You are on page 1of 8

© 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 expres-
sion 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 the FMR1 gene. Alleles with between 60 and 230 CGG repeats
are called premutation. They are generally unmethylated with
Fragile X syndrome is one of the most common forms of inher-
normal transcript and protein level, but are extremely unstable
ited mental retardation with the estimated incidence of 1 in during transmission to next generation (1,14). Expansion of
4000 males and 1 in 8000 females (1,2). The syndrome is premutation into full mutation can only occur by maternal
transmitted as an X-linked dominant trait and with reduced transmission and depends on the length of the maternal
penetrance (80% in males and 30% in females) (1,2). Fragile X premutation. Due to X-linkage, affected males have more
syndrome is associated with a fragile site, designated FRAXA severe phenotypes than affected females, whose phenotype is
(Fragile site, X chromosome, A site), at Xq27.3 near the end of modulated by the activation ratio of the normal X chromo-
the long arm. The clinical presentations of fragile X syndrome some. Identification of other mutations of the FMR1 gene,
include mild to severe mental retardation, with IQ between 20 such as deletions and point mutation among patients with usual
and 60, mildly abnormal facial features of a prominent jaw and phenotype but without fragile site expression, firmly estab-
large ears, mainly in males, and macroorchidism in post- lished that the FMR1 gene is the only gene involved in the
pubescent males. Many patients also display subtle connective pathogenesis of fragile X syndrome (15,16). Thus, the absence
tissue abnormalities, hyperactive and attention deficit disorder of the FMR1 gene product, fragile X mental retardation protein
and autistic-like behavior (1,2). In 1991, the molecular basis of (FMRP), is the typical cause of fragile X syndrome. The FMR1
fragile X syndrome was revealed by positioning cloning and gene is widely expressed in both human and murine tissues
shown to be associated with a massive trinucleotide repeat (17). Multiple FMRP isoforms, through extensive alternative
expansion within the gene fragile X mental retardation-1 splicing at the 3′ end, appear in a variety of tissues, and some
(FMR1) (3–6). This was one of the first identified human of them are present at different quantitative levels (7,18,19).
disorders caused by dynamic mutation, trinucleotide repeat FMRP has been implicated in RNA binding and is possibly
expansion. involved in translational control (20,21). An animal model of
FMR1 is a highly conserved gene that consists of 17 exons fragile X syndrome was created using gene targeting (22). The
and spans ∼38 kb (7,8). Within the 4.4 kb of FMR1 transcript, FMR1 knockout mice exhibit macroorchidism and a subtle
a CGG trinucleotide repeat is located at the 5′-untranslated deficit in learning and memory, which mimic the human
region (5′-UTR). Among normal individuals, this CGG repeat phenotype.
is highly polymorphic in length and content, often punctuated Recent research on fragile X syndrome has been focused on
by AGG interruptions (9–13). The normal repeat size ranges two areas. (i) Instability and methylation of CGG repeats when
from 7 to ∼60, with 30 repeats found on the most common and how does CGG repeat instability occur? What is the
allele. In most affected individuals, CGG repeats are massively molecular mechanism of DNA methylation of expanded CGG
expanded over 230 repeats (full mutation) and becomes repeats? (ii) Physiological function(s) of FMRP; what genes
abnormally hypermethylated, which results in the silence of are the targets of FMRP in vivo? As an RNA-binding protein,

+To whom correspondence should be addressed. Tel: +1 404 727 5979; Fax: +1 404 727 5408; Email: swarren@bimcore.emory.edu
902 Human Molecular Genetics, 2000, Vol. 9, No. 6 Review

what role(s) does FMRP play in translation? How does the exists post-zygotically (28). The supportive evidence is that
absence of FMRP lead to deficit in learning and memory full mutation carriers are found to be somatic mosaicisms of
during development? Here we will review the latest progress CGG repeats (28,29). However, the length of CGG repeats on
made in these two areas towards understanding the molecular a particular allele in a differentiated cell derived from these
basis of fragile X syndrome. carriers is generally mitotically stable although recent studies
show that fully expanded FMR1 CGG repeats exhibit a length-
and differentiation-dependent instability in cell hybrids (30,
INSTABILITY AND METHYLATION OF CGG 31). Analysis of CGG repeats in gametes of fetuses with full
REPEATS mutation concluded that the testes of a 13 week full-mutation
In the normal population, the CGG repeat is polymorphic but fetus showed no evidence of premutations, whereas a 17 week
inherited in a stable fashion. The repeat is cryptic with inter- full-mutation fetus exhibited some germ cells with attributes of
spersed AGG repeats most often found at repeat positions 10 premutations (32). Also only full-expansion alleles can be
and 20 (11). The CGG repeat length variation is found at the 3′ detected in the oocytes of full-mutation fetuses (32). These
end of the repeat and it appears that AGG triplets play a crucial results indicate that CGG repeat instability happens either
role in the maintenance of repeat stability (11). Most premuta- during premeiotic or meiotic division, or post-zygotically, but
tion alleles have either no AGG or only a single AGG interrup- at an extremely early developmental stage prior to germline
tion at the 5′ end of the repeat (10,13,23). Loss of these segregation, which happens on day 5 of development (33).
interruptions leads to alleles with longer perfect CGG repeat However, the simplest explanation, supported by modeling, is
tracts, which are prone to expansion into premutation, espe- that the full mutation is transmitted in the oocyte and reduc-
cially when tracts contain >30 perfect CGG repeats. In contrast tional instability accounts for the mosaicism. This awaits direct
to the normal allele, premutation is unstable and may expand to experimental confirmation and it remains possible that events
a full mutation or a different sized premutation (6). No new in the zygote promote expansion.
