizing antiviral defense and set the stage for the discovery of helicase-dependent functions in other Dicers.

| S-EPMC6154394 | biostudies-literature

Loquacious-PD facilitates <i>Drosophila</i> Dicer-2 cleavage through interactions with the helicase domain and dsRNA.

Project description:Loquacious-PD (Loqs-PD) is required for biogenesis of many endogenous siRNAs in Drosophila In vitro, Loqs-PD enhances the rate of dsRNA cleavage by Dicer-2 and also enables processing of substrates normally refractory to cleavage. Using purified components, and Loqs-PD truncations, we provide a mechanistic basis for Loqs-PD functions. Our studies indicate that the 22 amino acids at the C terminus of Loqs-PD, including an FDF-like motif, directly interact with the Hel2 subdomain of Dicer-2's helicase domain. This interaction is RNA-independent, but we find that modulation of Dicer-2 cleavage also requires dsRNA binding by Loqs-PD. Furthermore, while the first dsRNA-binding motif of Loqs-PD is dispensable for enhancing cleavage of optimal substrates, it is essential for enhancing cleavage of suboptimal substrates. Finally, our studies define a previously unrecognized Dicer interaction interface and suggest that Loqs-PD is well positioned to recruit substrates into the helicase domain of Dicer-2.

| S-EPMC5617286 | biostudies-literature

The helicase domain of Pol? counteracts RPA to promote alt-NHEJ.

Project description:Mammalian polymerase theta (Pol?) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Pol?-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Pol?-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Pol? and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.

| S-EPMC6047744 | biostudies-literature

Human DICER helicase domain recruits PKR and dsRNA binding proteins during viral infection

Project description:The innate immune response against viruses mainly involves type I interferon (IFN) in mammalian cells. The exact contribution of the RNA silencing machinery remains to be established, but several recent studies indicate that the type III ribonuclease DICER can generate viral siRNAs in specific conditions. In addition, it has been proposed that type I IFN and RNA silencing could be mutually exclusive responses. In order to decipher the implication of DICER during infection of human cells with the Sindbis virus, we determined its interactome by immunoprecipitation and mass spectrometry analysis. Our results show that human DICER specifically interacts with several double-stranded RNA binding proteins and helicases during viral infection. In particular, proteins such as DHX9, ADAR-1 and the protein kinase PKR are enriched with DICER in virus-infected cells. We validated the importance of the helicase domain of DICER in its interaction with PKR and showed that it has functional consequences for the cellular response to viral infection.

2021-04-15 | PXD019093 | Pride

Staufen1 dimerizes through a conserved motif and a degenerate dsRNA-binding domain to promote mRNA decay.

Project description:Staufen1 (STAU1)-mediated mRNA decay (SMD) degrades mammalian-cell mRNAs that bind the double-stranded RNA (dsRNA)-binding protein STAU1 in their 3' untranslated region. We report a new motif, which typifies STAU homologs from all vertebrate classes, that is responsible for human STAU1 (hSTAU1) homodimerization. Our crystal structure and mutagenesis analyses reveal that this motif, which we named the Staufen-swapping motif (SSM), and the dsRNA-binding domain 5 ('RBD'5) mediate protein dimerization: the two SSM ?-helices of one molecule interact primarily through a hydrophobic patch with the two 'RBD'5 ?-helices of a second molecule. 'RBD'5 adopts the canonical ?-?-?-?-? fold of a functional RBD, but it lacks residues and features required to bind duplex RNA. In cells, SSM-mediated hSTAU1 dimerization increases the efficiency of SMD by augmenting hSTAU1 binding to the ATP-dependent RNA helicase hUPF1. Dimerization regulates keratinocyte-mediated wound healing and many other cellular processes.

| S-EPMC4096160 | biostudies-literature

Crystal structure and mode of helicase binding of the C-terminal domain of primase from Helicobacter pylori.

