Current Biology, Vol. 13, 41–46, January 8, 2003, 2003 Elsevier Science Ltd. All rights reserved.

PII S0960-9822(02)01394-5

Short-Interfering-RNA-Mediated Gene Silencing
in Mammalian Cells Requires Dicer and eIF2C
Translation Initiation Factors
Noboru Doi,1 Shuhei Zenno,1 Ryu Ueda,2,3
Hiroko Ohki-Hamazaki,4 Kumiko Ui-Tei,5,6
and Kaoru Saigo1,*
1
Department of Biophysics and Biochemistry
Graduate School of Science
University of Tokyo
7-3-1 Hongo
Bunkyo-ku, Tokyo 113-0033
Japan
2
Mitsubishi-Kagaku Insititute of Life Sciences
11 Minami-ooya
Machida, Tokyo 194-8511
Japan
3
Genetic Strain Research Center
National Institute of Genetics
Mishima, Shizuoka 411-8540
Japan
4
Department of Molecular Neuroscience
Medical Reseach InstituteTokyo Medical and Dental University
1-5-45 Yushima
Bunkyo-ku, Tokyo 113-8519
Japan
5
Department of Pharmacology
Nippon Medical School
1-1-5 Sendagi
Bunkyo-ku, Tokyo 113-8602
Japan

Summary
RNA interference (RNAi) is the process of long, doublestranded (ds), RNA-dependent posttranscriptional genesilencing (PTGS) [1]. In lower eukaryotes, dsRNA introduced into the cytoplasm is cleaved by the RNaseIII-likeenzyme, Dicer, to 21–23 nt RNA (short interfering [si]RNA), which may serve as guide for target mRNA degradation [2]. In mammals, long-dsRNA-dependent PTGS
is applicable only to a limited number of cell types [3–7],
whereas siRNA synthesized in vitro is capable of effectively inducing gene silencing in a wide variety of cells[8]. Although biochemical and genetic analyses in lower
eukaryotes showed that Dicer and some PIWI family
member proteins are essential for long-dsRNA-dependent PTGS [9–11], little is known about the molecularmechanisms underlying siRNA-based PTGS. Here, weshow that Dicer and eIF2C translation initiation factors
belonging to the PIWI family (eIF2C1–4) play an essential
role in mammalian siRNA-mediated PTGS, most probably through synergistic interactions. Immunoprecipitation experiments suggest that, in human and mouse
cells, complex formation occurs between Dicer andeIF2C1 or 2 and that the PIWI domain of eIF2C is essential for the formation of this complex.
*Correspondence: saigo@biochem.s.u-tokyo.ac.jp
6
Present address: Undergraduate Program for Bioinformatics andSystems Biology, Department of Biophysics and Biochemistry,
Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Results and Discussion
As the first step toward clarifying the molecular mechanisms of mammalian RNAi, we searched for possible
human homologs of Caenorhabditis elegans RDE-1 [10]and Drosophila melanogaster ARGONAUTE2 (AGO2)[11]. Four components, highly similar in sequence torabbit eIF2C [12], were identified (Figure 1A), two of
which had been previously noted to correspond toeIF2C1 and eIF2C2 [13]. Genes encoding three of thefour (eIF2C1, FLJ12765 [eIF2C3], KIAA1567 [eIF2C4])
were clustered near Ip34-p35 on the first chromosome,with the other (eIF2C2) being situated on the eighth
chromosome ([13] and National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/). Fourmouse counterparts were also isolated by RT-PCR. Phylogenetic tree analysis (Figure 1A) shows that eIF2Cs
are not the counterparts of either AGO2 or RDE-1, suggesting that mammalian genomes lack AGO2 and RDE-1
orthologs. Unlike AGO2 and RDE-1, eIF2Cs along with
Drosophila AGO-1 and C. elegans ALGs share in common a new motif, PRP, in addition to the PAZ and PIWI
domains (Figures 1A and 1B).
To knock down human and mouse eIF2Cs and dicer,
we designed a series of siRNA, siECx (x ⫽ 1–4) andsiDCR, the former being for eIF2Cx (x ⫽ 1–4) knockdown
and the latter for dicer. We used long sequences that
had been conserved between human and mouse target
genes so that each siRNA was capable of simultaneously knocking down human and mouse target mRNAs(see Experimental Procedures section). siRNAs cognate
to the firefly luciferase gene (luc; siFL), DsRed (siRED),or EGFP (siGFP) were also prepared. Prior to systematically analyzing possible suppressor activity of siRNAs
for eIF2C or dicer, we wanted to discover whether mammalian cells are constitutively expressing eIF2C1–4 anddicer mRNA and, if so, whether these mRNAs and theirprotein counterparts would be specifically and effectively eliminated by transfection of cognate siRNA. HeLa
cells were transfected with various siRNAs at the concentration of 100 nM and possible change in mRNA
expression levels in cells were first examined via semiquantitative RT-PCR. Cells were collected 36 hr after
siRNA transfection. All four eIF2C genes, along withdicer, were found to be constitutively expressed, andexpression was effectively and specifically knocked
down by cognate siRNAs (Figure 2A). As with mRNA,
Dicer protein (Figure 2B) and eIF2C protein (our unpublished data) were significantly reduced in HeLa cells 36
hr after cognate siRNA treatment. Tubulin was used as
an internal control. F9 cells also exhibited similar siDCRdependent concentration reduction of Dicer protein(Figure 2B). Thus, dicer and eIF2C activities are concluded to be virtually completely eliminated in humanand mouse cells by cognate siRNA treatment and asubsequent 36 hr incubation.
We examined possible inhibitory effects arising fromsiRNA transfection by using the dual luciferase system[3], in which firefly luc provides target mRNA while Renilla luc serves as the control. luc constructs were trans-

