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

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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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

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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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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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Opposite actions of two dsRNA-binding proteins PACT and TRBP on RIG-I mediated signaling

Biochemical Journal ◽

10.1042/bcj20200987 ◽

2021 ◽

Vol 478 (3) ◽

pp. 493-510

Author(s):

Lauren S. Vaughn ◽

Evelyn Chukwurah ◽

Rekha C. Patel

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Cellular Protein ◽

Independent Manner ◽

Viral Origin ◽

Antiviral Signaling ◽

Downstream Signaling ◽

Viral Susceptibility ◽

Dsrna Binding ◽

Mitochondrial Antiviral Signaling

An integral aspect of innate immunity is the ability to detect foreign molecules of viral origin to initiate antiviral signaling via pattern recognition receptors (PRRs). One such receptor is the RNA helicase retinoic acid inducible gene 1 (RIG-I), which detects and is activated by 5′triphosphate uncapped double stranded RNA (dsRNA) as well as the cytoplasmic viral mimic dsRNA polyI:C. Once activated, RIG-I's CARD domains oligomerize and initiate downstream signaling via mitochondrial antiviral signaling protein (MAVS), ultimately inducing interferon (IFN) production. Another dsRNA binding protein PACT, originally identified as the cellular protein activator of dsRNA-activated protein kinase (PKR), is known to enhance RIG-I signaling in response to polyI:C treatment, in part by stimulating RIG-I's ATPase and helicase activities. TAR-RNA-binding protein (TRBP), which is ∼45% homologous to PACT, inhibits PKR signaling by binding to PKR as well as by sequestration of its’ activators, dsRNA and PACT. Despite the extensive homology and similar structure of PACT and TRBP, the role of TRBP has not been explored much in RIG-I signaling. This work focuses on the effect of TRBP on RIG-I signaling and IFN production. Our results indicate that TRBP acts as an inhibitor of RIG-I signaling in a PACT- and PKR-independent manner. Surprisingly, this inhibition is independent of TRBP's post-translational modifications that are important for other signaling functions of TRBP, but TRBP's dsRNA-binding ability is essential. Our work has major implications on viral susceptibility, disease progression, and antiviral immunity as it demonstrates the regulatory interplay between PACT and TRBP IFN production.

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Characterization of RNA Determinants Recognized by the Arginine- and Proline-Rich Region of Us11, a Herpes Simplex Virus Type 1-Encoded Double-Stranded RNA Binding Protein That Prevents PKR Activation

Journal of Virology ◽

10.1128/jvi.76.23.11971-11981.2002 ◽

2002 ◽

Vol 76 (23) ◽

pp. 11971-11981 ◽

Cited By ~ 44

Author(s):

David Khoo ◽

Cesar Perez ◽

Ian Mohr

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Rna Binding ◽

Binding Protein ◽

Binding Domain ◽

Double Stranded Rna ◽

Dsrna Binding ◽

Rna Ligands ◽

Pkr Kinase ◽

Simplex Virus

ABSTRACT The herpes simplex virus Us11 gene product inhibits activation of the cellular PKR kinase and associates with a limited number of unrelated viral and cellular RNA molecules via a carboxyl-terminal 68-amino-acid segment rich in arginine and proline. To characterize the determinants underlying the recognition of an RNA target by Us11, we employed an in vitro selection technique to isolate RNA ligands that bind Us11 with high affinity from a population of molecules containing an internal randomized segment. Binding of Us11 to these RNA ligands is specific and appears to occur preferentially on conformational isoforms that possess a higher-order structure. While the addition of unlabeled poly(I · C) reduced binding of Us11 to a selected radiolabeled RNA, single-stranded homopolymers were not effective competitors. Us11 directly associates with poly(I · C), and inclusion of an unlabeled selected RNA in the reaction reduces poly(I · C) binding, while single-stranded RNA homopolymers have no effect. Finally, Us11 binds to defined, double-stranded RNA (dsRNA) molecules that exhibit greater sequence complexity. Binding to these dsRNA perfect duplexes displays a striking dependence on length, as 39-bp or shorter duplexes do not bind efficiently. Furthermore, this interaction is specific for dsRNA as opposed to dsDNA, implying that the Us11 RNA binding domain can distinguish nucleic acid duplexes containing 2′ hydroxyl groups from those that do not. These results establish that Us11 is a dsRNA binding protein. The arginine- and proline-rich Us11 RNA binding domain is unrelated to known dsRNA binding elements and thus constitutes a unique recognition motif that interacts with dsRNA. The ability of Us11 to bind dsRNA may be important for inhibiting activation of the cellular PKR kinase in response to dsRNA.

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Double-Stranded RNA Binding May Be a General Plant RNA Viral Strategy To Suppress RNA Silencing

Journal of Virology ◽

10.1128/jvi.01963-05 ◽

2006 ◽

Vol 80 (12) ◽

pp. 5747-5756 ◽

Cited By ~ 202

Author(s):

Zsuzsanna Mérai ◽

Zoltán Kerényi ◽

Sándor Kertész ◽

Melinda Magna ◽

Lóránt Lakatos ◽

...

