Double-stranded RNA Is Internalized by Scavenger Receptor-mediated Endocytosis in Drosophila S2 Cells

Abstract

Double-stranded RNA (dsRNA) fragments are readily internalized and processed by Drosophila S2 cells, making these cells a widely used tool for the analysis of gene function by gene silencing through RNA interference (RNAi). The underlying mechanisms are insufficiently understood. To identify components of the RNAi pathway in S2 cells, we developed a screen based on rescue from RNAi-induced lethality. We identified Argonaute 2, a core component of the RNAi machinery, and three gene products previously unknown to be involved in RNAi in Drosophila: DEAD-box RNA helicase Belle, 26 S proteasome regulatory subunit 8 (Pros45), and clathrin heavy chain, a component of the endocytic machinery. Blocking endocytosis in S2 cells impaired RNAi, suggesting that dsRNA fragments are internalized by receptor-mediated endocytosis. Indeed, using a candidate gene approach, we identified two Drosophila scavenger receptors, SR-CI and Eater, which together accounted for more than 90% of the dsRNA uptake into S2 cells. When expressed in mammalian cells, SR-CI was sufficient to mediate internalization of dsRNA fragments. Our data provide insight into the mechanism of dsRNA internalization by Drosophila cells. These results have implications for dsRNA delivery into mammalian cells. Double-stranded RNA (dsRNA) fragments are readily internalized and processed by Drosophila S2 cells, making these cells a widely used tool for the analysis of gene function by gene silencing through RNA interference (RNAi). The underlying mechanisms are insufficiently understood. To identify components of the RNAi pathway in S2 cells, we developed a screen based on rescue from RNAi-induced lethality. We identified Argonaute 2, a core component of the RNAi machinery, and three gene products previously unknown to be involved in RNAi in Drosophila: DEAD-box RNA helicase Belle, 26 S proteasome regulatory subunit 8 (Pros45), and clathrin heavy chain, a component of the endocytic machinery. Blocking endocytosis in S2 cells impaired RNAi, suggesting that dsRNA fragments are internalized by receptor-mediated endocytosis. Indeed, using a candidate gene approach, we identified two Drosophila scavenger receptors, SR-CI and Eater, which together accounted for more than 90% of the dsRNA uptake into S2 cells. When expressed in mammalian cells, SR-CI was sufficient to mediate internalization of dsRNA fragments. Our data provide insight into the mechanism of dsRNA internalization by Drosophila cells. These results have implications for dsRNA delivery into mammalian cells. Many organisms mount specific defense responses to silence invading nucleic acid sequences before these sequences integrate into the host genome and disturb cellular processes. At the core of these sequence-directed immunity mechanisms is dsRNA, 2The abbreviations used are: dsRNA, double-stranded RNA; AcLDL, acetylated low density lipoprotein; Chc, clathrin heavy chain; CHO, Chinese hamster ovary; DEAD-box, Asp-Glu-Ala-Asp sequence; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; RISC, RNA-induced silencing complexes; RNAi, RNA interference; siRNA, small interfering RNA; PBS, phosphate-buffered saline; BSA, bovine serum albumin. which becomes processed and causes gene silencing, referred to as RNA interference (RNAi) (1Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Nature. 1998; 391: 806-811Crossref PubMed Scopus (11908) Google Scholar, 2Mello C.C. Conte Jr., D. Nature. 2004; 431: 338-342Crossref PubMed Scopus (779) Google Scholar). In addition to its defense function, RNAi guides endogenous developmental and regulatory processes and is used as a research tool to suppress the expression of cellular genes in numerous model organisms (2Mello C.C. Conte Jr., D. Nature. 