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The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing

Abstract

Many metazoan cells can take up exogenous double-stranded (ds) RNA and use it to initiate an RNA silencing response, however, the mechanism for this uptake is ill-defined. Here, we identify the pathway for dsRNA uptake in Drosophila melanogaster S2 cells. Biochemical and cell biological analyses, and a genome-wide screen for components of the dsRNA-uptake machinery, indicated that dsRNA is taken up by an active process involving receptor-mediated endocytosis. Pharmacological inhibition of endocytic pathways disrupted exogenous dsRNA entry and the induction of gene silencing. This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. Thus, this entry pathway is required for systemic RNA silencing in whole organisms. In Drosophila cells, pharmacological evidence suggests that dsRNA entry is mediated by pattern-recognition receptors. The possible role of these receptors in dsRNA entry may link RNA interference (RNAi) silencing to other innate immune responses.

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Figure 1: RNAi in Drosophila S2 cells is dependent on the length of the dsRNA.
Figure 2: An active mechanism for uptake of long dsRNA in Drosophila S2 cells.
Figure 3: RNAi screen for genes involved in the RNAi pathway.
Figure 4: Endocytic uptake of dsRNA into Drosophila S2 cells is mediated by scavenger receptors.
Figure 5: Orthologues of the Drosophila genes confer an RNAi phenotype in C. elegans.

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Acknowledgements

We are grateful to members of the Andino and O'Farrell labs for support and discussions; G. Yudowski, F. Martin and M. Strandh for help with equipment and reagents; C. Murphy and M. Van Gilst for discussions and advice on C. elegans experiments; M. Von Zastrow for advice on immunofluorescence microscopy; and J. Frydman for critical reading of the manuscript. We thank the Davis, Kenyon, Kornberg and Vale laboratories at UCSF for materials and advice on a number of experiments. J. Ahringer kindly provided RNAi clones for the C. elegans experiments. This work was supported by a European Molecular Biology Organization (EMBO) fellowship to R.P.V.R. and a National Institutes of Health (NIH) grant AI40085 to R.A.

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Correspondence to Raul Andino.

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Saleh, MC., van Rij, R., Hekele, A. et al. The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing. Nat Cell Biol 8, 793–802 (2006). https://doi.org/10.1038/ncb1439

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