mutations have been reported with all abnormal alleles ulti- The molecular mechanism of CGG repeat expansion is still
mately derived from premutations, at least in the most recent elusive so far. The replication-based model, in which slippage
generations. The risk of expansion of a premutation to a full of perfect repeat Okazaki fragments leads to repeat expansion,
mutation in the next generation exponentially increases with has been most favored in the literature, at least for premutation
the number of repeats between 65 and 100. This phenomenon to premutation variation. According to this model, for most
resolves anticipation in fragile X syndrome (also known as normal alleles, CGG repeats are interrupted by two AGGs and
‘Sherman Paradox’), which manifests clinically as an increase the long perfect repeat number is <30, which is no longer than
in the number and proportion of mentally retarded individuals the normal mammalian Okazaki fragment length of 25–300
in successive generations in fragile X families (24,25). nucleotides; the long perfect repeat can be anchored by AGG
Remarkably, premutation only expands to a full mutation interruptions. When the nascent strand contains >30 perfect
when it is transmitted by female carrier, but not male carrier. repeats without an interruption as reference, the Okazaki frag-
The reason for this is that full mutation males have only ment with CGG repeats only, will tend to slip during the repli-
premutation-size alleles in their sperm (see below) (26). In the cation, probably also influenced by unusual DNA structure
full mutation, the CGG repeat is massively expanded and assumed by the repeat (11,34,35). However, this model cannot
hypermethylated, which results in the repression of the FMR1 easily explain how slippage could generate the huge expansion
gene and leads to the loss of FMRP. However, mosaicism of during the transmission from premutation to full mutation.
both CGG repeat length and methylation status exists in fragile Recently the RAD27 endonuclease in the yeast, or FEN-1, the
X patients (1). In most affected individuals, somatic variation homolog in mammals, has been proposed to play a key role in
of repeat length is found in all tissues and is pronounced in repeat instability (36). This endonuclease activity is respon-
∼15% of patients, termed mosaics. In addition, some full muta- sible for the removal of the 5′ ribonucleotide, derived from the
tion carriers have unmethylated, premutation bands, which RNA primer and the 5′ end of an Okazaki fragment that is
presumably produce FMRP and which can be found in some displaced by the growing 3′ end of the preceding Okazaki frag-
high-functioning fragile X patients. Also some full-mutation ment. Deletion of RAD27 in yeast was found to destabilize
alleles are partially methylated or completely unmethylated. dinucleotide arrays, favoring repeat addition and also the accu-
Some evidence has been presented to indicate that FMR1 tran- mulation of duplication mutations, which indicate that RAD27
scripts with large expansions are not well translated (27). might therefore be involved in triplet repeat expansion (37–
These observations indicate that CGG repeat expansion and 39). The mammalian RAD27 homolog, FEN-1, functions both
methylation of expanded CGG repeats are two separate events, in the processing Okazaki fragments during lagging-strand
of which the association is still not clear. Both methylation synthesis and in the processing of branched structures which
status and the repeat length on the active X chromosome will arise in long-patch base excision repair, which indicates that
influence the amount of FMRP produced, which is important FEN-1 substrate must be single-stranded (40). Structural
during the cognition development. analysis suggested that triplet repeats could form an intramo-
Although the timing of CGG repeat expansion is still not lecular secondary structure (hairpin), which will be resistant to
clear, two time periods have been implicated during which FEN-1 endonuclease activity (36,41,42). In the absence of
premutation is expanded to full mutation: meiosis and early RAD27 in yeast, the observation that triplet repeats, including
embryonic development. The anticipation in each succeeding CGG, CAG and CTG, had much elevated frequency of expan-
generation of fragile X families indicates that expansion must sions supports this idea (43,44). Also, it was demonstrated that
occur in germ cells (either during premeiotic or meiotic divi- in yeast, secondary structure can indeed inhibit flap processing
sion). Also, it has been postulated that CGG repeat instability at triplet repeats in a length-dependent manner by concealing
Human Molecular Genetics, 2000, Vol. 9, No. 6 Review 903

the 5′ end of the flap that is necessary for both binding and deacetylation (53). In the cells from normal individuals, the 5′
cleavage by FEN-1 (45). So understanding the physiological end of FMR1, which contains CGG repeats, is associated with
significance of FEN-1 will be important to solve the mystery acetylated histones H3 and H4, but acetylation is reduced in
surrounding the triplet repeat expansion. In addition, DNA cells from hypermethylated full mutation (53). Histone
damage has been proposed to play a significant role in triplet deacetylation at the FMR1 locus, induced by the hypermethyl-
repeat expansion. In Escherichia coli, the deficiency of some ation of expanded CGG repeats, will alter the chromatin struc-
nucleotide excision repair functions can dramatically affect the ture of the 5′ end of the FMR1 gene, which will lead to
stability of long repeat inserts in plasmid (46). A single abasic transcriptional silence (Fig. 1). Treatment of fragile X cells
site analog at the 5′ end template of the triplet repeat tract can with 5-azadeoxycytidine (5-aza-dC), which induces DNA
induce massive triplet repeat expansion during DNA synthesis demethylation through the inhibition of DNA methyltrans-
in vitro (47). Indeed, DNA cleavage of the repeat, due to ferase, resulted in reassociation of acetylated histone H3 and
repair, may initiate strand invasion triggering expansion H4 with FMR1 and transcriptional reactivation (53,54). But
through an unscheduled DNA replication mechanism, such as treatment of fragile X cells with different inhibitors of histone
gene conversion-type events. deacetylases resulted in little reactivation of the FMR1 gene.
