MENTAL RETARDATION AND DEVELOPMENTAL DISABILITIESRESEARCH REVIEWS 10: 68–74 (2004)

RNA INTERFERENCE: A NEW MECHANISM BY
WHICH FMRP ACTS IN THE NORMAL BRAIN?
WHAT CAN DROSOPHILA TEACH US?
Haruhiko Siomi,* Akira Ishizuka, and Mikiko C. Siomi
Institute for Genome Research, University of Tokushima, Tokushima, Japan

Fragile X syndrome is the most common heritable form of mental
retardation caused by loss-of-function mutations in the FMR1 gene. The
FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. Recent work in
Drosophila melanogaster has shown that the ﬂy homolog of FMR1 (dFMR1)
plays an important role in regulating neuronal morphology, which may
underlie the observed deﬁcits in behaviors of dFMR1 mutant ﬂies. Biochemical analysis has revealed that dFMR1 forms a complex that includes ribosomal proteins and, surprisingly, Argonaute2 (AGO2), an essential component of the RNA-induced silencing complex (RISC) that mediates RNA
interference (RNAi) in Drosophila. dFMR1 also associates with Dicer, another essential processing enzyme of the RNAi pathway. Moreover, both amicro-RNA (miRNA) and short interfering RNAs (siRNAs) can coimmunoprecipitate with dFMR1. Together these ﬁndings suggest that dFMR1 functionsin an RNAi-related apparatus to regulate the expression of its target genes
at the level of translation. These ﬁndings raise the possibility that Fragile X
syndrome may be the result of a protein synthesis abnormality caused by adefect in an RNAi-related apparatus. Because the core mechanisms of complex behaviors such as learning and memory and circadian rhythms appear
to be conserved, studies of Fragile X syndrome using Drosophila as a model
provide an economy-of-scale for identifying biological processes that likely
underlie the abnormal morphology of dendritic spines and behavioral dis© 2004 Wiley-Liss, Inc.
turbances observed in Fragile X patients.
MRDD Research Reviews 2004;10:68 –74.

Key Words: FMR1; Drosophila; RNAi; miRNA; translational repressionIn most cases, fragile X syndrome is caused by a trinucleotide
repeat expansion in the 5⬘ untranslated region of the fragile
X mental retardation 1 gene (FMR1) [Imbert et al., 1998;
O’Donnell and Warren, 2002]. An expansion of the CGG
repeat is associated with abnormal DNA methylation of both anearby CpG island and the repeat itself. As a result, the FMR1
locus becomes silent at the transcriptional level and thus notranslation occurs [Verheij et al., 1993; Siomi et al., 1993]. It isclear that fragile X syndrome results from the lack of FMR1
expression. In other words, the cause of the disease is a loss-offunction of FMR1. This has been conﬁrmed by the Dutch–
Belgian Consortium [1994], which generated mouse models by
the targeted disruption (knockout) of the mouse Fmr1 gene. The
Fmr1 knockout mouse reproduces a subtle phenotype reminiscent of the human phenotype [Dutch–Belgian Consortium,1994]. It is therefore clear that the pathophysiological mechanisms leading to the symptoms in fragile X syndrome can beelucidated by studying the function of the FMR1 gene.
© 2004 Wiley-Liss, Inc.

