Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells
Thomas OhrtJörg MützeWolfgang StaroskeLasse WeinmannJulia HöckKarin CrellGunter MeisterPetra Schwille
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Abstract:
Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large approximately 3 MDa complex in the cytoplasm and a 20-fold smaller complex of approximately 158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments.Keywords:
RNA-induced silencing complex
Argonaute
A bstract : Transient gene silencing in mammalian cells can be mediated by double stranded RNA (dsRNAs) molecules of ∼20–25 nucleotides termed short interfering (siRNAs). Naturally occurring siRNAs in lower eukaryotes have characteristic structural elements; however, little is known about what features are critical for an exogenous siRNA to mediate RNAi in mammals. We have recently determined some of the critical parameters that influence the efficiency of siRNA‐mediated RNAi in mammalian cells and have been considering the use of RNAi as a functional genomics tool, particularly for high throughput analysis, and the potential use of RNAi as a therapeutic tool.
Trans-acting siRNA
RNA Silencing
Functional Genomics
RNA-induced transcriptional silencing
DNA-directed RNA interference
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RNA silencing plays an important role in plants in defence against viruses. To overcome this defence, plant viruses encode suppressors of RNA silencing. The most common mode of silencing suppression is sequestration of double-stranded RNAs involved in the antiviral silencing pathways. Viral suppressors can also overcome silencing responses through protein–protein interaction. The poleroviral P0 silencing suppressor protein targets ARGONAUTE (AGO) proteins for degradation. AGO proteins are the core component of the RNA-induced silencing complex (RISC). We found that P0 does not interfere with the slicer activity of pre-programmed siRNA/miRNA containing AGO1, but prevents de novo formation of siRNA/miRNA containing AGO1. We show that the AGO1 protein is part of a high-molecular-weight complex, suggesting the existence of a multi-protein RISC in plants. We propose that P0 prevents RISC assembly by interacting with one of its protein components, thus inhibiting formation of siRNA/miRNA–RISC, and ultimately leading to AGO1 degradation. Our findings also suggest that siRNAs enhance the stability of co-expressed AGO1 in both the presence and absence of P0.
Degradation
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Degradation of homologous RNA in RNA interference is carried out by functional RNA-induced silencing complex (RISC). RISC contains Dicer, Argonaute proein, siRNA and other components. Researching structures and functions of these components is primary important for understanding assembly and functional mechanism of RISC, as well as the whole RNAi pathway. Recent research works showed that Dicer, containing RNaseIII domain, is responsible for production of siRNA at the beginning of RNAi, and guarantees the stability of RISC intermediate in assembly process. As the core component of RISC, Argonaute protein functions as slicer to cleave target RNA and offers the binding site of siRNA in RISC assembly, which are depended on PIWI domain and PAZ domain separately. Although there is only one strand of siRNA that is the guider of RISC, the double stranded structural character of siRNA is determinant of RNAi. Except those, there are still other components with unknown functions in RISC. The knowledge about RISC components and assembly now, is basis of a presumed RISC assembly model.
Argonaute
Dicer
RNA-induced silencing complex
Piwi-interacting RNA
RNA Silencing
Trans-acting siRNA
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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).
Argonaute
RNA-induced silencing complex
Trans-acting siRNA
RNA Silencing
Dicer
RNA-induced transcriptional silencing
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Argonaute
Trans-acting siRNA
RNA-induced silencing complex
RNA Silencing
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Incorporation of chemical modifications into small interfering RNAs (siRNAs) increases their metabolic stability and improves their tissue distribution. However, how these modifications impact interactions with Argonaute-2 (Ago2), the molecular target of siRNAs, is not known. Herein we present the crystal structure of human Ago2 bound to a metabolically stable siRNA containing extensive backbone modifications. Comparison to the structure of an equivalent unmodified-siRNA complex indicates that the structure of Ago2 is relatively unaffected by chemical modifications in the bound siRNA. In contrast, the modified siRNA appears to be much more plastic and shifts, relative to the unmodified siRNA, to optimize contacts with Ago2. Structure-activity analysis reveals that even major conformational perturbations in the 3' half of the siRNA seed region have a relatively modest effect on knockdown potency. These findings provide an explanation for a variety of modification patterns tolerated in siRNAs and a structural basis for advancing therapeutic siRNA design.
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RNA interference is a process through which double-stranded RNA(dsRNA) induces the sequence-specific posttranscriptional silencing of gene. Argonaute proteins consists of a highly-conserved family with many members. They are the core component of RNA-induced silencing complex (RISC) essential for RNAi. Argonaute proteins selectively recruit miRNAs and siRNAs and also interact with Dicers. Their PIWI box interacts directly with the RNaseⅢ domain of Dicer. The interaction between PIWI and Dicer may facilitate the release of miRNA/siRNA. Furthermore, argonaute proteins may be the source of endonuclease activity in RNAi.
Argonaute
Dicer
RasiRNA
RNA-induced silencing complex
Piwi-interacting RNA
Trans-acting siRNA
RNA Silencing
RNA-induced transcriptional silencing
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A new era in genetics has started 15 years ago, when co-suppression in petunia has been discovered. Later, co-suppression was identified as RNA interference (RNAi) in many plant and lower eukaryote animals. Although an ancient antiviral host defense mechanism in plants, the physiologic role of RNAi in mammals is still not completely understood. RNAi is directed by short interfering RNAs (siRNAs), one subtype of short double stranded RNAs. In this review we summarize the history and mechanisms of RNAi. We also aim to highlight the correlation between structure and efficacy of siRNAs. Delivery is the most important obstacle for siRNA based gene therapy. Viral and nonviral deliveries are discussed. In vivo delivery is the next obstacle to clinical trials with siRNAs. Although hydrodynamic treatment is effective in animals, it cannot be used in human therapy. One possibility is organ selective catheterization. The known side effects of synthesized siRNAs are also discussed. Although there are many problems to face in this new field of gene therapy, successful in vitro and in vivo experiments raise hope for treating human disease with siRNA.
Trans-acting siRNA
RNA Silencing
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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.
Argonaute
RNA-induced silencing complex
Trans-acting siRNA
RNA Silencing
RNA-induced transcriptional silencing
Piwi-interacting RNA
RasiRNA
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Citations (34)
Argonaute
Piwi-interacting RNA
RNA Silencing
Trans-acting siRNA
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Citations (19)