A Drosophila fragile X protein interacts
with components of RNAiand ribosomal proteins
Akira Ishizuka,1,2,3 Mikiko C. Siomi,1,3 and Haruhiko Siomi1,4
1

Institute for Genome Research, 2Graduate School of Nutrition University of Tokushima, Tokushima 770-8503, Japan

Fragile X syndrome is a common form of inherited mental retardation caused by the loss of FMR1 expression.
The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as anegative translational regulator. In Drosophila, the fly homolog of the FMR1 protein (dFMR1) binds to andrepresses the translation of an mRNA encoding of the microtuble-associated protein Futsch. We have isolated
a dFMR1-associated complex that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The
dFMR1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68).
AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease
complex that mediates RNA interference (RNAi) in Drosophila. We show that Dmp68 is also required for
efficient RNAi. We further show that dFMR1 is associated with Dicer, another essential component of theRNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that dFMR1 is part of the RNAi-related
apparatus. Our findings suggest a model in which the RNAi and dFMR1-mediated translational control
pathways intersect in Drosophila. Our findings also raise the possibility that defects in an RNAi-related
machinery may cause human disease.
[Keywords: fragile X syndrome; FMR1; RNAi; RNA helicase; miRNA]
Received July 9, 2002; revised version accepted August 22, 2002.

Fragile X syndrome is the single most common form ofinherited disease causing mental retardation with aprevalence estimated at ∼ 1 in 4000 male births and 1 in
8000 female births (for review, see Hagerman 2002). Cognitive deficits reported in fragile X children range frommild to severe, and behavioral disturbances include social and attention deficits, autistic-like behaviors, unusual responses to sensory stimuli, hyperactivity, andabnormal sleep (Gould et al. 2000; Hagerman 2002). In
most cases, the syndrome is caused by a trinucleotide
repeat expansion in the 5⬘ untranslated region of the fragile X mental retardation 1 gene (FMR1; for review, see
Imbert et al. 1998; O’Donnell and Warren 2002). An expansion of the CGG repeat is associated with abnormal
DNA methylation of both a nearby CpG island and therepeat itself. As a result, the FMR1 locus becomes silent
at the transcriptional level and thus no translation occurs (Siomi et al. 1993; Verheij et al. 1993). It is therefore
clear that the pathophysiological mechanisms leading to

3

These authors contributed equally to this work.
Corresponding author.
E-MAIL siomi@genome.tokushima-u.ac.jp; FAX 81-88-633-9451.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/
gad.1022002.

4

the symptoms in fragile X syndrome can be elucidated bystudying the function of the FMR1 gene.
Pathology in the brains of fragile X patients and Fmr1
knockout mice show the presence of abnormal dendritic
spines reminiscent of a maturation delay (Greenough et
al. 2001; Irwin et al. 2001). Because the FMR1 protein(FMRP) is an RNA-binding protein that associates with
polyribosomes, it is thought to be related to posttranscriptional regulation of gene expression in a manner
critical for the correct development of neurons (Inoue etal. 2000; O’Donnell and Warren 2002). Indeed, biochemical studies suggest that FMRP acts as a negative regulator of translation (Laggerbauer et al. 2000; Li et al. 2000;
Schaeffer et al. 2001). These observations suggest that bymodulating mRNA translation and consequently protein
synthesis, FMRP is important for the formation andfunction of synapses. However, to what extent and how
it might effect translation in vivo is unknown. The fruit
fly Drosophila melanogaster has proven to be a powerfultool for the genetic dissection of biochemical pathways.
Because many genes are conserved between flies and humans, including genes that regulate complex behaviors
such as learning and memory, as well as entire pathwaysof development and oncogenesis (Patel 1994; Miklos andRubin 1996; Rubin et al. 2000), Drosophila could be
used, therefore, to define the molecular pathways lead-

