Cell, Vol. 109, 861–871, June 28, 2002, Copyright 2002 by Cell Press

The dsRNA Binding Protein RDE-4 Interacts
with RDE-1, DCR-1, and a DExH-Box Helicase
to Direct RNAi in C. elegans
Hiroaki Tabara,1,3,5 Erbay Yigit,1 Haruhiko Siomi,3and Craig C. Mello1,2,4
1
Program in Molecular Medicine
2
Howard Hughes Medical Institute
University of Massachusetts Medical School
Worcester, Massachusetts 01605
3
Institute for Genome Research
University of Tokushima
Tokushima 770-8503
Japan

Summary
Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early stepin RNAi, an RNaseIII-related enzyme, Dicer (DCR-1),
processes long-trigger dsRNA into small interfering
RNAs (siRNAs). DCR-1 is also required for processing
endogenous regulatory RNAs called miRNAs, but how
DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C.
elegans RNAi pathway gene, rde-4, encodes a dsRNA
binding protein that interacts during RNAi with RNA
identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved
DExH-box helicase. Our findings suggest a model inwhich RDE-4 and RDE-1 function together to detect
and retain foreign dsRNA and to present this dsRNA
to DCR-1 for processing.
Introduction
The introduction of dsRNA can induce sequence-specific posttranscriptional silencing of cognate genes in avariety of organisms (reviewed in Cogoni and Macino,
2000; Vance and Vaucheret, 2001; Waterhouse et al.,2001). The experimental application of dsRNA to induce
gene silencing has been termed RNA interference orRNAi (Fire et al., 1998). RNAi and related posttranscriptional gene silencing (PTGS) pathways have been implicated in silencing transposons (Tabara et al., 1999a;
Ketting et al., 1999; Wu-Scharf et al., 2000) and/or viruses (Ratcliff et al., 1997; Mourrain et al., 2000; Dalmay
et al., 2001), suggesting that these pathways may function as a form of sequence-directed immunity.
PTGS pathways share features with a developmental
gene regulatory pathway that involves natural dsRNAencoding genes, recently named micro-RNA (miRNA)
genes (Lau et al., 2001; Lee and Ambros, 2001; LagosQuintana et al., 2001). During PTGS, introduced dsRNAs,
or dsRNAs synthesized from aberrant transgene transcripts, are processed into small RNAs of approximately
22–26 nucleotides that have been termed small interfering RNAs (siRNAs) because they are thought to guide4

Correspondence: craig.mello@umassmed.edu
Present address: Genome Biology Laboratory, National Institute of
Genetics, Mishima 411-8540, Japan.
5

the destruction of complementary target mRNAs (reviewed in Baulcombe, 2002). Natural miRNA genes encode RNA products of approximately 70 nucleotides
which are predicted to fold into stable stem-loop structures that are processed into mature ⵑ22 nucleotide
miRNAs. The founding members of the miRNA genefamily, lin-4 and let-7, encode miRNA products that appear to regulate translation during C. elegans development by base pairing with complementary sequences
located in the 3⬘ UTRs of their target mRNAs (Lee et al.,
1993; Wightman et al., 1993; Reinhart et al., 2000).
Genetic and molecular studies have uncovered similarities in the processing and function of miRNA andsiRNA species. For example, both the RNAi pathway
and miRNA pathway require related genes. In C. elegans, RNAi requires the gene rde-1, which encodes amember of a functionally novel but highly conserved
eukaryotic gene family (Tabara et al., 1999a) whose
members have also been implicated in gene silencing
in fungi, plants, and Drosophila (Catalanotto et al., 2000;
Fagard et al., 2000; Hammond et al., 2001). Homologsof rde-1 have also been implicated in development in
Drosophila, C. elegans, and Arabidopsis (reviewed in
Benfey, 1999). A recent study has shown that two C.
elegans homologs of rde-1, named alg-1 and alg-2, arerequired for the processing and function of the lin-4 andlet-7 miRNAs (Grishok et al., 2001). Similarly, studies
in Drosophila, C. elegans, and in vertebrate cells haveshown that the multifunctional RNaseIII-related enzyme
Dicer is required for the proper processing of both thedsRNAs that trigger RNAi (Bernstein et al., 2001; Ketting
et al., 2001) and the miRNA precursors whose products
mediate developmental gene regulation (Grishok et al.,
2001; Hutvagner et al., 2001; Ketting et al., 2001).
The convergence between PTGS pathways and pathways that control developmental gene regulation raises
the questions of what other features these pathways
share and, importantly, what factors function uniquely
within each pathway to guide RNA recognition and targeting. In the present study we show that rde-4 encodesa dsRNA binding protein required for RNAi. Interestingly,
RDE-4 is not required for other PTGS pathways or for
developmental pathways, suggesting that RDE-4 activity may function at a step unique to the RNAi pathway.
We show that the stable interaction between RDE-4
and dsRNA requires rde-1(⫹) activity and that RDE-4
interacts with the RDE-1 protein in vivo. RDE-4 protein
also interacts in vivo with DCR-1 and with a conserved
DExH-box helicase that is required for RNAi in C. elegans. Interactions were not detected between RDE-4
and siRNAs, or miRNA precursors. RNA sequences
bound to RDE-4 were restricted to regions found within
the trigger dsRNA, and thus RDE-4 does not appear
to interact with the mRNA or with dsRNA sequences
amplified from the target mRNA. Our findings suggestthat RDE-4 functions during the initial steps of RNAi torecognize foreign dsRNA and to present this dsRNA to
DCR-1 for processing.

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Figure 1. Cloning and Sequence Analysis of
rde-4(A) Maps of the RDE-4 genetic and physicalintervals on chromosome III. The structure ofrde-4 gene is diagrammed with coding regions boxed. The positions of the dsRBM motifs are shown as striped boxes. The positions
and nature of lesions in the ne299, ne301,
and ne337 alleles are indicated.
(B and C) Alignments of the first (B) and second (C) dsRBM in RDE-4 and four related
proteins. The sequences are RDE-4 (C. elegans), an open reading frame from the related
nematode (C. briggsae), PACT (Homo sapiens), PRBP (Mus musculus), CG6866 (Drosophila melanogaster), and F21M12.9 (Arabidopsis thaliana). Identities with RDE-4 areshaded in black, and identities among therelated proteins are shaded in gray. Each ofthese proteins contains two or more dsRBMs
and no other recognizable motifs.

