Gene silencing by double stranded Ribonucleic acid (RNA)
Hafiz Ghulam Muhu‐Din AhmedAmna Amna AmnaShadab ShaukatIqra KousarMaria RafiqMaria IlyasAziz UllahKhuda Buksh
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Abstract:
Ribonucleic acid (RNA) silencing, RNA interference (RNAi) or post-transcriptional gene silencing takes place in a variety of eukaryotes and it was discovered firstly in the plants. The RNA silencing process is activated by a trigger from dsRNA predecessor. A very important step in the silencing pathways the conversion of dsRNA into small duplexes of RNA of the representative length and arrangement. Then these small dsRNA monitor RNA silencing by different mechanisms. Post transcriptional gene silencing mechanisms were initially identified as an anti-viral process that give protection to the organisms from the viruses or which inhibit the unsystematic incorporation of transposable components. The basic aim of this review article is to study the mechanism of gene silencing by dsRNA and the roles of certain proteins in cellular post transcriptional RNA silencing machinery and finally we also discuss the RNA silencing as an anti-viral defense mechanism in the plants.Keywords:
RNA Silencing
RNA-induced transcriptional silencing
RNA-induced silencing complex
Trans-acting siRNA
Argonaute
Piwi-interacting RNA
Plant RNA silencing machinery enlists four primary classes of proteins to achieve sequence-specific regulation of gene expression and mount an antiviral defense. These include Dicer-like ribonucleases (DCLs), Argonaute proteins (AGOs), dsRNA-binding proteins (DRBs), and RNA-dependent RNA polymerases (RDRs). Although at least four distinct endogenous RNA silencing pathways have been thoroughly characterized, a detailed understanding of the antiviral RNA silencing pathway is just emerging. In this report, we have examined the role of four DCLs, two AGOs, one DRB, and one RDR in controlling viral RNA accumulation in infected Arabidopsis plants by using a mutant virus lacking its silencing suppressor. Our results show that all four DCLs contribute to antiviral RNA silencing. We confirm previous reports implicating both DCL4 and DCL2 in this process and establish a minor role for DCL3. Surprisingly, we found that DCL1 represses antiviral RNA silencing through negatively regulating the expression of DCL4 and DCL3. We also implicate DRB4 in antiviral RNA silencing. Finally, we show that both AGO1 and AGO7 function to ensure efficient clearance of viral RNAs and establish that AGO1 is capable of targeting viral RNAs with more compact structures, whereas AGO7 and RDR6 favor less structured RNA targets. Our results resolve several key steps in the antiviral RNA silencing pathway and provide a basis for further in-depth analysis.
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RNA-induced silencing complex
Trans-acting siRNA
RNA Silencing
RNA-induced transcriptional silencing
Dicer
Piwi-interacting RNA
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Argonaute
Trans-acting siRNA
RNA-induced silencing complex
RNA Silencing
Dicer
RNA-induced transcriptional silencing
Piwi-interacting RNA
RasiRNA
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Plants utilize a variety of defense mechanisms against invading pathogens. RNA-interference (RNAi) is a defense mechanism that plants use to combat viruses1. Double-stranded virus RNA is recognized by host Dicer, an endonuclease that is part of the plant’s immune system, and cleaved at different positions creating 21-25 base pair small interfering RNAs (siRNAs)2. The siRNA becomes part of the RNA-induced silencing complex (RISC) with the protein Argonaute, and is used to locate complementary viral or host mRNAs. Once the complementary strand is located, it is cleaved by Argonaute, a protein component of RISC(Fig. 1), effectively silencing the gene. This phenomenon allows RNA viruses to be used as vectors for silencing host genes in a process termed virus-induced gene silencing (VIGS).