Although this disease is caused by the expansion of a
triplet repeat, it is not associated with neurodegeneration, as isoften the case for other “triplet repeat” diseases. Rather, thepathology of 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; see Beckel-Mitchener and Greenough, this issue]. Because spines are specialized regions where dendrites receive
synaptic input from other neurons, abnormal dendritic spines
observed in fragile X patients may be the basis of their mental
retardation. A link between abnormal dendritic spines (dendritic
spine “dysgenesis”) and mental retardation has been suggested
for many other mental retardation disorders, including trisomy
21 (Down syndrome) [Purpura, 1974; Nimchinsky et al., 2002].
Because the FMR1 protein (FMRP) is a cytoplasmic RNAbinding protein that associates with polyribosomes as part oflarge ribonucleoprotein complexes (mRNP), it is suspected thatit participates in the translational regulation of target mRNAs ina manner that is critical for the correct development of neurons[Inoue et al., 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 by modulating RNA translation, and consequently protein synthesis,
FMRP is important for the formation and function of synapses.
Recently, a number of mammalian FMRP mRNA targets havebeen identiﬁed [Schaeffer et al., 2001; Brown et al., 2001;
Darnell et al., 2001; Miyashiro et al., 2003; see Darnell, Warren,and Darnell, this issue]. Interestingly, a number of these RNA
molecules have peculiar RNA sequences capable of adopting aunique secondary structure forming G-quartets and code forimportant neuronal proteins [Schaeffer et al., 2001; Darnell etal., 2001; Brown et al., 2001]. However, to what extent andhow FMRP might affect translation in vivo is unknown. FurGrant sponsors: FRAXA Research Foundation; Ministry of Health, Labor and Welfareof Japan; Ministry of Education, Science, Sports, and Culture of Japan.
*Correspondence to: Haruhiko Siomi, Ph.D., Institute for Genome Research, University of Tokushima, 3–18-15 Kuramoto, Tokushima, Japan 770-8503. E-mail:siomi@genome.tokushima-u.ac.jp
Received 28 March 2003; Accepted 7 May 2003
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI: 10.1002/mrdd.10089

thermore, the discovery of the existenceof two FMR1-related genes, FXR1 andFXR2, has revealed an additional level ofcomplexity in the study of FMR1 functions in vertebrates [Siomi et al., 1995;
Zhang et al., 1995]. For many humandisorders, redundancy of gene function
due to gene duplication in the same species makes it difﬁcult to interpret clearly
the effects of loss-of-function mutations
on cellular physiology and biochemistry.
DROSOPHILA AS A MODELFOR FRAGILE X SYNDROME
With forward genetics or reverse
genetics, the fruit ﬂy D. melanogaster has
proven to be a powerful tool for the
genetic dissection of biochemical pathways. Many genes are conserved between ﬂies and humans, including genesthat regulate complex behaviors such aslearning and memory and circadianrhythms, as well as entire pathways ofdevelopment and oncogenesis [Patel,
1994; Miklos and Rubin, 1996; Rubin etal., 2000]. Although the ﬂy’s nervous system differs vastly from ours, it does work
via neurons, synapses, and transmittermolecules, and accumulating molecularbiological evidence suggests a staggeringlevel of evolutionary conservation, although there are, of course, variations indetail [Kammermeier and Reichert,
2001; Cayouette and Raff, 2002]. In addition, due to its small size, yet behavioral
complexity, the brain of the fruit ﬂy islargely accepted as an important model
for brain function. Drosophila could be
used, therefore, to deﬁne the molecularpathways leading to human neurologicaldiseases and to identify the genes involved [Fortini and Bonini, 2000; Muqit
and Feany, 2002]. Conversely, studies onhuman diseases using Drosophila as amodel offer new insights into normal developmental processes. In fact, Drosophila
has revealed much about the mysteries ofdevelopment and provided hints about
the elements directing complex behavior[Waddell and Quinn, 2001; Dubnau andTully, 1998; Dunlap, 1999; Hall, 1994].
Furthermore, building on years of experience, a number of ﬂy researchers arealso using Drosophila as a model for designing new therapeutic strategies fortreating human diseases [Steffan et al.,
2001; Kazantsev et al., 2002; Auluck etal., 2002].
The Drosophila genome contains asingle gene that is homologous to FMR1,
the Drosophila FMR1 related gene(dFMR1, also referred to as dfxr) [Wan etal., 2000]. Drosophila and vertebrate
FMR1 proteins share a number of topographical landmarks [Wan et al., 2000],

including two types of RNA-binding
motifs, namely two KH domains and an
RGG box. Moreover, they show similar
biochemical properties, such as RNA
binding and ribosome association [Wanet al., 2000], and similar expression patterns with high levels of expression inneurons [Zhang et al., 2001; Dockendorff et al., 2002; Morales et al., 2002].
dFMR1 also has a high afﬁnity for G-rich
RNA sequences (MC Siomi and H
Siomi, unpublished results). Importantly,
genetic studies have demonstrated thatdFMR1 has a role in the regulation ofneuronal morphology and functions[Zhang et al., 2001; Dockendorff et al.,
2002; Morales et al., 2002; Inoue et al.,2002]. Examining the role of FMRP in
the fruit ﬂy is, therefore, a promising
approach for providing signiﬁcant insights into the function of FMRP.