Results
Molecular Identification of rde-4
In previous genetic screens for RNAi-deficient mutants,
we found that many C. elegans mutants deficient in RNAialso exhibit additional phenotypes including activationof transposons in the germline, a low penetrance sterile
phenotype, and meiotic chromosome nondisjunction
phenotypes (Tabara et al., 1999a). These findings suggested that RNAi may play a role in transposon silencing
and in other essential functions in the germline. However, paradoxically, the two strongest mutants, rde-1
and rde-4, were completely deficient in RNAi but failed
to exhibit transposon activation or any other discernible
phenotypes. Subsequent genetic studies suggested
that rde-1 and rde-4 may function upstream in the initiation of RNAi (Grishok et al., 2000).
To shed more light on the role of the RDE-4 protein
in RNAi, we set out to clone the corresponding gene.
We first mapped the gene to a small genetic interval
and then assayed corresponding DNA sequences fromthe interval for rescue of rde-4 (see Experimental Procedures). rde-4 rescue was mapped to two overlapping
cosmid clones, T20G5 and E03F9, and to the single
corresponding open reading frame T20G5.11 (Figure1A). We next sequenced the T20G5.11 gene in eachof the rde-4 mutant strains and found point mutations
predicted to disrupt the open reading frame of T20G5.11
in each allele. Two alleles, ne299 and ne301, have an
identical lesion, a 1 base insertion creating a prematurestop codon at the end of the first exon of T20G5.11(Figure 1A). These two alleles were isolated from a single

large clonal population during a screen for spontaneous
mutants (Tabara et al., 1999a) and may thus reflect asingle spontaneous lesion (we will henceforth refer tothis allele as ne299). A second allele of rde-4, ne337,
was identified in a screen using chemical mutagenesis
and induces a premature stop codon at amino acid residue 229 of the T20G5.11 product (Figure 1A). The rde-4
gene is predicted to encode a 385 amino acid proteinthat contains two copies of a conserved motif found in
dsRNA binding proteins (Figures 1B and 1C). The genetic lesions in rde-4 appear likely to be null mutations,and consistent with this idea the ne299 allele, whenplaced in trans to a chromosomal deficiency, exhibited
an RNAi-deficient phenotype and no other additional
phenotypes (data not shown).
RDE-4 Interacts with dsRNA In Vitro and In Vivo
The RDE-4 protein contains two copies of a dsRNA
binding motif (dsRBM) that is found in numerous otherproteins that interact with dsRNA in a sequence-nonspecific manner (reviewed in Fierro-Monti and Mathews,
2000). Therefore, we first asked whether recombinant
RDE-4 could bind to dsRNA by using an in vitro gel shift
assay. Labeled dsRNA corresponding to a portion of a
GFP cDNA was incubated with GST-fused RDE-4, orwith the GST protein alone, and was then run on a nativepolyacrylamide gel. The GST-fused RDE-4 efficiently
bound to the dsRNA, causing a mobility shift, while the
GST protein did not (data not shown). In competition
assays, dsRNA but not single-stranded RNA efficiently
competed for RDE-4 binding (data not shown). Similarly,
we asked whether the labeled dsRNA could bind to theThe RDE-4 Protein and RNAi in C. elegans
863

Figure 2. RDE-4 Binds dsRNA In Vivo
(A and B) Wild-type or rde mutant animals were exposed to pos-1 dsRNA by feeding on E. coli expressing pos-1 dsRNA for 15 min or 2 days
(as indicated). The E. coli feeding strain used expresses a dsRNA, which corresponds to 85% of the pos-1 mRNA (as indicated in the
diagram in [E]). After immunoprecipitation (IP) with anti-RDE-4 antibody (⫹) or with the IgG fraction obtained from the pre-immune serum (⫺),
coprecipitated RNA was fractionated on 1.8% denaturing agarose gels (A) or on a 15% polyacrylamide sequencing gel (B). RNA species were
blotted and analyzed by Northern hybridization with sense RNA probes (W, Y) and antisense RNA probes (X, Z) as illustrated in (E). The
hybridizations in (A) and (B) were done with the same stringency. The probes Y and Z were prepared from a region not contained in thebacterial feeding vector. To remove single-stranded RNA, the RNA sample in lane 14 (asterisk) was treated with 10 ␮g/ml RNaseA and 0.5
␮g/ml RNaseT1. The blot in (B) was hybridized with the sense RNA probe (W).
(C) Northern analysis of RDE-4::IPs prepared from wild-type animals and transgenic animals expressing an unc-22-specific dsRNA segment.
The transgenic C. elegans line was designed to express a 1400 nt segment of unc-22, indicated by the black bar in the diagram (E), as bothsense and antisense RNA. After immunoprecipitation with the anti-RDE-4 antibody, coprecipitated RNA was fractionated on a 1.2% denaturing
agarose gel. Northern hybridization was performed with sense RNA probes U and T as illustrated in (E). The size of the unc-22 RNA associatedwith RDE-4 was approximately 2800 nt, suggesting that the transgenes express dsRNA with a hairpin structure.
(D) RDE-4 does not interact detectably with siRNA. Extracts prepared from wild-type animals either not exposed to dsRNA (lane 3) or exposed
to pos-1 dsRNA by feeding for 2 days (lanes 4, 5, and 6) were subjected to immunoprecipitations as described above. RNA species wererecovered from each fraction and were run on a 12% polyacrylamide sequencing gel. The RNA species were analyzed by Northern hybridization
with a sense RNA probe (W). Hybridization was done with a moderate stringency to allow detection of ⵑ25 nt RNA species. As controls, lane
1 contained a synthetic RNA oligo corresponding to 21 nt of antisense pos-1 sequence, and lane 2 contained size-marker RNA.
(E) Diagrams representing the pos-1 and unc-22 mRNAs are shown as gray bars. The regions targeted with dsRNA are indicated by the solid
black bars above each diagram. Probes used in the experiments are indicated by arrows beneath the diagrams.