To fully understand the timing and molecular mechanism of However, combining these histone deacetylase inhibitors with
CGG repeat expansion, the animal models, which display 5-aza-dC can lead to greater increase of FMR1 transcripts than
CGG repeat instability during germline transmission, will be 5-aza-dC alone, which suggests a synergistic effect of histone
essential. Only in animal models will it become possible to hyperacetylation and DNA demethylation in the reactivation of
easily study gametogenesis and early embryogenesis at FMR1 full mutation (55). This reactivation strategy may
specific time points. Unfortunately, no such animal model is provide a potential therapeutic approach for fragile X syn-
currently available. Creation of animal models with full- drome. However, the side effect of these drugs and the diffi-
mutation CGG repeats is currently technically challenging due culty in efficiently translating FMR1 transcripts with large
to the extreme instability of full mutation in E.coli. Transgenic repeats may limit this approach (27,56).
mice with FMR1 premutation allele were generated by two
separate groups; however, no instability of the CGG repeat was
PHYSIOLOGICAL FUNCTION(S) OF FMRP
observed in both cases (48,49). Recently, YAC transgenic
mice have been generated that carry the human FMR1 gene Since fragile X syndrome is a single gene disorder caused by
containing various sized CGG repeats and no instability of the absence of FMRP, the question becomes how the absence
CGG repeat was detected (A.M. Peier and D.L. Nelson, of FMRP induces the clinical phenotype. The answer to this
submitted for publication). In these transgenic mice, the inte- question requires a full understanding of the physiological
gration sites of either transgene or YAC are random, which function(s) of FMRP. The FMR1 gene is widely expressed in
may indicate that triplet repeat instability requires the cis- both human and murine tissues. In situ hybridization with the
acting element(s). This is further supported by association adult mouse tissues showed abundant expression in brain,
studies, although this remains controversial (50). It will be testes, ovary, esophageal epithelium, thymus, eye and spleen,
interesting to study the instability of CGG repeats with with moderate expression in colon, uterus, thyroid and liver,
premutation size at murine FMR1 or adjacent locus using a and no expression in the heart, aorta or muscle (17). Analysis
gene-targeting approach (knock-in). Furthermore, these of the amino acid sequence of FMRP revealed the presence of
knock-in mice will be tested in different genetic backgrounds two types of RNA-binding motif, two ribonucleoprotein K
and applied to mutagenesis to define the pathways that lead to homology domains (KH domains) and clusters of arginine and
triplet repeat instability. However, it is possible that there may glycine residues (RGG boxes), which suggested that FMRP is
exist interspecies differences between human and mouse in an RNA-binding protein (20,21). In addition, a patient carrying
this regard and that humans lack mechanisms, present in mice, a missense mutation was identified with unusually severe
which stabilize the repeats. mental retardation (15). The point mutation alters an isoleucine
The timing and molecular mechanism of methylation on residue to asparagine at position 304 (I304N), which is located
expanded CGG repeat, which lead to FMR1 transcriptional at the second KH domain of FMRP, and this isoleucine is well
silencing, are also under extensive study. As discussed earlier, conserved in almost every KH domain of different proteins
all alleles from intact ovaries of full-mutation fetuses are fully (57,58). This further suggested that the RNA-binding property
expanded, but unmethylated. However, chrorionic villi sam- of FMRP is critical for its functions. Also two autosomal
ples taken at different stages of development were shown to be homologs of the FMR1 gene, FXR1 and FXR2, have been iden-
methylated to various degrees (32). Also, the studies on human tified and the overall structures of the corresponding proteins
lymphoblastoids of full mutation showed that the methylation are very similar to that of FMRP (59,60). It has been proposed
of individual CpG cytosine is strikingly variable in hypermeth- that due to their similarities, FXR1P and FXR2P can compen-
ylated epigenotype obtained from a single individual (51). sate for the function(s) of FMRP in its absence, which leads to
These results indicate that the methylation of expanded CGG not lethal but very mild phenotype in both human and mouse.
repeats occurs within a broad window and is a dynamic However, cells from fragile X patients and the FMR1 knockout
process. Three distinct families of DNA methyltransferase mice lacking FMRP expression have a normal expression level
genes, named Dnmt1, Dnmt2 and Dnmt3, have been identified of both FXR1P and FXR2P (61). The functions of this FMRP
in mammalian cells, but which DNA methyltransferase(s) is family, including FMRP, FXR1P and FXR2P, have been
involved in the methylation of expanded CGG repeats is still extensively studied in the past several years (62).
unknown (52). Recently it has been shown that methylation of Studies of the RNA-binding specificity of FMRP showed
CGG repeats causes transcriptional silencing through histone that FMRP preferentially binds to poly(G) and poly(U) instead
904 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 exclu-
sion (step D).

of poly(C) and poly(A) in vitro. Moreover, FMRP can only the first KH domains. The RNA binding by the C-terminus,
bind to selective brain mRNAs in vitro, including the 3′-UTRs which contains an RGG box, appears non-specific, as it recog-
of myelin basic protein (MBP) and the FMR1 message itself nizes the bases with comparable affinity (64). Based on these
(63). However, the in vivo binding specificity and the native data, it has been proposed that FMRP is a protein with multiple
RNA targets of FMRP remain to be determined. This will be sites of interaction with RNA: sequence specificity is most
very critical for understanding the physiological functions of likely achieved by the whole block that compromises the first
FMRP. The other side of this story is which region(s) of FMRP ∼400 residues, whereas the C-terminus provides a non-specific
interact(s) with RNA. Due to the presence of KH domains and binding surface (64). The crystal structure of full-length FMRP
RGG box within FMRP, it would be assumed that these will ultimately address this question.
regions should be responsible for RNA interaction. However, FMRP has also been shown to associate with actively trans-
by creating deletion mutants of FMRP, the N-terminal stretch, lating polyribosomes in an RNA-dependent manner via
two KH regions and C-terminal stretch, it has been shown that messenger ribonucleoprotein (mRNP) particles (65,66). First,
only the first KH domain but not the second interacts with FMRP–ribosome association is sensitive to low levels of
RNA (64). This result is consistent with the observation that RNase, which removes the mRNA linking translating poly-
FMRP with a point mutation (I304N) at the second KH domain ribosomes without disturbing ribosome assembly (65–67).
can bind to RNA, although this mutation can disrupt the struc- Secondly, EDTA treatment, which dissociates ribosomes into
ture of the KH domain (63). More interestingly, both the N- subunits, releases FMRP into complexes with a similar size
and C-terminal regions show RNA-binding ability. Within the range to a large mRNP particle, which is ∼660 kDa (67,68).
