scholarly journals Defective Interfering RNA Hinders the Activity of a Tombusvirus-Encoded Posttranscriptional Gene Silencing Suppressor

2005 ◽  
Vol 79 (1) ◽  
pp. 450-457 ◽  
Author(s):  
Zoltán Havelda ◽  
Csaba Hornyik ◽  
Anna Válóczi ◽  
József Burgyán
Keyword(s):  
Gene Silencing ◽  
Viral Infections ◽  
Rna Viruses ◽  
Helper Virus ◽  
Small Interfering Rnas ◽  
Posttranscriptional Gene Silencing ◽  
Virus Rna ◽  
Defective Interfering Rna ◽  
Interfering Rna

ABSTRACT Defective interfering (DI) RNAs are subviral replicons originating from the viral genome and are associated with many plant RNA viruses and nearly all animal RNA viruses. The presence of DI RNAs in tombusvirus-infected plants reduces the accumulation of helper virus RNA and results in the development of attenuated symptoms similar to those caused by tombusviruses defective in p19, the posttranscriptional gene silencing (PTGS) suppressor. In situ analysis of infected plants containing DI RNAs revealed that the extent of virus infection was spatially restricted as was found for p19-defective tombusvirus. Previously, p19 was shown to suppress PTGS by sequestering the small interfering RNAs (siRNAs), which act as the specificity determinant for PTGS. Our results demonstrate that DI RNAs dramatically elevate the level of virus-specific siRNAs in viral infections, resulting in the saturation of p19 and the accumulation of unbound siRNAs. Moreover, we showed that, at low temperature, where PTGS is inhibited, DI RNAs are not able to efficiently interfere with virus accumulation and protect the plants. These data show that the activation of PTGS plays a pivotal role in DI RNA-mediated interference. Our data also support a role for 21-nucleotide siRNAs in PTGS signaling.

scholarly journals Small Interfering RNAs That Trigger Posttranscriptional Gene Silencing Are Not Required for the Histone H3 Lys9 Methylation Necessary for Transgenic Tandem Repeat Stabilization in Neurospora crassa

2005 ◽  
Vol 25 (9) ◽  
pp. 3793-3801 ◽  
Author(s):  
Agustin Chicas ◽  
Emma C. Forrest ◽  
Silvia Sepich ◽  
Carlo Cogoni ◽  
Giuseppe Macino
Keyword(s):  
Gene Silencing ◽  
Neurospora Crassa ◽  
Chromatin Immunoprecipitation ◽  
Histone H3 ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Posttranscriptional Gene Silencing ◽  
Endogenous Gene ◽  
Homologous Genes ◽  
Interfering Rna

ABSTRACT In Neurospora crassa, the introduction of a transgene can lead to small interfering RNA (siRNA)-mediated posttranscriptional gene silencing (PTGS) of homologous genes. siRNAs can also guide locus-specific methylation of Lys9 of histone H3 (Lys9H3) in Schizosaccharomyces pombe. Here we tested the hypothesis that transgenically derived siRNAs may contemporaneously both activate the PTGS mechanism and induce chromatin modifications at the transgene and the homologous endogenous gene. We carried out chromatin immunoprecipitation using a previously characterized albino-1 (al-1) silenced strain but detected no alterations in the pattern of histone modifications at the endogenous al-1 locus, suggesting that siRNAs produced from the transgenic locus do not trigger modifications in trans of those histones tested. Instead, we found that the transgenic locus was hypermethylated at Lys9H3 in our silenced strain and remained hypermethylated in the quelling defective mutants (qde), further demonstrating that the PTGS machinery is dispensable for Lys9H3 methylation. However, we found that a mutant in the histone Lys9H3 methyltransferase dim-5 was unable to maintain PTGS, with transgenic copies being rapidly lost, resulting in reversion of the silenced phenotype. These results indicate that the defect in PTGS of the Δdim-5 strain is due to the inability to maintain the transgene in tandem, suggesting a role for DIM-5 in stabilizing such repeated sequences. We conclude that in Neurospora, siRNAs produced from the transgenic locus are used in the RNA-induced silencing complex-mediated PTGS pathway and do not communicate with an RNAi-induced initiation of transcriptional gene silencing complex to effect chromatin-based silencing.

