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© 2000 Oxford University Press
Human Molecular Genetics, 2000, Vol. 9, No. 6 Review
901–908
Understanding the molecular basis of fragile X
syndrome
Peng Jin and Stephen T. Warren+
Howard Hughes Medical Institute and Departments of Biochemistry, Pediatrics and Genetics, Emory University
School of Medicine, Rollins Research Center, 1510 Clifton Road, Atlanta, GA 30322, USA
Received 13 January 2000; Accepted 1 February 2000
Fragile X syndrome, a common form of inherited mental retardation, is mainly caused by massive expansion
of CGG triplet repeats located in the 5′-untranslated region of the fragile X mental retardation-1 (FMR1) gene.
In patients with fragile X syndrome, the expanded CGG triplet repeats are hypermethylated and the expression of the FMR1 gene is repressed, which leads to the absence of FMR1 protein (FMRP) and subsequent
mental retardation. FMRP is an RNA-binding protein that shuttles between the nucleus and cytoplasm. This
protein has been implicated in protein translation as it is found associated with polyribosomes and the rough
endoplasmic reticulum. We discuss here the recent progress made towards understanding the molecular
mechanism of CGG repeat expansion and physiological function(s) of FMRP. These studies will not only help
to illuminate the molecular basis of the general class of human diseases with trinucleotide repeat expansion
but also provide an avenue to understand aspects of human cognition and intelligence.
INTRODUCTION
Fragile X syndrome is one of the most common forms of inherited mental retardation with the estimated incidence of 1 in
4000 males and 1 in 8000 females (1,2). The syndrome is
transmitted as an X-linked dominant trait and with reduced
penetrance (80% in males and 30% in females) (1,2). Fragile X
syndrome is associated with a fragile site, designated FRAXA
(Fragile site, X chromosome, A site), at Xq27.3 near the end of
the long arm. The clinical presentations of fragile X syndrome
include mild to severe mental retardation, with IQ between 20
and 60, mildly abnormal facial features of a prominent jaw and
large ears, mainly in males, and macroorchidism in postpubescent males. Many patients also display subtle connective
tissue abnormalities, hyperactive and attention deficit disorder
and autistic-like behavior (1,2). In 1991, the molecular basis of
fragile X syndrome was revealed by positioning cloning and
shown to be associated with a massive trinucleotide repeat
expansion within the gene fragile X mental retardation-1
(FMR1) (3–6). This was one of the first identified human
disorders caused by dynamic mutation, trinucleotide repeat
expansion.
FMR1 is a highly conserved gene that consists of 17 exons
and spans ∼38 kb (7,8). Within the 4.4 kb of FMR1 transcript,
a CGG trinucleotide repeat is located at the 5′-untranslated
region (5′-UTR). Among normal individuals, this CGG repeat
is highly polymorphic in length and content, often punctuated
by AGG interruptions (9–13). The normal repeat size ranges
from 7 to ∼60, with 30 repeats found on the most common
allele. In most affected individuals, CGG repeats are massively
expanded over 230 repeats (full mutation) and becomes
abnormally hypermethylated, which results in the silence of
+To
the FMR1 gene. Alleles with between 60 and 230 CGG repeats
are called premutation. They are generally unmethylated with
normal transcript and protein level, but are extremely unstable
during transmission to next generation (1,14). Expansion of
premutation into full mutation can only occur by maternal
transmission and depends on the length of the maternal
premutation. Due to X-linkage, affected males have more
severe phenotypes than affected females, whose phenotype is
modulated by the activation ratio of the normal X chromosome. Identification of other mutations of the FMR1 gene,
such as deletions and point mutation among patients with usual
phenotype but without fragile site expression, firmly established that the FMR1 gene is the only gene involved in the
pathogenesis of fragile X syndrome (15,16). Thus, the absence
of the FMR1 gene product, fragile X mental retardation protein
(FMRP), is the typical cause of fragile X syndrome. The FMR1
gene is widely expressed in both human and murine tissues
(17). Multiple FMRP isoforms, through extensive alternative
splicing at the 3′ end, appear in a variety of tissues, and some
of them are present at different quantitative levels (7,18,19).