Although the ﬂy’s
nervous system differs
vastly from ours, it does
work via neurons,
synapses, and transmittermolecules, andaccumulating molecularbiological evidence
suggests a staggeringlevel of evolutionary
conservation, although
there are, of course,
variations in detail.
PHENOTYPE
Fragile X patients exhibit elongated dendritic spines, a higher density ofspines along the dendrites, and a more
immature morphology compared with amatched sample of unaffected individuals[Greenough et al., 2001; Irwin et al.,2001]. Fmr1 knockout mice also show anincrease in spine density and spine lengthon pyramidal cell dendrites comparedwith wild-type mice [Kooy, 2003].
These ﬁndings suggest an impairment ofspine maturation and pruning in fragile X
patients [Greenough et al., 2001; Irwin etal., 2001].
Recently, several groups have independently generated dFMR1 null alleles. dFMR1 null mutants proceed into

MRDD RESEARCH REVIEWS ● RNA INTERFERENCE ● SIOMI ET AL.

adulthood and appear to be morphologically normal in external appearance.
However, dFMR1 has a role in the regulation of neuronal morphology andfunction as is the case for mammalian
FMR1. Zhang et al. [2001] found thatdFMR1 null mutant ﬂies display enlarged
synaptic terminals at the neuromuscularjunction (NMJ). In addition, the loss ofdFMR1 resulted in an altered neurotransmission with synapse type-speciﬁc regulation in the central and peripheral synapses. This ﬁnding suggests that dFMR1
is required for the differential regulationof synaptic neurotransmission. Interestingly, dFMR1 mutations caused elevatedspontaneous vesicle fusion at the NMJ,
suggesting that the role of dFMR1 is primarily presynaptic, mediating synaptic
vesicle fusion probability. Because spontaneous neurotransmitter release is crucial
for organizing sites of neuronal communication in the developing ﬂy NMJ, but
probably also at other synapses [Saitoe etal., 2001; Broadie and Richmond, 2002],
the possible role of dFMR1 in synaptic
vesicle fusion may underlie the observed
deﬁcits in the neuronal morphology ofdFMR1 mutant ﬂies. Because the role of
FMR1 in mammals appears to be primarily postsynaptic as judged by abnormal
dendritic spines observed in fragile X patients and Fmr1 knockout mice, the presynaptic deﬁcits observed in dFMR1 mutant ﬂies suggest that the cellularfunctions to which FMR1 contribute
may change through evolution, although
the molecular mechanisms may be conserved. Zhang et al. [2001] also foundthat, taking a clue from the considerable
similarity in phenotype between thedFMR1 mutants and futsch mutants, theintroduction of the futsch mutation rescued the structural and functional defects
at the NMJ observed in dFMR1 mutants.
Together with their ﬁnding that futsch
mRNA is associated with dFMR1, andthat Futsch expression is higher indFMR1 mutants, these results suggestthat dFMR1 regulates the synaptic structure and function, probably by acting as atranslational repressor of an mRNA encoding Futsch, which is the ﬂy homologof the microtubule-associated protein
MAP1B [Zhang et al., 2001].
Dockendorff et al. [2002] and Morales
et al. [2002] examined neurons in thebrain of dFMR1 mutants and found abnormal axon branching and an overgrowth of terminal arborizations in dorsal
cluster (DC) neurons and ventral lateral
neurons (LNv), but not in other neurons.
This suggests that different neuronal subtypes are affected differentially by the lossof dFMR1. Although observations of ax69

