Effect of RNA silencing suppression activity of chrysanthemum virus B p12 protein on small RNA species

Abstract Chrysanthemum virus B encodes a multifunctional p12 protein that acts as a transcriptional activator in the nucleus and as a suppressor of RNA silencing in the cytoplasm. Here, we investigated the impact of p12 on accumulation of major classes of small RNAs (sRNAs). The results show dramatic changes in the sRNA profiles characterised by an overall reduction in sRNA accumulation, changes in the pattern of size distribution of canonical siRNAs and in the ratio between sense and antisense strands, lower abundance of siRNAs with a U residue at the 5′-terminus, and changes in the expression of certain miRNAs, most of which were downregulated.

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Key Importance of Small RNA Binding for the Activity of a Glycine-Tryptophan (GW) Motif-containing Viral Suppressor of RNA Silencing

Journal of Biological Chemistry ◽

10.1074/jbc.m114.593707 ◽

2014 ◽

Vol 290 (5) ◽

pp. 3106-3120 ◽

Cited By ~ 28

Author(s):

Miryam Pérez-Cañamás ◽

Carmen Hernández

Keyword(s):

Small Rna ◽

Rna Silencing ◽

Rna Binding ◽

Viral Suppressor ◽

Suppressor Of Rna Silencing

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Characterization of Hypovirus-Derived Small RNAs Generated in the Chestnut Blight Fungus by an Inducible DCL-2-Dependent Pathway

Journal of Virology ◽

10.1128/jvi.02324-07 ◽

2008 ◽

Vol 82 (6) ◽

pp. 2613-2619 ◽

Cited By ~ 77

Author(s):

Xuemin Zhang ◽

Gert C. Segers ◽

Qihong Sun ◽

Fuyou Deng ◽

Donald L. Nuss

Keyword(s):

Small Rnas ◽

Rna Silencing ◽

Cryphonectria Parasitica ◽

Chestnut Blight ◽

Fungal Host ◽

Chestnut Blight Fungus ◽

Mutant Strains ◽

Suppressor Of Rna Silencing ◽

Dependent Pathway

ABSTRACT The disruption of one of two dicer genes, dcl-2, of the chestnut blight fungus Cryphonectria parasitica was recently shown to increase susceptibility to mycovirus infection (G. C. Segers, X. Zhang, F. Deng, Q. Sun, and D. L. Nuss, Proc. Natl. Acad. Sci. USA 104:12902-12906, 2007). We now report the accumulation of virus-derived small RNAs (vsRNAs) in hypovirus CHV1-EP713-infected wild-type and dicer gene dcl-1 mutant C. parasitica strains but not in hypovirus-infected dcl-2 mutant and dcl-1 dcl-2 double-mutant strains. The CHV1-EP713 vsRNAs were produced from both the positive and negative viral RNA strands at a ratio of 3:2 in a nonrandom distribution along the viral genome. We also show that C. parasitica responds to hypovirus and mycoreovirus infections with a significant increase (12- to 20-fold) in dcl-2 expression while the expression of dcl-1 is increased only modestly (2-fold). The expression of dcl-2 is further increased (∼35-fold) following infection with a hypovirus CHV1-EP713 mutant that lacks the p29 suppressor of RNA silencing. The combined results demonstrate the biogenesis of mycovirus-derived small RNAs in a fungal host through the action of a specific dicer gene, dcl-2. They also reveal that dcl-2 expression is significantly induced in response to mycovirus infection by a mechanism that appears to be repressed by the hypovirus-encoded p29 suppressor of RNA silencing.