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ing to human neurological diseases and to identify thegenes involved (Fortini and Bonini 2000). In the Drosophila genome, there is a single gene, dFMR1, that ishomologous to FMR1 (Wan et al. 2000). Drosophila andvertebrate FMR1 proteins share a number of topographical landmarks (Wan et al. 2000), including two typesof RNA-binding motifs, namely, two KH domains andan RGG box (Siomi and Dreyfuss 1997). Moreover,they show similar biochemical properties such as RNAbinding and ribosome-association (Wan et al. 2000).
Importantly, genetic studies demonstrate that dFMR1
has a role in the regulation of synapse growth and functions (Zhang et al. 2001; Dockendorff et al. 2002; Morales et al. 2002; Inoue et al. 2002), probably by acting asa translational repressor of an mRNA encoding Futsch,
which is the fly homolog of the microtubule-associatedprotein MAP1B (Zhang et al. 2001). Therefore, examining the role of FMRP in the fruit fly is a promising
approach providing significant insights into the functionof FMRP.
RNA interference (RNAi) is the process of sequencespecific posttranscriptional gene silencing (PTGS) in avariety of organisms, initiated by the introduction of
double-stranded (ds) RNA that is homologous in sequence to the silenced gene (Fire et al. 1998; for review,
see Cogoni and Macino 2000; Vance and Vaucheret 2001;
Waterhouse et al. 2001; Hannon 2002; Hutvagner andZamore 2002). During RNAi, introduced dsRNAs areprocessed into small RNAs of ∼ 21–22 nucleotides (nt)that have been termed small interfering RNAs (siRNAs)
because they direct the cleavage of complementarymRNA targets (Zamore et al. 2000; Elbashir et al. 2001).
RNAi pathways share features with a developmental
gene regulatory pathway that involves natural dsRNAencoding genes, recently named microRNA (miRNA)
genes (Lagos-Quintana et al. 2001; Lau et al. 2001; Lee
and Ambros 2001). Natural miRNA genes encode ∼ 70-nt
hairpin precursor RNAs that are processed into mature
22-nt miRNAs whose expression is often developmentally regulated. miRNAs are thought to regulate mRNAtranslation because the founding members of themiRNA gene family, lin-4 and let-7, encode miRNA
products that repress translation during Caenorhabditis
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). Although siRNA and miRNA pathways arethought to be distinct, studies in animals have revealed
that the multidomain RNase III-related enzyme Dicer isrequired for processing both siRNAs and miRNAs, therefore producing RNA species of similar sizes (Bernstein etal. 2001; Grishok et al. 2001; Hutvagner et al. 2001; Ketting et al. 2001). Furthermore, both the siRNA pathway
and miRNA pathway require members of the Argonautefamily [also referred to as the PAZ-and-Piwi domain(PPD) family, or the RDE-1 family; Tabara et al. 1999;
Catalanotto et al. 2000; Fagard et al. 2000; Hammond etal. 2001]. In C. elegans, RNAi requires the Argonautefamily member rde-1 (Tabara et al. 1999), whereas two
homologs of rde-1, named alg-1 and alg-2, are required

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for the processing and function of the lin-4 and let-7
miRNAs (Grishok et al. 2001). Argonaute family members have also been implicated in PTGS and development in fungi, plants, C. elegans, Arabidopsis, andDrosophila (Bohmert et al. 1998; Tabara et al. 1999;
Catalanotto et al. 2000; Fagard et al. 2000; Hammond etal. 2001; Harris and Macdonald 2001; Kennerdell et al.
2002; Pal-Bhadra et al. 2002; Williams and Rubin 2002).
Very recently, human eIL2C2, a human Argonaute protein, has been found in an RNA–protein complex, themiRNP, that contains miRNAs (Mourelatos et al. 2002).
Therefore, these observations suggest the speculationthat the distinct Argonaute family protein associatedwith siRNAs or miRNAs regulates the maturation andfunction of these small RNAs. In Drosophila, RNAi is
mediated by the RNA-induced silencing complex (RISC),a sequence-specific, multicomponent ribonucleoprotein complex that acts as an siRNA-directed endonuclease, recognizing and cleaving the complementary mRNA
(Hammond et al. 2001). Argonaute2 (AGO2), an Argonaute family protein, was found to be part of the
RISC nuclease and to copurify with siRNAs and Dicer(Hammond et al. 2001). However, other cellular components that associate with AGO2 have not been identified.
Here we report the identification of a novel ribonucleoprotein (RNP) complex that contains dFMR1,
AGO2, a Drosophila homolog of p68 RNA helicase(Dmp68), and two ribosomal proteins, L5 and L11, alongwith 5S ribosomal RNA (rRNA). dFMR1 interacts directly with L5 and L11, and forms a ternary complex
with the L5/5S rRNA. We show that Dmp68 is required
for efficient RNAi. dFMR1 also physically interacts with
Dicer and miRNAs; therefore, dFMR1 is in an RNAirelated apparatus. Our findings suggest a model in which
the RNAi and dFMR1-mediated translational control
pathways intersect in Drosophila.
Results
Isolation of a dFMR1-associated complex
To better understand the biological function of dFMR1,
we used a tandem affinity purification (TAP) method(Rigaut et al. 1999) to identify molecules that interact
with dFMR1 in vivo. dFMR1-TAP and associated molecules were recovered from a cytoplasmic lysate of S2cells expressing dFMR1-TAP. Protein contents in the tobacco etch virus (TEV) protease and the final extracts
were visualized on SDS-PAGE gels by staining with Coomassie Blue and SYPRO Ruby, respectively (Fig. 1A).
Several protein bands were observed specifically in thedFMR1-TAP plus lanes. Four proteins were found bymass spectrometric analysis to be two ribosomal proteins, L5 and L11, AGO2 (Hammond et al. 2001) and a
Drosophila homolog of p68 RNA helicase (Ford et al.
1988; Flybase annotation no. CG10279; we refer to theprotein as Dmp68). The presence of endogenous dFMR1
in both extracts supports our previous findings thatdFMR1 forms homodimers in vitro and in vivo (H. Siomi
and M.C. Siomi, in prep.; Fig. 1B).

dFMR1 in an RNAi-related apparatus in Drosophilawith L5, L11, AGO2, and Dmp68 are mediated by protein–protein interactions (Fig. 2A). We further investigated whether these proteins can bind to dFMR1 in vitro.
L5, L11, AGO2, and Dmp68 were produced by an in vitrotranslation system and used in binding assays with recombinant dFMR1 fused to glutathione S-transferase(GST; Fig. 2B). GST-dFMR1 interacted with all the proteins tested, whereas GST itself showed no detectablebindings. In addition, RNase A treatment did not affectthe in vitro interactions (Fig. 2B). Although the assay weused could not distinguish between direct and indirectinteractions among dFMR1, L5, L11, AGO2, and Dmp68(but also see below for L5 and L11), nevertheless, theseresults demonstrate that the association of dFMR1 with
L5, L11, AGO2, and Dmp68 occurs both in vivo and invitro and that RNA molecules do not mediate the association.