FMRP has been implicated in RNA binding and is possibly
involved in translational control (20,21). An animal model of
fragile X syndrome was created using gene targeting (22). The
FMR1 knockout mice exhibit macroorchidism and a subtle
deficit in learning and memory, which mimic the human
phenotype.
Recent research on fragile X syndrome has been focused on
two areas. (i) Instability and methylation of CGG repeats when
and how does CGG repeat instability occur? What is the
molecular mechanism of DNA methylation of expanded CGG
repeats? (ii) Physiological function(s) of FMRP; what genes
are the targets of FMRP in vivo? As an RNA-binding protein,
whom correspondence should be addressed. Tel: +1 404 727 5979; Fax: +1 404 727 5408; Email: [email protected]
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Human Molecular Genetics, 2000, Vol. 9, No. 6 Review
what role(s) does FMRP play in translation? How does the
absence of FMRP lead to deficit in learning and memory
during development? Here we will review the latest progress
made in these two areas towards understanding the molecular
basis of fragile X syndrome.
INSTABILITY AND METHYLATION OF CGG
REPEATS
In the normal population, the CGG repeat is polymorphic but
inherited in a stable fashion. The repeat is cryptic with interspersed AGG repeats most often found at repeat positions 10
and 20 (11). The CGG repeat length variation is found at the 3′
end of the repeat and it appears that AGG triplets play a crucial
role in the maintenance of repeat stability (11). Most premutation alleles have either no AGG or only a single AGG interruption at the 5′ end of the repeat (10,13,23). Loss of these
interruptions leads to alleles with longer perfect CGG repeat
tracts, which are prone to expansion into premutation, especially when tracts contain >30 perfect CGG repeats. In contrast
to the normal allele, premutation is unstable and may expand to
a full mutation or a different sized premutation (6). No new
mutations have been reported with all abnormal alleles ultimately derived from premutations, at least in the most recent
generations. The risk of expansion of a premutation to a full
mutation in the next generation exponentially increases with
the number of repeats between 65 and 100. This phenomenon
resolves anticipation in fragile X syndrome (also known as
‘Sherman Paradox’), which manifests clinically as an increase
in the number and proportion of mentally retarded individuals
in successive generations in fragile X families (24,25).
Remarkably, premutation only expands to a full mutation
when it is transmitted by female carrier, but not male carrier.
The reason for this is that full mutation males have only
premutation-size alleles in their sperm (see below) (26). In the
full mutation, the CGG repeat is massively expanded and
hypermethylated, which results in the repression of the FMR1
gene and leads to the loss of FMRP. However, mosaicism of
both CGG repeat length and methylation status exists in fragile
X patients (1). In most affected individuals, somatic variation
of repeat length is found in all tissues and is pronounced in
∼15% of patients, termed mosaics. In addition, some full mutation carriers have unmethylated, premutation bands, which
presumably produce FMRP and which can be found in some
high-functioning fragile X patients. Also some full-mutation
alleles are partially methylated or completely unmethylated.
Some evidence has been presented to indicate that FMR1 transcripts with large expansions are not well translated (27).
These observations indicate that CGG repeat expansion and
methylation of expanded CGG repeats are two separate events,
of which the association is still not clear. Both methylation
status and the repeat length on the active X chromosome will
influence the amount of FMRP produced, which is important
during the cognition development.
Although the timing of CGG repeat expansion is still not
clear, two time periods have been implicated during which
premutation is expanded to full mutation: meiosis and early
embryonic development. The anticipation in each succeeding
generation of fragile X families indicates that expansion must
occur in germ cells (either during premeiotic or meiotic division). Also, it has been postulated that CGG repeat instability
exists post-zygotically (28). The supportive evidence is that
full mutation carriers are found to be somatic mosaicisms of
CGG repeats (28,29). However, the length of CGG repeats on
a particular allele in a differentiated cell derived from these
carriers is generally mitotically stable although recent studies
show that fully expanded FMR1 CGG repeats exhibit a lengthand differentiation-dependent instability in cell hybrids (30,
31). Analysis of CGG repeats in gametes of fetuses with full
mutation concluded that the testes of a 13 week full-mutation
fetus showed no evidence of premutations, whereas a 17 week
full-mutation fetus exhibited some germ cells with attributes of
premutations (32). Also only full-expansion alleles can be
detected in the oocytes of full-mutation fetuses (32). These
results indicate that CGG repeat instability happens either
during premeiotic or meiotic division, or post-zygotically, but
at an extremely early developmental stage prior to germline
segregation, which happens on day 5 of development (33).