recombinant RDE-4 immobilized on a membrane. This
North-Western blot analysis also showed that thedsRNA binds efficiently to the GST-fused RDE-4 but not
to the GST protein alone (data not shown).
We next wished to ask if RDE-4 interacts with dsRNA
during RNAi in vivo. To address this question, polyclonal
antibodies were raised against a recombinant RDE-4
protein. We then exposed animals to dsRNA targeting
the gene pos-1 (Tabara et al., 1999b). We prepared extracts from these animals and from control animals not
exposed to dsRNA, and we used RDE-4-specific antibodies to precipitate the RDE-4 protein complex. The
precipitates were extracted to purify any associatedRNA and analyzed by agarose and acrylamide gel electrophoresis followed by Northern blotting using pos-1
sense and antisense radiolabeled RNA probes. Boththe sense and antisense probes detected approximately
equal amounts of pos-1 RNA that migrated below 350
bases on an agarose gel (Figure 2A, lanes 3 and 11)
and migrated in a size range of 50 to 200 bases on an

acrylamide gel (Figure 2B, lane 1). The coprecipitatedpos-1 RNA was resistant to treatment with a mixture of
the single-strand-specific RNaseA and RNaseT1 enzymes (Figure 2A, lane 14), suggesting that the pos-1
RNA is double stranded. Little or no pos-1 RNA coimmunoprecipitated with RDE-4, which was purified from
populations either not exposed to dsRNA or exposedfor only 15 min (Figure 2D, lane 3; Figure 2A, lane 1). As
expected, rde-4(ne299) mutants failed to exhibit pos-1
dsRNA in the precipitate (Figure 2A, lane 5). The RDE-4
immune complex recovered from the rde-1(ne300) andrde-3(ne298) mutant strains exhibited greatly reduced
quantities of pos-1 dsRNA (Figure 2A, lanes 7 and 9).
Target mRNA and siRNA Sequences Do Not
Coprecipitate with RDE-4
dsRNA is thought to exist and function at multiple steps
in RNAi. First, it functions in the initiation step when longdsRNA is recognized as foreign. Second, it functions in
the execution step when target mRNA is bound by siRNA

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Figure 3. Genetic Analysis of dsRNA Accumulation during RNAi(A) Genetic analyses of accumulated dsRNA species in total RNA extracts. Mixed-stage populations of wild-type (wt), rde-1, rde-2, rde-3,
rde-4, and mut-7 animals were cultured on E. coli expressing pos-1 dsRNA. Subsequently, RNA was extracted from each population andsubjected to Northern analyses. The design of probes W, X, and Z was as illustrated in Figure 2E. The RNA samples of lanes 18–20 weretreated with a mixture of RNaseA and T1. Ethidium bromide (EtBr) staining of 18S and 28S ribosomal RNA serves as a loading control.
(B) A large portion of the foreign dsRNA in total RNA extracts associates with RDE-4. Extracts from wild-type animals exposed to pos-1
dsRNA by feeding as described above were subjected to immunoprecipitation with anti-RDE-4 antibody (⫹) or with the IgG obtained fromthe preimmune serum (⫺). RNA species were recovered from each immune complex (beads) and each supernatant (sup) and were fractionatedon a 1.8% denaturing agarose gel. The RNA species were analyzed by Northern hybridization with a sense RNA probe (W).

and cleaved. And third, it functions in a potential amplification step in which target mRNA sequences are copied
by an RNA-dependent RNA polymerase activity. In theabove experiments, the probes and techniques usedwere designed to detect long dsRNA sequences contained within the trigger dsRNA, and thus could not
address the question of whether siRNAs or target mRNA
sequences might also coimmunoprecipitate with RDE-4.
To address these questions, we first probed for sequences in the 3⬘ UTR of pos-1 that were not includedin the dsRNA trigger (probes Y and Z, see Figure 2E).
These experiments failed to detect significant coprecipitating RNA (Figure 2A, lanes 15 and 17), indicating thatsequences within the target mRNA are not stable components of the RDE-4 immune complex.
Two recent studies suggest that RNAi may involve a
mechanism for the amplification of the dsRNA signal
and provide evidence that sequences in the target
mRNA that lie 5⬘ of the trigger sequence can be amplified(Lipardi et al., 2001; Sijen et al., 2001). In order to ask if
the RDE-4 immune complex contains antisense RNA
sequences derived from regions of the mRNA located5⬘ of the trigger dsRNA, we utilized transgenic animals
that express dsRNA corresponding to a portion of
unc-22 gene (see Figure 2E). RNA probes were thenused to assay for sequences in the RDE-4 immune complex that were derived either from the trigger region orfrom the region located just 5⬘ of the trigger. As expected, the RDE-4 immune complex contained unc-22
antisense RNA corresponding to the targeted region of
unc-22 (Figure 2C, lane 5). In contrast, no significant
hybridization was observed when a sequence derived
from the region located just 5⬘ of the trigger dsRNA
was used as a probe (Figure 2C, lane 1). As expected,
precipitates prepared from nontransgenic wild-type animals did not exhibit significant hybridization to the probes
designed to detect unc-22 antisense RNA (Figure 2C, lanes
3 and 7).
Finally, in order to ask if siRNA sequences precipitate

with RDE-4, we fractionated the RNA species in theRDE-4 immune complex and in the RDE-4-depleted supernatant by acrylamide gel electrophoresis. We thenanalyzed RNA species smaller than 150 bases by Northern blotting using a pos-1 sense probe. We found that
the RNA species in the supernatant included abundant
molecules of less than 150 bases, including a prominent
signal corresponding in size to siRNAs, that migrated
at approximately 24 to 25 bases (Figure 2D, lane 6). RNA
species of 24 to 25 bases were absent in the RDE-4
immune complex, suggesting that RDE-4 preferentiallyinteracts with longer dsRNAs (Figure 2D, lane 5). Taken
together, these studies indicate that RDE-4 binds tolong dsRNA sequences that are restricted to the region
present within the trigger dsRNA. These findings support a model in which RDE-4 interacts with the trigger
dsRNA and functions in the initiation step of RNAi.
dsRNA Bound to RDE-4 Accumulates during RNAi
In order to determine what fraction of the total dsRNA
was bound to RDE-4 and to examine the requirements
for the stability of the dsRNA bound to RDE-4, we next
analyzed RNA extracts from wild-type and rde mutant
strains. Wild-type animals showed an accumulation ofantisense and sense RNA corresponding to the pos-1
trigger dsRNA sequence that appeared to be very similar
to the dsRNA sequences found in the RDE-4 immune
complex (Figure 3A, lanes 3 and 12). This RNA species
was similar in size but slightly smaller, on average, thanthe major pos-1 RNA species detected in the bacterial
expression strain used to induce RNAi (Figure 3A, lane9), suggesting that some digestion of the bacteriallyexpressed dsRNA may have occurred during transit
through the intestine of the animals. Single-stranded
RNA-specific nucleases digested the endogenous
pos-1 mRNA but failed to digest the accumulated RNA
species (Figure 3A, lanes 19 and 20), suggesting that theaccumulated RNA species is indeed double-stranded.
Interestingly, the analysis of total RNA extracts