onal defects in dFMR1 mutants suggest apossible local function for dFMR1 in theregulation of axonal outgrowth and/or
presynaptic structure, it remains to be
determined whether dendritic (postsynaptic) abnormalities are found in dFMR1
mutants such as in fragile X patients andFmr1 knockout mice. Most multiple
dendritic (MD) neurons in the peripheral
nervous system in Drosophila larvae develop highly diverse dendritic branchingpatterns with spinelike structures andshare morphological similarities withdendrites of the mammalian central nervous system (CNS) [Sweeney et al.,2002]. These neurons thus could be used
as a model system for addressing this issue.
Although we state early in this article that dFMR1 is homologous to
FMR1, the fact that the role of dFMR1
appears to be primarily presynaptic may
also cast this in a new light. The aminoacid sequence of dFMR1 is equally similar, if not more so, to FXR1 and FXR2.
The loss of FMR1 in fragile X patientscannot be compensated by the presenceof FXR1 and FXR2. Therefore, there isa functionally unique region in FMR1 or
the protein is localized to an exclusive
region in the cell. In this regard, it will be
particularly interesting to ask the question of which one of the human FMR1/
FXR family genes can ameliorate or evenrescue neuronal morphology and/or behavioral abnormalities (see below) indFMR1-null ﬂies.
BEHAVIOR
Fragile X patients are known to
exhibit a more or less speciﬁc complex of
behavioral disturbances [Hagerman,
2002]. These include social and attention
deﬁcits, autisticlike behaviors, unusual
responses to sensory stimuli, hyperactivity, and abnormal sleep patterns. Fmr1
knockout mice show mild but consistentabnormalities, analogous to the clinicalsymptoms observed in human patients[Kooy, 2003].
Although in many behavioral tests
the dFMR1 mutant ﬂies did not differ
from wild-type ﬂies, three researchgroups found independently that a loss ofdFMR1 affects circadian behavior[Dockendorff et al., 2002; Morales et al.,
2002; Inoue et al., 2002]. In a 24-h light:
dark (LD) cycle, wild-type ﬂies are entrained to (or synchronized with) LD cycling and exhibit a substantial locomotor
activity rise during the second half of theday. Rhythmic behavior persists whenwild-type ﬂies proceed from LD into
constant darkness (DD). dFMR1 mutant
ﬂies were well entrained during LD cy70

cles and exhibited normal ⬃24-h periodicity under such conditions. However,the locomotor behavior of dFMR1 mutant ﬂies in DD was arrhythmic. The
arrhythmic locomotor activity phenotype in DD could be rescued by the
introduction of a genomic fragment containing the wild-type dFMR1 gene,demonstrating that the disruption of thedFMR1 gene is directly responsible forthe circadian phenotype in mutant ﬂies.
This ﬁnding is consistent with the observation that dFMR1 is required for thenormal extension and guidance of thelateral neurons in which the clock cells
that control the circadian rhythms are
known to be located in the brain of Drosophila [Kaneko et al., 1997]. However,
because the circadian locomotor defects
are not rescued by the introduction of afutsch loss-of-function mutation into thedFMR1 mutant background [Dockendorff et al., 2002], these behavioral defects are not due to the same synaptic
defects caused by Futsch misregulation at
the NMJ, suggesting that dFMR1 differentially regulates diverse targets in thebrain.
Among the behavioral disturbances
observed in fragile X children, sleep
problems are, according to parental reports, especially difﬁcult to manage[Gould et al., 2000]. These include
shorter sleep durations, greater variations
in sleep duration, longer night waking
episodes, and sleep timing problems. It is
tempting to speculate that these aspects ofsleep are attributable to alterations in circadian rhythmicity because sleep propensity is modulated by a circadian clock[Klein et al., 1991]. Most organisms contain molecular time-keepers known ascircadian clocks, which drive daily variations in many physiological and behavioral processes, such as the wake/sleep
rhythm and daily variations in body temperature, hormone levels, cognition, andmemory [Klein et al., 1991; Hall, 1998].
Drosophila has the best understood molecular clock [Dunlap, 1999; Young,1998]. In addition, recent experiments
have revealed that the molecular mechanisms involved in the generation of circadian rhythms are remarkably similar
between Drosophila and mammals [Dunlap, 1999]. Therefore, a Drosophila modelof fragile X syndrome provides insight
into the sleep/wake cycles of mammals.
The discovery of modiﬁers involved in
the dFMR1-mediated regulation of circadian rhythms reveals additional molecular mechanisms in the fragile X syndrome.