However, the simplest explanation, supported by modeling, is
that the full mutation is transmitted in the oocyte and reductional instability accounts for the mosaicism. This awaits direct
experimental confirmation and it remains possible that events
in the zygote promote expansion.
The molecular mechanism of CGG repeat expansion is still
elusive so far. The replication-based model, in which slippage
of perfect repeat Okazaki fragments leads to repeat expansion,
has been most favored in the literature, at least for premutation
to premutation variation. According to this model, for most
normal alleles, CGG repeats are interrupted by two AGGs and
the long perfect repeat number is <30, which is no longer than
the normal mammalian Okazaki fragment length of 25–300
nucleotides; the long perfect repeat can be anchored by AGG
interruptions. When the nascent strand contains >30 perfect
repeats without an interruption as reference, the Okazaki fragment with CGG repeats only, will tend to slip during the replication, probably also influenced by unusual DNA structure
assumed by the repeat (11,34,35). However, this model cannot
easily explain how slippage could generate the huge expansion
during the transmission from premutation to full mutation.
Recently the RAD27 endonuclease in the yeast, or FEN-1, the
homolog in mammals, has been proposed to play a key role in
repeat instability (36). This endonuclease activity is responsible for the removal of the 5′ ribonucleotide, derived from the
RNA primer and the 5′ end of an Okazaki fragment that is
displaced by the growing 3′ end of the preceding Okazaki fragment. Deletion of RAD27 in yeast was found to destabilize
dinucleotide arrays, favoring repeat addition and also the accumulation of duplication mutations, which indicate that RAD27
might therefore be involved in triplet repeat expansion (37–
39). The mammalian RAD27 homolog, FEN-1, functions both
in the processing Okazaki fragments during lagging-strand
synthesis and in the processing of branched structures which
arise in long-patch base excision repair, which indicates that
FEN-1 substrate must be single-stranded (40). Structural
analysis suggested that triplet repeats could form an intramolecular secondary structure (hairpin), which will be resistant to
FEN-1 endonuclease activity (36,41,42). In the absence of
RAD27 in yeast, the observation that triplet repeats, including
CGG, CAG and CTG, had much elevated frequency of expansions supports this idea (43,44). Also, it was demonstrated that
in yeast, secondary structure can indeed inhibit flap processing
at triplet repeats in a length-dependent manner by concealing
Human Molecular Genetics, 2000, Vol. 9, No. 6 Review
the 5′ end of the flap that is necessary for both binding and
cleavage by FEN-1 (45). So understanding the physiological
significance of FEN-1 will be important to solve the mystery
surrounding the triplet repeat expansion. In addition, DNA
damage has been proposed to play a significant role in triplet
repeat expansion. In Escherichia coli, the deficiency of some
nucleotide excision repair functions can dramatically affect the
stability of long repeat inserts in plasmid (46). A single abasic
site analog at the 5′ end template of the triplet repeat tract can
induce massive triplet repeat expansion during DNA synthesis
in vitro (47). Indeed, DNA cleavage of the repeat, due to
repair, may initiate strand invasion triggering expansion
through an unscheduled DNA replication mechanism, such as
gene conversion-type events.
To fully understand the timing and molecular mechanism of
CGG repeat expansion, the animal models, which display
CGG repeat instability during germline transmission, will be
essential. Only in animal models will it become possible to
easily study gametogenesis and early embryogenesis at
specific time points. Unfortunately, no such animal model is
currently available. Creation of animal models with fullmutation CGG repeats is currently technically challenging due
to the extreme instability of full mutation in E.coli. Transgenic
mice with FMR1 premutation allele were generated by two
separate groups; however, no instability of the CGG repeat was
observed in both cases (48,49). Recently, YAC transgenic
mice have been generated that carry the human FMR1 gene
containing various sized CGG repeats and no instability of
CGG repeat was detected (A.M. Peier and D.L. Nelson,
submitted for publication). In these transgenic mice, the integration sites of either transgene or YAC are random, which
may indicate that triplet repeat instability requires the cisacting element(s). This is further supported by association
studies, although this remains controversial (50). It will be
interesting to study the instability of CGG repeats with
premutation size at murine FMR1 or adjacent locus using a
gene-targeting approach (knock-in). Furthermore, these
knock-in mice will be tested in different genetic backgrounds
and applied to mutagenesis to define the pathways that lead to
triplet repeat instability. However, it is possible that there may
exist interspecies differences between human and mouse in
this regard and that humans lack mechanisms, present in mice,
which stabilize the repeats.