N-terminal stretch, no known RNA-binding motif is identifi- However, in the cells with the I304N mutant FMRP, despite
able and this region binds to poly(G) preferentially, similar to normal expression and cytoplasmic RNA association, the
Human Molecular Genetics, 2000, Vol. 9, No. 6 Review 905

mRNP particles are of smaller size, and do not associate with functions (73). This idea is further supported by the identifica-
actively translating polyribosomes (68). Thirdly, nearly all the tion of a novel RNA-binding nuclear protein (NUFIP) that
cytoplasmic FMRP can be co-captured with poly(A) RNA interacts with FMRP. NUFIP only interacts with FMRP and
(65,68). Taken together, these data suggest that FMRP associ- not FXR1P and FXR2P (70). It is therefore possible that these
ates with ribosomes as a component of mRNP particles. three homologous proteins interact with different proteins/sites
Considering the severe phenotype of patients with point muta- within the nucleus and may have specific rather than overlap-
tions, the association of FMRP with polyribosomes must be ping function(s) there but appear to coalesce, at least partially,
functionally important. Recent studies show that FMRP can in the cytoplasm, forming a common mRNP particle.
form dimers, and point-mutation proteins fail to do so, which Since the primary phenotype of fragile X syndrome, mental
suggests that dimerization may be critical for the association of retardation, occurs in the CNS, the question becomes what the
FMRP with polyribosomes (D.M. Absher and S.T. Warren, function of FMRP is in CNS, or how the absence of FMRP
manuscript in preparation). The proteins that make up the leads to cognition deficit. Based on the above findings, it was
FMRP-containing mRNPs remain largely unknown. Recently, hypothesized that in neurons FMRP may play a role in the
a cell culture system expressing epitope-tagged FMRP has transportation of its target mRNAs from nucleus to cytoplasm,
been developed. By immunoprecipitation, at least six other modulate the localization of these mRNAs, and further regu-
proteins are shown to form the mRNP particles along with late the local protein synthesis by being part of polyribosomes,
FMRP (69). Two of these proteins are FXR1P and FXR2P, which will be important for normal neuronal development and
which is consistent with previous findings (60). Additionally, functions (Fig. 2). Several observations support this idea. First,
nucleolin, a known component of other mRNPs, is also present immunogold studies showed that FMRP was localized in
in the FMRP-containing mRNP particles (69,70). Identifica- neuronal nucleoplasm and within the nuclear pore, which
tion of the remaining associated proteins will be important for suggests that FMRP indeed participates in the nuclear export
the analysis of FMRP function(s). The observation that FMRP of mRNA in the neuron (71). Secondly, ultrastructural studies
is associated with polyribosomes suggests that FMRP may in rat brain revealed high levels of FMRP immunoreactivity in
modulate mRNA translation and influence mRNA instability neuronal perikarya, where it is concentrated in regions rich in
in vivo. Indeed, recent studies have shown that FMRP can ribosomes, in particular near or between rough endoplasmic
suppress translation of bound messages in an in vitro transla- reticulum cisternae (71). Moreover, FMRP was also observed
tion assay (Z. Li and Y. Feng, manuscript in preparation). in large- and small-caliber dendrites, in dendritic branch
Although FMRP is predominantly localized in the cyto- points, at the origins of spine necks and in spine heads, all
plasm, both a functional nuclear localization signal (NLS) and known locations of neuronal polyribosomes (71). Thirdly,
nuclear export signal (NES) have been identified within FMRP FMRP can be co-fractionated with synaptosomal ribosomes,
using FMRP deletion constructs (67). Whereas NLS is located which indicates that FMRP may regulate the local protein
in the N-terminus of FMRP, the NES of FMRP, encoded by synthesis of synaptosomes (71). The regulation of protein
exon 14, closely resembles the NES motifs described for HIV- synthesis within the dendritic spine is important for synaptic
1 Rev and protein kinase inhibitor (PKI) and is sufficient to development and brain plasticity (74–77). Consistent with this
direct nuclear export of a microinjected protein conjugate (67). idea, abnormal dendritic spines were observed both in brains of
The presence of these localization signals suggests that FMRP fragile X patients and in FMR1 knockout mice (78,79). This
may shuttle between the nucleus and cytoplasm. Immunogold suggests that in most fragile X patients, due to the absence of
electron microscopy demonstrated that FMRP exists in the FMRP, the protein synthesis is misregulated during synapse
nucleus and has been observed in transit through the nuclear development, which may lead to mental retardation. In the
pore (71). Based on these observations, it has been proposed patient with a point mutation (I304N), FMRP was unable to
that FMRP plays a role in specific mRNA export from nucleus dimerize and FMRP-containing mRNP failed to associate with
to cytoplasm (67). Nascent FMRP enters the nucleus and there the polyribosome, which may sequester FMRP-bound mRNA
assembles into an mRNP, interacting with specific RNA tran- from translation, thus resulting in an unusually severe pheno-
scripts and other proteins. The mRNP particles containing type (68) (Fig. 2). The regulation of synaptic-activity-
FMRP and its target RNA will be transported to the cytoplasm. dependent protein synthesis by FMRP is further supported by
Alternatively a possible role in modulating the localization, the observation that the translation of FMRP was increased in
stability and/or translation of its target mRNA has also been rat synaptoneurosomes after stimulation by a specific mGluR
hypothesized. Recently, FMRP has been shown to be a phos- agonist (80). To further understand the neuronal functions of
phoprotein and a substrate of the Fes non-receptor tyrosine FMRP, the animal model will be important. However,
kinase (72). Interestingly, tyrosine phosphorylated FMRP is currently the only available animal model is the FMR1
chiefly located in the nucleus. The significance of phosphor- knockout mouse, whose behavioral abnormalities are
ylation status of FMRP in the shuttle between nucleus and extremely subtle, limiting widespread analysis (81; A.M. Peier
cytoplasm is unclear at present, but suggests regulation by and D.L. Nelson, submitted for publication). Development of
signal transduction. The two homologs of FMRP, FXR1P and new animal models including mice with regulatable FMRP
FXR2P, also contain an NLS and an NES signal motif at a expression will be very helpful to dissect the role(s) of FMRP
similar position to that seen in FMRP. Although FXR2P has a during neuronal development and synapse formation.