dFMR1 associates with ribosomes through interaction
with L5 and L11

Figure 1. Protein components of TAP-purified dFMR1 complex from S2 cells. (A) The protein components in the TEV andthe final extracts obtained from S2 cells expressing dFMR1-TAP
and the parental cells (dFMR1-TAP minus) were resolved on
SDS-PAGE and visualized by Coomassie Blue and SYPRO Ruby
staining, respectively. Four of the distinct bands observed onlyin the dFMR1-TAP plus lanes (indicated with an asterisk) wereanalyzed by mass spectrometry and were found to be AGO2,
Dmp68, and ribosomal proteins L5 and L11 as indicated at right.
The protein bands indicated with two asterisks correspond to
dFMR1-CBP (a converted form of dFMR1-TAP after TEV cleavage) and endogenous dFMR1 (see panel B). (B) Western blot
analysis on the TEV and the final extracts using anti-dFMR1
antibody. The bands corresponding to dFMR1-CBP and endogenous dFMR1 are indicated at right.

dFMR1 interacts with AGO2, Dmp68, and ribosomal
proteins in vivo and in vitro
To confirm the in vivo association with dFMR1, either
myc-L5, myc-L11, myc-Dmp68, or AGO2-His were coexpressed with dFMR1-TAP in S2 cells. Cytoplasmic lysates of the transfected cells were prepared and subjectedto IgG-bead binding. Western blots with anti-myc or
anti-His antibody revealed that each protein tested specifically copurified with dFMR1 (Fig. 2A). The interactions were not abolished by ribonuclease A (RNase A)
treatment, suggesting that the associations of dFMR1That the large ribosomal subunit proteins, L5 and L11,
associate with dFMR1 is consistent with our previous
finding that FMRP is associated with the 60S ribosomal
subunit (Siomi et al. 1996). To determine the regions in
dFMR1 that mediate its interactions with L5 and L11,
we made several dFMR1 deletion mutants and performed binding assays. Unexpectedly, GST-dFleu, whichcontains the region of dFMR1 equivalent to the ribosomal-binding domain in human FMRP, bound neither
L5 nor L11 (Fig. 3A). In contrast, dFC285, containing the
C-terminal 285 amino acids of dFMR1, did bind both ofthe proteins (Fig. 3A). A further delineation revealed that
dFC150 was capable of interacting with both L5 and L11,
but dFC120 was not, demonstrating that dFC150 contains the critical region for the interactions (Fig. 3A). To
address the question of whether L5 and/or L11 might act
as bridging molecules between dFMR1 and ribosomes,
dFMR1 lacking the dFC150 region (dFMR1⌬150) was expressed in S2 cells. Fractionating the cytoplasmic lysate
by sedimentation on a linear sucrose gradient revealed
that dFMR1⌬150 was less abundant in the monosome/polysome fractions, but accumulated near the top of thegradient, whereas endogenous dFMR1 were detected in
the monosome/polysome fractions as expected (Fig. 3B).
Although it is conceivable that other proteins within thedFMR1 complex interact with the ribosomal L5 and L11,
which interact with the N-terminal part of dFMR1, theseresults indicate that the dFC150 region in dFMR1, thebinding domain to L5 and L11, plays an important role in
the association of dFMR1 with ribosomes in vivo.

dFMR1 interacts directly with L5 bound to 5S RNA
L5 is a 5S rRNA-binding protein. Therefore, we tested if
the dFMR1 complex contains 5S rRNA. Northern blot
analysis on RNA molecules isolated from the TEV extract revealed that 5S rRNA is a component of the complex (Fig. 3C). We then used gel mobility shift assays to

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Figure 2. Confirmation of dFMR1 interactions with AGO2, Dmp68, and ribosomal proteins L5 and L11. (A) Cytoplasmic lysate
prepared from S2 cells expressing dFMR1-TAP with either AGO2-His, myc-Dmp68, myc-L5, or myc-L11 was incubated with IgG beads
and a Western blot performed on the IgG bound fractions using anti-myc and anti-His antibodies. All proteins found as dFMR1interacting proteins (Fig. 1A) were detected in the bound fractions. RNase A treatment showed no effect on the binding, indicating thatthe associations occur through protein–protein interactions. The presence of endogenous dFMR1 in the IgG bound fraction wasdetermined by the Western blot using anti-dFMR1 antibody. (B) 35S-labeled AGO2, Dmp68, and ribosomal proteins L5 and L11 were
produced by an in vitro transcription and translation system in the presence of [35S]methionine and incubated with either GST-dFMR1or GST itself immobilized on glutathione-Sepharose resin. After extensive washing, the bound fractions were resolved on a polyacrylamide gel and the proteins labeled with 35S visualized by autoradiography. All proteins tested were detected only in the bound fractionsof GST-dFMR1, demonstrating that they interact with dFMR1 in vitro. RNase A treatment showed no effect on the bindings.
Coomassie Blue stainings of GST and GST-dFMR1 used in this experiment are shown at left.