BIOCHEMISTRY
How might dFMR1 regulate synapse growth and function in the ﬂy brain?
Because dFMR1 is likely to function incytoplasmic mRNP particles associatedwith ribosomes, as is the case for mammalian FMR1 [Imbert et al., 1998; Inoueet al., 2000], Ishizuka et al. [2002] conducted afﬁnity puriﬁcation of a dFMR1associated complex from cultured Drosophila S2 cell lysates to ﬁnd what
proteins and RNAs are stably associatedwith it in the hope of applying genetic
analysis to the associated components.
Consistent with previous ﬁndings implicating FMRP in translation inhibition
and ribosomal association, two ribosomal
proteins, L5 and L11, along with 5S
ribosomal RNA (rRNA) were identiﬁed in the dFMR1-associated complex.
Surprisingly, Ishizuka et al. [2002] discovered that in addition to ribosomal
proteins, dFMR1 associates with components of RNA interference (RNAi).
These include AGO2, p68 RNA helicase(Dmp68), and Dicer.
RNAi/miRNA PATHWAYS
The RNAi pathway was ﬁrst discovered in the nematode worm Caenorhabditis elegans and is widely used as anexperimental tool for uncovering thefunction of genes of interest in a varietyof organisms. RNAi is the process ofsequence-speciﬁc posttranscriptional genesilencing (PTGS) in a variety of organisms, initiated by the introduction of
double-stranded (ds) RNA that is homologous in sequence to the silenced
gene [Fire et al., 1998; Cogoni andMacino, 2000; Vance and Vaucheret,
2001; Waterhouse et al., 2001; Hutvagner and Zamore, 2002a; Hannon, 2002].
During RNAi, the introduced dsRNAs
are processed into small dsRNAs of approximately 21–22 nucleotides (nt)through the action of Dicer, an RNase
III family enzyme [Hammond et al.,2000]. These processed small dsRNAs
have been termed small interfering
RNAs (siRNAs) because they assemble
into a multiprotein RNase enzyme called
an RNA-induced silencing complex(RISC) and act as guides for the complex
that uses the siRNA to identify and destroy complementary mRNA, thus silencing gene expression in a sequencespeciﬁc manner (Fig. 1) [Zamore et al.,
2000; Elbashir et al., 2001]. Although it
was initially thought to be an immune
system that protects against transposones
and viruses, RNAi is emerging as a fundamental regulatory process that is likely
to affect many layers of endogenous gene
expressions in eukaryotes. For example,

MRDD RESEARCH REVIEWS ● RNA INTERFERENCE ● SIOMI ET AL.

Fig. 1. RNAi and miRNA pathways. Two distinct pathways exist in eukaryotes in which ⬃22nucleotide (nt) RNAs function as posttranscriptional regulators of gene expression. RNA interference (RNAi) is the process by which doubl-stranded (ds) RNA triggers the destruction of cognate
mRNAs. An RNase III enzyme, Dicer, cleaves the dsRNA into duplexes of 21–23 nt, termed shortinterfering RNAs (siRNA), which then guide a multicomponent complex known as RISC to mRNAs
sharing perfect complementarity and target cleavage. A related short RNA species, microRNA(miRNA), is endogenously transcribed as a long RNA and then processed to a premiRNA of ⬃70 nt.
This premiRNA forms an imperfect hairpin structure, which is processed by Dicer to produce themature, single-stranded ⬃22-nt miRNA. Some of the miRNAs are known to regulate developmental timing by mediating sequence-speciﬁc repression of mRNA translation. This translational repression is achieved by partial base pairing between miRNAs and the 3⬘ UTR of target mRNAs. Although
the mechanism of translational repression remains to be elucidated, miRNAs are known to beincorporated into RISC. Thus a RISC core might interact with different combinations of short RNA
and accessory proteins to carry out either transcript cleavage or translational repression (see text).
[Color ﬁgure can be viewed in the online issue, which is available at www.interscience.wiley.com]

components of RNAi are involved inheterochromatin formation, thus suggesting that RNAi processes appear to be
associated with the chromatin-silencing
machinery [Volpe et al., 2002; Hall et al.,2002].
RNAi pathways share featureswith a developmental gene regulatory
pathway that involves natural dsRNAencoding genes, recently named microRNA (miRNA) genes (Fig. 1) [Lee andAmbros, 2001; Lau et al., 2001; LagosQuintana et al., 2001]. miRNA genes
encode approximately 70 nt hairpin precursor RNAs that are processed into mature 22 nt miRNAs. Dicer is also required for processing miRNAs, thus
making it essential for both the siRNA
pathway and the miRNA pathway[Bernstein et al., 2001; Ketting et al.,
2001; Grishok et al., 2001; Hutvagner etal., 2001]. miRNAs are thought to regulate mRNA translation because thefounding members of the miRNA genefamily, lin-4 and let-7, encode miRNA
products that repress translation during
C. elegans development by base pairingwith complementary sequences locatedin the 3⬘UTRs of their target mRNAs[Lee et al., 1993; Wightman et al., 1993;