similar signal motif to FMRP, it was shown that FMRP and
FXR2P have distinct nuclear localization (73). FMRP shuttles
CONCLUSION
between cytoplasm and nucleoplasm, whereas FXR2P shuttles
between cytoplasm and nucleolus, which suggests that they A great deal has been learned about fragile X syndrome since
may transport different RNAs or have different physiological the discovery in 1991 of the FMR1 gene and the molecular
906 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 under-
standing of the molecular mechanism of CGG repeat expan-
sion, 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 physio-
logical function(s) of FMRP is being extensively studied to
address how the absence of FMRP leads to the clinical pheno-
type 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 para-
digms of behavior testing have to be developed. These studies
will also provide us with insight into the mechanisms of cogni-
tion, memory and behavior in human. Moreover, discovery of
those genes whose mRNAs interact with FMRP will provide
candidate genes for other disorders limiting cognition develop-
ment.

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.

REFERENCES
1. Warren, S.T. and Sherman, S.L. (2000) The fragile X syndrome. In Scriver,
C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (eds), The Metabolic and
Molecular Basis of Inherited Disease, 8th edn. McGraw-Hill, New York,
NY, in press.
2. Warren, S.T. and Nelson, D.L. (1994) Advances in molecular analysis of
fragile X syndrome. J. Am. Med. Assoc., 271, 536–542.
3. Kremer, E.J., Pritchard, M., Lynch, M., Yu, S., Holman, K., Baker, E.,
Warren, S.T., Schlessinger, D., Sutherland, G.R. and Richards, R.I. (1991)
Mapping of DNA instability at the fragile X to a trinucleotide repeat
sequence p(CCG)n. Science, 252, 1711–1714.
4. Yu, S., Pritchard, M., Kremer, E., Lynch, M., Nancarrow, J., Baker, E.,
Holman, K., Mulley, J.C., Warren, S.T., Schlessinger, D. et al. (1991)
Fragile X genotype characterized by an unstable region of DNA. Science,
252, 1179–1181.
Figure 2. A speculative model for FMRP function(s). (A) In the normal indi- 5. Verkerk, A.J., Pieretti, M., Sutcliffe, J.S., Fu, Y.H., Kuhl, D.P., Pizzuti, A.,
vidual, nascent FMRP, dimerized in the cytoplasm, enters the nucleus and Reiner, O., Richards, S., Victoria, M.F., Zhang, F.P. et al. (1991)
there assembles into mRNP, interacting with specific RNA transcripts and Identification of a gene (FMR-1) containing a CGG repeat coincident with
other proteins, some of which are known. The mRNP particles containing a breakpoint cluster region exhibiting length variation in fragile X
FMRP and its target mRNA will be transported to cytoplasm and the mRNP syndrome. Cell, 65, 905–914.
presented to the ribosome. In the neurons, some of these FMRP-containing 6. Oberle, I., Rousseau, F., Heitz, D., Kretz, C., Devys, D., Hanauer, A., Boue,
mRNPs will be localized in the dendrites and participate in local protein syn- J., Bertheas, M.F. and Mandel, J.L. (1991) Instability of a 550-base pair
thesis. (B) In most fragile X patients, without FMRP, the target mRNAs of DNA segment and abnormal methylation in fragile X syndrome. Science,
FMRP may interact with alternative mRNP allowing partial translation but 252, 1097–1102.
abnormal in regulation, localization or abundance. (C) In the patient with a 7. Ashley, C.T., Sutcliffe, J.S., Kunst, C.B., Leiner, H.A., Eichler, E.E.,
point mutation (I304N), FMRP fails to dimerize in the cytoplasm and forms a Nelson, D.L. and Warren, S.T. (1993) Human and murine FMR-1:
partial mRNP, which can rapidly shuttle between the nucleus and cytoplasm. alternative splicing and translational initiation downstream of the CGG-
FMRP-containing mRNPs now fail to associate with the polyribosome, per- repeat. Nature Genet., 4, 244–251.
haps sequestering FMRP-bound mRNA from translation, resulting in an unu- 8. Eichler, E.E., Richards, S., Gibbs, R.A. and Nelson, D.L. (1993) Fine
sually severe phenotype through suppression of even partial translation. structure of the human FMR1 gene. Hum. Mol. Genet., 2, 1147–1153.
[Erratum (1994) Hum. Mol. Genet., 3, 684–685.]
9. Fu, Y.H., Kuhl, D.P., Pizzuti, A., Pieretti, M., Sutcliffe, J.S., Richards, S.,
Verkerk, A.J., Holden, J.J., Fenwick Jr, R.G., Warren, S.T. et al. (1991)
basis of this disorder. However, many questions remain. Variation of the CGG repeat at the fragile X site results in genetic instability:
resolution of the Sherman paradox. Cell, 67, 1047–1058.
Although CGG repeat expansion seems to happen during 10. Eichler, E.E., Holden, J.J., Popovich, B.W., Reiss, A.L., Snow, K.,
meiosis or early embryonic development, the exact timing of Thibodeau, S.N., Richards, C.S., Ward, P.A. and Nelson, D.L. (1994)
expansion has yet to be determined. Developing the animal Length of uninterrupted CGG repeats determines instability in the FMR1
gene. Nature Genet., 8, 88–94.