2004; 431: 338-342Crossref PubMed Scopus (779) Google Scholar). Although species variations exist, the silencing mechanisms in plants, fungi, worms, insects, and mammals share common features and conserved genes. Drosophila S2 cells are widely used to carry out large scale functional screens (3Rämet M. Manfruelli P. Pearson A. Mathey-Prevot B. Ezekowitz R.A. Nature. 2002; 416: 644-648Crossref PubMed Scopus (594) Google Scholar, 4Boutros M. Kiger A.A. Armknecht S. Kerr K. Hild M. Koch B. Haas S.A. Consortium H.F. Paro R. Perrimon N. Science. 2004; 6: 832-835Crossref Scopus (613) Google Scholar, 5Kleino A. Valanne S. Ulvila J. Kallio J. Myllymäki H. Enwald H. Stöven S. Poidevin M. Ueda R. Hultmark D. Lemaitre B. Rämet M. EMBO J. 2005; 24: 3423-3434Crossref PubMed Scopus (181) Google Scholar) because dsRNA fragments (usually about 500-700 bp) can be added directly to the cell culture medium, “soaking” the cells rather than transfecting them (6Clemens J.C. Worby C.A. Simonson-Leff N. Muda M. Maehama T. Hemmings B.A. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6499-6503Crossref PubMed Scopus (715) Google Scholar). This makes silencing in these cells easy and efficient, in contrast to mammalian cells that require small interfering RNAs (siRNAs) to be delivered by transfection. In contrast to Caenorhabditis elegans cells, which internalize dsRNA by the channel-forming transmembrane protein SID-1 (7Winston W.M. Molodowitch C. Hunter C.P. Science. 2002; 295: 2456-2459Crossref PubMed Scopus (630) Google Scholar), the mechanism of dsRNA uptake into Drosophila cells is unknown. In the cytosol, dsRNA fragments are processed into short 21-23-nucleotide dsRNA duplexes (siRNAs) by a dsRNA-specific RNase-III-type endonuclease called Dicer-2 (8Hammond S.M. Bernstein E. Beach D. Hannon G.J. Nature. 2000; 404: 293-296Crossref PubMed Scopus (2422) Google Scholar, 9Zamore P.D. Tuschl T. Sharp P.A. Bartel D.P. Cell. 2000; 101: 25-33Abstract Full Text Full Text PDF PubMed Google Scholar, 10Bernstein E. Caudy A.A. Hammond S.M. Hannon G.J. Nature. 2001; 409: 363-366Crossref PubMed Scopus (3819) Google Scholar). Dicer-2 is stably complexed with the dsRNA binding domain-containing protein R2D2 binding to siRNA and thereby, facilitating siRNA loading onto RNA-induced silencing complexes (RISC) (11Liu Q. Rand T.A. Kalidas S. Du F. Kim H.E. Smith D.P. Wang X. Science. 2003; 301: 1921-1925Crossref PubMed Scopus (562) Google Scholar). RISC contain a member of the Argonaute (Ago) protein family Ago-2, which has been shown to mediate siRNA-directed mRNA cleavage (12Hammond S.M. Boettcher S. Caudy A.A. Kobayashi R. Hannon G.J. Science. 2001; 293: 1146-1150Crossref PubMed Scopus (1194) Google Scholar) and degradation of the passenger strand in the siRNA duplex (13Matranga C. Tomari Y. Shin C. Bartel D.P. Zamore P.D. Cell. 2005; 123: 607-620Abstract Full Text Full Text PDF PubMed Scopus (849) Google Scholar, 14Miyoshi K. Tsukumo H. Nagami T. Siomi H. Siomi M.C. Genes Dev. 2005; 19: 2837-2848Crossref PubMed Scopus (316) Google Scholar). Although Dicer-2, R2D2, and Ago-2 can constitute “minimal” RISC under certain circumstances (15Rand T.A. Petersen S. Du F. Wang X. Cell. 2005; 123: 621-629Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar), many other proteins have been suggested to be involved in RNA silencing in vivo (16Meister G. Tuschl T. Nature. 2004; 431: 343-349Crossref PubMed Scopus (2002) Google Scholar, 17Tomari Y. Zamore P.D. Genes Dev. 2005; 19: 517-529Crossref PubMed Scopus (750) Google Scholar, 18Sontheimer E.J. Nat. Rev. Mol. Cell Biol. 2005; 6: 127-138Crossref PubMed Scopus (339) Google Scholar). In particular, it has been postulated that an RNA helicase is required for loading of the RISC with the guide strand of the siRNA duplex. In Drosophila, three putative RNA helicases (Armitage, Spindle E, and Dmp68) have been implicated in RNAi (16Meister G. Tuschl T. Nature. 