In C. elegans, High Levels of dsRNA Allow RNAi in the Absence of RDE-4

Jeffrey W Habig,Brenda L Bass,P Joseph Aruscavage,Thomas Preiss

doi:10.1371/journal.pone.0004052

Jeffrey W Habig, Brenda L Bass + Show 2 more

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https://doi.org/10.1371/journal.pone.0004052

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Journal: PloS one	Publication Date: Dec 29, 2008
Citations: 48	License type: CC BY 4.0

Affiliation: Howard Hughes Medical Institute, University of Utah

Abstract

C. elegans Dicer requires an accessory double-stranded RNA binding protein, RDE-4, to enact the first step of RNA interference, the cleavage of dsRNA to produce siRNA. While RDE-4 is typically essential for RNAi, we report that in the presence of high concentrations of trigger dsRNA, rde-4 deficient animals are capable of silencing a transgene. By multiple criteria the silencing occurs by the canonical RNAi pathway. For example, silencing is RDE-1 dependent and exhibits a decrease in the targeted mRNA in response to an increase in siRNA. We also find that high concentrations of dsRNA trigger lead to increased accumulation of primary siRNAs, consistent with the existence of a rate-limiting step during the conversion of primary to secondary siRNAs. Our studies also revealed that transgene silencing occurs at low levels in the soma, even in the presence of ADARs, and that at least some siRNAs accumulate in a temperature-dependent manner. We conclude that an RNAi response varies with different conditions, and this may allow an organism to tailor a response to specific environmental signals.

Highlights

The RNA interference (RNAi) and micro-RNA pathways employ small RNAs to modulate gene expression [1]
Results for adr;rde-1 animals were consistent with assays of GFP fluorescence in that sur-5::gfp mRNA levels were slightly lower in the adr;rde-1 strain after heat shock (Fig. 2B), but a statistically significant difference between the untreated and heat shocked samples was not observed (Fig. 2C). These findings suggested that when presented with high concentrations of trigger dsRNA, in this case provided by the GFP[IR] transgene, RDE-4 deficient strains were capable of silencing transgene expression through a process that involved loss of target mRNA, as expected in the canonical RNAi pathway
Our studies indicate that RNAi occurs in C. elegans deficient for RDE-4 if the trigger dsRNA is provided at high concentrations

Summary

Introduction

The RNA interference (RNAi) and micro-RNA (miRNA) pathways employ small RNAs to modulate gene expression [1]. In both pathways the small RNAs are ,21–25 nucleotides in length and are processed from dsRNA precursors by the RNase III enzyme Dicer. The dsRNA-binding protein (dsRBP) RDE-4 acts with Dicer (DCR-1) to facilitate processing of long dsRNA into primary (1u) siRNAs (Fig. 1; [2,3,4]). The Argonaute protein, RDE-1, interacts with RDE-4 [4,5], but is not necessary for processing long dsRNA by DCR-1 [3]. RDE-1 acts downstream of 1u siRNA production to facilitate a sequence specific interaction between the 1u siRNA and targeted mRNA. RRF-1 amplifies the RNAi response by using the mRNA as a template for producing secondary (2u) siRNAs, which direct the cleavage of the targeted mRNA by the enzyme, CSR-1 [7]

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