find whether dFMR1 interacts with the L5/5S rRNA
complex. The ability of bacterially produced L5 and L11
to interact directly with bacterially expressed recombinant dFMR1 had been ascertained by binding assays (Fig.
3D). In gel-shift assays, addition of dFMR1 to the preformed L5/5S rRNA yielded a new complex containing
both L5 and dFMR1, demonstrating that dFMR1 forms aternary complex with the L5/5S rRNA (Fig. 3E). L5 andL11 are eukaryotic counterparts of bacterial ribosomal
proteins L18 and L5, respectively, both of which are
shown to locate on the surface of the 50S large ribosomal

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subunit along with 5S rRNA (Nissen et al. 2000). Therefore, it can be envisioned that the dFMR1 interaction
with the ribosome takes place on its surface where L11and L5/5S rRNA are located.
A conserved DEAD-box helicase required for RNAi
The identification of AGO2 as a dFMR1-interacting protein is particularly striking. AGO2 is a member of the
Argonaute gene family and is an essential component for
the RNA-induced silencing complex (RISC), a sequence-

dFMR1 in an RNAi-related apparatus in Drosophila

Figure 3. Analysis of dFMR1 interaction with the ribosomal proteins L5 and L11. (A) Delineation of dFMR1 to determine the binding
domains with L5 and L11. A fragment, dFLeu, containing the region equivalent to the ribosome-binding domain in hFMR1 interactedwith neither L5 nor L11. A delineated fragment, dFC150, interacted with 35S-labeled L5 and L11 as in the case of dFC181, whereas
dFC120 did not, demonstrating that dFC150 is the L5 and L11 binding domain in dFMR1. (B) The binding domain to L5 and L11 confers
to dFMR1 the activity of interacting with ribosomes. A truncated mutant of dFMR1 lacking the binding domains with L5 and L11(dFMR1⌬150) was expressed in S2 cells and the cytoplasmic lysate was subjected to sedimentation on a linear density sucrose gradient.
Western blots were performed on the fractions using anti-dFMR1 antibody. (C) Northern blot on TEV extracts obtained from cytoplasmic lysates with and without dFMR1-TAP. After protease K treatment of the TEV extracts, RNA molecules were recovered andresolved on a 10% denaturing gel containing 6 M urea. A Northern blot was then performed using a riboprobe specific for 5S rRNA.
The total RNA lane contains the total RNA isolated from the parental S2 cells. Mass markers are indicated at left. (D) dFMR1 is able
to interact directly with L5 and L11 in vitro. GST pull-down assays were carried out using bacterially expressed His-L5 and His-L11,
and Western blots with anti-His antibody were performed. (E) The ternary complex formation of 5S rRNA/L5/dFMR1 in vitro. 5S rRNA
was labeled with [32P]UTP. When dFMR1 was incubated with preformed L5/5S rRNA, the RNA band was super-shifted. The migrationof free 5S rRNA, 5S rRNA plus BSA, or 5S rRNA plus L5 are shown.

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specific nuclease complex that mediates RNAi in Drosophila (Hammond et al. 2001). Therefore, the findingthat dFMR1 forms a complex in vivo with AGO2 suggests that dFMR1 may function in RNAi. To test this,we used RNAi to suppress the endogenous proteins,much as had been done previously to establish a role for
AGO2 in RNAi (Hammond et al. 2001). Suppression ofribosomal proteins L5 and L11 with specific dsRNAs
made S2 cells so sick that we could not assess their roles
in RNAi. However, treatment of S2 cells with dsRNAs
homologous to AGO2, dFMR1, or Dmp68 markedly reduced the levels of these proteins (Fig. 4A). We then assessed the ability of these cells to carry out RNAi by
transfection with enhanced green fluorescent protein(EGFP) expression plasmid in combination with an EGFP
dsRNA. Suppression of AGO2 expression correlatedwith a pronounced reduction in the ability of cells tosilence the reporter EGFP by RNAi as reported previously (Hammond et al. 2001; Fig. 4A). Interestingly,
RNAi targeting Dmp68 resulted in inhibition of RNAi in
S2 cells. These results suggest that the DEAD-box helicase Dmp68 not only interacts with dFMR1 but is also
required for efficient RNAi in S2 cells. Dmp68 is a Drosophila ortholog of human p68 (Fig. 4B), which has re-

Figure 4. AGO2 and Dmp68, but not dFMR1 alone, are essential for the RNAi pathway. (A) When AGO2 was suppressed byintroducing specific dsRNA in S2 cells expressing EGFP, theability of the cells to silence EGFP by RNAi was profoundly
reduced as reported previously (Hammond et al. 2001). In contrast, when dFMR1 expression was suppressed by dFMR1
dsRNA, the EGFP silencing effect was unaffected, indicatingthat dFMR1 is not essential for the RNAi pathway. Interestingly, when Dmp68 expression was repressed, EGFP silencing
by RNAi was completely abolished, indicating that Dmp68 is a
novel protein playing an essential role in the RNAi pathway. (B)The amino acid sequence alignment of Dmp68 with human
p68. Amino acids identical in Dmp68 and human p68 are indicated with black boxes and residues conserved within DEAD
box RNA helicases are underlined.