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865

showed that animals lacking rde-1, rde-3, and rde-4
activities exhibited markedly reduced levels of dsRNA.
In contrast, animals lacking rde-2 and mut-7 accumulated dsRNA to levels that were intermediate (rde-2) orsimilar to wild-type (mut-7) (Figure 3A). The finding thatthe accumulation of this dsRNA species requires some
but not all rde(⫹) activities supports the idea that thisRNA species is an intermediate in the RNAi process.
To ask what fraction of the accumulating dsRNA in
the total RNA extract from wild-type animals is associated with RDE-4, we repeated the immunoprecipitations
with RDE-4-specific antibodies or with control nonspecific IgG obtained from preimmune serum and compared
the amount of the dsRNA present in the immune complex and supernatant of each lysate. We found that approximately 60% of the pos-1 dsRNA species present
in the total RNA coprecipitates with the RDE-4 immune
complex (Figure 3B, compare lanes 3 and 4), indicatingthat a majority of the accumulating dsRNA is associatedwith RDE-4.
RDE-4 Interacts with RDE-1 In Vivo
Our previous studies have suggested that RDE-1 andRDE-4 function at an upstream step in the initiation ofRNAi (Grishok et al., 2000). This previous observation,and the finding that RDE-1 and RDE-4 are both required
for the accumulation of dsRNA, prompted us to ask if
RDE-4 forms a complex in vivo with RDE-1. For thesestudies we generated a transgenic strain expressing
RDE-1 tagged with the HA epitope and RDE-4 taggedwith the FLAG epitope. We first confirmed that thesetagged proteins were functional and able to rescue thecorresponding mutants (data not shown). We then
immunoprecipitated RDE-4 with RDE-4-specific polyclonal antibodies, and RDE-1 via the epitope tag, andanalyzed the precipitates by immunoblotting (Figure 4A).
In reciprocal assays, RDE-1 and RDE-4 were found tocoprecipitate. Based on a comparison of the RDE-1
present in total extracts and in the RDE-4 immune complex, we estimate that approximately 5% of the RDE-1
molecules stably associate with the RDE-4 complex.
The interaction between RDE-1 and RDE-4 occurredin animals that were not exposed to exogenous dsRNA,and the interaction was not abolished by treatment witheither double-stranded RNA-directed nuclease, RNaseV1,
or a mixture of RNaseA and T1 that are single-stranded
RNA-specific nucleases (data not shown). These findings suggest that RDE-1 and RDE-4 form a complex in
the absence of dsRNA, but cannot rule out the possibility
that dsRNA promotes the interaction or is required forthe interaction but is protected from digestion in the
immune complex.
RDE-4 Forms a Complex with DCR-1 In Vivo
In order to identify additional proteins that interact with
RDE-4 in vivo, we isolated large quantities of the RDE-4
immune complex and then resolved its components using SDS polyacrylamide gel electrophoresis (SDSPAGE). For this experiment we used rde-4(ne299) mutant animals that were rescued via an RDE-4::GFP fusion
protein. We then passed protein extracts prepared fromthese transgenic animals and from control wild-type animals over a column conjugated with anti-GFP mono-

Figure 4. RDE-4 Associates with RDE-1, DCR-1, and an RNA Helicase-Related Protein DRH-1(A) RDE-1 and RDE-4 form a complex in vivo. Protein extracts wereprepared from an rde-1(ne300) mutant strain that was rescued bya transgene expressing HA-tagged RDE-1 and FLAG-tagged RDE-4.
The total protein extract and immunoprecipitates prepared by incubating the extract with anti-RDE-4 or anti-HA and with control nonspecific sera (preimmune or normal IgG) were run on SDS-PAGE(10%). Western blots were prepared and immunostained, as indicated, with anti-HA (to detect HA::RDE-1) or anti-FLAG or antiRDE-4 (to detect RDE-4).
(B and C) Immunopurification of proteins associated with RDE-4 invivo. The rde-4(ne299) mutant was rescued by an integrated transgene expressing an RDE-4::GFP fusion protein. In (B), total protein
extracts prepared from this strain and from a wild-type nontransgenic strain were resolved on SDS-PAGE and immunostained with
anti-GFP polyclonal antibody (Molecular Probes). The expressionof RDE-4::GFP was detected as a single band. In (C), total protein
extracts were first precipitated with anti-GFP-specific antibodies,and proteins associated with the RDE-4::GFP immune complex wereresolved by SDS-PAGE (8%) followed by silver staining.

clonal antibody. After electrophoresis, the gel was silver
stained to detect components of the RDE-4::GFP immune complex (Figure 4C). These experiments identified
prominent bands migrating at 210 kilodaltons (kDa) and
110 kDa as well as several additional polypeptides thatwere present in the RDE-4::GFP immune complex but
absent in wild-type control animals. A minor band at
125 kDa was identified, corresponding in size to RDE-1,
but contained insufficient protein for further analysis(Figure 4C). The 210 kDa and 110 kDa bands were subjected to tryptic digestion, and the resulting fragments
were then analyzed by mass spectrometry. The peptide
mass fingerprints of p210 and p110 were compared within silico-digested sequences from protein databases(see Experimental Procedures). A total of 21 peptides
from the tryptic digestion of p210 matched DCR-1 (Table

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Table 1. Peptide Mass Fingerprinting of p210 (DCR-1) and p110 (DRH-1)
MH⫹
PeptideData of p210 Matched Sequences

Amino Acid Position
MH⫹
Peptidein K12H4.8 (DCR-1) Data of p110 Matched Sequences

Amino Acid Positionin F15B10.2

1049.59
1067.581085.67
1134.60

478–485
289–297695–703
6–14
1458–1468
1797–1806
1828–1836
1638–1648
1638–1648
957–967
660–670
1828–1828
416–428
429–441
346–357
1531–1542
649–659382–394780–793
49–64
1742–1755
1593–1608

996–1003
732–740
438–446
216–224
654–663759–768
654–663708–717527–538
264–275
686–697552–5641160.66
1214.72
1231.711248.75
1313.80
1317.791370.79
1380.791412.781428.91
1446.961490.841498.89
1638.87
1778.02
1807.89
1868.04