Reinhart et al., 2000]. Both the siRNA
pathway and the miRNA pathway require members of the Argonaute family(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 et al., 2001; Mourelatos et al.,2002]. Recently, it has been shown thatin addition to siRNAs, miRNAs can also
be incorporated into RISCs [Hutvagner
and Zamore, 2002b]. Thus these observations suggest that miRNA functions
through RNAi-related pathways to regulate the expression of target genes andthat the distinct Argonaute family protein
associated with siRNAs or miRNAs regulates the function of these small RNAs
in a variety of homology-dependent
mechanisms that involve base pairing between small guide RNAs and target
mRNAs.
Recently, Ishizuka et al. [2002]
found that a dFMR1-associated complex
contains AGO2, an RNA helicase
Dmp68, Dicer, and miRNAs. AGO2 isan Argonaute family protein that is an
essential component of the RISC nuclease in Drosophila and copurifys withsiRNAs and Dicer [Hammond et al.,MRDD RESEARCH REVIEWS ● RNA INTERFERENCE ● SIOMI ET AL.

2001]. Studies in other organisms havealso shown that Argonaute family proteins essential for RNAi are in complexes
that contain an RNA helicase(s), Dicer,
and small guide RNAs [Tabara et al.,
2002; Mourelatos et al., 2002; Doi et al.,2003]. Dmp68 belongs to a large familyof highly evolutionarily conserved proteins, the so-called DEAD-box family of
putative ATPases and helicases [de la
Cruz et al., 1999], and is required for
efﬁcient RNAi [Ishizuka et al., 2002].
Because ATP-dependent unwinding of
the siRNA duplex remodels the RISC togenerate an active RISC in the RNAi
pathway [Nykanen et al., 2002], Dmp68
may mediate the unwinding process.
Caudy et al. [2002] isolated theendogenous RISC complex from S2 cells
to identify the associated proteins andfound that dFMR1 and a novel RNAbinding protein termed VIG are such
associated factors. These, together withthe ﬁndings by Ishizuka et al. [2002],
suggest that dFMR1 may associate with
RISC and thus be involved in RNAi.
Supporting this idea, siRNAs can coimmunoprecipitate with dFMR1 [Caudy etal., 2002]. Moreover, Caudy et al. [2002]
were able to show that immunoprecipitates of dFMR1 from dsRNA-challenged cells have RISC activity. Theseobservations strongly suggest thatdFMR1 is part of RISC in Drosophila.
Given the observed defects in neuronalmorphology and behaviors of dFMR1
mutant ﬂies, these new ﬁndings further
suggest the possibility that RNAi-related
processes may allow neurons to ﬁne-tune
the expression of mRNAs that are criticalfor morphology and the function of neurons.
POSSIBLE MECHANISM OF
ACTIONAt present, the role of dFMR1 in
RISC is not clear. Two groups performed similar experiments to determine
the role of dFMR1 in mRNA degradation by RNAi and found that dFMR1 is
not an essential factor for RNAi-mediated degradation, at least in cultured S2cells [Caudy et al., 2002, Ishizuka et al.,2002]. However, the association ofdFMR1 with miRNA, as well as with
RISC, may provide a means of linking
the RNAi pathways to dFMR1-mediated translational control pathways, asdFMR1 and FMRP have been shown torepress the translation of select mRNAs,
as do some miRNAs. These new ﬁndings
suggest a model in which dFMR1is targeted to its mRNA substrates as part of
RISC, which are guided by miRNAs(Fig. 2). Thus, RISC could provide a71