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cently been demonstrated to unwind short but not longdsRNAs in an ATP-dependent manner (Huang and Liu
2002). We conclude that at least two of the dFMR1-interacting proteins, AGO2 and Dmp68, are required for
RNAi in cultured Drosophila S2 cells. In contrast, depletion of dFMR1 did not appear to affect the EGFP silencing (Fig. 4A). Therefore, although dFMR1 interacts
strongly with AGO2 and Dmp68 in vivo, dFMR1 does
not appear to be essential for efficient RNAi.
dFMR1 is associated with an RNAi-related apparatus
Recent work in numerous organisms has shown that
RNAi shares features with a developmental gene regulatory mechanism that involves miRNAs (Lagos-Quintana
et al. 2001; Lau et al. 2001; Lee and Ambros 2001). These
small RNAs (siRNAs and miRNAs) are thought to beincorporated into silencing complexes that mediatemRNA destruction during RNAi and translational control during development, respectively. Therefore, it issuggested that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation (Hutvagner and Zamore 2002).
AGO2 and Dmp68 are essential for RNAi in Drosophila(Fig. 4A). On the other hand, dFMR1 appears to be atranslation repressor (Zhang et al. 2001). Because dFMR1
interacts with AGO2 and Dmp68 in vivo, we wanted to
examine whether dFMR1 is also present in an AGO2and/or Dmp68-associated complex. To do this, we expressed TAP-tagged AGO2 (AGO2-TAP) or Dmp68(Dmp68-TAP) in S2 cells. Cytoplasmic lysate of the cells
expressing AGO2-TAP or Dmp68-TAP was prepared andsubjected to TAP purifications. In reciprocal assays, endogenous dFMR1 and AGO2 were found to associatewith each other (Fig. 5A). In addition, endogenous AGO2
was copurified with AGO2-TAP. Endogenous dFMR1
and AGO2 were also found to be present in the Dmp68associated complex (Fig. 5B). Because AGO2 can be coimmunoprecipitated with Dicer (Hammond et al. 2001),
which initiates RNAi by processing dsRNA silencing
triggers into siRNAs, and also processes miRNA precursors into mature miRNAs (Bernstein et al. 2001; Grishok
et al. 2001; Hutvagner et al. 2001; Ketting et al. 2001);
therefore, we considered the possibility that dFMR1
might also interact physically with Dicer. Indeed, endogenous Dicer can be copurified not only with AGO2-TAP
but also with dFMR1-TAP (Fig. 5C). Next, we tested
whether the interaction between AGO2 and dFMR1 ischanged upon RNAi induction. Extracts prepared from
S2 cells expressing AGO2-TAP, either not exposed todsRNA or exposed to dsRNA targeting the casein kinase
II (CKII) ␤ subunit (Saxena et al. 1987), were subjected to
TAP purifications. Cells introduced with the CKII␤
dsRNA by soaking showed diminished levels of endogenous CKII␤ protein (data not shown). As shown in Figure 5D, dFMR1 remains associated with AGO2 after
RNAi induction. It is well established that siRNAs associate with AGO2 during RNAi in S2 cells (Hammondet al. 2001). Therefore, these results indicate that dFMR1
may be a part of RISC. Finally, analogous to the humandFMR1 in an RNAi-related apparatus in Drosophila

Figure 5. dFMR1 is associated with RNAi-related complexes. (A) An AGO2 complex formed in S2 cells contains dFMR1. The TEV
extracts prepared from S2 cells expressing AGO2-TAP or dFMR1-TAP, and extracts from the parental cells were subjected to Western
blots using anti-AGO2 and anti-dFMR1 antibodies. As expected, endogenous dFMR1 was found in both fractions of AGO2-TAP anddFMR1-TAP, confirming that AGO2 interacts with dFMR1 in vivo. Endogenous AGO2 was also copurified with AGO2-TAP. (B) A
Dmp68 complex formed in S2 cells contains both dFMR1 and AGO2. The TEV extracts prepared from S2 cells expressing Dmp68-TAP,and extracts from the parental cells were subjected to Western blots using anti-dFMR1 or AGO2 antibodies. Endogenous dFMR1 andAGO2 were found in the Dmp68-associated complex, confirming that Dmp68 interacts with dFMR1 and AGO2 in vivo. (C) Dicer
physically interacts with dFMR1. The TEV extracts prepared from S2 cells expressing AGO2-TAP or dFMR1-TAP, and extracts fromthe parental cells were subjected to Western blot using anti-Dicer antibodies (a kind gift of S. Hammond and G. Hannon; Bernstein etal. 2001). Endogenous Dicer was found in both fractions of AGO2-TAP and dFMR1-TAP, confirming that Dicer interacts with dFMR1
in vivo. The S2 control lane (indicated as total) contains cytoplasmic lysates of parental S2 cells. (D) dFMR1 remains associated with
AGO2 during RNAi. AGO2-TAP TEV extracts prepared from S2 cells treated with and without dCKII ␤ dsRNA were subjected toWestern blot using anti-dFMR1 antibodies. The amount of dFMR1 bound to AGO2 was not affected by RNAi treatment, suggesting
the possibility that dFMR1 is part of RISC. (E) An miRNA, miR-2b, is associated with dFMR1 and AGO2 in vivo. TAP-purifications
were performed using cytoplasmic lysates of S2 cells expressing dFMR1-TAP or AGO2-TAP. RNAs were isolated from the purifiedcomplexes and analyzed by Northern blot as described (Lagos-Quintana et al. 2001; Lee and Ambros 2001). The blot was probed for themiR-2b (Lagos-Quintana et al. 2001).