Human Molecular Genetics, 2000, Vol. 9, No. 6 Review 907

11. Kunst, C.B. and Warren, S.T. (1994) Cryptic and polar variation of the fragile 32. Malter, H.E., Iber, J.C., Willemsen, R., de Graaff, E., Tarleton, J.C., Leisti,
X repeat could result in predisposing normal alleles. Cell, 77, 853–861. J., Warren, S.T. and Oostra, B.A. (1997) Characterization of the full fragile
12. Snow, K., Doud, L.K., Hagerman, R., Pergolizzi, R.G., Erster, S.H. and X syndrome mutation in fetal gametes. Nature Genet., 15, 165–169.
Thibodeau, S.N. (1993) Analysis of a CGG sequence at the FMR-1 locus in 33. Larsen, W.J. (1993) Human Embryology. Churchill Livingstone, New
fragile X families and in the general population. Am. J. Hum. Genet., 53, York, NY.
1217–1228. 34. Eichler, E.E., Macpherson, J.N., Murray, A., Jacobs, P.A., Chakravarti, A.
13. Snow, K., Tester, D.J., Kruckeberg, K.E., Schaid, D.J. and Thibodeau, S.N. and Nelson, D.L. (1996) Haplotype and interspersion analysis of the FMR1
(1994) Sequence analysis of the fragile X trinucleotide repeat: implications CGG repeat identifies two different mutational pathways for the origin of
for the origin of the fragile X mutation. Hum. Mol. Genet., 3, 1543–1551. the fragile X syndrome. Hum. Mol. Genet., 5, 319–330.
14. Feng, Y., Lakkis, L., Devys, D. and Warren, S.T. (1995) Quantitative 35. Chung, M.Y., Ranum, L.P., Duvick, L.A., Servadio, A., Zoghbi, H.Y. and
comparison of FMR1 gene expression in normal and premutation alleles. Orr, H.T. (1993) Evidence for a mechanism predisposing to
Am. J. Hum. Genet., 56, 106–113. intergenerational CAG repeat instability in spinocerebellar ataxia type I.
15. De Boulle, K., Verkerk, A.J., Reyniers, E., Vits, L., Hendrickx, J., Van Roy, Nature Genet., 5, 254–258.
B., Van den Bos, F., de Graaff, E., Oostra, B.A. and Willems, P.J. (1993) A 36. Gordenin, D.A., Kunkel, T.A. and Resnick, M.A. (1997) Repeat
point mutation in the FMR-1 gene associated with fragile X mental expansion—all in a flap? Nature Genet., 16, 116–118.
retardation. Nature Genet., 3, 31–35. 37. Johnson, R.E., Kovvali, G.K., Prakash, L. and Prakash, S. (1995)
16. Wohrle, D., Kotzot, D., Hirst, M.C., Manca, A., Korn, B., Schmidt, A., Requirement of the yeast RTH1 5′ to 3′ exonuclease for the stability of
Barbi, G., Rott, H.D., Poustka, A., Davies, K.E. and Steinbach, P. (1992) A simple repetitive DNA. Science, 269, 238–240.
microdeletion of less than 250 kb, including the proximal part of the FMR- 38. Kokoska, R.J., Stefanovic, L., Tran, H.T., Resnick, M.A., Gordenin, D.A.
I gene and the fragile-X site, in a male with the clinical phenotype of fragile- and Petes, T.D. (1998) Destabilization of yeast micro- and minisatellite
X syndrome. Am. J. Hum. Genet., 51, 299–306. DNA sequences by mutations affecting a nuclease involved in Okazaki
17. Hinds, H.L., Ashley, C.T., Sutcliffe, J.S., Nelson, D.L., Warren, S.T., fragment processing (rad27) and DNA polymerase delta (pol3-t). Mol. Cell.
Housman, D.E. and Schalling, M. (1993) Tissue specific expression of Biol., 18, 2779–2788.
FMR-1 provides evidence for a functional role in fragile X syndrome. 39. Tishkoff, D.X., Filosi, N., Gaida, G.M. and Kolodner, R.D. (1997) A novel
Nature Genet., 3, 36–43. [Erratum (1993) Nature Genet., 5, 312.] mutation avoidance mechanism dependent on S. cerevisiae RAD27 is
18. Khandjian, E.W., Fortin, A., Thibodeau, A., Tremblay, S., Cote, F., Devys, distinct from DNA mismatch repair. Cell, 88, 253–263.
D., Mandel, J.L. and Rousseau, F. (1995) A heterogeneous set of FMR1 40. Bambara, R.A., Murante, R.S. and Henricksen, L.A. (1997) Enzymes and
proteins is widely distributed in mouse tissues and is modulated in cell reactions at the eukaryotic DNA replication fork. J. Biol. Chem., 272, 4647–
culture. Hum. Mol. Genet., 4, 783–789. 4650.
19. Verheij, C., de Graaff, E., Bakker, C.E., Willemsen, R., Willems, P.J., 41. Gacy, A.M., Goellner, G., Juranic, N., Macura, S. and McMurray, C.T.
Meijer, N., Galjaard, H., Reuser, A.J., Oostra, B.A. and Hoogeveen, A.T. (1995) Trinucleotide repeats that expand in human disease form hairpin
(1995) Characterization of FMR1 proteins isolated from different tissues. structures in vitro. Cell, 81, 533–540.
Hum. Mol. Genet., 4, 895–901. 42. Chen, X., Mariappan, S.V., Catasti, P., Ratliff, R., Moyzis, R.K., Laayoun,
20. Ashley Jr, C.T., Wilkinson, K.D., Reines, D. and Warren, S.T. (1993) A., Smith, S.S., Bradbury, E.M. and Gupta, G. (1995) Hairpins are formed
FMR1 protein: conserved RNP family domains and selective RNA binding. by the single DNA strands of the fragile X triplet repeats: structure and
Science, 262, 563–566. biological implications. Proc. Natl Acad. Sci. USA, 92, 5199–5203.
