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ARTICLES
Biochemical and genetic interaction between
the fragile X mental retardation protein and the
microRNA pathway
Peng Jin1, Daniela C Zarnescu2, Stephanie Ceman1,3, Mika Nakamoto1, Julie Mowrey1, Thomas A Jongens4,
David L Nelson5, Kevin Moses2 & Stephen T Warren1
Fragile X syndrome is caused by a loss of expression of the fragile X mental retardation protein (FMRP). FMRP is a selective
RNA-binding protein which forms a messenger ribonucleoprotein (mRNP) complex that associates with polyribosomes. Recently,
mRNA ligands associated with FMRP have been identified. However, the mechanism by which FMRP regulates the translation of
its mRNA ligands remains unclear. MicroRNAs are small noncoding RNAs involved in translational control. Here we show that
in vivo mammalian FMRP interacts with microRNAs and the components of the microRNA pathways including Dicer and the
mammalian ortholog of Argonaute 1 (AGO1). Using two different Drosophila melanogaster models, we show that AGO1 is critical
for FMRP function in neural development and synaptogenesis. Our results suggest that FMRP may regulate neuronal translation
via microRNAs and links microRNAs with human disease.
Fragile X syndrome, a common form of inherited mental retardation,
is caused by the loss of the fragile X mental retardation protein
(FMRP)1. FMRP, and its autosomal paralogs, the fragile X–related
proteins FXR1P and FXR2P, constitute a small family of RNA-binding
proteins (fragile X–related gene family)2,3. These proteins share >60%
amino acid identity and contain two types of RNA-binding motifs:
two ribonucleoprotein K homology domains (KH domains) and a
cluster of arginine and glycine residues (RGG box).
FMRP is a selective RNA-binding protein that forms a messenger
ribonucleoprotein (mRNP) complex that associates with polyribosomes1. It has been shown that FMRP could suppress protein translation in vitro4,5. The brains of both human patients with fragile
X syndrome and Fmr1 knockout mice show abnormal dendritic
spines6–8. Interestingly, FMRP is apparently associated with polyribosomes within and at the base of dendritic spines of wild-type
neurons9. Based on these observations, it has been proposed that
FMRP is a translation suppressor that is involved in synaptic plasticity through regulating local protein synthesis of specific mRNAs
in response to synaptic stimulation signals10. Consistent with this
model, the long-term depression (LTD) that depends on protein
synthesis and is triggered by activation of metabotropic glutamate
receptors is markedly increased in the hippocampus of Fmr1
knockout mice11. Some of the mRNAs associated with FMRP have
recently been identified, and a G-quartet/stem structure in these
mRNAs has been shown to be involved in the FMRP-mRNA interaction12–17. However, the mechanism by which FMRP regulates
translation of its mRNA ligands remains unclear, although it has
recently been suggested that the removal of a phosphate from the
Ser499 residue of FMRP may release the translational suppression
mediated by FMRP18.
MicroRNAs (miRNAs) are a new class of noncoding RNAs that are
believed to control translation of specific target mRNAs by complementing with antisense sequences in the 3′ untranslated region of
these messages19–22. However, most target mRNAs that interact with
miRNAs remain to be identified. Mature miRNAs are single-stranded
and composed of 20–25 nucleotides that are processed from ∼70 or
longer nucleotide stem-loop precursors by Dicer, a dsRNA-specific
RNaseIII19,21. The same activity is also involved in the generation of
short interfering RNAs (siRNA), widely used to experimentally
degrade RNA targets20,23. Members of the PIWI/PAZ-domain protein
(Argonaute) family facilitate processing and downstream functions of
miRNAs and siRNAs24. Previous biochemical studies show that the
D. melanogaster gene dFmr1, which encodes the ortholog of FMRP,
associates with Argonaute 2 (AGO2) and the RNA-induced silencing
complex (RISC) that mediates RNA interference (RNAi) in
D. melanogaster25,26. The significance of these observations relative to
fragile X syndrome is unclear, as there is no evidence of abnormal
mRNA degradation, despite exhaustive microarray analyses, in either
cells from fragile X patients or the Fmr1 knockout mouse brain that
show no substantial deviation from normal steady-state expression
levels for most genes27. Considering that miRNA and siRNA pathways
share common components, we reasoned that FMRP may alterna-
1Department of Human Genetics and 2Department of Cell Biology, Emory University, 615 Michael Street, Atlanta, Georgia 30322, USA. 3Present address:
Department of Cell and Structural Biology, University of Illinois, 601 S. Goodwin Avenue, Urbana, Illinois 61801, USA. 4Department of Genetics, University of
Pennsylvania School of Medicine, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. 5Department of Molecular and Human Genetics, Baylor College
of Medicine, One Baylor Plaza, Houston, Texas 77030, USA. Correspondence should be addressed to S.T.W. ([email protected]).