1049.52
1067.541085.651134.541134.541160.61
1214.63
1231.631248.66
1313.74
1317.721370.731380.69
1412.721428.80
1446.801490.701498.80
1638.76
1777.92
1807.86
1867.95

FDRPLDMRQLGPWAAWR
IPPFPVFLRADLQCFNPRTGWDIGGDVSKVTVEVVNNMRYLHQIEQQR
QYSPGVLTDLRa
QYSPGVLTDLRa
VNQLLLTDELRFREAELTLNPKYLHQIEQQRR
FVNPDYVVGASGRNLASSDSQGLHKRSIEALRPYVPQR
VMNWMGLKVIQKECFIYAFELER
AEHLSAIIFVDQRYVSEVVANMENMPR
EYGVQLFAPLDQGGKRNLDTTWQVIFHMMR
AYLVQAFTHASYINNR

1026.53
1067.631081.511111.66
1192.631195.43
1208.661295.671371.68
1490.80
1536.841026.53
1067.481081.511111.591192.611195.61
1208.61
1208.661295.61
1371.69
1490.80
1536.82YFTPTEIRSEWMSGLNKNSPYSNIMRFYQIINASRTPNFTVNMIRbLKMFADGEIRTPNFTVNMIRbTRYEATILNKSTDGPIGMFTNR
VDNIVQENSTPRTVQYIVEQNLQRTALRNEHIGIEQR

The masses of the tryptic peptides were measured with a MALDI-TOF mass spectrometer. The values for mass/charge ratio of the protonatedmolecular ions corresponding to 21 fragments of p210 were matched to the sequence of K12H4.8 (DCR-1) with mass errors of 0.002%–0.011%.
Eleven fragments of p110 were matched to the sequence of F15B10.2 (DRH-1) with mass errors of 0.001%–0.015%.
a
The difference between these two originates in a possible modification of glutamine (Q).
b
The difference between these two originates in a possible modification of methionine (M).

1), while 11 peptides from p110 matched a DExH-box
RNA helicase-related protein F15B10.2 (Table 1, see
below).
The finding that RDE-4 forms a complex in vivo with
DCR-1 suggests that RDE-4 may function to present theforeign trigger dsRNA to DCR-1 for processing. DCR-1
also acts to process the stem-loop precursors of thedevelopmental regulators lin-4 and let-7 (Grishok et al.,
2001; Ketting et al., 2001). However, rde-4 mutants do
not exhibit developmental defects (Tabara et al., 1999a),and thus are not required for the activities of lin-4 orlet-7. Furthermore, we did not observe lin-4 or let-7 precursor RNAs associated with the RDE-4 immune complex (data not shown). Thus, although RDE-4 interacts
strongly with DCR-1 in vivo, RDE-4 is not essential forthe developmental functions of DCR-1 and may rather
serve as an adaptor protein that recruits DCR-1 to theRNAi pathway.
A Conserved DExH-Box Helicase Requiredfor RNAi
The RDE-4 protein interacts with a 110 kDa protein,which as described in the previous section corresponds
to the predicted gene F15B10.2 (Table 1), one of two
closely related C. elegans DExH-box RNA helicase
genes. F15B10.2, along with its homolog C01B10.1, map
directly adjacent to one another on chromosome IV andappear likely to be expressed together from a single
promoter. Consistent with this idea, we found that some
F15B10.2 cDNAs were spliced to the trans-spliced
leader sequence (sl2), which is often spliced onto the5⬘ end of transcripts encoded by downstream genes inan operon (reviewed in Blumenthal, 1995). We foundthat the C01B10.1 gene and a cDNA clone (yk226c6)
both appear to encode a frame-shifted open reading
frame predicted to terminate the protein prior to thehelicase domain, raising the question of whether or not
this represents a functional gene. An alternative conceptual splicing of C01B10.1, or utilization of an alternative
initial methionine, could encode a protein with overall
74% identity to F15B10.2 (Figures 5A and 5B).
These C. elegans genes have close homologs in mammals, including a gene named RHIV-1 induced in response to viral infection in pigs (Figures 5A and 5B;
Zhang et al., 2000). Notably, these helicase proteins are
more similar to the DCR-1 helicase domain than to any
other helicase-related proteins in C. elegans or otherorganisms (Figures 5A and 5B). Consequently, we havenamed these genes drh-1 and drh-2 (for F15B10.2 andC01B10.1, respectively) which stands for dicer-related
helicase.
To ask if these genes might function in RNAi, we used
RNAi to target their inactivation. Previous work on dcr-1
has shown that RNAi targeting dcr-1 causes developmental defects and can also partially inhibit RNAi (Grishok et al., 2001). Although the helicase domain of
DCR-1 is similar to DRH-1 and DRH-2 at the protein level,
the dcr-1 gene does not contain regions of extended
nucleotide identity required for crossinterference in an
RNAi assay. The drh-1 and drh-2 genes, on the other
hand, are more than 81% identical at the nucleotide level
and contain several regions of 40 to 200 nucleotides with
100% identity, and are thus expected to crossinterfere
in the RNAi assay. There are no extensive regions predicted to be unique to either helicase gene and thus it
is doubtful that effective single RNAi is possible. We

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867

Figure 5. DRH-1 and DRH-2 Are Helicase-Related Proteins Required for RNAi
(A and B) Alignment of DRH-1 (F15B10.2), DRH-2 (C01B10.1), D2005.5, RHIV-1, and DCR-1. (A) Alignment of DExH/D domains, and (B) alignmentof the helicase conserved C-terminal domains. Identities shared with DRH-1 are shaded in black, and identities among the other proteins areshaded in gray. Asterisks above residues in (A) indicate the location of DExH/D motif.
(C) Reduced sensitivity to RNAi following drh-1 and drh-2(RNAi).

