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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    • "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.
    Nucleic Acids Research 08/2013; 41(20). DOI:10.1093/nar/gkt717 · 9.11 Impact Factor
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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.
    PLoS ONE 05/2013; 8(5):e64804. DOI:10.1371/journal.pone.0064804 · 3.23 Impact Factor
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    • "In worms, the biogenesis and stability of 5′-polyphosphate small RNAs are dependent on RdRPs and WAGOs respectively, with these small RNAs being a component of the siRNA amplification pathway [33]. Signal amplification is controlled by using primary trigger siRNAs to instigate secondary siRNAs through RdRP for the enforced silencing, but limiting secondary siRNAs from doing further signal amplification [58]. Although small RNAs that mapped sense to coding regions were found in C. elegans and Ascaris suum 5'-monoP independent libraries, their existence and functionality were not confirmed; instead they were generally treated as non-specific degradation products [30,33]. "
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    ABSTRACT: Background: Small RNA mediated gene silencing is a well-conserved regulatory pathway. In the parasite Entamoeba histolytica an endogenous RNAi pathway exists, however, the depth and diversity of the small RNA population remains unknown. Results: To characterize the small RNA population that associates with E. histolytica Argonaute-2 (EhAGO2-2), we immunoprecipitated small RNAs that associate with it and performed one full pyrosequencing run. Data analysis revealed new features of the 27nt small RNAs including the 5'-G predominance, distinct small RNA distribution patterns on protein coding genes, small RNAs mapping to both introns and exon-exon junctions, and small RNA targeted genes that are clustered particularly in sections of genome duplication. Characterization of genomic loci to which both sense and antisense small RNAs mapped showed that both sets of small RNAs have 5'-polyphosphate termini; strand-specific RT-PCR detected transcripts in both directions at these loci suggesting that both transcripts may serve as template for small RNA generation. In order to determine whether small RNA abundance patterns account for strain-specific gene expression profiles of E. histolytica virulent and non-virulent strains, we sequenced small RNAs from a non-virulent strain and found that small RNAs mapped to genes in a manner consistent with their regulation of strain-specific virulence genes. Conclusions: We provided a full spectrum analysis for E. histolytica AGO2-2 associated 27nt small RNAs. Additionally, comparative analysis of small RNA populations from virulent and non-virulent amebic strains indicates that small RNA populations may regulate virulence genes.
    BMC Genomics 01/2013; 14(1):53. DOI:10.1186/1471-2164-14-53 · 3.99 Impact Factor
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