2004; 431: 343-349Crossref PubMed Scopus (2002) Google Scholar, 17Tomari Y. Zamore P.D. Genes Dev. 2005; 19: 517-529Crossref PubMed Scopus (750) Google Scholar, 18Sontheimer E.J. Nat. Rev. Mol. Cell Biol. 2005; 6: 127-138Crossref PubMed Scopus (339) Google Scholar, 19Preall J.B. Sontheimer E.J. Cell. 2005; 123: 543-545Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). To gain a better understanding of the mechanisms of dsRNA uptake, transfer, and processing in S2 cells, we carried out an RNAi-based screen to identify gene products required for RNAi. 2,000 dsRNAs were analyzed for their protective effect from lethality induced by RNAi against an essential gene for cell viability, the Drosophila homologue of ubiquitin (Ubi-p63E) (3Rämet M. Manfruelli P. Pearson A. Mathey-Prevot B. Ezekowitz R.A. Nature. 2002; 416: 644-648Crossref PubMed Scopus (594) Google Scholar, 4Boutros M. Kiger A.A. Armknecht S. Kerr K. Hild M. Koch B. Haas S.A. Consortium H.F. Paro R. Perrimon N. Science. 2004; 6: 832-835Crossref Scopus (613) Google Scholar). In addition, the mechanism and two receptors for dsRNA internalization in S2 cells were identified. Synthesis of the dsRNAs—A cDNA library derived from S2 cells (20Pearson A. Lux A. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4056-4060Crossref PubMed Scopus (202) Google Scholar) was used as a source for templates for RNA synthesis essentially as described earlier (3Rämet M. Manfruelli P. Pearson A. Mathey-Prevot B. Ezekowitz R.A. Nature. 2002; 416: 644-648Crossref PubMed Scopus (594) Google Scholar). Targeted dsRNA treatments were carried out as described (21Kallio J. Leinonen A. Ulvila J. Valanne S. Ezekowitz R.A. Rämet M. Microbes Infect. 2005; 7: 811-819Crossref PubMed Scopus (85) Google Scholar) using primers presented in a supplemental table. GFP dsRNA was produced using pMT/BiP/V5-His/GFP plasmid (Invitrogen) as a template. FITC-labeled GFP dsRNA was synthesized using a T7 MegaScript kit according to the manufacturer's instructions, except that half of the UTP was replaced with 1.5 mm FITC-UTP (Enzo). Ubi-p63E Survival Assay—S2 cells were grown as described (20Pearson A. Lux A. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4056-4060Crossref PubMed Scopus (202) Google Scholar). dsRNAs corresponding to indicated or random genes were introduced into S2 cells by soaking. On a 24-well plate, a total of 5 × 105 cells/well were treated with 10 μg of dsRNA. After 72 h, one-sixth of the cells treated with the first dsRNA was transferred into a 48-well plate, and Ubi-p63E dsRNA (5 μg) was introduced. 72 h later, the ability of the S2 cells to phagocytose FITC-labeled Escherichia coli was measured using flow cytometry (3Rämet M. Manfruelli P. Pearson A. Mathey-Prevot B. Ezekowitz R.A. Nature. 2002; 416: 644-648Crossref PubMed Scopus (594) Google Scholar). Luciferase Reporter Assay—S2 cells were transferred to 24-well plates, and 5 μg of dsRNA was added. After 48 h, the cells were transfected with 0.5 μg of constitutively active form of Toll (Toll10b) together with 0.5 μg of Drosomycin-luciferase firefly reporter, 0.1 μg of control plasmid encoding Renilla luciferase (Promega), and either 0.5 μg of Myd88 dsRNA or 0.5 μg of GFP dsRNA using FuGENE transfection reagent (Roche Applied Science). 