Double-stranded RNA-binding artificial cationic oligosaccharides stabilizing siRNAs with a low N/P ratio

Novel double-stranded RNA (dsRNA)-binding molecules were developed for the effective thermodynamic and biological stabilization of nucleic acids including short interfering RNAs (siRNAs).

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CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis

10.1101/376319 ◽

2018 ◽

Author(s):

Qin Yu ◽

Kun Qu ◽

Yorgo Modis

Keyword(s):

Rna Binding ◽

Atp Hydrolysis ◽

Type I ◽

List Type ◽

Base Pairs ◽

Nucleotide Analogs ◽

Double Stranded Rna ◽

Transition State Analogs ◽

Helical Twist ◽

Dsrna Binding

SummaryDouble-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection. Long cytosolic dsRNA is recognized by MDA5. The cooperative assembly of MDA5 into helical filaments on dsRNA nucleates the assembly of a multiprotein type-I-interferon signaling platform. Here, we determined cryoEM structures of MDA5-dsRNA filaments with different helical twists and bound nucleotide analogs, at resolutions sufficient to build and refine atomic models. The structures identify the filament forming interfaces, which encode the dsRNA binding cooperativity and length specificity of MDA5. The predominantly hydrophobic interface contacts confer flexibility, reflected in the variable helical twist within filaments. Mutation of filament-forming residues can result in loss or gain of signaling activity. Each MDA5 molecule spans 14 or 15 RNA base pairs, depending on the twist. Variations in twist also correlate with variations in the occupancy and type of nucleotide in the active site, providing insights on how ATP hydrolysis contributes to MDA5-dsRNA recognition.eTOCStructures of MDA5 bound to double-stranded RNA reveal a flexible, predominantly hydrophobic filament forming interface. The filaments have variable helical twist. Structures determined with ATP and transition state analogs show how the ATPase cycle is coupled to changes in helical twist, the mode of RNA binding and the length of the RNA footprint of MDA5.HighlightsCryoEM structures of MDA5-dsRNA filaments determined for three catalytic statesFilament forming interfaces are flexible and predominantly hydrophobicMutation of filament-forming residues can cause loss or gain of IFN-β signalingATPase cycle is coupled to changes in filament twist and size of the RNA footprint

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RNA-Regulated Interaction of Transportin-1 and Exportin-5 with the Double-Stranded RNA-Binding Domain Regulates Nucleocytoplasmic Shuttling of ADAR1

Molecular and Cellular Biology ◽

10.1128/mcb.01519-08 ◽

2009 ◽

Vol 29 (6) ◽

pp. 1487-1497 ◽

Cited By ~ 72

Author(s):

Jutta Fritz ◽

Alexander Strehblow ◽

Andreas Taschner ◽

Sandy Schopoff ◽

Pawel Pasierbek ◽

...

Keyword(s):

Nuclear Export ◽

Rna Binding ◽

Nuclear Export Signal ◽

Nucleocytoplasmic Transport ◽

Nucleocytoplasmic Shuttling ◽

Double Stranded Rna ◽

Binding Domains ◽

Dsrna Binding ◽

Localization Signal ◽

Editing Enzyme

ABSTRACT Double-stranded RNA (dsRNA)-binding proteins interact with substrate RNAs via dsRNA-binding domains (dsRBDs). Several proteins harboring these domains exhibit nucleocytoplasmic shuttling and possibly remain associated with their substrate RNAs bound in the nucleus during nuclear export. In the human RNA-editing enzyme ADAR1-c, the nuclear localization signal overlaps the third dsRBD, while the corresponding import factor is unknown. The protein also lacks a clear nuclear export signal but shuttles between the nucleus and the cytoplasm. Here we identify transportin-1 as the import receptor for ADAR1. Interestingly, dsRNA binding interferes with transportin-1 binding. At the same time, each of the dsRBDs in ADAR1 interacts with the export factor exportin-5. RNA binding stimulates this interaction but is not a prerequisite. Thus, our data demonstrate a role for some dsRBDs as RNA-sensitive nucleocytoplasmic transport signals. dsRBD3 in ADAR1 can mediate nuclear import, while interaction of all dsRBDs might control nuclear export. This finding may have implications for other proteins containing dsRBDs and suggests a selective nuclear export mechanism for substrates interacting with these proteins.