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Figure 1. Phylogenetic Tree and Domain
Structure of PIWI Family(A) The phylogenetic tree of PIWI domains
was constructed by the UPGMA method. Sequence data of PIWI family members otherthan those examined here were collectedfrom a published database (National Center
for Biotechnology Information). Abbreviations are as follows: eIF2C(O), rabbit (Oryctolagus cuniculus) eIF2C; D, D. melanogaster;A, Arabidopsis thaliana; N, Neurospora
crassa; C, Caenorhabditis.elegans; H, Homosapiens; and M, Mus musculus. Corresponding domain structures are shown on the rightside. PAZ domains are shown as open boxes
labeled with PAZ, and filled boxes represent
PIWI domains. polyQ, Glutamine-rich sequences; the polyQ domain of D. melanogaster AGO2 includes eight repeats of 20–21
amino acids. PRP is a motif newly found inall 2C-type translation initiation factors so far
examined. The amino acid sequence alignment of PRP is shown in (B), in which invariant
amino acids are colored in red. Note that PRP
in rabbit eIF2C is present in an N-terminal
extension of the sequence published by Zou
et al. (1998) [12]. Phylogenetic tree analysis(A) along with domain analysis (B) indicatesthat the Drosophila counterpart of eIF2Cs is
not AGO2 but AGO1, whereas the C. elegans
counterparts are ALGs.

