Current Biology, Vol. 12, R852–R854, December 23, 2002, ©2002 Elsevier Science Ltd. All rights reserved. PII S0960-9822(02)01352-0

RNA Interference: The Fragile XSyndrome Connection
Richard W. Carthew

Fragile X syndrome is caused by loss of expressionof FMRP, a protein proposed to act as a regulator ofmRNA translation which promotes synaptic maturation and function. Now FMRP has been found toassociate with the RNP complex that mediates posttranscriptional silencing by RNAi.

Fragile X syndrome is one of the most common forms
of inherited mental retardation. In most cases, thedisease is caused by the methylation-induced transcriptional silencing of the FMR1 gene which occurs asa result of the expansion of a CGG repeat in thegene’s 5′′ untranslated region (reviewed in [1]). Thisleads to the loss of production of gene product FMRP,an RNA-binding protein that contains several
structural motifs implicated in RNA interactions — twoKH domains and one RGG motif. FMRP associateswith translating polyribosomes as part of a large
ribonucleoprotein (RNP) complex [2], suggesting arole in regulating translation. Indeed, hundreds ofmRNA transcripts exhibit abnormal polyribosome
profiles in cells derived from patients with fragile X
syndrome [3], and biochemical studies of FMRP
suggest that it inhibits translation [1].
Pathological studies of the brains from fragile X
syndrome patients and Fmr1 knockout mice haverevealed abnormal dendritic spines on neurons,
implicating FMRP in synapse formation and function[4]. Both in vitro and in vivo neuronal studies haveshown that FMRP is located at the synapse, and theloss of FMRP alters synaptic plasticity [1], which isthought to underlie learning and memory. Interestingly, there is increasing evidence that synaptic efficacy is controlled in part by the regulated translationof mRNAs stored in dendrites [5]. It is tempting tospeculate on a potential link between FMRP-mediatedtranslation control and synapse function, more sosince FMRP is associated with ribosomes localized topostsynaptic dendritic spines [6].
Ishizuka et al. [7] purified the Drosophila homologof FMRP (dFMR1) as a complex from cell lysates tofind what proteins stably associate with it. They useddFMR1 in the hope of applying genetic analysis toassociated proteins, a fortunate choice. Drosophila hasa single FMR1 homolog, and the dFMR1 gene producthas similar biochemical properties to mammalian
FMRP [8]. Moreover, mutation studies indicated thatdFMR1 regulates synapse growth and function in thefly [9,10]. Ishizuka et al. [7] purified from Drosophila celllysates a dFMR1 complex that contained two ribosomal
Department of Biochemistry, Molecular Biology and Cell
Biology, Northwestern University, Evanston, Illinois 60208,
USA. E-mail: r-carthew@northwestern.edu