AGO2 ortholog (eIF2C2)-associated complex that contains a DEAD-box type RNA helicase and miRNAs(Mourelatos et al. 2002), we wanted to test whether miRNAs are also found in AGO2- and/or dFMR1-associatedcomplexes. RNA molecules copurified with AGO2-TAP
or dFMR1-TAP were recovered, dissolved on a 15% polyacrylamide denaturing gel, and subjected to Northernblot analysis. A known miRNA, miR-2b (Lagos-Quintana et al. 2001), in Drosophila S2 cells can be detectedboth in the AGO2- and dFMR1-associated complexes(Fig. 5E). Together, our data show that dFMR1 is present
in a complex with components of RNAi and miRNAs in
cultured Drosophila S2 cells.
Discussion
dFMR1 is thought to have important roles in the translation of some specific mRNAs such as futsch mRNA
(Zhang et al. 2001). Although it is unclear how dFMR1
regulates translation of such mRNAs, our findings may

hold some clues. Because dFMR1 interacts with ribosomal L5/5S rRNA and L11, all of which are located at
the top of the 60S ribosomal subunit (Nissen et al. 2000),
it is likely that this interaction brings dFMR1 to the 60S
ribosomal subunit. Therefore, the association of dFMR1with the 60S ribosomal subunit through direct interactions with ribosomal L5/5S rRNA and L11 may inhibittranslation by preventing the assembly of initiation
complexes or by giving rise to structural change of theribosomes, which, in turn, influences a step(s) after
translation initiation. Alternatively, because 5S rRNA is
the only known RNA species that binds ribosomal proteins, including L5, and forms a 5S RNP before it is incorporated into the ribosomes (Steitz et al. 1988; Szymanski et al. 2000), dFMR1 may interact with the cytoplasmic nonribosome-associated 5S RNP (Steitz et al.
1988), which, in turn, influences the formation of themature 60S ribosomal subunit. It is interesting to note inthis context that only about half of the 5S rRNA molecules in mammalian cells are associated with the 60S

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ribosomal subunit (Knight and Darnell 1967) and that
although 5S rRNA enhances ribosomal activity, it is not
absolutely essential for it (Moore 1996; Nissen et al.
2000).
dFMR1 is present in a complex isolated from S2 cells,
which also contains AGO2 and Dicer. AGO2 and Dicer
are essential components of RNAi (Hammond et al.
2001). The interaction between dFMR1 and AGO2 remains constant before and after RNAi induction (Fig.
5D), suggesting that dFMR1 is part of RISC during RNAi.
However, there is no evidence to support the notion that
RISC formation is induced by treatment of S2 cells withdsRNA. As one of the functions of the RNAi apparatus is
to silence transposons and repetitive sequences residing
naturally in the Drosophila genome (Hannon 2002;
Hutvagner and Zamore 2002), these cells are therefore
likely to be full of pre-formed RISC complexes, irrespective of dsRNA treatment. Therefore, it is possible thatdFMR1 is part of the pre-formed RISC complexes andremains to be part of the active RISC after ATP-dependent siRNA unwinding (Nykanen et al. 2001; Hutvagner
and Zamore 2002).
The involvement of another dFMR1-interacting protein, Dmp68, in RNAi further suggests the close association of dFMR1 with RNAi. The p68 RNA helicase
was first identified by cross-reaction with a monoclonal
antibody that was originally raised against SV40 large T
antigen two decades ago (Lane and Hoeffler 1980). The
helicase plays important roles in cell proliferation andorgan maturation (Iggo et al. 1991; Stevenson et al. 1998)
and belongs to a large family of highly evolutionarily
conserved proteins, the so-called DEAD-box family of
putative ATPases and helicases (for review, see de la
Cruz et al. 1999). Recent studies have revealed several
RNA helicases including mut6 (Wu-Scharf et al. 2000),
SDE3 (Dalmay et al. 2001), mut14 (Tijsterman et al.
2002), drh-1 (Tabara et al. 2002), and spindle-E (Kennerdell et al. 2002) required for RNAi and related posttranscriptional gene silencing (PTGS) pathways. Dmp68
is similar to, but clearly not an ortholog of these proteins. Therefore, Dmp68 is a novel component of RNAi.
Because ATP-dependent unwinding of the siRNA duplex
remodels the RISC to generate an active RISC in theRNAi pathway (Nykanen et al. 2001), Dmp68 may mediate the unwinding process. It is also conceivable that
Dmp68 may be involved in downstream events such as
target RNA recognition, as an RNA chaperone (Lorsch
2002) or an RNPase (Linder et al. 2001).
The connection that we have established between
dFMR1, components of RNAi, miRNAs, and the generaltranslation machinery is of considerable significance because they provide intriguing clues and possible connections to the function of dFMR1 and the pathways withwhich it may intersect. Recent work in numerous organisms has shown that RNAi shares features with a developmental gene regulatory mechanism that involves
miRNAs (Lagos-Quintana et al. 2001; Lau et al. 2001;
Lee and Ambros 2001). For example, both the foreigndsRNAs that trigger RNAi and the endogenous miRNA
precursors that function in development are processed