fected simultaneously with siRNA, and the effect ofsiRNA treatment was examined 36 hr after transfection.
As shown in Figure 2D, about 80% of firefly luc activity,
normalized with Renilla luc activity, was abolished bysiFL at the concentration of 20 nM, whereas 100 nM
noncognate siRNAs (siGFP or siEC3) had only slight, if
any, effects on firefly and Renilla luc expression in both
NIH3T3 and HeLa cells (Figure 2C). One hundred nM
siRNA for EGFP, each of the eIF2Cs, or dicer was cotransfected with 20 nM siFL, and the effects on siFL-dependent knockdown of firefly luc activity in human (HeLa,
P19) and mouse (NIH3T3, F9) cells were examined (Figure 2D). The RNAi effect due to siFL was almost completely abolished in all four cell lines subsequent to cotransfection of siDCR or siEC1, and the effect, if any, ofsiGFP (noncognate siRNA) was subtle. siRNAs corresponding to eIF2Cs other than eIF2C1 gave intermediate
suppression effects.
siDCR and siEC1-dependent suppression of RNAi
was also examined via systems other than the dual luciferase assay. Mouse F9 cells were doubly transfectedwith plasmids harboring EGFP and DsRed genes, andgene activity was knocked down by siGFP. Transfection
efficiency was assessed as 80% and 40% after EGFPand DsRed-positive cells, respectively, were counted;
more than 99% of DsRed-positive cells were noted as
being EGFP positive (Figure 2Ea–a″). Because of delayed
DsRed fluorescence response, gene expression was examined 48 hr after transfection. Figure 2Eb–b″ indicatesthat effective and specific gene silencing is induced by
siGFP. That cotransfection of siFL (noncognate siRNA)
has little or no effect on siGFP-dependent gene silencing
is evident from Figure 2Ec–c″. The effect of siGFP-dependent gene silencing was significantly abolished in thepresence of siDCR (Figure 2Ed–d″) or siEC1 (Figure 2Ee-e″).
siRED-dependent silencing of DsRed was also significantly abolished by siDCR or siEC1 (our unpublished data).
Based on these observations, we conclude that Dicer,
eIF2C1, and probably other eIF2Cs are components essential for siRNA-mediated gene silencing in mammaliancells and hence that they are involved in the effecter
step of mammalian RNAi. Recently, as with Dicer in(Figure 2 continued from page 43)
siDCR, or siEC1–4 (100 nM each). As in (A), firefly luc activity relative to that of Renilla luc was normalized. As a normalization control, thefirefly luc activity relative to that of Renilla luc in cells transfected with firefly and Renilla luc plasmids one of the following was used: siGFP,
siDCR, or siEC1–4 (100 nM each). All four cell lines exhibited almost identical results so that they were averaged, and the average values areshown by thick horizontal bars.
(E) Suppression of siGFP-dependent posttranscriptional gene silencing by reduction of Dicer or eIF2C1 activity. F9 cells were transfectedwith plasmids directing the expression of target genes, EGFP and DsRed, in the presence (b–e) or absence (a) of various siRNA. (a–e) EGFP
fluorescence signals are colored in green. (a⬘–e⬘) DsRed fluorescence signals are colored in red. (a″–e″) Merged pictures, in which yellow
corresponds to cells strongly expressing both EGFP and DsRed. (a–a″) Control without siRNA transfection. About 80% and 40% of cells,
respectively, were positive to EGFP (a) and DsRed (a⬘). Nearly all DsRed-positive cells appeared capable of expressing EGFP. (b–b″) Cells weretransfected in the presence of siGFP. Virtually all EGFP signals disappered (b and b″), but DsRed signals were normal (b⬘). Panels in (c–c″) showthat there is no substantial effect of cotransfection with siFL on siGFP-dependent posttranscriptional gene silencing. Compare (c) with (b) and (c″)
with (b″). In contrast, siDCR (d–d″) and siEC1 (e–e″) exhibited strong suppression effects on siGFP-dependent posttranscriptional gene silencing.