Dispatchproteins, L5 and L11, 5S rRNA and, surprisingly, AGO2,a protein implicated in RNA interference (RNAi).
RNAi is a conserved gene-silencing response todouble-stranded RNA (dsRNA), which mediates resistance to both endogenous and exogenous parasitic
nucleic acids, encoded by transposons and viruses,respectively (reviewed in [11]). RNAi also regulates theexpression of cellular protein-coding genes. A signature feature of RNAi is the formation of small RNAs,
21–23 nucleotides in length, which are generated by aclass of conserved RNase III enzymes called theDICER family (Figure 1).
DICER recognizes and cleaves dsRNA molecules toproduce 21–23 nucleotide fragments [12]. It has beenshown that these fragments, called short interfering
RNAs (siRNAs), can substitute for dsRNA in stimulating degradation of homologous mRNAs, the primarymechanism of gene silencing in RNAi [13]. Thus,
siRNAs are thought to be the direct guides that identify homologous mRNA substrates for degradation.
DICER also processes short hairpin RNAs transcribedfrom endogenous genes [11], producing 21 nucleotide
single-stranded micro-RNAs (miRNAs). In contrast tothe siRNA pathway, miRNAs can silence gene expression by repressing translation of complementarymRNA transcripts [14,15]. There are estimated to bepossibly hundreds of miRNA-encoding genes in thegenome [16].
Biochemical analysis using Drosophila extracts hasrevealed how siRNAs interact with mRNA substrates(Figure 1A). It was observed that siRNAs associate into
a nuclease complex dubbed RISC which rec-ognizes
and cleaves homologous mRNAs [17]. The cleavage
reaction is endonucleolytic and occurs at a central site
in the region homologous to the siRNA [13]. One of thesubunits of RISC is AGO2 [18], the same protein foundby Ishizuka et al. [7] to associate with dFMR1. At thesame time, Caudy et al. [19] were identifying proteinsassociated with RISC activity from partially purified
Drosophila preparations, and found that dFMR1 is one
such associated factor. Together, these initial observations suggested that dFMR1 may be involved in RNAi.
The two groups [7,19] report a variety of experimental evidence in support of this idea. dFMR1 coimmunoprecipitates with AGO2 and another RISC
protein (VIG) from Drosophila cell lysates. Moreover,both a miRNA (miR-2b) and siRNAs can co-immunoprecipitate with dFMR1, consistent with the view thatdFMR1 associates with RISC. The most compelling
biochemical evidence, provided by Caudy et al. [19],is that immunoprecipitates of dFMR1 from dsRNAchallenged cells have RISC activity.
All this suggests that dFMR1 is intimately associated with RISC in Drosophila cells; but is it required
for mRNA degradation? The matter is unclear. Bothgroups [7,19] performed a similar experiment to determine the function of dFMR1. They used RNAi tosilence the endogenous dFMR1 gene in cells, andCurrent Biology
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Figure 1. A model for dFMR1’s role in RNAi.
(A) The mRNA cleavage/degradation
pathway is initiated by dsRNA conversion
to siRNAs, which associate with RISC
complexes. The proteins AGO2, VIG anddFMR1 also associate with RISC. dFMR1,and possibly VIG, are not found in all
RISC complexes, as they appear not to beabsolutely essential for the degradation
reaction. These assembled complexes arecompetent to carry out target mRNArecognition and cleavage from immunoprecipitates, though it is unclear whether
the complexes remain assembled during
the cleavage reaction. (B) The translation
repression pathway is initiated by miRNA
precursor processing to mature miRNAs,
which associate with related RISC complexes. AGO2, VIG, and dFMR1 are also
associated with these complexes. The
relationship between assembled miRNA
complexes and mRNA translation control
is surmised by a variety of circumstantial
evidence implicating some complex components — miRNAs and dFMR1 — intranslation control.

dsRNAABmiRNA precursor

DICERATPADP+PisiRNA
21-23 nt
AGO2
DICER

RISC
dFMR1

Mature miRNA

VIGmRNA

then challenged these cells with a heterologous
dsRNA complementary to a reporter gene as a way ofmeasuring RNAi activity in the absence of dFMR1. An
earlier application of this approach established a role
for AGO2 in mRNA degradation by RNAi [18], suggesting it can detect strong functional interactions.
However, Caudy et al. [19] detected only a weakrequirement for dFMR1, and Ishizuka et al. [7] detected
none. But each group measured RNAi activity semiquantitatively by reporter-protein levels. Direct analysis of reporter mRNA levels might have revealed similarities or differences that were not detected at theprotein level. Clearly more work is needed to resolve
this issue.
What is clear is that dFMR1 is not an essential
factor for RNAi-mediated degradation. Rather thanbeing a constitutive component of transcript-cleaving
RISC, dFMR1 might be an accessory factor that associates with RISC but uses another mechanism to regulate its targets. This interpretation fits nicely with theother mechanism being translational repression,
as dFMR1 and FMRP have been shown to repress
translation of select mRNAs, as do some miRNAs[3,9,14,15,20]. Because miRNAs and dFMR1 interact
with each other as well as with RISC, it is quite possible that dFMR1 is targeted to substrate transcripts aspart of a RISC complex that is guided by miRNAs.
Thus, a RISC core might interact with different combinations of RNA and protein accessory factors to carryout either transcript cleavage or translation control.
An alternative scenario is that dFMR1-associated
RISC complexes may only use a subset of trigger
siRNAs or miRNAs available. dFMR1 itself may contribute to such RNA specificity, as FMRP was found toselectively bind RNA oligonucleotides with G-quartet
structures adjacent to short RNA helices [3,20]. Whileit was originally proposed that FMRP directly binds tomRNA substrates with these structural features,
FMRP may instead recognize specific miRNAs withCleavage
Degradation
Translation block
Current Biology

these features, which then guide FMRP to its correct
targets. It is possible that some, but not all, siRNAs
associate with FMRP and guide RISC complexes thatare competent to degrade mRNA as well.
The connection between the RNAi and fragile X fields
is likely to grow as we find out which small RNAs
mediate translational repression by dFMR1 and FMRP,
and how the repressive mechanism operates. Nevertheless, it is worth pondering that fragile X syndrome
may be the result of a defect in the RNAi activity withinneurons, making its molecular nature a RISCy business.
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