Fig. 2. A model for dFMR1’s role in RISC. The association of dFMR1 with miRNA as well as with
RISC suggests a model in which dFMR1is targeted to its mRNA substrates as part of RISC, which are
guided by miRNAs. Thus, RISC could provide a platform through which dFMR1 can act. dFMR1
could act as a chaperone or selector for recruiting speciﬁc miRNAs onto RISC. The interaction
between the dFMR1-associated RISC and components of ribosome such as L5 and L11 may inhibittranslation at one or more postinitiation steps (see text). [Color ﬁgure can be viewed in the online
issue, which is available at www.interscience.wiley.com]

platform through which dFMR1 can act.
Because dFMR1 appears to have an intrinsic ability to discriminate among different RNAs [Wan et al., 2000, MC
Siomi unpublished results], as is the casefor mammalian FMRP, it is also possiblethat dFMR1 may act as a chaperone or
selector for recruiting speciﬁc miRNAs
onto RISC. This scenario further envisions that a RISC core might interact
with different combinations of RNA andprotein accessory factors to carry out either mRNA degradation or translation
control.
How then might dFMR1 repress
the translation of its mRNA targets? lin-4
and let-7, prototypes of miRNAs, regulate endogenous genes involved in developmental timing in C. elegans by partiallybase pairing to the 3⬘ UTR of target
mRNAs such as lin-14 and lin-41, respectively [Lee et al., 1993; Wightman etal., 1993; Ha et al., 1996; Reinhart et al.,
2000; Slack et al., 2000]. This interaction
does not affect the stability of the target
mRNA but rather represses gene expression through an unknown mechanism
known as translational repression. The
polysome proﬁle of the target mRNA
does not change on gene silencing [Ol72son and Ambros, 1999; Seggerson et al.,2002], suggesting a block at a step after
translation initiation. dFMR1 may form
a RISC complex containing speciﬁc
miRNAs on its mRNA targets, and theassociation of this complex with components of ribosome such as L5 and L11
may inhibit translation at one or more
postinitiation steps, including elongation,
termination, or the release of functional
proteins (Fig. 2). The formation of the
RISC on its mRNA targets could be
regulated by phosphorylation of dFMR1
because the protein/protein interaction
and RNA-binding activities of dFMR1
are drastically modulated by phosphorylation [Siomi et al., 2002], thereby making possible the on-and-off regulation oftranslational repression of target mRNAs.
Although dFMR1 has multiple
RNA-binding domains, it appears to be
in RISC complexes through protein/protein interactions [Ishizuka et al.,2002]. So what is the role of RNA binding of FMRP? Although it was originallyproposed that FMRP binds directly to its
mRNA targets with G-quartet motifs[Schaeffer et al., 2001; Brown et al.,
2001; Darnell et al., 2001], FMRP may
instead recognize speciﬁc miRNAs withthese features, which then guide FMRP
to its targets. It is also conceivable thatthe recognition of target mRNAs by the
FMRP-associated RISC takes place in
two steps. Initially, the RISC may target
mRNAs, which are guided by miRNAs.
This binding may then be stabilized by
the attachment of FMRP to the G-quartet motif found in these target mRNAs.
Alternatively, there could be two pathways in which FMRP recognizes target
mRNAs: one is the direct binding of
FMRP to the G-quartet motif that isfound in a number of potential target
mRNAs, and the other is the indirectbinding of FMRP to the non-G-quartet
mRNAs via complementary miRNAs.
Whatever the mechanism of actionof FMRP in translation, the associationof dFMR1 with components of RNAiand miRNAs [Caudy et al., 2002; Ishizuka et al., 2002] has the implication that
defects in an RNAi-related process may
cause human disease. Of course, onemust be cautious in making connections
such as this, because, needless to say, a ﬂy
is not a human. An important step toward establishing the importance of an
FMRP/RNAi link in humans will be todetermine if any or all of the FMR1/
FXR family members interact with a
RISC and function in an RNAi-related
process in mammalian cells. Studies of
disease-causing proteins have often provided the ﬁrst glimpses into hitherto-unknown cellular regulatory mechanisms. If
history repeats itself, FMRP and dFMR1
are the harbingers of regulatory mechanisms of gene expression waiting to bediscovered. Further studies of dFMR1
aimed at, for example, the identiﬁcationof miRNAs that speciﬁcally associatewith a dFMR1-associated RISC in theﬂy brain will shed light on the mechanism of action of dFMR1 and provide
further clues about how the lack of
FMRP expression leads to fragile X syndrome. f
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