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into small RNAs by Dicer (Bernstein et al. 2001; Grishok
et al. 2001; Hutvagner et al. 2001; Ketting et al. 2001).
Members of the Argonaute gene family are also involvedin both the siRNA and miRNA pathways (Grishok et al.
2001). In C. elegans, Tabara et al. (2002) have shown that
Dicer, the dsRNA-binding protein RDE-4, and a conserved DExH-box RNA helicase (DRH-1) are in a complex with RDE-1, an AGO2 ortholog. Furthermore, thehuman AGO2 ortholog, eIF2C2, is in a complex, themiRNP, that contains the DEAD-box RNA helicase
Gem3 (Mourelatos et al. 2002). Therefore, Argonaute
proteins appear to be in a complex that contains an RNA
helicase(s), Dicer and small guide RNAs, and function ina variety of homology-dependent mechanisms that involve base-pairing between small guide RNAs and target
mRNAs. Our findings that dFMR1 interacts with AGO2,
Dmp68, Dicer, miRNAs, and the general translation machinery, provide a means to link RNAi enzymes to translational control pathways, and are also consistent withthe fact that the RISC nuclease fractionates with ribosomes (Hammond et al. 2000, 2001).
It appears that dFMR1 is important for translational
control, possibly mediated by RNAi-related components
and miRNAs. Although recent studies have identified alist of mRNAs that are potential FMRP targets (Brown etal. 2001; Darnell et al. 2001), our results further suggest
a model in which FMRP may not directly bind its mRNAtargets but rather it is targeted to its mRNA substrates aspart of RNAi-related apparatus, which are guided bymiRNAs. How then might FMRP regulate translation ofits mRNA targets? In the case of lin-4, a prototype of
miRNAs, its mRNA targets (lin-14 and lin-28) are translationally repressed yet remain associated with polyribosomes (Olsen and Ambros 1999; Seggerson et al. 2002),
suggesting a block at a step after translation initiation.
FMRP may form an miRNP complex on its mRNA targets and the association of this complex with ribosomal
L5/5S rRNA and L11 may inhibit translation at one ormore postinitiation steps, including elongation, termination, or the release of functional protein as discussed
above. Finally, we propose that fragile X syndrome may
be the result of protein synthesis abnormality caused bya defect(s) in an RNAi-related apparatus.

Materials and methods
TAP purification from S2 cells
An expression vector containing genes encoding dFMR1,
AGO2, or Dmp68 and a TAP tag (Rigaut et al. 1999) under thecontrol of a metallothionein promoter (pRmHa vector) wastransfected into S2 cells, and the expression of dFMR1-TAP,AGO2-TAP, or Dmp68-TAP fusion protein was induced by adding copper ion to the medium (Lafont et al. 1998). After incubation overnight, a cytoplasmic lysate of the cells was preparedin a buffer containing 10 mM Tris-HCl (pH 8.0), 150 mM NaCl,
0.5% Triton X-100, 2 µg/mL leupeptin, 2 µg/mL pepstatin, and
0.5% aprotinin. dFMR1-TAP, AGO2-TAP, or Dmp68-TAP and
the associated materials within the lysate were purified withIgG beads (Amersham Bioscience). After extensive washing, thedFMR1 in an RNAi-related apparatus in Drosophila

bound fraction was eluted by treating with TEV protease (Invitrogen). The TEV extract was then incubated with calmodulincoated beads (Stratagene) and the final extract was obtained byadding EGTA to the beads.
Protein identification by peptide mass fingerprinting
Specific bands shown in Figure 1A were excised from the gel anddigested in gel with trypsin. The masses of the tryptic peptides
were measured with matrix-assisted laser desorption ionization-reflectron time-of-flight (MALDI-TOF) mass spectrometry(Shimazu Biotech, Japan).
Protein–protein interaction assays
To obtain cDNAs encoding ribosomal proteins L5 and L11,
Dmp68 and AGO2, polyA(+) RNAs were purified from S2 cells
and RT–PCR carried out using primers specifically designed foreach clone. For the expression in S2 cells by transfection, thecDNAs were inserted into the pRmHa3 vector (Lafont et al.
1998). After coexpression of either myc-L5, myc-L11, mycDmp68, or AGO2-His with dFMR-TAP in S2 cells, the cytoplasmic lysate was prepared and incubated with IgG beads. Afterextensive washing, the bound materials were subjected to Western blots using anti-myc, anti-His (Santa Cruz), and anti-dFMR1
antibodies. RNase A treatment of the lysate was carried out for
30 min prior to the IgG bindings. To produce [35S]methioninelabeled proteins by a TnT in vitro transcription and translation
kit (Promega), the cDNAs of L5 and L11, Dmp68 and AGO2
were inserted into an expression vector, pET28 (Novagen). The
GST pull-down assays were carried out using GST-dFMR1 and
GST itself that were bound to glutathione-Sepharose 4B resins(Amersham Bioscience) in a binding buffer containing 10 mM
Tris-HCl (pH 8.0), 150 mM NaCl, and 0.5% Triton X-100. Afterincubation with the TnT products and extensive washing, thebound fractions were separated on a polyacrylamide gel andproteins labeled with 35S were visualized by autoradiography
after treatment with Amplify Fluorographic Reagent (Amersham Bioscience). RNase A treatment was carried out by adding
the enzyme to the binding mixture. To produce GST fusion
proteins, either full-length or truncated dFMR1 cDNA was subcloned into a pGEX-5X expression vector (Amersham Bioscience). The fusion proteins, as well as GST itself, were induced
and purified as described by the manufacturer. cDNAs of all thedeletion mutants were produced by performing PCR using primers specifically designed for the truncation.
Sucrose gradient centrifugation
Cytoplasmic lysate of the cells expressing dFMR1⌬150 was resolved on a linear sucrose density gradient (5%–30%). The gradient was centrifuged at 4°C in a Beckman MLS-50 rotor at
40,000 rpm for 90 min. Following centrifugation, fractions werecollected and subjected to Western blots using anti-dFMR1 andanti-P0 ribosomal protein antibodies (Uchiumi and Kominami
1997).
Northern blot analysis and gel mobility shift assay
TEV extracts plus/minus dFMR1-TAP were treated with proteinase K (0.5 mg/mL; Roche) at 37°C for 20 min, followed by aphenol/chloroform extraction and isopropanol precipitation.
Resultant RNA molecules were resolved on a 10% acrylamide/6
M urea denaturing gel and subjected to a Northern blot using a
specific riboprobe for 5S rRNA. Gel retardation assay was carried out using 5S rRNA labeled with [32P]UTP and His-tagged L5