Published online 4 January 2004; doi:10.1038/nn1174
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ARTICLES
Figure 1 FMRP associates with endogenous microRNAs and Dicer activity.
(a) Immunoprecipitating antibodies specific to FXR2, FXR1 and human
FMRP were used to immunoprecipitate their respective proteins from an
EBV-transformed human B cell line. The efficiency of the
immunoprecipitations was calculated by determining how many cells were
required to immunoprecipitate 5 × 105 cell equivalents, shown as the
lysate (input) in the first lane. (b) Immunoprecipitations were performed
with anti-FMRP, anti-FXR1P and anti-FXR2P antibodies using human
lymphoblastoid cell line from a normal male. As a negative control,
immunoprecipitation was also performed on a fragile X cell line, using
antibody to FMRP (αFMRP). The bands for miRNAs and their precursors
are indicated. (c) The dicer activity is specifically associated with the
immunoprecipitation complexes with anti-FMRP and anti-FXR2P, but not
with anti-FXR1P. IgG only indicates negative controls. Processed short
RNAs (∼20 nt) are indicated.
tively regulate the translation of its target genes through miRNAs in a
fashion analogous to let-7 and lin-4 in C. elegans21.
Here we show that in vivo mammalian FMRP interacts with
miRNAs and the components of the miRNA pathways including
Dicer and the mammalian ortholog of AGO1. Furthermore, using
D. melanogaster as a model system, we demonstrate that AGO1 is critical for FMRP function in neural development and synaptogenesis.
These results suggest that FMRP may regulate the translation of its
mRNA ligands via miRNA involvement and that the absence of
FMRP may disrupt this regulatory process.
RESULTS
FMRP associates with miRNAs and miRNA components
Given that FMRP belongs to a small family of three RNA-binding proteins with a high degree of similarity, we developed non-crossreacting
antibodies against FMRP, FXR1P and FXR2P (see Methods) and
determined the immunoprecipitation efficiency for each antibody
(Fig. 1a). Using these antibodies, we first determined whether or not
endogenous miRNAs are associated with the FMRP-mRNP complex.
We performed immunoprecipitation from a normal human cell line
and, as a negative control, from a fragile X patient cell line using the
specific antibodies that recognize FMRP, FXR1P and FXR2P. For each
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reaction, the immunoprecipitated RNA was isolated, radioactively
labeled and resolved by electrophoresis. RNAs migrating at ∼20
nucleotides (nt) were coimmunoprecipitated with FMRP, FXR1P and
FXR2P from normal cells. No such RNA was observed when FMRP
was immunoprecipitated from patient cells (Fig. 1b). The size of these
RNAs suggests that they represent the mature form of endogenous
miRNAs found associated with the FMRP mRNP complex.
Furthermore, RNAs at ∼80 nt were also coimmunoprecipitated
with FMRP and FXR2P, suggesting that FMRP and FXR2P may also
be associated with the miRNA precursor transcript (Fig. 1b). Indeed,
two bands of approximately 40 nt are also observed, which may represent the initial Dicer cleavage products of the precursor transcripts.
Since this implies that Dicer may also be found with the FMRP
mRNP complex, we measured Dicer activity associated with
immunoprecipitated complexes using the FMRP, FXR1P and FXR2P
specific antibodies. Fluorescently-labeled double-stranded RNAs
(dsRNAs) were incubated with either immunoprecipitated complexes or IgG only. The immunoprecipitated complexes using FMRP
or FXR2P antibody could cleave the long dsRNAs into small RNAs of
approximately 20 nt (Fig. 1c). In addition, no long dsRNAs could be
cleaved into small RNAs when incubating with the complex coimmunoprecipitated with FMRP antibody from fragile X cell lines (data
not shown). These data suggest that FMRP and FXR2P indeed associate with Dicer activity in vivo. Surprisingly, the absence of Dicer
activity suggests that FXR1P may not fully participate in the miRNA
pathway, despite 60% identity with the other paralogs.