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Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless

10.1101/466763 ◽

2018 ◽

Author(s):

Pravin Kumar Ankush Jagtap ◽

Marisa Müller ◽

Pawel Masiewicz ◽

Sören von Bülow ◽

Nele Merret Hollmann ◽

...

Keyword(s):

Rna Binding ◽

Binding Motifs ◽

Double Stranded Rna ◽

Binding Domains ◽

Dsrna Binding ◽

Rna Binding Domains ◽

Human Orthologue ◽

Rox Rna

ABSTRACTMaleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAK RNA binding motifs. NMR titrations combined with filter binding experiments document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.

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The cytoplasm of Xenopus oocytes contains a factor that protects double-stranded RNA from adenosine-to-inosine modification.

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5425 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5425-5432 ◽

Cited By ~ 41

Author(s):

L Saccomanno ◽

B L Bass

Keyword(s):

Adenosine Deaminase ◽

Rna Binding ◽

Binding Protein ◽

Protein S ◽

Meiotic Maturation ◽

Protection Factor ◽

Independent Manner ◽

Double Stranded Rna ◽

Dsrna Binding

Here we describe studies of double-stranded RNA (dsRNA) adenosine deaminase in Xenopus laevis, in particular during meiotic maturation, the period during which a stage VI oocyte matures to an egg. We show that dsRNA adenosine deaminase is in the nuclei of stage VI oocytes. Most importantly, we demonstrate that the cytoplasm of stage VI oocytes contains a factor that protects microinjected dsRNA from deamination when dsRNA adenosine deaminase is released from the nucleus during meiotic maturation. Our data suggest that the protection factor is a cytoplasmic dsRNA-binding protein or proteins that bind to dsRNA in a sequence-independent manner to occlude dsRNA from binding to dsRNA adenosine deaminase. The cytoplasmic double-stranded RNA-binding protein(s) does not bind to other nucleic acids and can be titrated at high concentrations of dsRNA. These studies raise the question of whether all dsRNA-binding proteins share endogenous substrates and also suggest potential means of regulating dsRNA adenosine deaminase in vivo.

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Double-Stranded-RNA-Binding Protein 2 Participates in Antiviral Defense

Journal of Virology ◽

10.1128/jvi.00017-20 ◽

2020 ◽

Vol 94 (11) ◽

Cited By ~ 3

Author(s):

Károly Fátyol ◽

Katalin Anna Fekete ◽

Márta Ludman

Keyword(s):

Rna Interference ◽

Virus Infection ◽

Nicotiana Benthamiana ◽

Rna Binding ◽

Binding Protein ◽

Systemic Necrosis ◽

Double Stranded Rna ◽

Content Type ◽

Dsrna Binding ◽

Dsrna Binding Protein

ABSTRACT Double-stranded RNA (dsRNA) is a common pattern formed during the replication of both RNA and DNA viruses. Perception of virus-derived dsRNAs by specialized receptor molecules leads to the activation of various antiviral measures. In plants, these defensive processes include the adaptive RNA interference (RNAi) pathway and innate pattern-triggered immune (PTI) responses. While details of the former process have been well established in recent years, the latter are still only partially understood at the molecular level. Nonetheless, emerging data suggest extensive cross talk between the different antiviral mechanisms. Here, we demonstrate that dsRNA-binding protein 2 (DRB2) of Nicotiana benthamiana plays a direct role in potato virus X (PVX)-elicited systemic necrosis. These results establish that DRB2, a known component of RNAi, is also involved in a virus-induced PTI response. In addition, our findings suggest that RNA-dependent polymerase 6 (RDR6)-dependent dsRNAs play an important role in the triggering of PVX-induced systemic necrosis. Based on our data, a model is formulated whereby competition between different DRB proteins for virus-derived dsRNAs helps establish the dominant antiviral pathways that are activated in response to virus infection. IMPORTANCE Plants employ multiple defense mechanisms to restrict viral infections, among which RNA interference is the best understood. The activation of innate immunity often leads to both local and systemic necrotic responses, which confine the virus to the infected cells and can also provide resistance to distal, noninfected parts of the organism. Systemic necrosis, which is regarded as a special form of the local hypersensitive response, results in necrosis of the apical stem region, usually causing the death of the plant. Here, we provide evidence that the dsRNA-binding protein 2 of Nicotiana benthamiana plays an important role in virus-induced systemic necrosis. Our findings are not only compatible with the recent hypothesis that DRB proteins act as viral invasion sensors but also extends it by proposing that DRBs play a critical role in establishing the dominant antiviral measures that are triggered during virus infection.