The timing and molecular mechanism of methylation on
expanded CGG repeat, which lead to FMR1 transcriptional
silencing, are also under extensive study. As discussed earlier,
all alleles from intact ovaries of full-mutation fetuses are fully
expanded, but unmethylated. However, chrorionic villi samples taken at different stages of development were shown to be
methylated to various degrees (32). Also, the studies on human
lymphoblastoids of full mutation showed that the methylation
of individual CpG cytosine is strikingly variable in hypermethylated epigenotype obtained from a single individual (51).
These results indicate that the methylation of expanded CGG
repeats occurs within a broad window and is a dynamic
process. Three distinct families of DNA methyltransferase
genes, named Dnmt1, Dnmt2 and Dnmt3, have been identified
in mammalian cells, but which DNA methyltransferase(s) is
involved in the methylation of expanded CGG repeats is still
unknown (52). Recently it has been shown that methylation of
CGG repeats causes transcriptional silencing through histone
903
deacetylation (53). In the cells from normal individuals, the 5′
end of FMR1, which contains CGG repeats, is associated with
acetylated histones H3 and H4, but acetylation is reduced in
cells from hypermethylated full mutation (53). Histone
deacetylation at the FMR1 locus, induced by the hypermethylation of expanded CGG repeats, will alter the chromatin structure of the 5′ end of the FMR1 gene, which will lead to
transcriptional silence (Fig. 1). Treatment of fragile X cells
with 5-azadeoxycytidine (5-aza-dC), which induces DNA
demethylation through the inhibition of DNA methyltransferase, resulted in reassociation of acetylated histone H3 and
H4 with FMR1 and transcriptional reactivation (53,54). But
treatment of fragile X cells with different inhibitors of histone
deacetylases resulted in little reactivation of the FMR1 gene.
However, combining these histone deacetylase inhibitors with
5-aza-dC can lead to greater increase of FMR1 transcripts than
5-aza-dC alone, which suggests a synergistic effect of histone
hyperacetylation and DNA demethylation in the reactivation of
FMR1 full mutation (55). This reactivation strategy may
provide a potential therapeutic approach for fragile X syndrome. However, the side effect of these drugs and the difficulty in efficiently translating FMR1 transcripts with large
repeats may limit this approach (27,56).
PHYSIOLOGICAL FUNCTION(S) OF FMRP
Since fragile X syndrome is a single gene disorder caused by
the absence of FMRP, the question becomes how the absence
of FMRP induces the clinical phenotype. The answer to this
question requires a full understanding of the physiological
function(s) of FMRP. The FMR1 gene is widely expressed in
both human and murine tissues. In situ hybridization with the
adult mouse tissues showed abundant expression in brain,
testes, ovary, esophageal epithelium, thymus, eye and spleen,
with moderate expression in colon, uterus, thyroid and liver,
and no expression in the heart, aorta or muscle (17). Analysis
of the amino acid sequence of FMRP revealed the presence of
two types of RNA-binding motif, two ribonucleoprotein K
homology domains (KH domains) and clusters of arginine and
glycine residues (RGG boxes), which suggested that FMRP is
an RNA-binding protein (20,21). In addition, a patient carrying
a missense mutation was identified with unusually severe
mental retardation (15). The point mutation alters an isoleucine
residue to asparagine at position 304 (I304N), which is located
at the second KH domain of FMRP, and this isoleucine is well
conserved in almost every KH domain of different proteins
(57,58). This further suggested that the RNA-binding property
of FMRP is critical for its functions. Also two autosomal
homologs of the FMR1 gene, FXR1 and FXR2, have been identified and the overall structures of the corresponding proteins
are very similar to that of FMRP (59,60). It has been proposed
that due to their similarities, FXR1P and FXR2P can compensate for the function(s) of FMRP in its absence, which leads to
not lethal but very mild phenotype in both human and mouse.