Suppression of endogenous gene silencing by bidirectional cytoplasmic RNA decay in Arabidopsis

Science ◽  
2015 ◽  
Vol 348 (6230) ◽  
pp. 120-123 ◽  
Author(s):  
Xinyan Zhang ◽  
Ying Zhu ◽  
Xiaodan Liu ◽  
Xinyu Hong ◽  
Yang Xu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Plant Immunity ◽  
Rna Decay ◽  
Foreign Gene ◽  
Small Interfering Rnas ◽  
Developmental Defects ◽  
Posttranscriptional Gene Silencing ◽  
Endogenous Gene ◽  
Cytoplasmic Rna ◽  
Cognate Gene

Plant immunity against foreign gene invasion takes advantage of posttranscriptional gene silencing (PTGS). How plants elaborately avert inappropriate PTGS of endogenous coding genes remains unclear. We demonstrate in Arabidopsis that both 5′-3′ and 3′-5′ cytoplasmic RNA decay pathways act as repressors of transgene and endogenous PTGS. Disruption of bidirectional cytoplasmic RNA decay leads to pleiotropic developmental defects and drastic transcriptomic alterations, which are substantially rescued by PTGS mutants. Upon dysfunction of bidirectional RNA decay, a large number of 21- to 22-nucleotide endogenous small interfering RNAs are produced from coding transcripts, including multiple microRNA targets, which could interfere with their cognate gene expression and functions. This study highlights the risk of unwanted PTGS and identifies cytoplasmic RNA decay pathways as safeguards of plant transcriptome and development.

scholarly journals Defective Interfering RNA Viruses and the Host-Cell Response

1980 ◽  
pp. 137-192 ◽  
Author(s):  
John J. Holland ◽  
S. Ian T. Kennedy ◽  
Bert L. Semler ◽  
Charlotte L. Jones ◽  
Laurent Roux ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Response ◽  
Rna Viruses ◽  
Host Cell Response ◽  
Defective Interfering Rna ◽  
Interfering Rna

scholarly journals Investigation of the effects of P1 on HC-Pro-mediated gene silencing suppression through genetics and omics approaches

2020 ◽  
Author(s):  
Sin-Fen Hu ◽  
Wei-Lun Wei ◽  
Syuan-Fei Hong ◽  
Ru-Ying Fang ◽  
Hsin-Yi Wu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Rna Degradation ◽  
Novel Genes ◽  
Posttranscriptional Gene Silencing ◽  
Comparative Network Analysis ◽  
New Findings ◽  
A Subunit ◽  
Silencing Suppression ◽  
Viral Suppressors ◽  
Interfering Rna

Abstract Background: Posttranscriptional gene silencing (PTGS) is one of the most important mechanisms for plants during viral infection. However, viruses have also developed viral suppressors to negatively control PTGS by inhibiting microRNA (miRNA) and short-interfering RNA (siRNA) regulation in plants. The first identified viral suppressor, P1/HC-Pro, is a fusion protein. Upon infecting plants, the P1 protein itself is released from HC-Pro by the self-cleaving activity of P1. P1 has an unknown function in enhancing HC-Pro-mediated PTGS suppression. We performed proteomics to identify P1-interacting proteins. We also performed transcriptomics that were generated from Col-0 and various P1/HC-Pro-related transgenic plants to identify novel genes. The results showed several novel genes were identified through the comparative network analysis that might be involved in P1/HC-Pro-mediated PTGS suppression. Results: First, we demonstrated that P1 enhances HC-Pro function and that the mechanism might work through P1 binding to VERNALIZATION INDEPENDENCE 3/SUPERKILLER 8 (VIP3/SKI8), a subunit of the exosome, to interfere with the 5'-fragment of the PTGS-cleaved RNA degradation product. Second, specifically the AGO1 was specifically posttranslationally degraded in transgenic Arabidopsis expressing P1/HC-Pro of turnip mosaic virus (TuMV) (P1/HCTu plant). Third, the comparative network highlighted potentially critical genes in PTGS, including miRNA targets, calcium signaling, hormone (JA, ET, and ABA) signaling, and defense response. Conclusion: Through these genetic and omics approaches, we revealed an overall perspective to identify many critical genes involved in PTGS. These new findings significantly impact in our understanding of P1/HC-Pro-mediated PTGS suppression.

scholarly journals In Situ Characterization of Cymbidium Ringspot Tombusvirus Infection-Induced Posttranscriptional Gene Silencing in Nicotiana benthamiana

2003 ◽  
Vol 77 (10) ◽  
pp. 6082-6086 ◽  
Author(s):  
Zoltán Havelda ◽  
Csaba Hornyik ◽  
Aniello Crescenzi ◽  
József Burgyán
Keyword(s):  
Viral Infection ◽  
Gene Silencing ◽  
Nicotiana Benthamiana ◽  
Defense Mechanism ◽  
Vascular Bundles ◽  
Wild Type ◽  
Posttranscriptional Gene Silencing ◽  
Defective Mutant ◽  
Ptgs Suppressor