and dFMR1 recombinant proteins produced in bacteria. Formation of L5/5S rRNA complex was carried out on ice for 30 min
in the presence of 0.5 µg of BSA and 0.5 µg of unlabeled yeast
RNA. His-dFMR1 was subsequently added to the L5/5S rRNA
complex and further incubation carried out on ice for 30 min.
The samples were then separated on a 6% nondenaturing gel
and autoradiography was performed to visualize the complexes.
For miRNA Northern blots (Lagos-Quintana et al. 2001; Lee
and Ambros 2001), RNA samples were run on 15% acrylamide
denaturing urea gels and then transferred to Zeta-Probe GT
membranes by electrophoresis. After transfer, they were UVcrosslinked and baked at 80°C for 1 h. Oligonucleotide probes
were labeled with T4 polynucleotide kinase and [␥-32P]ATP andhybridized to the membranes at 42°C in 7% SDS, 0.2 M Na2PO4(pH 7.0) overnight. Membranes were washed at 42°C twice with
2xSSPE, 0.1% SDS. The blots were exposed on BAS-MS 2040
imaging plate and signals were quantified using BAS-2500 (FUJIFILM).
Detection of Dicer in an AGO2 complex
or a dFMR1 complex
dFMR1-TAP or GO2-TAP and the associated materials were
purified with IgG beads (Amersham Bioscience). After extensive
washing, the bound fractions were subjected to Western blots
using anti-Dicer antibodies (a kind gift of S. Hammond and G.
Hannon; Bernstein et al. 2001).
RNAi
Full-length EGFP cDNA and portions of cDNAs of AGO2,
Dmp68, and dFMR1, each about 700 bp in size, were subcloned
into pBluescript, and dsRNA were produced by in vitro T7 andT3 transcriptions, followed by annealing in water. dsRNAs of
AGO2, Dmp68, or dFMR1 were introduced to S2 cells by soaking (Clemens et al. 2000). After 3 d, the cells were harvested and
EGFP dsRNAs were then introduced into half of the cells. Bothcell pools treated with and without EGFP dsRNA were cultured
for another 4 d and subjected to Western blots using anti-AGO2,
anti-dFMR1, MAD1 [anti-human p68 cross-reacting to Dmp68;
a gift of F. Fuller-Pace (Department of Molecular and Cellular
Pathology, University of Dundee, Ninewells Medical School,
Dundee, U.K.)] and anti-EGFP antibodies. Anti-dFMR1 antibody was a gift of G. Dreyfuss (Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, PA). We also produced polyclonal anti-dFMR1 antibodies
by immunizing mice with bacterially produced dFMR1 fragments. Anti-AGO2 polyclonal antibodies were raised in mice byimmunizing with bacterially produced AGO2 fragments.
dsRNA of CKII ␤ subunit was produced by in vitro transcription, followed by annealing. CKII ␤ dsRNA was then introduced
by soaking into S2 cells that express AGO2-TAP fusion.

Acknowledgments
We thank G. Dreyfuss and L. Wan for dfmr1 cDNA and antidFMR1 antibodies; F. Fuller-Pace for anti-human p68 antibody
MAD1; S. Hammond and G. Hannon for anti-Dicer antibodies;
T. Uchiumi for anti-ribosomal protein P0 antibodies; B.
Seraphin for the TAP plasmids; and F. Lafont for pRmHa plasmid. We thank Y. Nakahori and members of the Siomi laboratory for discussions and A. Azuma and Y. Kawamura for technical assistance. This work was supported by grants from the
FRAXA Research Foundation; the Japan Society for the Promotion of Science (JSPS); the Ministry of Health, Labor and Welfare

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Ishizuka et al.

of Japan; and the Ministry of Education, Science, Sports, andCulture of Japan.
The publication costs of this article were defrayed in part by
payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 USC section
1734 solely to indicate this fact.

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