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Incorporating 2-Thiouracil into Short Double-Stranded RNA-Binding Peptide Nucleic Acids for Enhanced Recognition of A-U Pairs and for Targeting a MicroRNA Hairpin Precursor

Biochemistry ◽

10.1021/acs.biochem.9b00521 ◽

2019 ◽

Vol 58 (32) ◽

pp. 3444-3453 ◽

Cited By ~ 4

Author(s):

Alan Ann Lerk Ong ◽

Desiree-Faye Kaixin Toh ◽

Manchugondanahalli S. Krishna ◽

Kiran M. Patil ◽

Katsutomo Okamura ◽

...

Keyword(s):

Nucleic Acids ◽

Rna Binding ◽

Peptide Nucleic Acids ◽

Binding Peptide ◽

Double Stranded Rna ◽

Hairpin Precursor

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Trafficking of siRNA precursors by the dsRBD protein Blanks in Drosophila

Nucleic Acids Research ◽

10.1093/nar/gkaa072 ◽

2020 ◽

Vol 48 (7) ◽

pp. 3906-3921 ◽

Cited By ~ 1

Author(s):

Volker Nitschko ◽

Stefan Kunzelmann ◽

Thomas Fröhlich ◽

Georg J Arnold ◽

Klaus Förstemann

Keyword(s):

Small Rnas ◽

Rna Binding ◽

Small Interfering Rnas ◽

Double Stranded Rna ◽

Binding Domains ◽

Convergent Transcription ◽

Dsrna Binding ◽

Functional Screens

Abstract RNA interference targets aberrant transcripts with cognate small interfering RNAs, which derive from double-stranded RNA precursors. Several functional screens have identified Drosophila blanks/lump (CG10630) as a facilitator of RNAi, yet its molecular function has remained unknown. The protein carries two dsRNA binding domains (dsRBD) and blanks mutant males have a spermatogenesis defect. We demonstrate that blanks selectively boosts RNAi triggered by dsRNA of nuclear origin. Blanks binds dsRNA via its second dsRBD in vitro, shuttles between nucleus and cytoplasm and the abundance of siRNAs arising at many sites of convergent transcription is reduced in blanks mutants. Since features of nascent RNAs - such as introns and transcription beyond the polyA site – contribute to the small RNA pool, we propose that Blanks binds dsRNA formed by cognate nascent RNAs in the nucleus and fosters its export to the cytoplasm for dicing. We refer to the resulting small RNAs as blanks exported siRNAs (bepsiRNAs). While bepsiRNAs were fully dependent on RNA binding to the second dsRBD of blanks in transgenic flies, male fertility was not. This is consistent with a previous report that linked fertility to the first dsRBD of Blanks. The role of blanks in spermatogenesis appears thus unrelated to its role in dsRNA export.

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Exportin-5, a novel karyopherin, mediates nuclear export of double-stranded RNA binding proteins

The Journal of Cell Biology ◽

10.1083/jcb.200110082 ◽

2002 ◽

Vol 156 (1) ◽

pp. 53-64 ◽

Cited By ~ 108

Author(s):

Amy M. Brownawell ◽

Ian G. Macara

Keyword(s):

Nuclear Export ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Dependent Manner ◽

Intact Cells ◽

Double Stranded Rna ◽

Permeabilized Cells ◽

Dsrna Binding ◽

Rna Export

We have identified a novel human karyopherin (Kap)β family member that is related to human Crm1 and the Saccharomyces cerevisiae protein, Msn5p/Kap142p. Like other known transport receptors, this Kap binds specifically to RanGTP, interacts with nucleoporins, and shuttles between the nuclear and cytoplasmic compartments. We report that interleukin enhancer binding factor (ILF)3, a double-stranded RNA binding protein, associates with this Kap in a RanGTP-dependent manner and that its double-stranded RNA binding domain (dsRBD) is the limiting sequence required for this interaction. Importantly, the Kap interacts with dsRBDs found in several other proteins and binding is blocked by double-stranded RNA. We find that the dsRBD of ILF3 functions as a novel nuclear export sequence (NES) in intact cells, and its ability to serve as an NES is dependent on the expression of the Kap. In digitonin-permeabilized cells, the Kap but not Crm1 stimulated nuclear export of ILF3. Based on the ability of this Kap to mediate the export of dsRNA binding proteins, we named the protein exportin-5. We propose that exportin-5 is not an RNA export factor but instead participates in the regulated translocation of dsRBD proteins to the cytoplasm where they interact with target mRNAs.