also targeted D2005.5, which, other than DCR-1, is thenext most similar C. elegans helicase-related gene (Figures 5A and 5B).
We did not observe any developmental abnormalities
in animals exposed to drh-1 or drh-2 dsRNA, injected
either singly or in combination. However, we found that
injection of either drh-1 or drh-2 (or both) dsRNAs rendered the injected animals resistant to RNAi targetinga second gene (Figure 5C). Both germline and somatic
RNAi was dramatically reduced in the helicase RNAianimals. RNAi targeting D2005.5 did not induce developmental or RNAi defects (Figure 5C and data not shown).
These results suggest that the DExH-box helicase
DRH-1 (and perhaps DRH-2) not only interact with
RDE-4 but are also required for RNAi in C. elegans.
Discussion
The RDE-4 Protein and the Initiation of RNAi
Recent work in numerous organisms has shown that
RNAi and other forms of sequence-directed immunity
share features both with each other and with naturaldevelopmental regulatory pathways. For example, boththe foreign dsRNAs that trigger RNAi and the endogenous miRNA precursors that function in development
are processed into small RNAs by a multifunctional
RNaseIII-related enzyme, Dicer (DCR-1). These small
RNAs (siRNAs and miRNAs) are thought to be incorporated into silencing complexes that mediate, respectively, mRNA destruction during RNAi or translational
control during development. These findings raise twokinds of interesting question. First, what additional
genes and mechanisms do these pathways share? And
second, what factors function uniquely within each pathway to guide RNA recognition and targeting? In thepresent study we have described the dsRNA binding
protein RDE-4, which appears to be dedicated to dsRNA

recognition within the RNAi gene-silencing pathway. We
have shown that RDE-4 forms a complex in vivo with
DCR-1. However, the RDE-4 protein is not required fordevelopment and does not interact detectably with
miRNAs in vivo. These findings suggest a model in which
RDE-4 functions as a sensor that recognizes foreigndsRNA and brings this RNA to DCR-1 for processing(Figure 6A).
The RDE-4 protein contains two copies of the dsRNA
binding motif (dsRBM) and no other recognizable functional domains. The crystal structure of a dsRBM boundto dsRNA reveals extensive contacts between this protein motif and the backbone of the double-helical RNA(Ryter and Schultz, 1998). Thus, dsRBMs are expected
to interact nonspecifically with dsRNAs of any sequence. Several groups of dsRBM-containing proteinscan be distinguished by the presence of additional conserved motifs (reviewed in Fierro-Monti and Mathews,
2000). Members of one such group, the dsRNA-activated protein kinases (PKRs) in mammals, contain akinase motif and two dsRBMs. PKRs have previously
been shown to be involved in the response to foreigndsRNA (reviewed in Proud, 1995). PKRs are activatedby binding dsRNA, and they trigger signal transduction
pathways that direct a general repression of translation
and ultimately induce apoptosis. Thus, PKRs arethought to provide a first line of defense for clearing
viral infection in mammals. Like PKRs, RDE-4 appears
to play an upstream role in the recognition of foreigndsRNA. However, instead of a general translation arrest,
RDE-4 initiates the sequence-specific destruction ofcomplementary mRNAs.
The RDE-1 Protein and the Initiation of RNAi
We have shown that the RDE-4 protein interacts with
RDE-1 in vivo. The RDE-1 protein has numerous homologs in C. elegans and in other organisms, and members

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868

Figure 6. Model(A) RNAi.
(B) Cosuppression and transposon silencing.
(C) miRNA-mediated translational regulation.
The unlabeled shapes in the diagram indicate
unknown proteins. The yellow arrows andbrackets in (A) indicate potential interactions.

of the RDE-1 family have been implicated in gene silencing and developmental pathways in plants, fungi, andanimals. RDE-1 homologs in Drosophila and Neurospora
are found in complexes that contain siRNA molecules(Hammond et al., 2001; Catalanotto et al., 2002). Furthermore, RDE-1 homologs are required for miRNA function
in C. elegans (Grishok et al., 2001) and have recently
been found in a complex with miRNAs in vertebrate cells(Mourelatos et al., 2002). Thus, RDE-1-related proteinsappear to function in a variety of homology-dependent
mechanisms that involve pairing between small guide
RNAs and mRNAs.
The interaction we detect between RDE-4 and RDE-1
could reflect a transfer of siRNA products from theRDE-4/DCR-1 complex to an RNA-induced silencing
complex (RISC) that contains RDE-1 (Figure 6A). However, we have shown that RDE-1 activity is required forthe accumulation of dsRNA bound to RDE-4. Thus, if
RDE-1 does function downstream in the execution stepof RNAi, it must also function upstream at a step required
for the retention of dsRNA (Figure 6A). It is also possiblethat both the initiation of RNAi and the subsequent destruction of target mRNA occurs within a single complex
and that completion of the targeting steps may be required for the continued retention and accumulation of
the trigger dsRNA molecules engaged by RDE-4.
The activities of RDE-1 and RDE-4 appear to protect
the foreign dsRNA from entering a nonspecific dsRNA
destruction pathway (Figure 6A). This finding suggeststhat the initiation complex containing RDE-4 may collectdsRNA faster than DCR-1 or other downstream factors
can process the dsRNA. Conceivably, RDE-1 and RDE-4
function as adaptor proteins that regulate the entry ofdsRNA into the RNAi pathway so as not to saturate thecell’s essential dsRNA-processing machinery (Figure 6A).
A DExH-Box RNA Helicase Involved in RNAi
We found a 110 kDa RNA helicase-related protein,
DRH-1, in a protein complex with RDE-4 in vivo. Our
analysis, using peptide mass fingerprinting, specifically
identified DRH-1, but we cannot rule out the possibility
that a small portion of the 110 kDa band contains asecond very similar predicted protein named DRH-2.
While numerous proteins related to DRH-1 are present