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Abstract RNA interference/silencing mechanisms triggered by double‐stranded RNA (dsRNA) have been described in many eukaryotes, including fungi. These mechanisms have in common small RNA molecules (siRNAs or microRNAs) originating from dsRNAs that, together with the effector protein Argonaute, mediate silencing. The genome of the fungal pathogen Candida albicans harbours a well‐conserved Argonaute and a non‐canonical Dicer, essential members of silencing pathways. Prototypical siRNAs are detected as members of the C. albicans transcriptome, which is potential evidence of RNA interference/silencing pathways in this organism. Surprisingly, expression of a dsRNA a hairpin ADE2 dsRNA molecule to interfere with the endogenous ADE2 mRNA did not result in down‐regulation of the message or produce adenine auxotrophic strains. Cell free assays showed that the hairpin dsRNA was a substrate for the putative C. albicans Dicer, discounting the possibility that the nature of the dsRNA trigger affects silencing functionality. Our results suggested that unknown cellular events govern the functionality of siRNAs originating from transgenes in RNA interference/silencing pathways in C. albicans . Copyright © 2010 John Wiley & Sons, Ltd.
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AGO/RISC-mediated antiviral RNA silencing, an important component of the plant's immune response against RNA virus infections, was recapitulated in vitro. Cytoplasmic extracts of tobacco protoplasts were applied that supported Tombusvirus RNA replication, as well as the formation of RNA-induced silencing complexes (RISC) that could be functionally reconstituted with various plant ARGONAUTE (AGO) proteins. For example, when RISC containing AGO1, 2, 3 or 5 were programmed with exogenous siRNAs that specifically targeted the viral RNA, endonucleolytic cleavages occurred and viral replication was inhibited. Antiviral RNA silencing was disabled by the viral silencing suppressor p19 when this was present early during RISC formation. Notably, with replicating viral RNA, only (+)RNA molecules were accessible to RISC, whereas (-)RNA replication intermediates were not. The vulnerability of viral RNAs to RISC activity also depended on the RNA structure of the target sequence. This was most evident when we characterized viral siRNAs (vsiRNAs) that were particularly effective in silencing with AGO1- or AGO2/RISC. These vsiRNAs targeted similar sites, suggesting that accessible parts of the viral (+)RNA may be collectively attacked by different AGO/RISC. The in vitro system was, hence, established as a valuable tool to define and characterize individual molecular determinants of antiviral RNA silencing.
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RNA-induced transcriptional silencing
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Argonaute
Dicer
Trans-acting siRNA
RNA Silencing
RNA-induced silencing complex
RNA-induced transcriptional silencing
RasiRNA
DNA-directed RNA interference
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Heterochromatic regions of the genome are epigenetically regulated to maintain a heritable '"silent state"'. In fission yeast and other organisms, epigenetic silencing is guided by nascent transcripts, which are targeted by the RNA interference pathway. The key effector complex of the RNA interference pathway consists of small interfering RNA molecules (siRNAs) associated with Argonaute, assembled into the RNA-induced transcriptional silencing (RITS) complex. This review focuses on our current understanding of how RITS promotes heterochromatin formation, and in particular on the role of Argonaute-containing complexes in many other functions such as quelling, release of RNA polymerases, cellular quiescence and genome defense.
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In most eukaryotes, RNA silencing is an adaptive immune system regulating key biological processes including antiviral defense. To evade this response, viruses of plants, worms and insects have evolved viral suppressors of RNA silencing proteins (VSRs). Various VSRs, such as P1 from Sweet potato mild mottle virus (SPMMV), inhibit the activity of RNA-induced silencing complexes (RISCs) including an ARGONAUTE (AGO) protein loaded with a small RNA. However, the specific mechanisms explaining this class of inhibition are unknown. Here, we show that SPMMV P1 interacts with AGO1 and AGO2 from Arabidopsis thaliana, but solely interferes with AGO1 function. Moreover, a mutational analysis of a newly identified zinc finger domain in P1 revealed that this domain could represent an effector domain as it is required for P1 suppressor activity but not for AGO1 binding. Finally, a comparative analysis of the target RNA binding capacity of AGO1 in the presence of wild-type or suppressor-defective P1 forms revealed that P1 blocks target RNA binding to AGO1. Our results describe the negative regulation of RISC, the small RNA containing molecular machine.