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Studies of the Interaction of the Viral Suppressor of RNA Silencing Protein p19 with Small RNAs Using Fluorescence Polarization†

Biochemistry ◽

10.1021/bi800401y ◽

2008 ◽

Vol 47 (31) ◽

pp. 8130-8138 ◽

Cited By ~ 18

Author(s):

Jenny Cheng ◽

Selena M. Sagan ◽

Zygmunt J. Jakubek ◽

John Paul Pezacki

Keyword(s):

Fluorescence Polarization ◽

Small Rnas ◽

Rna Silencing ◽

Viral Suppressor ◽

Suppressor Of Rna Silencing

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Comparative profiling of small RNAs of pig seminal plasma and ejaculated and epididymal sperm

Reproduction ◽

10.1530/rep-17-0014 ◽

2017 ◽

Vol 153 (6) ◽

pp. 785-796 ◽

Cited By ~ 13

Author(s):

Cai Chen ◽

Han Wu ◽

Dan Shen ◽

Saisai Wang ◽

Li Zhang ◽

...

Keyword(s):

Seminal Plasma ◽

Small Rna ◽

Small Rnas ◽

Target Genes ◽

Large Population ◽

Small Nuclear Rna ◽

Epididymal Sperm ◽

Zygote Development ◽

Similarities And Differences ◽

Small Rna Species

The similarities and differences of small RNAs in seminal plasma, epididymal sperm and ejaculated sperm remain largely undefined. We conducted a systematic comparative analysis of small RNA profiles in pig ejaculated sperm, epididymal sperm and seminal plasma and found that the diversity distribution of small RNA species was generally similar, whereas the abundance of small RNAs is dramatically different across the three libraries; miRNAs and small RNAs derived from rRNA, tRNA, small nuclear RNA, 7SK RNA, NRON RNA and cis-regulatory RNA were enriched in the three libraries, but piRNA was absent. A large population of small RNAs from ejaculated sperm are ejaculated sperm specific, and only 8–30% of small RNAs overlapped with those of epididymal sperm or seminal plasma and a small proportion (5–18%) of small RNAs were shared in the three libraries, suggesting that, in addition to the testes, sperm RNAs may also originate from seminal plasma, epididymis as well as other resources. Most miRNAs were co-distributed but differentially expressed across the three libraries, with epididymal sperm exhibiting the highest abundance, followed by ejaculated sperm and seminal plasma. The prediction of target genes of the top 10 highly expressed miRNAs across the three libraries revealed that these miRNAs may be involved in spermatogenesis, zygote development and the interaction between the environment and animals. Our study provides the first description of the similarities and differences of small RNA profiles in ejaculated sperm, epididymal sperm and seminal plasma and indicates that sperm RNA may have origins other than the testes.

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Translocation of Tomato Bushy Stunt Virus P19 Protein into the Nucleus by ALY Proteins Compromises Its Silencing Suppressor Activity

Journal of Virology ◽

10.1128/jvi.00953-06 ◽

2006 ◽

Vol 80 (18) ◽

pp. 9064-9072 ◽

Cited By ~ 67

Author(s):

Tomas Canto ◽

Joachim F. Uhrig ◽

Maud Swanson ◽

Kathryn M. Wright ◽

Stuart A. MacFarlane

Keyword(s):

Rna Silencing ◽

Tomato Bushy Stunt Virus ◽

Bimolecular Fluorescence Complementation ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Long Distance ◽

Suppressor Activity ◽

Recognition Motif ◽

Silencing Suppression ◽

Suppressor Of Rna Silencing

ABSTRACT The P19 protein of Tomato bushy stunt virus is a potent suppressor of RNA silencing and, depending on the host species, is required for short- and long-distance virus movement and symptom production. P19 interacts with plant ALY proteins and relocalizes a subset of these proteins from the nucleus to the cytoplasm. Here we showed that coexpression by agroinfiltration in Nicotiana benthamiana of P19 and the subset of ALY proteins that are not relocalized from the nucleus interfered with the ability of P19 to suppress RNA silencing. We demonstrated that this interference correlates with the relocation of P19 from the cytoplasm into the nucleus, and by constructing and analyzing chimeric ALY genes, we showed that the C-terminal part of the central, RNA recognition motif of ALY is responsible for interaction with P19, relocalization or nonrelocalization of ALY, and inhibition of silencing suppression by P19. We studied the interaction of ALY and P19 by using the technique of bimolecular fluorescence complementation to show that these proteins associate physically in the nucleus but not detectably in the cytoplasm, and we present a model to explain the dynamics of this interaction.