in eukaryotes, DRH-1 is most similar to an unstudied
human protein and to a porcine protein, RHIV-1. Interestingly, the rhiv-1 gene was identified as a locus whose
expression is activated in pigs infected with porcine
reproductive and respiratory syndrome virus (PPRSV)(Zhang et al., 2000). PPRSV is a positive-strand RNA
virus that forms dsRNA structures during its life cycle.
The finding that DRH-1 is most similar to a family ofvertebrate helicase proteins that include a protein activated in response to viral infection suggests that thisfamily of proteins may have ancestral functions in the
response to foreign dsRNA.
Up to the present, studies in other organisms haverevealed several RNA helicases required for PTGS pathways. Mut6 in Chlamydomonas is a DEAH-box RNA
helicase involved in PTGS and is most similar to the C.
elegans MOG-1 helicase required for sex determinationin the germline (Wu-Scharf et al., 2000). SDE3 in Arabidopsis is required for PTGS and is similar to Upf1-type
RNA helicases (Dalmay et al., 2001). A biochemical studyof RNAi in Drosophila suggests that RNA helicase activity may be required downstream of siRNA formation(Nykanen et al., 2001). And another recent study in C.
elegans has identified MUT-14 as a DEAD-box helicase
required downstream of siRNA formation for target
mRNA destruction during RNAi (Tijsterman et al., 2002).
The Dicer protein also contains an RNA helicase domain,which notably is most similar to the helicase domain of
DRH-1.
We have found that RNAi targeting drh-1 results ininhibition of RNAi in both the soma and germline but
causes no other phenotypes. In contrast, mutations in
dcr-1 or RNAi targeting dcr-1 inhibit RNAi but also resultin lethal phenotypes (Grishok et al., 2001; Knight andBass, 2001; Ketting et al., 2001). Thus, DRH-1 does notappear to be required for developmental functions of
DCR-1 and may instead function closely with RDE-4 ina step specifically required for RNAi (Figure 6A). There
are a several possibilities for the role of DRH-1. For
example, the helicase activity of DRH-1 may change
the conformation of the dsRNA in order to facilitate thetranslocation of the protein complex along the dsRNA.
Or it may facilitate transfer of the dsRNA from RDE-4
to the active site of DCR-1. It is conceivable that thisadditional helicase activity is necessary in order to pro-The RDE-4 Protein and RNAi in C. elegans869

cess the long-contiguous helices of the foreign dsRNAs
that initiate RNAi. This activity may be less critical inprocessing miRNA precursors in which only short-contiguous helices are present (Lau et al., 2001; Lee andAmbros, 2001; Lagos-Quintana et al., 2001).
Distinct Initiation Steps in Related Gene
Silencing Pathways
In C. elegans, the RNAi pathway overlaps geneticallywith the transposon-silencing and cosuppression pathways (Tabara et al., 1999a; Ketting et al., 1999; Ketting
and Plasterk, 2000; Dernburg et al., 2000). Cosuppression is a PTGS silencing phenomenon that occurs when
extra copies of a gene are introduced into an organism,
resulting in the simultaneous silencing of both the transgene and homologous endogenous genes (reviewed in
Cogoni and Macino, 2000). Transposon silencing may
represent one in vivo function of the cosuppression
pathway, as no genetic or experimental data provides
evidence for a distinction between these pathways.
A previous study on the genetics of RNAi pathway
genes suggested that rde-1 and rde-4 function upstream in a step that is unique to the RNAi pathway, whilerde-2 and mut-7 function together at a downstream step
common to the RNAi, transposon silencing, and cosuppression pathways (Grishok et al., 2000). Consistent withthese findings, we have shown that during the initiationof RNAi, foreign dsRNA is recognized by RDE-4 andaccumulates in tissues in a process that requires both
RDE-4 and RDE-1. Furthermore, we have shown thatRDE-2 and MUT-7, which appear to function in multiple
silencing pathways, are not required for the accumulation of dsRNA during RNAi, consistent with a downstream function for these gene products (Figure 6).
The finding that RDE-4 is a dsRNA binding proteinthat interacts with foreign dsRNAs raises the questionof why this protein does not also recognize and initiatesilencing in response to dsRNAs that are postulated tofunction in the transposon silencing and cosuppression
pathways (reviewed in Plasterk and Ketting, 2000; Vance
and Vaucheret, 2001; Waterhouse et al., 2001). One attractive possibility is that transposon silencing and cosuppression are triggered when transcription occurs
within partially repressed chromatin (Figure 6B). Conceivably, abnormal transcripts produced from the repressed chromatin are converted into dsRNAs throughthe activity of an RNA polymerase dedicated to thissilencing pathway. Perhaps DCR-1 resides in a complex
with this polymerase and thus binds the nascentdsRNAs efficiently without the need for RDE-4. Alternatively, the initiation of RNAi might occur when dsRNA
is encountered in the cytoplasm, while the initiation oftransposon silencing and cosuppression could involve
nuclear dsRNA triggers. However, this latter modelseems unlikely, as RDE-1 and RDE-4 are both absolutely
required for dsRNA-induced silencing, even when thetrigger dsRNA is expressed from a transgene directly
within the nuclei of target tissues (Tabara et al., 1999a).
RDE-1 and RDE-4 are required for the initiation ofRNAi in animals exposed to dsRNA, but not for interference inherited by progeny (Grishok et al., 2000). Thus,
if an amplification mechanism functions downstream in
the RNAi pathway (Lipardi et al., 2001; Sijen et al., 2001),

RDE-4 is not likely to function in the recognition of theamplified dsRNAs. Perhaps amplification in the RNAi
pathway involves the same hypothetical RNA polymerase complex postulated to function in the productionof dsRNA transcripts from partially repressed chromatin(Figure 6). A good candidate for such a polymerase has
been described as a factor required for cosuppression
in both fungi and plants (Cogoni and Macino, 1999;
Mourrain et al., 2000; Dalmay et al., 2000). However,surprisingly, members of this protein family are also
required for RNAi in C. elegans (Smardon et al., 2000;
Sijen et al., 2001; D. Conte and C.C.M., unpublished).
These findings could indicate that amplification is required for RNAi even when abundant dsRNA is present.
Alternatively, it is possible that members of this protein
family do not function in dsRNA synthesis, or function
at multiple steps, both in producing dsRNAs that trigger
cosuppression and in later steps that occur downstreamof the convergence between these silencing pathways.
Our study sheds light on the upstream events that
distinguish RNAi from related gene-silencing pathways.
A future challenge will be to elucidate the events uniqueto the initial steps in the cosuppression and microRNA
pathways. These studies will no doubt lead to new insights into the world of small regulatory RNAs andshould in turn lead to improved methods for controllinggene expression.
Experimental Procedures
Strains, Genetic Analysis, and Mapping
The Bristol strain N2 was used as a standard wild-type C. elegans
strain. Genetic mapping placed rde-4(ne299) mutation to the rightend of the interval between sqv-3(n2842) and unc-69(e587) mutations on chromosome III. Rescue experiments were performed bycoinjection of the rescuing construct with a mixture of plasmids that
express sense and antisense unc-22 RNA as well as rol-6 transformation marker pRF4 as described in Tabara et al. (1999a).
rde-1(ne300), rde-2(ne221), rde-3(ne298), rde-4(ne299), and mut7(pk204) mutants were used to analyze the accumulation of dsRNA
species during feeding RNAi. rde-4(ne337) was isolated by this workand utilized for the detection of a rde-4 mutation.
RNA Interference Assays
RNAi by microinjection was performed as described in Fire et al.
(1998) and Rocheleau et al. (1997). RNAi by feeding was performed
as described in Timmons and Fire (1998) and Tabara et al. (1999a).
Cosmid and cDNA Clones
Cosmid clones around rde-4 locus were obtained from A. Coulson.
rde-4 cDNA clone yk333g4, drh-1 cDNA clone yk447b12, and drh-2
cDNA clone yk226c6 were obtained from Y. Kohara and used in thiswork.
Transgenic Worms Used for Immunoprecipitationrde-1 and rde-4 genes were tagged with epitope sequences andcloned into plasmids. Instead of 145 lysine in RDE-1, two copies of
HA-tag sequences were inserted into the rde-1 coding region. FLAGtag or GFP-tag sequences were introduced at the carboxyl terminusof rde-4 coding region.
Transgenic lines were produced with microinjection as describedin Mello and Fire (1995). The HA-tagged rde-1 gene, the FLAGtagged rde-4 gene, and pRF4 were coinjected into rde-1(ne300)
mutant. The GFP-tagged rde-4 gene and pRF4 were coinjected
into rde-4(ne299) mutant. Animals whose transgenes were stably
integrated into chromosomes were selected following UV irradiation
or identified as a spontaneous event.