We then examined whether or not human FMRP associates with
Argonaute proteins, which have been shown to have important roles
in both siRNA and miRNA pathways28,29. Since the mammalian
Argonaute protein eIF2C2 is the only protein that has previously been
shown to associate with the endogenous miRNAs, we tested whether
or not eIF2C2 interacts in vivo with human FMRP or its two paralogs
FXR1P or FXR2P by immmunoprecipitation29. We found that
endogenous eIF2C2 could indeed be coimmunoprecipitated with
FMRP, FXR1P and FXR2P (Fig. 2a). These results were confirmed by
immunoprecipitating eIF2C2 from cell lysate and probing with antibodies against the three human FMR paralogs (Fig. 2b). As a negative
control, an unrelated RNA-binding protein also found on polysomes,
polyA-binding protein, was shown not to associate with eIF2C2
(Fig. 2b). These data suggest that in human cells, eIF2C2 interacts
with the FMRP mRNP complex. Because this complex includes all
three FMR1 paralogs, it is unclear whether the association of eIF2C2
is solely via interaction with FXR2P. Using mouse fibroblasts derived
from either Fmr1-knockout or Fxr2-knockout mice30,31, we show that
eIF2C2 can still be coimmunoprecipitated with mammalian FMRP in
the absence of Fxr2P (Fig. 2c). These results suggest that the fragile X
mental retardation protein associates with miRNA and the components of microRNA pathway in vivo.
Loss of AGO1 suppresses dFmr1 overexpression apoptosis
To determine the functional significance of the in vivo association
between FMRP and eIF2C2, a component of microRNA pathway,
we examined their genetic interaction using D. melanogaster as a
model system. First, we determined that the mammalian eIF2C2
translation initiation factor is the bona fide mammalian AGO1
ortholog despite previous reports that eIF2C2 is the ortholog of
AGO2 in the mammalian genome (Supplementary Methods
online)24,32. Indeed, there appears to be no clear ortholog of AGO2
in the mammalian genome. It has been shown that overexpression
of dFMR1 causes neuronal cell death and leads to apoptosis33,34.
Here we used this established D. melanogaster model where a
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ARTICLES
Figure 2 The fragile X–related protein family associates with eIF2C2, a
member of the Argonaute protein family. (a) Total HeLa cell lysates were
used for immunoprecipitation with antibodies specific to FMRP (αFMRP),
FXR1P (αFXR1P) and FXR2P (αFXR2P). IgG only indicates negative
controls. The immunoprecipitates were analyzed by immunoblotting with a
monoclonal antibody to eIF2C2. We ran, in parallel, 5% of the input used
for IPs. (b) Immunoprecipitation (IP) was performed from total HeLa cell
lysates with a monoclonal antibody to eIF2C2. IgG alone was used as a
negative control. The same blot was probed with different antibodies:
monoclonal anti-FMRP, polyclonal anti-FXR1P, monoclonal-FXR2P and
monoclonal anti-polyA binding protein (PABP). (c) Immunoprecipitation
(IP) was performed on wild-type mouse fibroblasts, Fmr1 knockout
fibroblasts and Fxr2 knockout fibroblasts using anti-FMRP with IgG as a
negative control. The immunoprecipitates were analyzed by immunoblotting
with a monoclonal antibody specific to eIF2C2. 10% of the input used for
IPs was run in parallel.
sevenless promoter driving dFmr1 overexpression in the eye produces a consistently mild rough eye (Fig. 3a)33 (D.C.Z and K.M.,
unpublished data). Three P-element insertions (l(2)04845,
l(2)k00208 and l(2)k08121) at the AGO1 locus have been found to
greatly reduce the expression of AGO1 and lead to recessive lethality35,36. By crossing these P insertion lines to a sevenless-dFmr1
transgenic line, we found that the reduction of AGO1 could
markedly suppress the mild rough eye phenotype due to the over
expression of dFmr1. The l(2)k00208 insertion produced the
strongest suppression, whereas l(2)04845 only showed weak suppression (Fig. 3a and data not shown). This corresponds to the
allelic strength of P insertions and l(2)04845 only leads to semilethality35,36. Internally, the disruption of ommatidia caused by the
overexpression of dFmr1 was also suppressed by the reduction of
AGO1 as revealed by tangential sections (Fig. 3a). As a control for
genetic background effects, the AGO1 revertant generated by precise P-element excision did not suppress the rough eye phenotype.