21. Siomi, H., Siomi, M.C., Nussbaum, R.L. and Dreyfuss, G. (1993) The 43. Freudenreich, C.H., Stavenhagen, J.B. and Zakian, V.A. (1997) Stability of
protein product of the fragile X gene, FMR1, has characteristics of an RNA- a CTG/CAG trinucleotide repeat in yeast is dependent on its orientation in
binding protein. Cell, 74, 291–298. the genome. Mol. Cell. Biol., 17, 2090–2098.
22. The Dutch–Belgian Fragile X Consortium (1994) Fmr1 knockout mice: a 44. White, P.J., Borts, R.H. and Hirst, M.C. (1999) Stability of the human
model to study fragile X mental retardation. Cell, 78, 23–33. fragile X (CGG)(n) triplet repeat array in Saccharomyces cerevisiae
23. Zhong, N., Yang, W., Dobkin, C. and Brown, W.T. (1995) Fragile X gene deficient in aspects of DNA metabolism. Mol. Cell. Biol., 19, 5675–5684.
instability: anchoring AGGs and linked microsatellites. Am. J. Hum. Genet., 45. Spiro, C., Pelletier, R., Rolfsmeier, M.L., Dixon, M.J., Lahue, R.S., Gupta,
57, 351–361. G., Park, M.S., Chen, X., Mariappan, S.V.S. and McMurray, C.T. (1999)
24. Sherman, S.L., Morton, N.E., Jacobs, P.A. and Turner, G. (1984) The Inhibition of FEN-1 processing by DNA secondary structure at trinucleotide
marker (X) syndrome: a cytogenetic and genetic analysis. Ann. Hum. repeats. Mol. Cell, 4, 1079–1085.
Genet., 48, 21–37. 46. Parniewski, P., Bacolla, A., Jaworski, A. and Wells, R.D. (1999) Nucleotide
25. Sherman, S.L., Jacobs, P.A., Morton, N.E., Froster-Iskenius, U., Howard- excision repair affects the stability of long transcribed (CTG*CAG) tracts in
Peebles, P.N., Nielsen, K.B., Partington, M.W., Sutherland, G.R., Turner, an orientation-dependent manner in Escherichia coli. Nucleic Acids Res.,
G. and Watson, M. (1985) Further segregation analysis of the fragile X 27, 616–623.
syndrome with special reference to transmitting males. Hum. Genet., 69, 47. Lyons-Darden, T. and Topal, M.D. (1999) Abasic sites induce triplet-repeat
289–299. expansion during DNA replication in vitro. J. Biol. Chem., 274, 25975–
26. Reyniers, E., Vits, L., De Boulle, K., Van Roy, B., Van Velzen, D., de 25978.
Graaff, E., Verkerk, A.J., Jorens, H.Z., Darby, J.K., Oostra, B. and Willems, 48. Lavedan, C., Grabczyk, E., Usdin, K. and Nussbaum, R.L. (1998) Long
P.J. (1993) The full mutation in the FMR-1 gene of male fragile X patients uninterrupted CGG repeats within the first exon of the human FMR1 gene
is absent in their sperm. Nature Genet., 4, 143–146. are not intrinsically unstable in transgenic mice. Genomics, 50, 229–240.
27. Feng, Y., Zhang, F., Lokey, L.K., Chastain, J.L., Lakkis, L., Eberhart, D. 49. Bontekoe, C.J., de Graaff, E., Nieuwenhuizen, I.M., Willemsen, R. and
and Warren, S.T. (1995) Translational suppression by trinucleotide repeat Oostra, B.A. (1997) FMR1 premutation allele (CGG)81 is stable in mice.
expansion at FMR1. Science, 268, 731–734. Eur. J. Hum. Genet., 5, 293–298.
28. Devys, D., Biancalana, V., Rousseau, F., Boue, J., Mandel, J.L. and Oberle, 50. Brock, G.J., Anderson, N.H. and Monckton, D.G. (1999) Cis-acting
I. (1992) Analysis of full fragile X mutations in fetal tissues and modifiers of expanded CAG/CTG triplet repeat expandability: associations
monozygotic twins indicate that abnormal methylation and somatic with flanking GC content and proximity to CpG islands. Hum. Mol. Genet.,
heterogeneity are established early in development. Am. J. Med. Genet., 43, 8, 1061–1067.
208–216. 51. de Vries, B.B., Halley, D.J., Oostra, B.A. and Niermeijer, M.F. (1998) The
29. Wohrle, D., Hirst, M.C., Kennerknecht, I., Davies, K.E. and Steinbach, P. fragile X syndrome. J. Med. Genet., 35, 579–589.
(1992) Genotype mosaicism in fragile X fetal tissues. Hum. Genet., 89, 52. Okano, M., Xie, S. and Li, E. (1998) Cloning and characterization of a
114–146. family of novel mammalian DNA (cytosine-5) methyltransferases. Nature
30. Burman, R.W., Popovich, B.W., Jacky, P.B. and Turker, M.S. (1999) Fully Genet., 19, 219–220.
expanded FMR1 CGG repeats exhibit a length- and differentiation- 53. Coffee, B., Zhang, F., Warren, S.T. and Reines, D. (1999) Acetylated
dependent instability in cell hybrids that is independent of DNA histones are associated with FMR1 in normal but not fragile X-syndrome
methylation. Hum. Mol. Genet., 8, 2293–2302. cells. Nature Genet., 22, 98–101. [Erratum (1999) Nature Genet., 22, 209.]
31. Wohrle, D., Hennig, I., Vogel, W. and Steinbach, P. (1993) Mitotic stability 54. Chiurazzi, P., Pomponi, M.G., Willemsen, R., Oostra, B.A. and Neri, G.
of fragile X mutations in differentiated cells indicates early post- (1998) In vitro reactivation of the FMR1 gene involved in fragile X
conceptional trinucleotide repeat expansion. Nature Genet., 4, 140–142. syndrome. Hum. Mol. Genet., 7, 109–113.