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Trans-acting siRNA
RNA Silencing
RNA-induced transcriptional silencing
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CONTENTS 13.1 Introduction ............................................................................................................................. 197 13.2 Experimental RNA Silencing ................................................................................................. 19813.2.1 Aspergillus fumigatus ................................................................................................ 198 13.2.2 Aspergillus fl avus and Aspergillus parasiticus ......................................................... 198 13.2.3 Experimental RNA Silencing During Infection ........................................................ 19913.3 Genetic Analysis of Experimental RNA Silencing ................................................................ 199 13.3.1 Aspergillus nidulans RNA Silencing Model ............................................................. 199 13.3.2 RNA Silencing Proteins in Aspergillus nidulans ...................................................... 20013.4 Aspergillus RNA Silencing Gene Evolution ........................................................................... 200 13.4.1 Dicers and Argonautes ............................................................................................... 201 13.4.2 RNA-Dependent RNA Polymerases .......................................................................... 20113.5 Possible Roles of Aspergillus RNA Silencing in Nature ........................................................ 203 13.5.1 Meiotic Silencing ....................................................................................................... 203 13.5.2 Quelling ..................................................................................................................... 204 13.5.3 RNAi-Mediated Heterochromatic Silencing ............................................................. 20413.6 Future Directions .................................................................................................................... 205 References .......................................................................................................................................... 20613.1 Introduction Small noncoding RNAs have numerous biological functions, mediating processes such as post-transcriptional gene silencing, heterochromatic silencing, antiviral defense, and transposable element control.1-3 The proteins involved in small RNA use and production are called RNA silencing proteins. Core RNA silencing proteins are Dicer, Argonaute, and RNA-dependent RNA polymerase (RDRP). Dicer is an RNAseIII-containing protein responsible for processing dsRNA into various small RNA species, typically 21-25 nt in length.4,5 Dicer-processed small RNAs are incorporated into Argonaute - containing effector complexes, such as RISC (RNA-induced silencing complex), which uses the incorporated small RNA to fi nd and cleave complementary mRNA.6 Argonaute proteins are made up of two major domains, a PAZ domain and a Piwi domain.7,8 The PAZ domain has small RNA binding activity 9,10 and the Piwi domain, at least in RISC complexes, contains a "slicer" activity that degrades target mRNAs.11-13 RDRPs are thought to participate in RNA silencing processes by forming dsRNA for Dicer processing or by directly forming small RNAs for incorporation into effector complexes.2,14-16In the last 10 years a few specifi c biological phenomena in fungi have been linked to RNA silencing. These include cosuppression in Neurospora crassa (quelling),17 meiotic silencing of unpaired DNA in N. crassa,18 and some types of heterochromatic silencing in Schizosaccharomyces pombe (RNAi-mediated heterochromatic silencing).19 Most of what is known about fungal RNA silencing stems from work on these specifi c phenomena. How common these processes are across the Fungal Kingdom is unknown, but analysis of available fungal genomes suggests that they cannot be fundamentally conserved.
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JMJ14 is a histone H3 Lys4 (H3K4) trimethyl demethylase that affects mobile RNA silencing in an Arabidopsis transgene system. It also influences CHH DNA methylation, abundance of endogenous transposon transcripts, and flowering time. JMJ14 acts at a point in RNA silencing pathways that is downstream from RNA-dependent RNA polymerase 2 (RDR2) and Argonaute 4 (AGO4). Our results illustrate a link between RNA silencing and demethylation of histone H3 trimethylysine. We propose that JMJ14 acts downstream from the Argonaute effector complex to demethylate histone H3K4 at the target of RNA silencing.
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RNA-induced transcriptional silencing
RNA-induced silencing complex
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RNA Silencing
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Demethylase
RasiRNA
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