To further understand how the reduction of AGO1 could suppress
the rough eye phenotype caused by the overexpression of dFmr1, we
NATURE NEUROSCIENCE VOLUME 7 | NUMBER 2 | FEBRUARY 2004
Figure 3 Loss of AGO1 suppresses the rough eye phenotype caused by the
overexpression of dFmr1. Genotypes were indicated on the top. (a) Adult
compound eyes with different genotypes are shown. The first two rows:
SEM images. Magnification: first row, 150×; second row, 400×. The
tangential sections were shown on the third row. (b) Larval eye discs of the
indicated genotypes stained for dFmr1 protein, anterior right.
examined the expression of dFmr1 in the eye discs of the two different
genotypes. As expected, dFmr1 protein was elevated posterior to the
morphogenetic furrow in the Sev:dFmr1/+ eye disc, and no significant difference was observed when AGO1 expression was reduced
(Fig. 3b). This suggests that AGO1 does not affect the expression or
stability of dFmr1 protein but rather is required for the biological
functions of dFmr1.
AGO1 is required for dFmr1 regulation of synaptic plasticity
To further study this dominant interaction between AGO1 and dFmr1,
we examined whether AGO1 could modulate the synaptic growth regulated by dFmr1 using the loss-of-function dFmr1 mutants
(dFmr13/TM6C)37. It has been shown that dFmr1 regulates synaptic
structure at the larval neuromuscular junction (NMJ), a well-defined
system for studying synaptic structure and neurotransmission in
D. melanogaster34. In the absence of dFmr1, pronounced synaptic overgrowth was observed at NMJ, which is reminiscent of the dendritic spine
overgrowth observed in both human patients and Fmr1 knockout
mice34. We examined the NMJs from dFmr1 heterozygotes or homozy-
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© 2004 Nature Publishing Group http://www.nature.com/natureneuroscience
dFmr13/dFmr13) were expected, but only
46 flies with the correct genotype were recovered. This reduced viability of the dFmr1 null
mutant caused by the reduction of AGO1 further highlights the importance of the interaction of AGO1 with dFmr1.
DISCUSSION
FMRP has been proposed to function as a
translation suppressor that is involved in
synaptic plasticity through translational regulation of its mRNA ligands27. Although some
of FMRP mRNA ligands have been identified,
the mechanism by which FMRP regulates
translation of these mRNAs remains obscure1.
The data reported here, that FMRP shows both
biochemical and genetic interactions with certain components of the miRNA pathway,
immediately suggest a mechanism by which
FMRP may exert translational suppression. We
propose that the previously identified G-quartet/stem structure is required for an initial lowspecificity scanning and capture of its mRNA
ligands by FMRP. Once FMRP binds to
mRNA, the associated RISC complex, particularly eIF2C2, the ortholog of AGO1, uses
miRNAs for specificity interrogation of
the mRNA and suppression of translation
(Supplementary Fig. 1 online). The functional
interaction between dFMR1 and AGO1 is evident from the suppression of the overexpression phenotype; since dFMR1 is considered a
translational suppressor, overexpression could
lead to apoptosis via translational oversuppression of bona fide target mRNAs or promiscuFigure 4 AGO1 dominantly modulates dFmr1 function in synaptic growth and structure. (a) Shown
ous translational suppression of non-target
are the neuromuscular junctions (segment 3, muscle 6/7) with the three genotypes indicated on the
mRNAs. If AGO1 works in stoichiometric protop. Left-hand insets are high-magnification pictures. The reduction of AGO1 expression markedly
portion with dFMR1, limiting AGO1 could
altered the synaptic structure of NMJs in dFmr1 heterozygote. (b) Histogram showing number of
reduce or nullify the functional excess of
boutons per junction with different genotypes. Error bars indicate standard error. Genotypes are
dFMR1. The model proposed here is also conindicated on the top of each bar.
sistent with a proposed use of miRNAs as a
mechanism to generate synaptic tags that trangotes with and without the reduction of AGO1. Surprisingly, we found siently mark a synapse after activation, allowing for local protein synthethat AGO1 dominantly regulates dFmr1 function. Compared to AGO1 sis–dependent synaptic strengthening38, a cascade of events for which
or dFmr1 heterozygotes, which appear to have normal NMJ and similar FMRP has been strongly implicated39. Finally, disruption of this mechanumbers of synaptic boutons to wild-type larvae, the trans-heterozygote nism, via the loss of FMRP in fragile X syndrome, would signify the
(l(2)k00208/+;dFmr13/+) showed a strong synaptic overgrowth and direct involvement of the miRNA pathway in human disease.