Keyword(s):

Rna Silencing ◽

Rna Binding ◽

Binding Protein ◽

Turnip Crinkle Virus ◽

Double Stranded Rna ◽

Dsrna Binding ◽

Beet Yellows Virus ◽

Dsrna Binding Protein ◽

Silencing Suppression ◽

Suppression Strategy

ABSTRACT In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a general silencing suppression strategy. Indeed, it was shown that the related aureusvirus P14 and tombusvirus P19 suppressors are dsRNA-binding proteins. Interestingly, P14 is a size-independent dsRNA-binding protein, while P19 binds only 21-nucleotide ds-sRNAs (small dsRNAs having 2-nucleotide 3′ overhangs), the specificity determinant of the silencing system. Much evidence supports the idea that P19 inhibits silencing by sequestering silencing-generated viral ds-sRNAs. In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy. Here we show that many plant viral silencing suppressors bind dsRNAs. Beet yellows virus Peanut P21, clump virus P15, Barley stripe mosaic virus γB, and Tobacco etch virus HC-Pro, like P19, bind ds-sRNAs size-selectively, while Turnip crinkle virus CP is a size-independent dsRNA-binding protein, which binds long dsRNAs as well as ds-sRNAs. We propose that size-selective ds-sRNA-binding suppressors inhibit silencing by sequestering viral ds-sRNAs, whereas size-independent dsRNA-binding suppressors inactivate silencing by sequestering long dsRNA precursors of viral sRNAs and/or by binding ds-sRNAs. The findings that many unrelated silencing suppressors bind dsRNA suggest that dsRNA binding is a general silencing suppression strategy which has evolved independently many times.

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Mammalian Staufen Is a Double-Stranded-RNA- and Tubulin-Binding Protein Which Localizes to the Rough Endoplasmic Reticulum

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.2220 ◽

1999 ◽

Vol 19 (3) ◽

pp. 2220-2230 ◽

Cited By ~ 161

Author(s):

Louise Wickham ◽

Thomas Duchaîne ◽

Ming Luo ◽

Ivan R. Nabi ◽

Luc DesGroseillers

Keyword(s):

Endoplasmic Reticulum ◽

Rough Endoplasmic Reticulum ◽

Molecular Mechanisms ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Mrna Transport ◽

Double Labeling ◽

Double Stranded Rna ◽

Dsrna Binding

ABSTRACT Staufen (Stau) is a double-stranded RNA (dsRNA)-binding protein involved in mRNA transport and localization in Drosophila.To understand the molecular mechanisms of mRNA transport in mammals, we cloned human (hStau) and mouse (mStau)staufen cDNAs. In humans, four transcripts arise by differential splicing of the Stau gene and code for two proteins with different N-terminal extremities. In vitro, hStau and mStau bind dsRNA via each of two full-length dsRNA-binding domains and tubulin via a region similar to the microtubule-binding domain of MAP-1B, suggesting that Stau cross-links cytoskeletal and RNA components. Immunofluorescent double labeling of transfected mammalian cells revealed that Stau is localized to the rough endoplasmic reticulum (RER), implicating this RNA-binding protein in mRNA targeting to the RER, perhaps via a multistep process involving microtubules. These results are the first demonstration of the association of an RNA-binding protein in addition to ribosomal proteins, with the RER, implicating this class of proteins in the transport of RNA to its site of translation.

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A Novel Murine Staufen Isoform Modulates the RNA Content of Staufen Complexes

Molecular and Cellular Biology ◽

10.1128/mcb.20.15.5592-5601.2000 ◽

2000 ◽

Vol 20 (15) ◽

pp. 5592-5601 ◽

Cited By ~ 22

Author(s):

Thomas Duchaîne ◽

Hui-Jun Wang ◽

Ming Luo ◽

Sergey V. Steinberg ◽

Ivan R. Nabi ◽

...

Keyword(s):

Mammalian Cells ◽

Rna Binding ◽

Binding Domain ◽

Binding Ability ◽

Double Stranded Rna ◽

Transfected Cells ◽

Rna Content ◽

Dsrna Binding ◽

In Vivo Analysis

ABSTRACT Mouse Staufen (mStau) is a double-stranded RNA-binding protein associated with polysomes and the rough endoplasmic reticulum (RER). We describe a novel endogenous isoform of mStau (termed mStaui) which has an insertion of six amino acids within dsRBD3, the major double-stranded RNA (dsRNA)-binding domain. With a structural change of the RNA-binding domain, this conserved and widely distributed isoform showed strongly impaired dsRNA-binding ability. In transfected cells, mStaui exhibited the same tubulovesicular distribution (RER) as mStau when weakly expressed; however, when overexpressed, mStaui was found in large cytoplasmic granules. Markers of the RER colocalized with mStaui-containing granules, showing that overexpressed mStaui could still be associated with the RER. Cotransfection of mStaui with mStau relocalized overexpressed mStaui to the reticular RER, suggesting that they can form a complex on the RER and that a balance between these isoforms is important to achieve proper localization. Coimmunoprecipitation demonstrated that the two mStau isoforms are components of the same complex in vivo. Analysis of the immunoprecipitates showed that mStau is a component of an RNA-protein complex and that the association with mStaui drastically reduces the RNA content of the complex. We propose that this new isoform, by forming a multiple-isoform complex, regulates the amount of RNA in mStau complexes in mammalian cells.

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