The phenomenon of RNAi silencing is widely conserved among all higher eukaryotes. Exploiting this process is becoming increasingly important as an experimental tool, as well as for therapeutic applications. Although most cells possess the basic RNAi core machinery, some cell types have the intriguing ability to naturally take up exogenous dsRNA and use it to initiate RNAi silencing2,3,5–7
. Furthermore, some organisms, such as plants, C. elegans
and planaria (Girardia tigrina
) can transmit the RNA silencing signal from cell to cell, resulting in the systemic spread of the RNAi response8,26,29,30
. It is currently believed that insects lack a pathway for the systemic spreading of RNAi. Nevertheless, injected dsRNA elicits cell non-autonomous RNAi in adult Drosophila
, juvenile grasshopper, Tribolium castaneum
(flour beetle) and Anopheles gambiae9,31–33
. In plants, it seems that systemic spread relies on the plasmodesmal channel system, which connects all the cells in the plant34,35
. However, this system is absent from animal organisms. Despite the importance of RNAi processes, little is known about the machineries that mediate either dsRNA uptake or systemic spread of the RNAi signal in animal cells. A number of genetic screens using C. elegans
have identified components required for systemic spread of an RNAi signal. Because systemic RNA silencing is a multistep process that requires uptake, amplification and spread of the silencing signal, the specific functions of these components within this complex process have not been precisely defined. Here, we sought to specifically identify the machinery that mediates uptake of exogenous dsRNA to induce an RNAi response using a less complex model system. As Drosophila
S2 cells can efficiently take up exogenous dsRNA they provided with a well-defined system to identify the mechanism and components of dsRNA entry. Using biochemical, genomic and pharmacological approaches we found that dsRNA enters the RNAi pathway through an active and specific pathway that involves clathrin-mediated endocytosis. Furthermore, biochemical and pharmacological analyses implicate scavenger-like pattern-recognition receptors in dsRNA entry. We also examined whether C. elegans
homologues of components of the Drosophila
dsRNA entry pathway function in systemic spread of an ingested dsRNA signal. Whereas downregulation of core endocytosis components (such as clathrin and V-H-ATPase) was lethal in C. elegans,
downregulation of several components of vesicular intracellular transport and lipid metabolism blocked systemic spread of the RNAi signal. It thus seems that RNAi spread is an active process that involves vesicle-mediated intracellular trafficking and depends on lipid modifications and cytoskeleton guidance. Based on these experiments, we hypothesize that the dsRNA entry pathway we have identified in Drosophila
is conserved in other animal cells. The severity of the phenotype observed for downregulation of the endocytic pathway may account for the inability to detect this pathway of entry in screens carried out in whole C. elegans
The identification of components of the endocytic pathway required for dsRNA entry to initiate an RNAi response raises a number of interesting questions. Several lines of evidence, including the requirements of clathrin, ARF72A, V-H-ATPase and Rab 7 for exogenous dsRNA-initiated silencing ( and ), suggest that endocytic vesicles are critical in the entry pathway. However, the RNAi uptake pathway would need to deviate from standard endocytic uptake at some point if it is to deliver dsRNA to the cytoplasm. It is tempting to speculate that the RNAi signal may be directly translocated, perhaps through SID-1-like channels, from specialized entry vesicles to the RNAi machinery. Intriguingly, several components of the RNAi machinery, including dicer and ago-2, are membrane associated or have membrane-anchoring domains (Saleh, M.C., University of California San Francisco and Joachimiak, M., University of California Berkley; unpublished observations and ref. 36
). Our observation that in cells defective for V-H-ATPase, dsRNA still accumulates in vesicles () but does not initiate an RNAi response ( and ) suggests that the V-H-ATPase activity controls progression of the dsRNA through the RNAi entry pathway. Future studies should determine the mechanisms by which dsRNA is loaded onto the RNAi apparatus. This, in turn, may explain why some cells are uniquely able to take up exogenous dsRNA to initiate an RNAi response.
The observation that members of the scavenger-receptor family act as receptors of dsRNA may provide insight into the physiological role of this pathway, as these proteins have well-known roles in the ancestral innate immune response24,37
. For example, scavenger receptors participate in the uptake of bacterial pathogens and have also been implicated in the uptake of chaperone-peptide complexes38
. It is thought that the chaperone-bound peptide is translocated from vesicles to the cytosol to enter the antigen-presentation pathway in a process that bears some similarities to dsRNA uptake39,40
. The pathway for dsRNA uptake may thus serve a protective role to prevent the spread of viral infections by uptake of viral replicative intermediate dsRNAs that are released on cell lysis.
RNAi has tremendous potential for specific and effective therapeutic applications but the main obstacle to achieving in vivo therapies by RNAi technologies is delivery. Our observations that the pathway of dsRNA entry utilizes components of the endocytic machinery may provide a starting point to develop novel strategies for RNAi delivery. The identification and exploitation of this natural RNAi entry pathway may provide more effective and non-toxic strategies of dsRNA delivery.