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Two basic motifs of reovirus σ3 protein are involved in double-stranded RNA binding

Biochemistry and Cell Biology ◽

10.1139/o95-017 ◽

1995 ◽

Vol 73 (3-4) ◽

pp. 137-145 ◽

Cited By ~ 24

Author(s):

T. Mabrouk ◽

C. Danis ◽

G. Lemay

Keyword(s):

Rna Binding ◽

Inhibitory Effect ◽

Site Directed Mutagenesis ◽

General Mechanism ◽

Virus Protein ◽

Double Stranded Rna ◽

Kinase Activation ◽

Dsrna Binding ◽

Dsrna Binding Protein ◽

Cellular Antiviral Response

It has been reported that the σ3 protein of reovirus can exert an inhibitory effect on the cellular double-stranded RNA (dsRNA) activated protein kinase. Activation of this kinase is thought to be a general mechanism mediating a cellular antiviral response. This enzyme can also be activated upon transfection, resulting in translational inhibition of plasmid-encoded mRNAs. σ3 has an affinity for dsRNA postulated to be responsible for antikinase activity. In the present study, site-directed mutagenesis was performed on two basic regions previously suggested as dsRNA-binding motifs and the mutant σ3 proteins were then expressed in COS cells. These experiments revealed that both motifs are involved in σ3 attachment to RNA. Expression of the mutants lacking RNA-binding capability is stimulated by coexpression of another dsRNA-binding protein, the E3L vaccinia virus protein. These results support a model in which the attachment to dsRNA is directly responsible for the trans-stimulating effect of σ3 on expression of cotransfected genes.Key words: reovirus, PKR, protein synthesis, RNA binding.

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Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus

Journal of Virology ◽

10.1128/jvi.00009-07 ◽

2007 ◽

Vol 81 (11) ◽

pp. 5449-5459 ◽

Cited By ~ 27

Author(s):

Beau J. Fenner ◽

Winnie Goh ◽

Jimmy Kwang

Keyword(s):

Rna Silencing ◽

Rna Binding ◽

Amino Acid Residues ◽

Alanine Scanning Mutagenesis ◽

Marine Aquaculture ◽

Double Stranded Rna ◽

Dsrna Binding ◽

Fish Cells ◽

Interfering Rna

ABSTRACT Betanodaviruses are small RNA viruses that infect teleost fish and pose a considerable threat to marine aquaculture production. These viruses possess a small protein, termed B2, which binds to and protects double-stranded RNA. This prevents cleavage of virus-derived double-stranded RNAs (dsRNAs) by Dicer and subsequent production of small interfering RNA (siRNA), which would otherwise induce an RNA-silencing response against the virus. In this work, we have performed charged-to-alanine scanning mutagenesis of the B2 protein in order to identify residues required for dsRNA binding and protection. While the majority of the 19 mutated B2 residues were required for maximal dsRNA binding and protection in vitro, residues R53 and R60 were essential for both activities. Subsequent experiments in fish cells confirmed these findings by showing that mutations in these residues abolished accumulation of both the RNA1 and RNA2 components of the viral genome, in addition to preventing any significant induction of the host interferon gene, Mx. Moreover, an obvious positive correlation was found between dsRNA binding and protection in vitro and RNA1, RNA2, and Mx accumulation in fish cells, further validating the importance of the selected amino acid residues. The same trend was also demonstrated using an RNA silencing system in HeLa cells, with residues R53 and R60 being essential for suppression of RNA silencing. Importantly, we found that siRNA-mediated knockdown of Dicer dramatically enhanced the accumulation of a B2 mutant. In addition, we found that B2 is able to induce apoptosis in fish cells but that this was not the result of dsRNA binding.

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