Cell
870

Antibody Production and Immunoprecipitation
A carboxy-terminal region (amino acid residues 218–385) of RDE-4
was tagged with 6⫻ His and expressed in bacteria. Polyclonal rabbit
antibodies were raised against the purified recombinant protein.
The antibody was affinity purified using the recombinant protein
immobilized to metal-chelated resin.
Before immunoprecipitation, animals were separated from thebacteria with sucrose flotation method and agitated in M9 buffer
for 30 min to digest the bacteria remaining in guts. The animals
were homogenized in lysis buffer (25 mM HEPES-NaOH [pH 7.4],
150 mM NaCl, 0.2 mM DTT, 10% glycerol, 1% Triton X-100 andcomplete protease inhibitors [Roche]). For RNA coprecipitation experiments, 2% Superasein (Ambion) was included in the lysis buffer.
The affinity-purified anti-RDE-4 antibody, anti-HA antibody 3F10
(Roche), and anti-FLAG antibody M2 (Sigma) were used for immunoprecipitation and immunoblotting. To recover RNA from RDE-4 immunoprecipitates, the precipitates were incubated in 0.2 mg/ml Proteinase K, 0.7⫻ TBS, 5 mM EDTA, and 0.1% SDS for 30 min at 50⬚C
and were extracted with phenol and chloroform. The recovered
nucleic acids were analyzed by Northern hybridization essentiallyas described below.
Protein Identification by Peptide Mass Fingerprinting
To identify proteins interacting with RDE-4, the transgenic animals
expressing RDE-4::GFP fusion protein were homogenized in thelysis buffer as described above. Anti-GFP monoclonal antibody 3E6(Q-BIOgene) was crosslinked onto Protein G-Sepharose beads withdimethyl pimelimidate. RDE-4::GFP immune complex was precipitated with the anti-GFP beads and was analyzed with SDS-PAGE.
The gels were visualized by silver staining or Coomassie staining.
After Coomassie staining, 210 kDa and 110 kDa bands were excised from the gel and digested in gel with trypsin. The massesof the tryptic peptides were measured with matrix-assisted laser
desorption ionization-reflectron time-of-flight (MALDI-TOF) mass
spectrometry (Shimazu Biotech, Inc., Japan). Searching of the mass
values against the database was performed with the program MSFit at http://prospector.ucsf.edu (Clauser et al., 1999).
Northern Blot Analysis
For analyses of total RNA, animals exposed to pos-1 dsRNA byfeeding were purified from the plates and homogenized in guanidine
solution (4 M guanidine thiocyanate, 50 mM Tris [pH 7.4], 10 mM
EDTA, 0.65% lauroylsarcosine, and 1% 2-mercaptoethanol). Nucleic
acids were extracted from the homogenates with phenol and chloroform and were precipitated with sodium acetate and isopropanol.
The precipitates were resuspended in water and were subsequently
acidified with sodium acetate (330 mM [pH 4]). DNA was removed
from the nucleic acids by extraction with phenol and chloroform
under this acidic condition.
RNA samples were diluted with formamide/EDTA gel-loading
buffer and were heat denatured before loading. Approximately 20
␮g of total RNA was run per each lane on agarose/formaldehydegels. RNA species were transferred onto plus-charged nylon membranes from the gels with capillary blotting. Strand-specific riboprobes were synthesized using T7 or T3 RNA polymerase and[␣-32P]UTP (380 Ci/mmol). Hybridization was performed at 57⬚C in
50% formamide, 2⫻ SSC, 1% SDS, 5% dextran sulfate, and 150
␮g/ml Torula yeast RNA. The blots were washed basically with 0.1⫻
SSC/0.1% SDS at 65⬚C.
To detect low-molecular-weight RNA, RNA species were run onpolyacrylamide/urea gels and transferred to membranes via semidryelectroblotting. For riboprobes used for the detection of 25 nt RNA
species, the radiolabeled probe was partially fragmented by boilingfor 60 min in a hybridization buffer containing formamide. Subsequently, blots were hybridized with riboprobe in 50% formamide,3⫻ SSC, 1% SDS, 5% dextran sulfate, and 150 ␮g/ml yeast RNA
at 50⬚C and washed with 0.8⫻ SSC/0.1% SDS at 50⬚C. To detectlin-4 and let-7 RNAs, DNA oligonucleotides were end-labeled andused for the hybridization as described in Reinhart et al. (2000).

for reagents and helpful discussions; T.H. Shin, A.E. Pasquinelli,and H. Sakamoto for several technical suggestions; J.F. Mello forcomments on the manuscript; Y. Kohara and A. Coulson for strains
and reagents; the C. elegans Genetics Center (funded by National
Institutes of Health National Center for Research Support) for strains;
and the PMM core for sequence analysis and peptide mass spectrometry. Research support was provided in part by Japan Society
for the Promotion of Science grants to H.T. and H.S. and a National
Institutes of Health grant to C.C.M. (GM58800). C.C.M. is a Howard
Hughes Medical Institute assistant investigator.
Received: September 28, 2001
Revised: May 22, 2002
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Accession Number
The GenBank accession numbers are as follows: rde-4, AY071926;
drh-1, AF480439; and drh-2, AF480440.