Project description:To better understand the poor conservation of the helicase binding domain of primases (DnaGs) among the eubacteria, we determined the crystal structure of the Helicobacter pylori DnaG C-terminal domain (HpDnaG-CTD) at 1.78 Å. The structure has a globular subdomain connected to a helical hairpin. Structural comparison has revealed that globular subdomains, despite the variation in number of helices, have broadly similar arrangements across the species, whereas helical hairpins show different orientations. Further, to study the helicase-primase interaction in H. pylori, a complex was modeled using the HpDnaG-CTD and HpDnaB-NTD (helicase) crystal structures using the Bacillus stearothermophilus BstDnaB-BstDnaG-CTD (helicase-primase) complex structure as a template. By using this model, a nonconserved critical residue Phe534 on helicase binding interface of DnaG-CTD was identified. Mutation guided by molecular dynamics, biophysical, and biochemical studies validated our model. We further concluded that species-specific helicase-primase interactions are influenced by electrostatic surface potentials apart from the critical hydrophobic surface residues.

| S-EPMC3697261 | biostudies-literature

The RNA helicase Dhh1p cooperates with Rbp1p to promote porin mRNA decay via its non-conserved C-terminal domain.

Project description:The yeast RNA helicase Dhh1p has been shown to associate with components of mRNA decay and is involved in mRNA decapping and degradation. An RNA-binding protein, Rbp1p, is known to bind to the 3'-UTR of porin (POR1) mRNA, and induces mRNA decay by an uncharacterized mechanism. Here, we show that Dhh1p can associate with POR1 mRNA and specifically promote POR1 mRNA decay via its interaction with Rbp1p. As compared to its mammalian homolog RCK/p54/DDX6, Dhh1p has a unique and long extension at its C-terminus. Interestingly, this non-conserved C-terminal region of Dhh1p is required for interaction with Rbp1p and modulating Rbp1p-mediated POR1 mRNA decay. Notably, expression of a C-terminal 81-residue deleted Dhh1p can fully complement the growth defect of a dhh1? strain and retains its function in regulating the mRNA level of an RNA-binding protein Edc1p. Moreover, mammalian DDX6 became capable of interacting with Rbp1p and could confer Rbp1p-mediated POR1 mRNA decay in the dhh1? strain upon fusion to the C-terminal unique region of Dhh1p. Thus, we propose that the non-conserved C-terminus of Dhh1p plays a role in defining specific interactions with mRNA regulatory factors that promote distinct mRNA decay.

| S-EPMC3273804 | biostudies-literature

A New BRCT Binding Mode in TopBP1-BLM Helicase Interaction.

Project description:Tandem BRCT domains are phophoprotein binding modules. In this issue of Structure, Sun et al. (2017) show that a single BRCT domain in TopBP1 binds tightly and specifically to phosphorylated Bloom syndrome helicase (BLM). This work reveals a novel BRCT binding mode and suggests a similar mechanism for TopBP1 interaction with 53BP1.

| S-EPMC5642270 | biostudies-literature

Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein.

Project description:Double-stranded RNA (dsRNA) plays essential functions in many biological processes, including the activation of innate immune responses and RNA interference. dsRNA also represents the genetic entity of some viruses and is a hallmark of infections by positive-sense single-stranded RNA viruses. Methods for detecting dsRNA rely essentially on immunological approaches and their use is often limited to in vitro applications, although recent developments have allowed the visualization of dsRNA in vivo. Here, we report the sensitive and rapid detection of long dsRNA both in vitro and in vivo using the dsRNA binding domain of the B2 protein from Flock house virus. In vitro, we adapted the system for the detection of dsRNA either enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. In vivo, we produced stable transgenic Nicotiana benthamiana lines allowing the visualization of dsRNA by fluorescence microscopy. Using these techniques, we were able to discriminate healthy and positive-sense single-stranded RNA virus-infected material in plants and insect cells. In N. benthamiana, our system proved to be very potent for the spatio-temporal visualization of replicative RNA intermediates of a broad range of positive-sense RNA viruses, including high- vs. low-copy number viruses.

| S-EPMC5799278 | 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