over-elaboration of synaptic terminals (Fig. 4a). This was confirmed
quantitatively by counting the number of synaptic boutons, which was METHODS
increased two-fold in the trans-heterozygote (Fig. 4b). Indeed, the Generation of antibodies specific to FMRP, FXR1P and FXR2P. Anti-FMRP was
synaptic overgrowth phenotype in the trans-heterozygote was stronger rabbit polyclonal sera directed against amino acids 359–370 (KENSTHFSQPNS).
than the phenotype found in the dFmr1 null mutants (Fig. 4b). This The peptide and affinity purified antisera were prepared by New England
observation suggests that AGO1 is a limiting factor for dFmr1 function. Peptide. Anti-FXR1P was chicken polyclonal sera directed against amino acids
Intriguingly, the NMJs from the dFmr1 null mutant on an AG01 het- 482–501 (NTESDQTADTDASESHHSTN). Anti-FXR2P was chicken polyclonal
erozygote background did not show an expected more severe phenotype sera directed against amino acids 406–429 SDKAGYSTDESSSSSLHAT. The
FXR1 and FXR2 peptides were synthesized by Aves Labs, Inc., who also perthan those of trans-heterozygote (Fig. 4b). It is very likely that the
formed the immunizations and affinity purifications. Immunoprecipitations
synapses of the NMJ cannot overgrow indefinitely, as overgrowth
were performed essentially as described13. The chicken antisera were coupled to
beyond a certain threshold is likely to lead to lethality. Indeed, in our immobilized anti-chicken Igγ (Promega) per manufacturer’s instructions.
attempts to generate the dFmr1 null mutant flies that were heterozygous
for AGO1, fewer than 50% of the expected number of progeny Mammalian cell culture, immunoprecipitation and RNA isolation. Both HeLa
were recovered. With 489 flies examined, 108 flies (l(2)k00208/+; cells and human lymphoblastoid cell lines (normal male and fragile X male not
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© 2004 Nature Publishing Group http://www.nature.com/natureneuroscience
expressing FMRP) were cultured as described previously13. Immunoprecipitation
and western blot using different antibody were performed as previously
described13. To detect miRNAs, the immunoprecipitated RNA was isolated by
phenol/chloroform extraction and ethanol precipitation. The immunoprecipitated RNA was 3′-end-labeled with [5′-32P]-pCp and resolved by electrophoresis
on 15% denaturing polyacrylamide gel. The 32P-label RNA size marker (Ambion)
for 10–100 nt was run in parallel.
Dicer activity assay. The Dicer activity assay was performed as previously
described23. Briefly, fluorescently labeled 500-nt double-stranded luciferase
RNAs (dsRNAs) were generated by MEGAscript RNAi kit (Ambion). For
cleavage of dsRNAs, the whole cell lysate or immunoprecipitated complex
from 107 HeLa cells was incubated with dsRNA for 1 h at 37 °C in the presence
of ATP. After incubation, the RNAs were isolated by phenol/chloroform
extraction, then ethanol precipitation, and then separated by electrophoresis
on 12% denaturing polyacrylamide gels. The gels were scanned using Typhoon
9200 (Amersham).
Drosophila melanogaster genetics. The transgenic fly w1118; sev:dFmr1 CyO
and dFmr1 null allele dFmr3/TM6C was published previously33,37. The
P-element insertions, l(2)04845, l(2)k00208 and l(2)k08121, were obtained
from the Bloomington Stock Center. The AGO1 revertant was generated by
precise excision of the P element in the l(2)k00208 line that mutates the AGO1
gene. Flies were maintained and crossed using standard procedures.
Microscopy and immunohistochemistry. Scanning Electron Microscopy
(SEM) and eye sections were done as described34. Antibodies specific to
dFMR1 6A15 and CSP were used as previously described33,34. Eye discs and
NMJs were prepared as described34. Analysis of boutons was adapted from a
previous study34.
Note: Supplementary information is available on the Nature Neuroscience website.
ACKNOWLEDGMENTS
The authors would like to thank G. Dreyfuss for anti-eIF2C2, anti-dFMR1
monoclonal 6A15 and anti-polyA binding protein antibodies, and J. Taylor and
R. Apkarian for technical assistance. This work was supported, in part, by grants
from the Rett Syndrome Research Foundation (P.J.), the FRAXA Research
Foundation (D.Z.) and National Institute of Health grants to D.C.Z., S.C., T.A.G.,
D.L.N., K.M. and S.T.W.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Received 28 October; accepted 12 December 2003
Published online at http://www.nature.com/natureneuroscience/
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