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Multiple Domains of the Tobacco mosaic virus p126 Protein Can Independently Suppress Local and Systemic RNA Silencing

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-06-11-0155 ◽

2012 ◽

Vol 25 (5) ◽

pp. 648-657 ◽

Cited By ~ 25

Author(s):

Li-Ya Wang ◽

Shih-Shun Lin ◽

Ting-Hsuan Hung ◽

Tsai-Kun Li ◽

Nai-Chun Lin ◽

...

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Rna Silencing ◽

Large Size ◽

Silencing Suppression ◽

Systemic Rna ◽

Multiple Domains ◽

Region Ii ◽

Suppressor Of Rna Silencing ◽

Antiviral Mechanism

Small RNA-mediated RNA silencing is a widespread antiviral mechanism in plants and other organisms. Many viruses encode suppressors of RNA silencing for counter-defense. The p126 protein encoded by Tobacco mosaic virus (TMV) has been reported to be a suppressor of RNA silencing but the mechanism of its function remains unclear. This protein is unique among the known plant viral silencing suppressors because of its large size and multiple domains. Here, we report that the methyltransferase, helicase, and nonconserved region II (NONII) of p126 each has silencing-suppressor function. The silencing-suppression activities of methyltransferase and helicase can be uncoupled from their enzyme activities. Specific amino acids in NONII previously shown to be crucial for viral accumulation and symptom development are also crucial for silencing suppression. These results suggest that some viral proteins have evolved to possess modular structural domains that can independently interfere with host silencing, and that this may be an effective mechanism of increasing the robustness of a virus.

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Functional analysis of virus and host components that mediate potyvirus-induced diseases

10.32747/2008.7591732.bard ◽

2008 ◽

Author(s):

Steven A. Whitham ◽

Amit Gal-On ◽

Tzahi Arazi

Keyword(s):

Small Rna ◽

Small Rnas ◽

Binding Activity ◽

Symptom Expression ◽

Wild Type ◽

Disease Symptoms ◽

Virus Accumulation ◽

Plant Genes ◽

Small Rna Species ◽

Host Genes

The mechanisms underlying the development of symptoms in response to virus infection remain to be discovered in plants. Insight into symptoms induced by potyviruses comes from evidence implicating the potyviral HC-Pro protein in symptom development. In particular, recent studies link the development of symptoms in infected plants to HC-Pro's ability to interfere with small RNA metabolism and function in plant hosts. Moreover, mutation of the highly conserved FRNK amino acid motif to FINK in the HC-Pro of Zucchini yellow mosaic virus (ZYMV) converts a severe strain into an asymptomatic strain, but does not affect virus accumulation in cucurbit hosts. The ability of this FINK mutation to uncouple symptoms from virus accumulation creates a unique opportunity to study symptom etiology, which is usually confounded by simultaneous attenuation of both symptoms and virus accumulation. Our goal was to determine how mutations in the conserved FRNK motif affect host responses to potyvirus infection in cucurbits and Arabidopsis thaliana. Our first objective was to define those amino acids in the FRNK motif that are required for symptoms by mutating the FRNK motif in ZYMV and Turnip mosaic virus (TuMV). Symptom expression and accumulation of resulting mutant viruses in cucurbits and Arabidopsis was determined. Our second objective was to identify plant genes associated with virus disease symptoms by profiling gene expression in cucurbits and Arabidopsis in response to mutant and wild type ZYMV and TuMV, respectively. Genes from the two host species that are differentially expressed led us to focus on a subset of genes that are expected to be involved in symptom expression. Our third objective was to determine the functions of small RNA species in response to mutant and wild type HC-Pro protein expression by monitoring the accumulation of small RNAs and their targets in Arabidopsis and cucurbit plants infected with wild type and mutant TuMV and ZYMV, respectively. We have found that the maintenance of the charge of the amino acids in the FRNK motif of HC-Pro is required for symptom expression. Reduced charge (FRNA, FRNL) lessen virus symptoms, and maintain the suppression of RNA silencing. The FRNK motif is involved in binding of small RNA species including microRNAs (miRNA) and short interfering RNAs (siRNA). This binding activity mediated by the FRNK motif has a role in protecting the viral genome from degradation by the host RNA silencing system. However, it also provides a mechanism by which the FRNK motif participates in inducing the symptoms of viral infection. Small RNA species, such as miRNA and siRNA, can regulate the functions of plant genes that affect plant growth and development. Thus, this binding activity suggests a mechanism by which ZYMVHC-Pro can interfere with plant development resulting in disease symptoms. Because the host genes regulated by small RNAs are known, we have identified candidate host genes that are expected to play a role in symptoms when their regulation is disrupted during viral infections. As a result of this work, we have a better understanding of the FRNK amino acid motif of HC-Pro and its contribution to the functions of HC-Pro, and we have identified plant genes that potentially contribute to symptoms of virus infected plants when their expression becomes misregulated during potyviral infections. The results set the stage to establish the roles of specific host genes in viral pathogenicity. The potential benefits include the development of novel strategies for controlling diseases caused by viruses, methods to ensure stable expression of transgenes in genetically improved crops, and improved potyvirus vectors for expression of proteins or peptides in plants.

