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Two classes of silencing RNAs move between Caenorhabditis elegans tissues

Abstract

Organism-wide RNA interference (RNAi) is due to the transport of mobile silencing RNA throughout the organism, but the identities of these mobile RNA species in animals are unknown. Here, we present genetic evidence that both the initial double-stranded RNA (dsRNA), which triggers RNAi, and at least one dsRNA intermediate produced during RNAi can act as or generate mobile silencing RNA in C. elegans. This dsRNA intermediate requires the long dsRNA-binding protein RDE-4, the endonuclease DCR-1, which cleaves long dsRNA into double-stranded short-interfering RNA (ds-siRNA), and the putative nucleotidyltransferase MUT-2 (RDE-3). However, single-stranded siRNA and downstream secondary siRNA produced upon amplification by the RNA-dependent RNA polymerase RRF-1 do not generate mobile silencing RNA. Restricting intertissue transport to long dsRNA and directly processed siRNA intermediates rather than amplified siRNA may serve to modulate the extent of systemic silencing in proportion to available dsRNA.

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Figure 1: RNAi-independent biogenesis of mobile RNA from expressed dsRNA.
Figure 2: Restricted expression of RDE-4 and MUT-2 but not RDE-1 enables non–cell-autonomous RNA silencing.
Figure 3: RDE-4- and MUT-2- but not RDE-1–processed ingested dsRNA is mobile.
Figure 4: Biogenesis of mobile RNA in C. elegans.

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Acknowledgements

We thank K. Ragkousi, S. Mango and members of the Hunter lab, particularly K. Pang, J. Brooks and D. Schott for comments on the manuscript; the C. elegans Genetics Center for some strains; H. Zhang, National Institute of Biological Sciences, for HZ202; S. Ekman (Harvard University) for two constructs; and the US National Institutes of Health (grant K99-GM085200 to A.M.J. and GM089795 to C.P.H.) and the National Science Foundation (MCB-0744029 to C.P.H.) for funding.

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A.M.J. conducted the experiments and G.A.G. generated most of the DNA constructs; A.M.J. and C.P.H. designed the study, analyzed the data and wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Craig P Hunter.

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The authors declare no competing financial interests.

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Supplementary Figures 1–4, Supplementary Tables 1 and 2, and Supplementary Methods (PDF 5014 kb)

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Jose, A., Garcia, G. & Hunter, C. Two classes of silencing RNAs move between Caenorhabditis elegans tissues. Nat Struct Mol Biol 18, 1184–1188 (2011). https://doi.org/10.1038/nsmb.2134

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