ABSTRACT In plants, posttranscriptional gene silencing (PTGS) is an ancient and effective defense mechanism against viral infection. A number of viruses encode proteins that suppress virus-activated PTGS. The p19 protein of tombusviruses is a potent PTGS suppressor which interferes with the onset of PTGS-generated systemic signaling and is not required for viral replication or for viral movement in Nicotiana benthamiana. This unique feature of p19 suppressor allowed us to analyze the mechanism of PTGS-based host defense and its viral suppression without interfering with other viral functions. In contrast to the necrotic symptoms caused by wild-type tombusvirus, the infection of p19-defective mutant virus results in the development of a typical PTGS-associated recovery phenotype in N. benthamiana. In this report we show the effect of PTGS on the viral infection process for N. benthamiana infected with either wild-type Cymbidium Ringspot Tombusvirus (CymRSV) or a p19-defective mutant (Cym19stop). In situ analyses of different virus-derived products revealed that PTGS is not able to reduce accumulation of virus in primary infected cells regardless of the presence of p19 PTGS suppressor. We also showed that both CymRSV and Cym19stop viruses move systemically in the vasculature, with similar efficiencies. However, in contrast to the uniform accumulation of CymRSV throughout systemically infected leaves, the presence of Cym19stop virus was confined to and around the vascular bundles. These results suggest that the role of p19 is to prevent the onset of mobile signal-induced systemic PTGS ahead of the viral infection front, leading to generalized infection.

An Automated High-Throughput Fluorescence In Situ Hybridization (FISH) Assay Platform for Use in the Identification and Optimization of siRNA-Based Therapeutics

2020 ◽  
pp. 247255522096004
Author(s):  
Hui H. Dou ◽  
Rommel Mallari ◽  
Andrew Pipathsouk ◽  
Amrita Das ◽  
Mei-Chu Lo
Keyword(s):  
In Situ Hybridization ◽  
Gene Silencing ◽  
Fluorescence In Situ Hybridization ◽  
High Throughput ◽  
Messenger Rna ◽  
Great Promise ◽  
Small Interfering Rnas ◽  
Response Curves ◽  
Vitro Assay

Since the revolutionary discovery of RNA interference (RNAi) more than 20 years ago, synthetic small interfering RNAs (siRNAs) have held great promise as therapeutic agents for treating human diseases by the specific knockdown of disease-causing gene products. To facilitate the development of siRNA therapeutics, a robust, high-throughput in vitro assay for measuring gene silencing is imperative during the initial siRNA lead sequence identification and, later, during the lead optimization with chemically modified siRNAs. There are several potential assays for measuring gene expression. Quantitative reverse transcription PCR (qRT-PCR) has been widely used to quantitate messenger RNA (mRNA). This method has a few disadvantages, however, such as the requirement for RNA isolation, complementary DNA (cDNA) generation, and PCR reaction, which are labor-intensive, limit the assay throughput, and introduce variability. We chose a high-content imaging assay, bDNA FISH, that combines the branched DNA (bDNA) technology with fluorescence in situ hybridization (FISH) to measure gene silencing by siRNAs because it is sensitive and robust with a short reagent procurement and assay development time. We also built a fully automated liquid-handling platform for executing bDNA FISH assays to increase throughput, and the system has a capacity of generating 192 concentration–response curves in a single run. We have successfully developed and executed the bDNA FISH assays for multiple targets using this automated platform to identify and optimize siRNA candidate molecules. Examples of the bDNA FISH assay for selected targets are presented.

scholarly journals Plant Virus-Derived Small Interfering RNAs Originate Predominantly from Highly Structured Single-Stranded Viral RNAs

2005 ◽  
Vol 79 (12) ◽  
pp. 7812-7818 ◽  
Author(s):  
Attila Molnár ◽  
Tibor Csorba ◽  
Lóránt Lakatos ◽  
Éva Várallyay ◽  
Christophe Lacomme ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Positive Strand ◽  
Posttranscriptional Gene Silencing ◽  
System A ◽  
Rna Duplexes ◽  
Positive Strand Rna ◽  
Dicer Cleavage ◽  
Pds Gene

ABSTRACT RNA silencing is conserved in a broad range of eukaryotes and includes the phenomena of RNA interference in animals and posttranscriptional gene silencing (PTGS) in plants. In plants, PTGS acts as an antiviral system; a successful virus infection requires suppression or evasion of the induced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected with positive-strand RNA viruses and provide specificity to this RNA-mediated defense. We present here the results of a survey of virus-specific siRNAs characterized by a sequence analysis of siRNAs from plants infected with Cymbidium ringspot tombusvirus (CymRSV). CymRSV siRNA sequences have a nonrandom distribution along the length of the viral genome, suggesting that there are hot spots for virus-derived siRNA generation. CymRSV siRNAs bound to the CymRSV p19 suppressor protein have the same asymmetry in strand polarity as the sequenced siRNAs and are imperfect double-stranded RNA duplexes. Moreover, an analysis of siRNAs derived from two other nonrelated positive-strand RNA viruses showed that they displayed the same asymmetry as CymRSV siRNAs. Finally, we show that Tobacco mosaic virus (TMV) carrying a short inverted repeat of the phytoene desaturase (PDS) gene triggered more accumulation of PDS siRNAs than the corresponding antisense PDS sequence. Taken together, these results suggest that virus-derived siRNAs originate predominantly by direct DICER cleavage of imperfect duplexes in the most folded regions of the positive strand of the viral RNA.