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Figure 2. Effects of the Reduction of dicer or eIF2C1-4 Activity on siRNA-Dependent Post-Transcriptional Gene Silencing(A) eIF2C1-4 and dicer are constitutively expressed in HeLa cells and that siRNA designed here are capable of cleaving target mRNA specifically.
RT-PCR was carried out with RNA preparations without any substantial contamination of genomic DNA. The concentration of siRNA used fortransfection was 100 nM.
(B) Dicer protein is specifically eliminated in HeLa and F9 cells by transfection of siDCR; irrelevant siRNA, siGFP, has no effect. Cells werelysed and analyzed by Western blotting with anti-Dicer and anti-Tubulin (control) antibodies.
(C) NIH3T3 and HeLa cells were cotransfected with plasmids directing the expression of firefly luc and Renilla luc in the presence or absenceof siRNA. Note that there are no appreciable effects of 100 nM siRNA (siGFP or siEC3) unrelated in sequence to firefly luc on firefly luc
expression. The value obtained in the absence of siRNA was used for normalizing Renilla luc activity. “Norm. F. luc/R. luc” is the normalized
ratio of firefly and Renilla luc activity.
(D) Suppression of siFL-mediated posttranscriptional gene silencing upon cotransfection of siDCR or siEC1-4. Mouse (NIH3T3, F9) or human(HeLa, P19) cells were cotransfected with firefly and Renilla luc plasmids and a combination of siFL (20 nM) and one of the following: siGFP,(legend continues on page 42)

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Figure 3. Genetic and Biochemical Analysis of Dicer/eIF2C1, 2 Interactions(A) Possible synergistic interactions between Dicer and eIF2C1. F9 cells were cotransfected with luc expression plasmids and the indicatedsiRNAs. Relative luc activity was normalized as in Figure 2D. Note that siDCR or siEC1 at 30 nM gave only slight suppression effects (seehatched areas) on siFL-dependent gene silencing; siGFP serves as a negative control. In contrast, the suppression effect of double knockdown
appears much greater than the sum of effects of single knockdown of dicer and eIF2C1.
(B and C) Coimmunoprecipitation experiments of Dicer and either eIF2C1 or eIF2C2 with anti-Dicer or anti-Myc antibodies. In (B), 293 cells
transfected with the indicated constructs were lysed and analyzed by immunoprecipitation (IP) with anti-Myc followed by Western blotting(W) with anti-Dicer and anti-Myc. In (C), similar cell lysates were analyzed by IP with anti-Dicer or total IgG followed by Western blotting.
These results strongly suggest that Dicer and eIF2C1 or 2 form complexes within 293 cells. In the lower panels labeled Lys, lysates wereprobed with anti-Dicer or anti-Myc.
(D) Diagrams of Myc-tagged deletion constructs of eIF2C1 are depicted. In A-Myc, the PIWI domain is deleted, whereas in B-Myc, PAZ and
PRP are deleted. “WT-Myc” is Myc-tagged intact eIF2C1.
(E) 293 cells transfected with constructs depicted in (D) or an empty vector encoding only the Myc tag peptide were lysed and analyzed byanti-Myc IP followed by Western blot analysis (W) (left panels). The B-Myc/Dicer interaction appears much stronger than the interaction
between WT-Myc and Dicer. In contrast, the A-Myc/Dicer interaction, if any, is very weak. Crude lysates (Lys) were also probed by Western
blotting (right panels).

Drosophila [2], mouse Dicer was found to be required
for long dsRNA cleavage in F9 cells [4]. Thus, the roleof Dicer in mammalian RNAi may be 2-fold. It is required
not only for the initial long dsRNA cleavage but also

for subsequent effecter steps, which may include theformation of long dsRNA (potential Dicer substrate) by
an RNA-dependent RNA polymerase [14, 15].
In Drosophila S2 cells, AGO2, a PIWI family member,

