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.

Figure 1

RNAi in Drosophila S2 cells is dependent on the length of the dsRNA. (a, b). Silencing of luciferase expression after exposure of S2 cells to dsRNA by transfection (a) or by adding dsRNA in the culture supernatant (soaking; b). S2 cells were cotransfected with expression plasmids encoding firefly and Renilla luciferase. Specific dsRNA targeting firefly luciferase was either transfected into the cell in conjunction with the expression plasmids or was added to the culture supernatant one day after transfection. Luciferase activity was monitored after 48 h and it is expressed as firefly:Renilla ratio. Luciferase activity after treatment with specific dsRNA of different sizes was compared with treatment with a non-specific dsRNA (control dsRNA). Results represent averages and s.d. from four independent experiments. The asterisk indicates P<0.01 with respect to untreated control (−). (c) Silencing of firefly luciferase expression after transfection or soaking pools of specific 21 bp siRNAs generated by cleavage of long dsRNA by recombinant Dicer (esiRNA) compared with luciferase activity in the absence of dsRNA (−). Results represent averages and s.d. from three independent experiments. The asterisk indicates P<0.01 with respect to untreated control (−). (d) Time course comparing silencing of luciferase after soaking of S2 cells in dsRNA of 21 or 1000 bp. The experiment was performed as described in a and b but dsRNA was washed away after the indicated incubation times. Results represent averages and s.d. from three independent experiments. The asterisk indicates P<0.01 with respect to no dsRNA control, (e) Silencing of luciferase expression after soaking cells with specific dsRNA at 4 °C or 25 °C. S2 cells were transfected with expression plasmids encoding firefly and Renilla luciferase and exposed to dsRNA at 4 °C or 25 °C. After 1 h incubation, the cells were washed and further cultured at 25 °C for 48 h, and luciferase activity was monitored. Results represent averages and s.d. from three independent experiments. The asterisk indicates P<0.01 with respect to no dsRNA control, (f) Cellular localization of Cy3-labelled dsRNA over time. Shortly after incubation, dsRNA seemed to bind in a punctate pattern to the cell surface (5 min). Over the course of 60 min the dsRNA was internalized but remained in small punctate structures. Optical sections were deconvolved and flattened into a two-dimensional projection for presentation. The 60 min time point corresponds to an independent experiment. The scale bar in f represents 2 μM.

Figure 2

An active mechanism for uptake of long dsRNA in Drosophila S2 cells. (a) Subcellular localization of Cy3-labelled 500 bp dsRNA, siRNA and 500 bp DNA. (b) Association of radiolabelled dsRNA or DNA with S2 cells over time. S2 cells were incubated with radiolabelled dsRNA or DNA of the same size and sequence and cell-bound radioactivity was determined in a scintillation counter. Results represent averages and s.d. from two independent experiments. (c) Subcellular localization of radiolabelled dsRNA or DNA with S2 cells over time. S2 cells were incubated with dsRNA or DNA for the indicated times, lysed and fractionated by ultracentrifugation. The data are expressed as the ratio of radioactivity in pellet to supernatant. Results represent averages and s.d. from two independent experiments. The scale bar in a represents 2 μm.

Figure 3

RNAi screen for genes involved in the RNAi pathway. (a) Schematic representation of the screening approach. S2 cells stably transfected with GFP under the inducible metallothionein promoter are treated with dsRNA from the RNAi library for three days. The cells are then split and refed with dsRNA from the RNAi library and with dsRNA targeting GFP. After a further three day incubation, GFP expression is induced by the addition of CuSO₄ to the culture supernatant and GFP expression is monitored. (b) Induction of GFP expression and RNAi in the reporter cell line. GFP expression is monitored on a FACS Calibur flow cytometer and analysed using Cellquest software. GFP expression is induced on addition of CuSO₄ in the culture medium. (c) Validation of the RNAi of RNAi approach. S2–GFP cells were pretreated with dsRNA targeting the RNAi-associated genes Dicer-1, Dicer-2, Ago-1 and Ago-2, followed by RNAi of the GFP marker gene.(d) Examples of three genes identified in the RNAi screen. Knockdown of V-H-ATPase subunit {Vha l6), CG3248 anti Clathrin heavy chain (chc) inhibits subsequent RNAi of GFP, as evident by a high GFP expression.

Figure 4

Endocytic uptake of dsRNA into Drosophila S2 cells is mediated by scavenger receptors, (a) Silencing of luciferase expression in the presence of inhibitors of the endocytic pathway. S2 cells were transfected with expression plasmids encoding firefly and Renilla luciferase. One day after transfection, the cells were exposed to different concentrations of methylβ-cyclodextran (MβCD), Cytochalasin D (CytoD) or Bafilomycin A (Baf A) for 30 min and dsRNA was added in the presence of these inhibitors. Controls include incubations of dsRNA in the presence of the solvent dimethylsulfoxide (DMSO) and in the absence of inhibitor (untreated). Results represent averages and s.d. from three independent experiments. The asterisk indicates P<0.01 with respect to untreated control.(b) Silencing of luciferase expression in the presence of competitive inhibitors of scavenger receptors. The experiment was performed as described in a, using the inhibitors of scavenger receptors poly-inosinic acid (Poly I) and Fucoidin. Controls include incubation of S2 cells with dsRNA in the presence of compounds that are not known to inhibit scavenger receptors, poly-adenosinic acid, galactose and lipopolysaccharide (LPS) and in the absence of inhibitors (−). Results represent averages and s.d. from three independent experiments. The asterisk indicates P<0.01 with respect to untreated control (−). (c) Subcellular localization of Cy3-labelled dsRNA after incubation in the presence of inhibitors of the endocytic pathway and of scavenger receptors. (d) Inhibitors of scavenger receptors do not affect RNAi when added after incubation of S2 cells with dsRNA. S2 cells were transfected with plasmids encoding firefly and Renilla luciferase. Cells were then incubated with dsRNA for 1 h, washed and fucoidin or poly I was added to the culture. The bars labelled ‘0 min control’ indicate incubations of Fucoidin in the presence of dsRNA and confirm the data in Fig. 4b. Results represent averages and s.d. from three independent experiments. No significant statistical difference was observed (asterisk), P>0.05, with respect to untreated control (−). The scale bar in c represents 2 μm.

Figure 5

Orthologues of the Drosophila genes confer an RNAi phenotype in C. elegans. (a) Schematic representation of the experimental design. Worms were grown on bacteria expressing dsRNA targeting specific genes. L2–L3 progeny was subsequently plated on bacteria expressing dsRNA specific for unc-52 and unc phenotype was monitored after 48 hours. (b) C. elegans grown on bacteria expressing dsRNA control targeting Dicer are incapable of processing unc-52 dsRNA and do not display the unc52 RNAi phenotype. The scale bars represent 0.2 mm.