However, cells from fragile X patients and the FMR1 knockout
mice lacking FMRP expression have a normal expression level
of both FXR1P and FXR2P (61). The functions of this FMRP
family, including FMRP, FXR1P and FXR2P, have been
extensively studied in the past several years (62).
Studies of the RNA-binding specificity of FMRP showed
that FMRP preferentially binds to poly(G) and poly(U) instead
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Human Molecular Genetics, 2000, Vol. 9, No. 6 Review
Figure 1. Mechanism of FMR1 transcriptional suppression via CGG repeat expansion. The normal chromatin structure around CGG repeats is shown at the top.
The expanded CGG repeats (800) are first methylated (step A). Then a methyl-binding domain protein (MeCP2) will bind to hypermethylated CGG repeats and
form a complex with histone deacetylases (HDAC) via the transcription repressor (Sin3) (step B). HDACs will deacetylate histones H3 and H4 of chromatin around
CGG repeats (step C). Histone deacetylation will lead to chromatin remodeling (chromatin condensation) and repress transcription, presumably by activator exclusion (step D).
of poly(C) and poly(A) in vitro. Moreover, FMRP can only
bind to selective brain mRNAs in vitro, including the 3′-UTRs
of myelin basic protein (MBP) and the FMR1 message itself
(63). However, the in vivo binding specificity and the native
RNA targets of FMRP remain to be determined. This will be
very critical for understanding the physiological functions of
FMRP. The other side of this story is which region(s) of FMRP
interact(s) with RNA. Due to the presence of KH domains and
RGG box within FMRP, it would be assumed that these
regions should be responsible for RNA interaction. However,
by creating deletion mutants of FMRP, the N-terminal stretch,
two KH regions and C-terminal stretch, it has been shown that
only the first KH domain but not the second interacts with
RNA (64). This result is consistent with the observation that
FMRP with a point mutation (I304N) at the second KH domain
can bind to RNA, although this mutation can disrupt the structure of the KH domain (63). More interestingly, both the Nand C-terminal regions show RNA-binding ability. Within the
N-terminal stretch, no known RNA-binding motif is identifiable and this region binds to poly(G) preferentially, similar to
the first KH domains. The RNA binding by the C-terminus,
which contains an RGG box, appears non-specific, as it recognizes the bases with comparable affinity (64). Based on these
data, it has been proposed that FMRP is a protein with multiple
sites of interaction with RNA: sequence specificity is most
likely achieved by the whole block that compromises the first
∼400 residues, whereas the C-terminus provides a non-specific
binding surface (64). The crystal structure of full-length FMRP
will ultimately address this question.
FMRP has also been shown to associate with actively translating polyribosomes in an RNA-dependent manner via
messenger ribonucleoprotein (mRNP) particles (65,66). First,
FMRP–ribosome association is sensitive to low levels of
RNase, which removes the mRNA linking translating polyribosomes without disturbing ribosome assembly (65–67).
Secondly, EDTA treatment, which dissociates ribosomes into
subunits, releases FMRP into complexes with a similar size
range to a large mRNP particle, which is ∼660 kDa (67,68).
However, in the cells with the I304N mutant FMRP, despite
normal expression and cytoplasmic RNA association, the
Human Molecular Genetics, 2000, Vol. 9, No. 6 Review
mRNP particles are of smaller size, and do not associate with
actively translating polyribosomes (68). Thirdly, nearly all the
cytoplasmic FMRP can be co-captured with poly(A) RNA
(65,68). Taken together, these data suggest that FMRP associates with ribosomes as a component of mRNP particles.