96 h after transfections, cells were lysed, and luciferase activity was measured. The ratio of firefly luciferase to Renilla luciferase activities was calculated, and the values were normalized to the values of GFP dsRNA-treated controls. Endocytosis Assay—For the S2 cells, 2 × 105 cells/well were seeded into 48-well plates for flow cytometry. After 72 h, 7-20 μg/ml FITC-labeled dsRNA (500 bp) or 4 μg/ml Alexa-acetylated low density lipoprotein (AcLDL) (Molecular Probes) was added to the growth medium and incubated for 20 min. The extracellular fluorescence was quenched by adding trypan blue just prior to the actual measurement. Thereafter, the cells were collected and analyzed by flow cytometry. For the Chinese hamster ovary (CHO) cells, 105 cells/well were seeded into 6-well plates for flow cytometry. 36-48 h later, 2 μg/ml Alexa-AcLDL or 7.5 μg/ml FITC-dsRNA (500 bp) was added to the growth medium and incubated for 2 h. Thereafter, the cells were washed with 1× PBS, detached by trypsinization, pelleted by centrifugation, resuspended in ice-cold 1× PBS, and analyzed by fluorescence-activated cell sorting. Confocal and Fluorescence Microscopy—106 S2 cells/well were seeded into two chamber slides (Nunc) and, when indicated, treated with a total of 20 μg of indicated dsRNA. 72 h later, cells were allowed to internalize FITC-labeled dsRNA (500 bp, 12 μg/ml) for 5, 20, or 60 min at 26 °C. Cells were then washed with 1× PBS and thereafter fixed for 20 min with 4% paraformaldehyde. Fixed cells were washed again with 1× PBS, and the cell nuclei were stained for 20 min with 1 μg/ml 4′-6-diamidino-2-phenylindole. After the staining, slides were washed twice with 1× PBS and once with distilled H2O and mounted on Aquamount. For the CHO cells, 105 cells/well were seeded into 6-well plates and acid-washed onto 22 × 22-mm coverslips for microscopy. 36-48 h later, 2 μg/ml Alexa488-AcLDL or 7.5 μg/ml FITC-dsRNA was added to the growth medium and incubated for 4 h. Cells were washed with 1× PBS and fixed for 20 min with 3% formaldehyde. Fixed coverslips were washed with 1× PBS, unreacted aldehyde groups were saturated with 50 mm ammonium chloride in 1× PBS, and coverslips were mounted on 15 μl of moviol. Pictures were taken on an inverted Nikon TE2000-U epifluorescence microscope equipped with a cooled CCD camera (Hamatsu Orca ER) using a 60× Plan-Apochromat oil objective. Images were acquired and enhanced using Openlab software (Improvision). Immunoelectron Microscopy—2 × 106 cells were seeded on 10-cm dishes, and 10 μg/ml dsRNA was added. After 72 h, cells were allowed to internalize FITC-dsRNA (8.5 μg/ml) for 0, 5, or 60 min at 26 °C. The cells were fixed in 4% paraformaldehyde in 0.2 with for min. The fixed cells were pelleted and in in 1× PBS, after which the were in in 1× PBS and in For the were first incubated in in 1× PBS by in with in 1× and the were in in 1× were in in 1× The were incubated with by protein for min. The were in and in microscope Images were with a CCD camera equipped with from and To identify components of the RNAi in S2 cells, we an based on the that RNAi against the essential gene Ubi-p63E S2 cells M. Kiger A.A. Armknecht S. Kerr K. Hild M. Koch B. Haas S.A. Consortium H.F. Paro R. Perrimon N. Science. 2004; 6: 832-835Crossref Scopus (613) Google Scholar) and their ability to phagocytose fluorescent (3Rämet M. Manfruelli P. Pearson A. Mathey-Prevot B. Ezekowitz R.A. Nature. 2002; 416: 644-648Crossref PubMed Scopus (594) Google Scholar). This to as a for the rescue from the of Ubi-p63E dsRNA. When the synthesis of an essential component of RNAi is by RNAi, the cells are from Ubi-p63E and their ability to phagocytose is shown in S2 cells the ability to phagocytose E. coli 48 h after addition of Ubi-p63E dsRNA into the cell culture of cells with Dicer-2 dsRNAs the effect of Ubi-p63E dsRNA in a and We 2,000 dsRNA fragments from random S2 cDNA (3Rämet M. Manfruelli P. Pearson A. Mathey-Prevot B. Ezekowitz R.A. Nature. 