scholarly journals Investigation of the Effects of P1 on Hc-Pro-Mediated Gene Silencing Suppression Through Genetics and Omics Approaches

2020 ◽  
Author(s):  
Sin-Fen Hu ◽  
Wei-Lun Wei ◽  
Syuan-Fei Hong ◽  
Ru-Ying Fang ◽  
Hsin-Yi Wu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Transgenic Arabidopsis ◽  
Rna Degradation ◽  
Interacting Proteins ◽  
Viral Suppressor ◽  
Posttranscriptional Gene Silencing ◽  
New Findings ◽  
A Subunit ◽  
Silencing Suppression ◽  
Interfering Rna

Abstract Background: Viral suppressor negatively controls posttranscriptional gene silencing (PTGS) byinhibiting microRNA (miRNA) and short-interfering RNA (siRNA)regulation in plants. The first identified viral suppressor-P1/HC-Pro is afusion protein. Upon infecting plants, theP1 protein itself gets released from HC-Pro bythe self-cleaving activity of P1. P1 has an unknown function in enhancing HC-Pro-mediated PTGS suppression.We performed proteomics to identifyP1-interacting proteins and observedwhole gene correlationsin P1/HC-Pro-mediated PTGS suppression through transcriptomic studied comparative networks.Results: First, we demonstrated that P1 enhances HC-Pro function and that the mechanism might workthrough the P1 binding to VERNALIZATION INDEPENDENCE 3/ SUPERKILLER8 (VIP3/SKI8),a subunit of the exosome, to interferewith the 5'-fragment of the PTGS-cleaved RNA degradation product.Second,specifically the AGO1 wasposttranslationaldegraded in transgenic Arabidopsis expressing P1/HC-Pro of turnip mosaic virus (TuMV) (P1/HCTu plant).Third, transcriptomic comparative networkshighlighted critical genes inPTGS, including miRNA targets,calcium signaling, hormone (JA, ET, and ABA) signaling, and defense response.Conclusion: Through thesetransgenic and omics approaches, we revealed an overall perspective and new findings in our understandingof the mechanism of P1/HC-Pro-mediated PTGS suppression. Many of the criticalgenes that weresignificantlyimpacted in the omics profiles will be further investigated by CRISPR-knockoutor gain-of-function to understand PTGS in plant better.

scholarly journals Posttranscriptional gene silencing in nuclei

2010 ◽  
Vol 108 (1) ◽  
pp. 409-414 ◽  
Author(s):  
Paul Hoffer ◽  
Sergey Ivashuta ◽  
Olga Pontes ◽  
Alexa Vitins ◽  
Craig Pikaard ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Target Genes ◽  
Rna Degradation ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Posttranscriptional Gene Silencing ◽  
Subcellular Compartmentation ◽  
Specific Degradation ◽  
Precursor Mrna ◽  
Specific Sequences

In plants, small interfering RNAs (siRNAs) with sequence homology to transcribed regions of genes can guide the sequence-specific degradation of corresponding mRNAs, leading to posttranscriptional gene silencing (PTGS). The current consensus is that siRNA-mediated PTGS occurs primarily in the cytoplasm where target mRNAs are localized and translated into proteins. However, expression of an inverted-repeat double-stranded RNA corresponding to the soybeanFAD2-1Adesaturase intron is sufficient to silenceFAD2-1, implicating nuclear precursor mRNA (pre-mRNA) rather than cytosolic mRNA as the target of PTGS. SilencingFAD2-1using intronic or 3′-UTR sequences does not affect transcription rates of the target genes but results in the strong reduction of target transcript levels in the nucleus. Moreover, siRNAs corresponding to pre-mRNA–specific sequences accumulate in the nucleus. In Arabidopsis, we find that two enzymes involved in PTGS, Dicer-like 4 and RNA-dependent RNA polymerase 6, are localized in the nucleus. Collectively, these results demonstrate that siRNA-directed RNA degradation can take place in the nucleus, suggesting the need for a more complex view of the subcellular compartmentation of PTGS in plants.

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