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is a component of RNA-induced silencing complexes(RISC) and is capable of interacting with Dicer [11]. Thus,
we investigated whether suppression of siFL-dependent
gene silencing is enhanced in F9 cells doubly knocked
down with dicer and eIF2C1 (Figure 3A). Irrelevant
siRNA, siGFP, was used to balance siRNA concentration. The concentration of siDCR and siEC1 for transfection was 30 nM, at which siFL-dependent gene silencing
would only be slightly suppressed in single-knockdown
cells. The occurrence of synergistic interactions between Dicer and eIF2C1 in siRNA-dependent mammalian gene silencing would be indicated by the result of
double-knockdown cells (Figure 3A).
To further clarify this point, we carried out immunoprecipitation experiments by using the lysates of 293 cells
transfected with Myc tag, Myc-tagged human eIF2C1,
or mouse eIF2C2 constructs. Myc-tagged eIF2C1 andeIF2C2 were immunoprecipitated with anti-Myc antibody,and a clear association was found with Dicer in both cases(Figure 3B). Similarly, anti-Dicer antibody immunoprecipitated endogenous Dicer associated with Myc-tagged
eIF2C1 or eIF2C2 (Figure 3C). Similar coimmunoprecipitation of Dicer and Myc-tagged eIF2C1 or eIF2C2 was also
evident in HeLa and F9 cells (our unpublished data). It
may thus follow that Dicer forms complexes with eIF2C1,
eIF2C2, or possibly other eIF2Cs in mammalian cells.
Immunoprecipitation experiments were also carriedout with the extracts of F9 and 293 cells cotransfectedwith siFL (an irrelevant siRNA) and constructs of Myctagged eIF2C1 or 2. That no apparent difference in thecoimmunoprecipitation pattern of Dicer and Myctagged eIF2C proteins was found (our unpublished data)suggests that Dicer/eIF2C complex formation may occur independently of exogenous siRNA. A recent experiment indicated that miRNP, a ribonucleoprotein complex containing numerous micro RNAs, contains eIF2C2
as a protein constituent [16]. However, at present we
do not know whether our putative Dicer/eIF2C complex
is relevant to miRNP. Our experiment also could notreveal whether eIF2C proteins (eIF2C1–4) directly or indirectly interact with Dicer.
Finally, we assessed which domain of eIF2C proteinsis essential for Dicer/eIF2C complex formation. The PAZ(with PRP) and PIWI domains of eIF2C1 were labeledwith the Myc tag (Figure 3D), transiently expressed in
293 cells, and immunoprecipitated. Figure 3E showsthat the PIWI domain (B-Myc) coimmunoprecipitatedDicer much more effectively than did intact eIF2C1 (WTMyc), whereas only a marginal level of Dicer precipitation was induced by the PAZ domain (A-Myc). Note that
production of B-Myc in cells was much less effective
than that of A-Myc, whereas there was no apparent
difference in Dicer production (see right panels). Thus,
the PIWI domain is tentatively concluded to play a keyrole in Dicer/eIF2C interactions.
In conclusion, we found that Dicer and eIF2C1-4, capable of forming complexes in mammalian cells, areessential for siRNA-based gene silencing in mammals.
Experimental Procedures
Cell Culture
HeLa and NIH3T3 cells were cultured at 37⬚C in Earle’s minimum
essential medium (GibcoBRL) supplemented with 10% fetal bovine

serum and Dulbecco’s modified Eagle medium (DMEM, GibcoBRL)
with 10% calf serum, respectively, whereas F9, 293, and P19 cells
were cultured in DMEM supplemented with 10% fetal bovine serum.
All mediums were supplemented with penicillin (30 ␮g/ml) and streptomycin (0.1 mg/ml). F9 and P19 were from IDAC at Tohoku University, and HeLa, 293, and NIH3T3 were from our laboratory stocks.
Cells were regularly transferred to maintain exponential growth. A
day before transfection, cells were trypsinized, diluted with fresh
medium without antibiotics, and transferred to 1.03 cm wells of the
48-well plate (Falcon).

Anti-Dicer Antibody Preparation
Anti-Dicer antibody was prepared with a C-terminal portion of human Dicer. A cDNA fragment of human dicer was PCR-amplified,digested with BclI and NotI, and a BclI/NotI fragment (position 4957–
5772) was inserted into the BamHI/NotI site of pET-33b (Novagen).
Bacterially expressed protein was isolated by Ni-NTA agarose (Qiagen) and used for immunizing rabbits. Antibody was used after
affinity purification. Anti-Dicer antibody thus prepared was used for
detection of human and mouse Dicer.