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Comparative Analysis of Biochemical Biases by Ligation- and Template-Switch-Based Small RNA Library Preparation Protocols

Clinical Chemistry ◽

10.1373/clinchem.2019.305045 ◽

2019 ◽

Vol 65 (12) ◽

pp. 1581-1591 ◽

Cited By ~ 2

Author(s):

Morgane Meistertzheim ◽

Tobias Fehlmann ◽

Franziska Drews ◽

Marcello Pirritano ◽

Gilles Gasparoni ◽

...

Keyword(s):

Small Rna ◽

Small Rnas ◽

Regulation Of Gene Expression ◽

Biochemical Properties ◽

Library Preparation ◽

Key Players ◽

Rna Fragments ◽

Template Switch ◽

Small Rna Species ◽

Small Rna Library

Abstract BACKGROUND Small RNAs are key players in the regulation of gene expression and differentiation. However, many different classes of small RNAs (sRNAs) have been described with distinct biogenesis pathways and, as a result, with different biochemical properties. To analyze sRNAs by deep sequencing, complementary DNA synthesis requires manipulation of the RNA molecule itself. Thus, enzymatic activities during library preparation bias the library content owing to biochemical criteria. METHODS We compared 4 different manipulations of RNA for library preparation: (a) a ligation-based procedure allowing only 5′-mono-phosphorylated RNA to enter the library, (b) a ligation-based procedure allowing additional 5′-triphosphates and Cap structures, (c) a ligation-independent, template-switch-based library preparation, and (d) a template-switch-based library preparation allowing 3′-phosphorylated RNAs to enter the library. RESULTS Our data show large differences between ligation-dependent and ligation-independent libraries in terms of their preference for individual sRNA classes such as microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and transfer RNA fragments. Moreover, the miRNA composition is different between both procedures, and more microRNA isoforms (isomiRs) can be identified after pyrophosphatase treatment. piRNAs are enriched in template-switch libraries, and this procedure apparently includes more different RNA species. CONCLUSIONS Our data indicate that miRNAomics from both methods will hardly be comparable. Ligation-based libraries enrich for canonical miRNAs, which thus may be suitable methods for miRNAomics. Template-switch libraries contain increased numbers and different compositions of fragments and long RNAs. Following different interests for other small RNA species, ligation-independent libraries appear to show a more realistic sRNA landscape with lower bias against biochemical modifications.

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