Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing
210
Citation
20
Reference
10
Related Paper
Citation Trend
Keywords:
Dicer
Trans-acting siRNA
RNA Silencing
Argonaute
RNA-induced transcriptional silencing
Cite
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.
Argonaute
RNA-induced silencing complex
Trans-acting siRNA
RNA Silencing
RNA-induced transcriptional silencing
Dicer
Piwi-interacting RNA
Cite
Citations (410)
Dicer
Argonaute
Trans-acting siRNA
RNA Silencing
RNA-induced silencing complex
RNA-induced transcriptional silencing
Cite
Citations (78)
Virus infection in plants is limited by RNA silencing. In turn, viruses can counter RNA silencing with silencing suppressors. Viral suppressors of RNA silencing have been shown to play a role in symptom development in plants. We here study four different strategies employed by silencing suppressors: small interfering RNA (siRNA) binding, double-strand RNA (dsRNA) binding and degrading or inactivating Argonaute. We study the effect of the suppressors on viral accumulation within the cell as well as its spread on a tissue with mathematical and computational models. We find that suppressors which target Argonaute are very effective in a single cell, but that targeting dsRNA or siRNA is much more effective at the tissue level. Although targeting Argonaute can be beneficial for viral spread, it can also cause hindrance in some cases owing to raised levels of siRNAs that can spread to other cells.
Argonaute
RNA Silencing
Trans-acting siRNA
RNA-induced silencing complex
Piwi-interacting RNA
RNA-induced transcriptional silencing
Cite
Citations (10)
Argonaute
Dicer
Trans-acting siRNA
RasiRNA
RNA Silencing
RNA-induced silencing complex
Small nucleolar RNA
Cite
Citations (716)
Small RNA molecules regulate eukaryotic gene expression during development and in response to stresses including viral infection. Specialized ribonucleases and RNA-binding proteins govern the production and action of small regulatory RNAs. After initial processing in the nucleus by Drosha, precursor microRNAs (pre-miRNAs) are transported to the cytoplasm, where Dicer cleavage generates mature microRNAs (miRNAs) and short interfering RNAs (siRNAs). These double-stranded products assemble with Argonaute proteins such that one strand is preferentially selected and used to guide sequence-specific silencing of complementary target mRNAs by endonucleolytic cleavage or translational repression. Molecular structures of Dicer and Argonaute proteins, and of RNA-bound complexes, have offered exciting insights into the mechanisms operating at the heart of RNA-silencing pathways.
Dicer
Argonaute
Drosha
Trans-acting siRNA
RNA-induced silencing complex
RNA Silencing
RasiRNA
Cite
Citations (982)
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.
Dicer
Argonaute
RNA Silencing
Trans-acting siRNA
RNA-induced transcriptional silencing
RNA-induced silencing complex
Cite
Citations (17)
ABSTRACT RNA interference (RNAi) is a eukaryotic gene-silencing mechanism that functions in antiviral immunity in diverse organisms. To combat RNAi-mediated immunity, viruses encode viral suppressors of RNA silencing (VSRs) that target RNA and protein components in the RNAi machinery. Although the endonuclease Dicer plays key roles in RNAi immunity, little is known about how VSRs target Dicer. Here, we show that the B2 protein from Wuhan nodavirus (WhNV), the counterpart of Flock House virus (FHV), suppresses Drosophila melanogaster RNAi by directly interacting with Dicer-2 (Dcr-2) and sequestering double-stranded RNA (dsRNA) and small interfering RNA (siRNA). Further investigations reveal that WhNV B2 binds to the RNase III and Piwi-Argonaut-Zwille (PAZ) domains of Dcr-2 via its C-terminal region, thereby blocking the activities of Dcr-2 in processing dsRNA and incorporating siRNA into the RNA-induced silencing complex (RISC). Moreover, we uncover an interrelationship among diverse activities of WhNV B2, showing that RNA binding enhances the B2–Dcr-2 interaction by promoting B2 homodimerization. Taken together, our findings establish a model of suppression of Drosophila RNAi by WhNV B2 targeting both Dcr-2 and RNA and provide evidence that an interrelationship exists among diverse activities of VSRs to antagonize RNAi.
Dicer
RNA Silencing
RNA-induced silencing complex
RNA-induced transcriptional silencing
Trans-acting siRNA
Argonaute
Cite
Citations (49)
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.
RNA-induced silencing complex
Argonaute
Trans-acting siRNA
RNA Silencing
RNA-induced transcriptional silencing
Cite
Citations (96)
Argonaute
Dicer
Trans-acting siRNA
RNA Silencing
RNA-induced silencing complex
RNA-induced transcriptional silencing
RasiRNA
DNA-directed RNA interference
Cite
Citations (1)
Double-stranded RNA (dsRNA) induces a sequence-specific silencing in eukaryotic cells. This silencing process beggins when long dsRNA is cleaved to 21 to 26 long small RNA by means of the RNAse III-type enzyme Dicer. These small dsRNA are included into silencing effector complexes, that are targeted to complementary sequences. Small RNA dependent gene silencing can be achieved by distinct mechanisms based depending mainly on the nature of target sequences and on the proteins present in the effector complex. The route of interference RNA (RNAi) begins when Dicer yields small interference RNA (siRNA) that bind to complementary mRNA for its degradation, forming the RISC complex. siRNA are naturally formed from transposons and dsRNA viruses during its replication, as well as from other bidirectional transcribed repetitive sequences. Some of the enzymes thar are part of the RNAi machinery, including Dicer, are encoded by multigene families in many species, that also play a role in other mechanisms of RND-dependent gene silencing. MicroRNA's (miRNA) are other small RNA's that can induce gene silencing at the mRNA level. These are formed in a general manner when Dicer process hairpin structures resulting from the transcription of non-coding sequences from plant and animal genomes. miRNA's are integrated into a RISC-like complex, after which, depending on their degree of complementarity with target mRNA, can either repress translation or induce mRNA degradation. miRNA-dependent silencing is essential for the development of multicellular organisms. Artificial RNAi induction by means of siRNA or miRNA is being used as a tool to inactivate gene expression in culture cells and in living organisms. This review focuses on the progress in the understanding of the mechanisms involved in gene regulation by RNA in animals and details some current efforts to apply theses phenomena as a tool in research and in the therapeutic of human diseases.
Dicer
RNA Silencing
RNA-induced silencing complex
Trans-acting siRNA
Argonaute
RNA-induced transcriptional silencing
Cite
Citations (10)