Considering the severe phenotype of patients with point mutations, the association of FMRP with polyribosomes must be
functionally important. Recent studies show that FMRP can
form dimers, and point-mutation proteins fail to do so, which
suggests that dimerization may be critical for the association of
FMRP with polyribosomes (D.M. Absher and S.T. Warren,
manuscript in preparation). The proteins that make up the
FMRP-containing mRNPs remain largely unknown. Recently,
a cell culture system expressing epitope-tagged FMRP has
been developed. By immunoprecipitation, at least six other
proteins are shown to form the mRNP particles along with
FMRP (69). Two of these proteins are FXR1P and FXR2P,
which is consistent with previous findings (60). Additionally,
nucleolin, a known component of other mRNPs, is also present
in the FMRP-containing mRNP particles (69,70). Identification of the remaining associated proteins will be important for
the analysis of FMRP function(s). The observation that FMRP
is associated with polyribosomes suggests that FMRP may
modulate mRNA translation and influence mRNA instability
in vivo. Indeed, recent studies have shown that FMRP can
suppress translation of bound messages in an in vitro translation assay (Z. Li and Y. Feng, manuscript in preparation).
Although FMRP is predominantly localized in the cytoplasm, both a functional nuclear localization signal (NLS) and
nuclear export signal (NES) have been identified within FMRP
using FMRP deletion constructs (67). Whereas NLS is located
in the N-terminus of FMRP, the NES of FMRP, encoded by
exon 14, closely resembles the NES motifs described for HIV1 Rev and protein kinase inhibitor (PKI) and is sufficient to
direct nuclear export of a microinjected protein conjugate (67).
The presence of these localization signals suggests that FMRP
may shuttle between the nucleus and cytoplasm. Immunogold
electron microscopy demonstrated that FMRP exists in the
nucleus and has been observed in transit through the nuclear
pore (71). Based on these observations, it has been proposed
that FMRP plays a role in specific mRNA export from nucleus
to cytoplasm (67). Nascent FMRP enters the nucleus and there
assembles into an mRNP, interacting with specific RNA transcripts and other proteins. The mRNP particles containing
FMRP and its target RNA will be transported to the cytoplasm.
Alternatively a possible role in modulating the localization,
stability and/or translation of its target mRNA has also been
hypothesized. Recently, FMRP has been shown to be a phosphoprotein and a substrate of the Fes non-receptor tyrosine
kinase (72). Interestingly, tyrosine phosphorylated FMRP is
chiefly located in the nucleus. The significance of phosphorylation status of FMRP in the shuttle between nucleus and
cytoplasm is unclear at present, but suggests regulation by
signal transduction. The two homologs of FMRP, FXR1P and
FXR2P, also contain an NLS and an NES signal motif at a
similar position to that seen in FMRP. Although FXR2P has a
similar signal motif to FMRP, it was shown that FMRP and
FXR2P have distinct nuclear localization (73). FMRP shuttles
between cytoplasm and nucleoplasm, whereas FXR2P shuttles
between cytoplasm and nucleolus, which suggests that they
may transport different RNAs or have different physiological
905
functions (73). This idea is further supported by the identification of a novel RNA-binding nuclear protein (NUFIP) that
interacts with FMRP. NUFIP only interacts with FMRP and
not FXR1P and FXR2P (70). It is therefore possible that these
three homologous proteins interact with different proteins/sites
within the nucleus and may have specific rather than overlapping function(s) there but appear to coalesce, at least partially,
in the cytoplasm, forming a common mRNP particle.
