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.RNAi is a highly conserved dsRNA-guided mechanism that mediates sequence-specific gene silencing 1 . A number of animal cells can naturally take up exogenous dsRNA and use it to initiate RNAi silencing 2,3 . In some organisms, such as Drosophila, certain cells can efficiently take up dsRNA but seem to be unable to transmit this dsRNA to other cells in the body 4 . dsRNA uptake without further transmission to other cells has also been reported for some mammalian cell types [5][6][7] . Other organisms (such as C. elegans or juvenile grasshoppers) can both take up dsRNA and spread it systemically to elicit an RNAi response throughout the entire animal 8,9 . The mechanisms of uptake and spread of dsRNA are poorly understood. It is unclear whether dsRNA enters cells through passive, non-specific mechanisms, or whether there is an active mechanism that controls entry. Genetic analysis to identify genes involved in systemic spread of dsRNA in C. elegans isolated several mutants unable to distribute an ingested dsRNA signal from the gut throughout the body 8,10,11 . One of these, SID-1 (also known as RSD-8) is a putative transmembrane protein required for systemic spread 8 . When expressed ectopically in Drosophila cells, SID-1 enhanced the RNAi response observed at low dsRNA 5Correspondence should be addressed to R.A. (e-mail: Raul.Andino@UCSF.edu). 3 Current address: Canada Research Chair in Innate Immunity, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada 4 These authors contributed equally to this work.Note: Supplementary Information is available on the Nature Cell Biology website. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. concentrations 12 , raising the possibility that SID-1 may function as a channel on the cell surface for uptake of dsRNA. However, endogenous sid-1 homologues have not been found in the Drosophila genome, yet Drosophila S2 cells...

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“…Incubation of S2 cells with FITC-dsRNA fragments ranging from 21-700 bp showed a positive correlation between fragment size and cell uptake of dsRNA as measured by intracellular fluorescence, with maximum internalization of dsRNA recorded at 700 bp (Ulvila et al, 2006). A similar trend was observed when S2 cells were incubated with 21-592 bp dsRNA targeting firefly luciferase, with maximum knockdown achieved using 211 and 592 bp dsRNA (Saleh et al, 2006). In T. castaneum, injection of long dsRNA fragments (85-425 bp)…”

“…The second mechanism implicates involvement of receptor-mediated endocytosis in dsRNA uptake. Screening of a dsRNA library performed by Saleh et al (2006) in D. melanogaster S2 cells identified 23 genes coding for components of endocytosis and vesicle-mediated trafficking that were necessary for uptake and processing of exogenous dsRNA. These included genes for clathrin heavy chain and its adaptor AP-50, which mediate early endocytic uptake, and vacuolar H + -ATPase (V-H-ATPase), involved in controlling endocytic vesicle trafficking and protein sorting.…”

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“…87,137 A second mechanism for taking sRNA into cells involves clathrin-mediated endocytosis and was first discovered in D. melanogaster. 101,102 Here, the scavenger receptor is responsible for recognizing the sRNA and initiating endocytosis.…”