2002; 416: 644-648Crossref PubMed Scopus (594) Google Scholar). treatments or RNAi treatments from RNAi-induced lethality to an to Dicer-2 RNAi was used as a These were dsRNAs the endonuclease Ago-2, a component of the RNAi in Drosophila (12Hammond S.M. Boettcher S. Caudy A.A. Kobayashi R. Hannon G.J. Science. 2001; 293: 1146-1150Crossref PubMed Scopus (1194) Google Scholar, T.A. K. Wang X. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: PubMed Scopus Google Scholar, K. A. Siomi H. Siomi M.C. Genes Dev. 2004; PubMed Scopus Google Scholar), and three genes that been implicated in RNAi in Drosophila a DEAD-box RNA the regulatory subunit 8 of the 26 S and clathrin heavy a component of the endocytic machinery. The that Chc, a component of the endocytic machinery, is required for RNAi in S2 cells that it a in the uptake of dsRNA, Ago-2, and are more to at in the silencing To we used an that dsRNA internalization by transfection. In of a gene by a constitutively active form of the cell Toll is by RNAi a component of the Toll pathway When an essential component of the RNAi is by RNAi, silencing of the Toll pathway is and the gene is shown in RNAi Ago-2, and as a Dicer-2 with Myd88 silencing of Toll pathway as measured by luciferase dsRNA silencing of gene These data on the the of and in RNA silencing and on the other that of silencing To the of endocytic pathway in RNAi, we used a of the gene as described We S2 cells with an of required for before the cells with dsRNA against shown in with the effect of Myd88 dsRNA on Drosomycin-luciferase that endocytosis of dsRNA fragments is for silencing in S2 cells. We used and to the uptake of FITC-dsRNA fragments into S2 cells dsRNA uptake is after 5 a of the FITC-dsRNA was at the cell just the or internalized the in small dsRNA be in together with the functional analysis described these data that dsRNA fragments are taken into S2 cells by receptor-mediated endocytosis. receptors are a of to mediate the endocytosis of certain nucleic in S. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The Drosophila genome scavenger receptors, of which have been in (20Pearson A. Lux A. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4056-4060Crossref PubMed Scopus (202) Google Scholar, P. Ezekowitz R.A. K. Science. Scopus Google Scholar, M. Pearson A. Manfruelli P. X. H. E. Krieger M. Ezekowitz R.A. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, C. N. Ulvila J. Pearson M. C. C. T. D. Rämet M. Ezekowitz R.A. Cell. 2005; 123: Full Text Full Text PDF PubMed Scopus Google Scholar). We RNAi of these receptors endocytosis of dsRNA RNAi the scavenger receptors and as as the and effect on RNAi the scavenger and the growth scavenger to a in the endocytosis of dsRNA fragments (500 bp) with SR-CI RNAi the endocytosis of AcLDL, RNAi a effect and M. Pearson A. Manfruelli P. X. H. E. Krieger M. Ezekowitz R.A. 2001; Full Text Full Text PDF PubMed Scopus Google and C. N. Ulvila J. Pearson M. C. C. T. D. Rämet M. Ezekowitz R.A. Cell. 2005; 123: Full Text Full Text PDF PubMed Scopus Google Scholar). of SR-CI and RNAi in in the internalization of dsRNA when with GFP dsRNA-treated controls. These data were by RNAi silencing of SR-CI and together to a internalization these receptors internalization of dsRNA, the that these scavenger receptors to fluorescein and to dsRNA These results that and SR-CI together for a of the internalized dsRNA in S2 cells. In contrast to Drosophila cells, mammalian cells readily internalize dsRNA fragments. we expression of a Drosophila scavenger can dsRNA internalization into mammalian cells. shown in 5, CHO cells stably transfected with SR-CI (20Pearson A. Lux A. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4056-4060Crossref PubMed Scopus (202) Google Scholar) a in dsRNA uptake when with control cells. expression of a Drosophila scavenger is sufficient to uptake of dsRNA into mammalian cells. We used an RNAi-based screen to genes required for RNAi in Drosophila S2 cells. We identified a RNAi component and three genes previously unknown to be involved in RNAi in The of Ago-2, the core component of the RISC (12Hammond S.M. Boettcher S. Caudy A.A. Kobayashi R. Hannon G.J. Science. 2001; 293: 1146-1150Crossref PubMed Scopus (1194) Google Scholar), screen and that it is for genes involved in RNAi. In addition, three other genes were and we that RNAi against Ago-2, or to control RNAi against Dicer-2, from the RNAi-induced effect of Ubi-p63E it that of these genes that of these genes be required for RNAi. Belle, three putative RNA helicases (Armitage, Spindle E, and Dmp68) have been to be for RNAi in S2 cells S. Genes Dev. 2002; PubMed Scopus Google Scholar, A. Siomi M.C. Siomi H. Genes Dev. 2002; PubMed Scopus Google Scholar, Y. Du T. B. R. Zamore P.D. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), and it is that or of these for it is that it be to silence the expression of a gene encoding for a protein that is for RNAi. DEAD-box proteins are RNA helicases that function at of RNA processing S. P. Nat. Rev. Mol. Cell Biol. 2004; PubMed Scopus Google Scholar). a Drosophila DEAD-box protein that is conserved from to R. R. P. P. Dev. Biol. 2004; Scopus Google Scholar). in lethality by growth and and is involved in RNA and of Science. 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Full Text Full Text PDF PubMed Scopus Google Scholar). because the proteasome before been implicated in RNAi, and RNAi screen was we used an RNAi to that is required for RNAi Our results that be involved in RNAi, either directly or and of its in RNA Our results that endocytosis a in the uptake of dsRNA in Drosophila cells. a candidate approach, we were to identify two endocytic receptors that together for a of the dsRNA uptake in S2 cells, the previously Drosophila scavenger receptors SR-CI and SR-CI and are two proteins that have a in the of and of mammalian scavenger receptors M. Pearson A. Manfruelli P. X. H. E. Krieger M. Ezekowitz R.A. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, C. N. Ulvila J. Pearson M. C. C. T. D. Rämet M. Ezekowitz R.A. Cell. 2005; 123: Full Text Full Text PDF PubMed Scopus Google Scholar). The binding activity of SR-CI has been to its two control protein and The of is to contain binding growth C. N. Ulvila J. Pearson M. C. C. T. D. Rämet M. Ezekowitz R.A. Cell. 2005; 123: Full Text Full Text PDF PubMed Scopus Google Scholar), the of which have been shown to in Our results that dsRNA be for these be to the of analysis of the of to provide insight into the of in the expression of SR-CI and has been shown to be to a cell of Drosophila M. Pearson A. Manfruelli P. X. H. E. Krieger M. Ezekowitz R.A. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, C. N. Ulvila J. Pearson M. C. C. T. D. Rämet M. Ezekowitz R.A. Cell. 2005; 123: Full Text Full Text PDF PubMed Scopus Google Scholar). For in vivo silencing which dsRNA into the of the A. S. J. 2003; PubMed Scopus Google Scholar), that a of the dsRNA fragments is by This have implications for of gene silencing in the The that mammalian cells internalization of dsRNA after expression of a Drosophila scavenger for delivery of silencing into mammalian cells. as be with scavenger facilitating their uptake and the for in cellular RNAi that SR-CI that are used for RNAi in mammalian cells rather fragments of dsRNA and data it is to delivery of dsRNA fragments into mammalian cells that carry of and protein a defense for dsRNA that is to with A. J. PubMed Scopus Google Scholar, 1998; PubMed Scopus Google Scholar). This mammalian RNAi screens with cell that are to by making screens and more We for for with the and other of and for with