Since the primary phenotype of fragile X syndrome, mental
retardation, occurs in the CNS, the question becomes what the
function of FMRP is in CNS, or how the absence of FMRP
leads to cognition deficit. Based on the above findings, it was
hypothesized that in neurons FMRP may play a role in the
transportation of its target mRNAs from nucleus to cytoplasm,
modulate the localization of these mRNAs, and further regulate the local protein synthesis by being part of polyribosomes,
which will be important for normal neuronal development and
functions (Fig. 2). Several observations support this idea. First,
immunogold studies showed that FMRP was localized in
neuronal nucleoplasm and within the nuclear pore, which
suggests that FMRP indeed participates in the nuclear export
of mRNA in the neuron (71). Secondly, ultrastructural studies
in rat brain revealed high levels of FMRP immunoreactivity in
neuronal perikarya, where it is concentrated in regions rich in
ribosomes, in particular near or between rough endoplasmic
reticulum cisternae (71). Moreover, FMRP was also observed
in large- and small-caliber dendrites, in dendritic branch
points, at the origins of spine necks and in spine heads, all
known locations of neuronal polyribosomes (71). Thirdly,
FMRP can be co-fractionated with synaptosomal ribosomes,
which indicates that FMRP may regulate the local protein
synthesis of synaptosomes (71). The regulation of protein
synthesis within the dendritic spine is important for synaptic
development and brain plasticity (74–77). Consistent with this
idea, abnormal dendritic spines were observed both in brains of
fragile X patients and in FMR1 knockout mice (78,79). This
suggests that in most fragile X patients, due to the absence of
FMRP, the protein synthesis is misregulated during synapse
development, which may lead to mental retardation. In the
patient with a point mutation (I304N), FMRP was unable to
dimerize and FMRP-containing mRNP failed to associate with
the polyribosome, which may sequester FMRP-bound mRNA
from translation, thus resulting in an unusually severe phenotype (68) (Fig. 2). The regulation of synaptic-activitydependent protein synthesis by FMRP is further supported by
the observation that the translation of FMRP was increased in
rat synaptoneurosomes after stimulation by a specific mGluR
agonist (80). To further understand the neuronal functions of
FMRP, the animal model will be important. However,
currently the only available animal model is the FMR1
knockout mouse, whose behavioral abnormalities are
extremely subtle, limiting widespread analysis (81; A.M. Peier
and D.L. Nelson, submitted for publication). Development of
new animal models including mice with regulatable FMRP
expression will be very helpful to dissect the role(s) of FMRP
during neuronal development and synapse formation.
CONCLUSION
A great deal has been learned about fragile X syndrome since
the discovery in 1991 of the FMR1 gene and the molecular
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Human Molecular Genetics, 2000, Vol. 9, No. 6 Review
models that display similar CGG repeat instability during
transmission through generations will be critical to the understanding of the molecular mechanism of CGG repeat expansion, which remains very elusive. These studies will not only
help us to understand the fragile X syndrome but also be
important in understanding the molecular basis of at least
another 12 human disorders with similar dynamic mutation,
trinucleotide repeat expansion. On the other hand, the physiological function(s) of FMRP is being extensively studied to
address how the absence of FMRP leads to the clinical phenotype of fragile X syndrome. To ultimately answer this question
and develop effective intervention to fragile X syndrome, both
molecular genetic and neurobiological approaches have to be
combined to bring closer molecular abnormalities to the
neurobehavioral phenotypes. New animal models and paradigms of behavior testing have to be developed. These studies
will also provide us with insight into the mechanisms of cognition, memory and behavior in human. Moreover, discovery of
those genes whose mRNAs interact with FMRP will provide
candidate genes for other disorders limiting cognition development.
ACKNOWLEDGEMENTS
P.J. is an associate and S.T.W. is an investigator with the
Howard Hughes Medical Institute. This work is supported, in
part, by NIH grants HD20521 and HD35576 to S.T.W.
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Figure 2. A speculative model for FMRP function(s). (A) In the normal individual, nascent FMRP, dimerized in the cytoplasm, enters the nucleus and
there assembles into mRNP, interacting with specific RNA transcripts and
other proteins, some of which are known. The mRNP particles containing
FMRP and its target mRNA will be transported to cytoplasm and the mRNP
presented to the ribosome. In the neurons, some of these FMRP-containing
mRNPs will be localized in the dendrites and participate in local protein synthesis. (B) In most fragile X patients, without FMRP, the target mRNAs of
FMRP may interact with alternative mRNP allowing partial translation but
abnormal in regulation, localization or abundance. (C) In the patient with a
point mutation (I304N), FMRP fails to dimerize in the cytoplasm and forms a
partial mRNP, which can rapidly shuttle between the nucleus and cytoplasm.
FMRP-containing mRNPs now fail to associate with the polyribosome, perhaps sequestering FMRP-bound mRNA from translation, resulting in an unusually severe phenotype through suppression of even partial translation.
basis of this disorder. However, many questions remain.
Although CGG repeat expansion seems to happen during
meiosis or early embryonic development, the exact timing of
expansion has yet to be determined. Developing the animal
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