Cloning of Mouse and Human dicer and eIF2C1-4 Genes
Total RNA was isolated with Trizol (Life Technologies) treatment
from NIH3T3 or HeLa cells cultured in a 90 mm dish to a late log
phase, treated with RQI DNase (Promega), and used for cDNA synthesis with Superscript II first strand synthesis systems. The nucleotide sequences of PCR primers used for amplification of mouse andhuman dicer and eIF2C1-4 are available upon request. Amplified
fragments were blunt-ended with T4 DNA polymerase and inserted
into the EcoRV site of pBluescript II KS(⫺).

Plasmids and Recombinant ProteinspGL3-Control and pRL-SV40 (Promega) encode firefly and Renillaluciferases, respectively. pEGFP-N1 encoding EGFP (enhanced
green fluorescent protein) was from Clontech. pCAGGS-DsRed, an
expression plasmid of DsRed, was a derivative of pDsRed1-1 (Clontech); the DsRed fragment of pDsRed1-1 was inserted into the EcoRI
site of pCAGGS [17]. In pCAGGS-DsRed, gene expression is under
the control of the CAG promoter. To produce Myc-epitope-tagged
proteins in mammalian cells, pcDNAM (N.D., S.Z., and K.S., unpublished data), a derivative of pVP22/Myc-His (Invitrogen), was used.
PCR-amplified eIF2C1–4 genes or their derivatives were inserted
into the BamHI/NotI site of pcDNAM.

siRNA Preparation
siRNAs were designed as 21 nt sense and antisense strands (dsRNA
region: 19 bp) or 22 nt sense and antisense strands (dsRNA: 20 bp).
siGFP corresponds in nucleotide sequence to position 290–312 of
the EGFP coding sequence; siRED, 239–261 (DsRed coding sequence); siFL, 36–58 (firefly luc); siDCR, 60–82 (human dicer) or
30–52 (mouse dicer); siEC1, 262–285 (human eIF2C1) or 262–285(mouse eIF2C1); siEC2, 746–769 (mouse eIF2C2) or 31–54 (humaneIF2C2 (XM_050334)); siEC3, 238–260 (human eIF2C3) or 238–260(mouse eIF2C3); and siEC4, 201–224 (human eIF2C4) or 201–224(mouse eIF2C4). Note that XM_050334 represents a truncated formof human eIF2C2. For siRNA annealing, sense and anti-sense singlestranded RNA (25 mM each) molecules were incubated in 10 mM
Tris-HCl (pH 8.0) with 20 mM NaCl for 2 min at 90⬚C followed by 1
hr at 37⬚C. The concentration of resulting double-stranded siRNAwas estimated by electrophoresis followed by ethidium bromide
staining. The concentration of siRNA was presented based on thatof single-stranded RNA.

Transfection
A day before transfection, cells (2 ⫻ 104 for HeLa and NIH3T3, 3 ⫻104 for F9 and P19, and 4 ⫻ 104 for 293) were trypsinized, diluted
with fresh medium without antibiotics, and transferred to a 1.03 cm
well. HeLa, 293, or NIH3T3 cells were transfected with plasmid DNA
and/or siRNA by the use of LipofectAMINE PLUS (Invitrogen), andLipofectAMINE 2000 (Invitrogen) was used for F9 and P19.

