Protection from Feed-Forward Amplification in an Amplified RNAi Mechanism

Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Cell (Impact Factor: 32.24). 11/2012; 151(4):885-99. DOI: 10.1016/j.cell.2012.10.022
Source: PubMed


The effectiveness of RNA interference (RNAi) in many organisms is potentiated through the signal-amplifying activity of a targeted RNA-directed RNA polymerase (RdRP) system that can convert a small population of exogenously-encountered dsRNA fragments into an abundant internal pool of small interfering RNA (siRNA). As for any biological amplification system, we expect an underlying architecture that will limit the ability of a randomly encountered trigger to produce an uncontrolled and self-escalating response. Investigating such limits in Caenorhabditis elegans, we find that feed-forward amplification is limited by biosynthetic and structural distinctions at the RNA level between (1) triggers that can produce amplification and (2) siRNA products of the amplification reaction. By assuring that initial (primary) siRNAs can act as triggers but not templates for activation, and that the resulting (secondary) siRNAs can enforce gene silencing on additional targets without unbridled trigger amplification, the system achieves substantial but fundamentally limited signal amplification.

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    • "RRF-1 is a somatic RNA-dependent RNA polymerase acting upstream but necessary to feed the nuclear RNAi pathway. RRF-1 is guided by the RDE-1/siRNA complex to complementary mRNA targets to generate secondary siRNAs (Pak et al. 2012), which bind to NRDE-3 to be escorted to the nucleus (Guang et al. 2008). Although near perfect complementarity between siRNAs and their target RNA sequences is required for efficient gene silencing, the exposure of eukaryotes to siRNA frequently results in the unintended silencing of genes exhibiting ,100% sequence identity to the trigger siRNA, a phenomenon termed off-target gene silencing (reviewed by Svoboda 2007). "
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    ABSTRACT: Small RNAs recognize, bind, and regulate other complementary cellular RNAs. The introduction of small RNAs to eukaryotic cells frequently results in unintended silencing of related, but not identical, RNAs: a process termed off-target gene silencing. Off-target gene silencing is one of the major concerns during the application of small RNA-based technologies for gene discovery and the treatment of human disease. Off-target gene silencing is commonly thought to be due to inherent biochemical limitations of the RNAi machinery. Here we show that, following the introduction of exogenous sources of dsRNA, the nuclear RNAi pathway, but not its cytoplasmic counterparts, is the primary source of off-target silencing in Caenorhabditis elegans. In addition, we show that during the normal course of growth and development the nuclear RNAi pathway regulates repetitive gene families. Therefore, we speculate that RNAi off-target effects might not be "mistakes" but, rather, an intentional and genetically programmed aspect of small RNA-mediated gene silencing, which might allow small RNAs to silence rapidly evolving parasitic nucleic acids. Finally, reducing off-target effects by manipulating the nuclear RNAi pathway in vivo might improve the efficacy of small RNA-based technologies.
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    • "We have previously identified that sRNAs in E. histolytica are enriched on coding regions, rather than intergenic regions and that they are likely templated on both nascent and mature transcript (16). Structural features indicate that these AS sRNAs are generated independent of Dicer processing, and instead are likely from an RdRP-dependent pathway similar to the secondary siRNA pathway in C. elegans (9,12,37,39). In C. elegans, the generation of secondary sRNAs is RdRP dependent and depends on the presence of mRNA and the precedent generation of Dicer-derived primary siRNAs (37). "
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    ABSTRACT: RNA interference uses small RNAs (sRNA), which target genes for sequence-specific silencing. The parasite Entamoeba histolytica contains an abundant repertoire of 27 nt antisense (AS) sRNA with 5′-polyphosphate termini, but their roles in regulating gene expression have not been well established. We demonstrate that a gene-coding region to which large numbers of AS sRNAs map can serve as a ‘trigger’ and silence the gene fused to it. Silencing is mediated by generation of AS sRNAs with 5′-polyphosphate termini that have sequence specificity to the fused gene. The mechanism of silencing is independent of the placement of the trigger relative to the silenced gene but is dependent on the sRNA concentration to the trigger. Silencing requires transcription of the trigger-gene fusion and is maintained despite loss of the trigger plasmid. We used this approach to silence multiple amebic genes, including an E. histolytica Myb gene, which is upregulated during oxidative stress response. Silencing of the EhMyb gene decreased parasite viability under oxidative stress conditions. Thus, we have developed a new tool for genetic manipulation in E. histolytica with many advantages over currently available technologies. Additionally, these data shed mechanistic insights into a eukaryotic RNA interference pathway with many novel aspects.
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    • "This 5′ spreading has been observed operational in C. elegans in investigations of chimeric unc-22/gfp transgenes, from which the term transitive silencing was coined [17]. Recent work has provided evidence for a substantial difference in the effects of primary and secondary siRNAs, since the former can act as triggers but not as templates for activation, while the resulting secondary siRNAs can enforce gene silencing on additional targets without uncontrolled trigger amplification, leading to substantial but fundamentally limited signal amplification [18]. "
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    ABSTRACT: RNA interference (RNAi) is a gene-regulatory mechanism in eukarya that is based on the presence of double stranded RNA and that can act on both, the transcription or post-transcriptional level. In many species, RNA-dependent RNA polymerases (RdRPs) are required for RNAi. To study the function of the three RdRPs in the amoeba Dictyostelium discoideum, we have deleted the encoding genes rrpA, rrpB and rrpC in all possible combinations. In these strains, expression of either antisense or hairpin RNA constructs against the transgene lacZ resulted in a 50% reduced β-Galactosidase activity. Northern blots surprisingly revealed unchanged lacZ mRNA levels, indicative of post-transcriptional regulation. Only in rrpC knock out strains, low levels of β-gal small interfering RNAs (siRNAs) could be detected in antisense RNA expressing strains. In contrast to this, and at considerably higher levels, all hairpin RNA expressing strains featured β-gal siRNAs. Spreading of the silencing signal to mRNA sequences 5' of the original hairpin trigger was observed in all strains, except when the rrpC gene or that of the dicer-related nuclease DrnB was deleted. Thus, our data indicate that transitivity of an RNA silencing signal exists in D. discoideum and that it requires the two enzymes RrpC and DrnB.
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