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luc Assay
The dual luciferase assay was performed as described previously[3]. In the case of HeLa, 293, or NIH3T3 transfection, 90 ng of pGL3Control DNA and 10 ng of pRL-SV40 were used per well, whereas
in F9 or P19 transfection, 160 ng of pGL3-Control and 40 ng of pRLSV40 were used. Cell lysates were prepared 36 hr after transfection.
Immunoblotting of Endogenous Dicer
HeLa or F9 cells were collected 36 hr after cotransfection with 100
nM siDCR and irrelevant plasmid DNA (carrier DNA; 100 and 200
ng for HeLa and F9, respectively), washed with phosphate-bufferedsaline (PBS), and treated with 10% trichloroacetic acid. Cells werecollected by centrifugation at 7,000 g for 20 min at 4⬚C. Pellets
were suspended in SDS-polyacrylamide gel electrophoresis (PAGE)
sample buffer, sonicated, clarified by centrifugation, neutralized with
1 M Tris, and subjected to SDS-PAGE and Western blotting. Monoclonal anti-Tubulin was purchased from ICN.
EGFP/DsRed Fluorescence Assay
F9 cells were transfected with 100 ng of pEGFP-N1 (Clontech) and
100 ng of pCAGGS-DsRed along with the indicated amounts ofsiRNA. Because of the delayed fluorescence response of DsRed,
this assay was carried out 48 hr after transfection. Cells were trypsinized and loaded on a hemocytometer, and fluorescence was detected under a confocal microscope (Bio-Rad).
RT-PCR Assay of Endogenous eIF2C1-4 and dicer
HeLa cells (2 ⫻ 104 cells/well) were transfected with 100 nM of agiven siRNA and 100 ng of pEGFP-N1 encoding EGFP. The transfection efficiency of pEGFP-N1 was estimated at 60%–80%. After cells
were collected from five wells and lysed with Trizol, a total RNA
was prepared. The RNA solution was treated with RQI DNase andthen with phenol-chroloform-isoamylalcohol. RNA was precipitatedwith ethanol and dissolved in RNase-free water. Half of the RNA
sample thus obtained was used for first-strand cDNA synthesis,
and the remaining sample was used for a negative control. The
nucleotide sequences of RT-PCR primers are available on request.
Immunoprecipitation Assay
For coimmunoprecipitations, 293 cells (1.5 ⫻ 106 cells/60 mm dish)transfected with Myc constructs and collected 42 hr after transfection were lysed in 1% NP-40 lysis buffer (with 20 mM Hepes [pH 7.3],
1 mM CaCl2, 1 mM Mg(OAc)2, 125 mM KOAc, 5 mM EGTA, 1 mM
dithiothreitol, and Complete Protease Inhibitor [Roche]). After being
clarified by a centrifugation at 15,000 g for 20 min at 4⬚C, lysates were
immunoprecipitated with anti-Dicer antibody, total IgG, or anti-c-Myc(Ab-1; oncogene) antibody followed by protein G sepharose (for antic-Myc, SIGMA) or proteinA agarose (for anti-Dicer and IgG, Gibco
BRL). Pellets were washed four times with lysis buffer. After SDSPAGE and Immobilon P (Millipore) transfer, proteins were visualized via
immunoblotting with indicated antibodies and subsequent secondaryantibody treatment (AP-conjugated-goat-affinity-purified antibody to
rabbit (or mouse) IgG [Cappel]) and chemiluminescence (CDP-Stardetection reagent [Amersham Pharmacia Biotech]).
Acknowledgments
We thank J. Miyazaki and F. Takahashi for supplying pCAGGS andfor critical discussion, respectively. This work was supported in partby grants from the Ministry of Education, Culture, Sport, Science,
and Technology of Japan to K.S.
Received: April 2, 2002
Revised: September 16. 2002
Accepted: October 17, 2002
Published: January 8, 2003
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Accession Numbers
GenBank accession numbers are as follows: human dicer (helicaseMOI), AB028449 [18]; mouse dicer, AB081470 (this work); humaneIF2C1 (eIF2C1), NM_012199; human eIF2C3 (FLJ12765), XM_029051;
human eIF2C4 (KIAA1567), AB046787; mouse eIF2C1, AB081471(this work); mouse eIF2C2, AB081472 (this work); mouse eIF2C3,
AB081473 (this work); mouse eIF2C4, AB081474 (this work).