908 Human Molecular Genetics, 2000, Vol. 9, No. 6 Review

55. Chiurazzi, P., Pomponi, M.G., Pietrobono, R., Bakker, C.E., Neri, G. and 69. Ceman, S., Brown, V. and Warren, S.T. (1999) Isolation of an FMRP-
Oostra, B.A. (1999) Synergistic effect of histone hyperacetylation and DNA associated messenger ribonucleoprotein particle and identification of
demethylation in the reactivation of the FMR1 gene. Hum. Mol. Genet., 8, nucleolin and the fragile X-related proteins as components of the complex.
2317–2323. Mol. Cell. Biol., 19, 7925–7932.
56. Tassone, F., Hagerman, R.J., Taylor, A.K., Gane, L.W., Godfrey, T.E. and 70. Bardoni, B., Schenck, A. and Mandel, J.L. (1999) A novel RNA-binding
Hagerman, P.J. (2000) Elevated levels of FMR1 messenger RNA in carrier nuclear protein that interacts with the fragile X mental retardation (FMR1)
males: a new mechanism of involvement in the fragile X syndrome. Am. J. protein. Hum. Mol. Genet., 8, 2557–2566.
Hum. Genet., 66, 6–15. 71. Feng, Y., Gutekunst, C.A., Eberhart, D.E., Yi, H., Warren, S.T. and Hersch,
57. Musco, G., Stier, G., Joseph, C., Castiglione Morelli, M.A., Nilges, M., S.M. (1997) Fragile X mental retardation protein: nucleocytoplasmic
Gibson, T.J. and Pastore, A. (1996) Three-dimensional structure and shuttling and association with somatodendritic ribosomes. J. Neurosci., 17,
stability of the KH domain: molecular insights into the fragile X syndrome. 1539–1547.
Cell, 85, 237–245. 72. DiMarco, S.P., Ceman, S., Torre, E. and Warren, S.T. (1999) FMRP is a
58. Siomi, H., Choi, M., Siomi, M.C., Nussbaum, R.L. and Dreyfuss, G. (1994) phosphoprotein and a substrate of the Fes non-receptor tryosine kinase. Am.
Essential role for KH domains in RNA binding: impaired RNA binding by J. Hum. Genet., 65(suppl.), A269.
a mutation in the KH domain of FMR1 that causes fragile X syndrome. Cell, 73. Tamanini, F., Bontekoe, C., Bakker, C.E., van Unen, L., Anar, B.,
77, 33–39. Willemsen, R., Yoshida, M., Galjaard, H., Oostra, B.A. and Hoogeveen,
59. Siomi, M.C., Siomi, H., Sauer, W.H., Srinivasan, S., Nussbaum, R.L. and A.T. (1999) Different targets for the fragile X-related proteins revealed by
Dreyfuss, G. (1995) FXR1, an autosomal homolog of the fragile X mental their distinct nuclear localizations. Hum. Mol. Genet., 8, 863–869.
retardation gene. EMBO J., 14, 2401–2408. 74. Steward, O. and Falk, P.M. (1986) Protein-synthetic machinery at
60. Zhang, Y., O’Connor, J.P., Siomi, M.C., Srinivasan, S., Dutra, A., postsynaptic sites during synaptogenesis: a quantitative study of the
Nussbaum, R.L. and Dreyfuss, G. (1995) The fragile X mental retardation association between polyribosomes and developing synapses. J. Neurosci.,
syndrome protein interacts with novel homologs FXR1 and FXR2. EMBO 6, 412–423.
J., 14, 5358–5366.
75. Steward, O. and Reeves, T.M. (1988) Protein-synthetic machinery beneath
61. Hoogeveen, A.T. and Oostra, B.A. (1997) The fragile X syndrome. J.
postsynaptic sites on CNS neurons: association between polyribosomes and
Inherit. Metab. Dis., 20, 139–151.
other organelles at the synaptic site. J. Neurosci., 8, 176–184.
62. Khandjian, E.W. (1999) Biology of the fragile X mental retardation protein,
an RNA-binding protein. Biochem. Cell Biol., 77, 331–342. 76. Miyashiro, K., Dichter, M. and Eberwine, J. (1994) On the nature and
63. Brown, V., Small, K., Lakkis, L., Feng, Y., Gunter, C., Wilkinson, K.D. and differential distribution of mRNAs in hippocampal neurites: implications
Warren, S.T. (1998) Purified recombinant Fmrp exhibits selective RNA for neuronal functioning. Proc. Natl Acad. Sci. USA, 91, 10800–10804.
binding as an intrinsic property of the fragile X mental retardation protein. 77. Kang, H. and Schuman, E.M. (1996) A requirement for local protein
J. Biol. Chem., 273, 15521–15527. synthesis in neurotrophin-induced hippocampal synaptic plasticity. Science,
64. Adinolfi, S., Bagni, C., Musco, G., Gibson, T., Mazzarella, L. and Pastore, 273, 1402–1406.
A. (1999) Dissecting FMR1, the protein responsible for fragile X syndrome, 78. Comery, T.A., Harris, J.B., Willems, P.J., Oostra, B.A., Irwin, S.A., Weiler,
in its structural and functional domains. RNA, 5, 1248–1258. I.J. and Greenough, W.T. (1997) Abnormal dendritic spines in fragile X
65. Corbin, F., Bouillon, M., Fortin, A., Morin, S., Rousseau, F. and Khandjian, knockout mice: maturation and pruning deficits. Proc. Natl Acad. Sci. USA,
E.W. (1997) The fragile X mental retardation protein is associated with 94, 5401–5404.
poly(A)+ mRNA in actively translating polyribosomes. Hum. Mol. Genet., 79. Hinton, V.J., Brown, W.T., Wisniewski, K. and Rudelli, R.D. (1991)
6, 1465–1472. Analysis of neocortex in three males with the fragile X syndrome. Am. J.
66. Tamanini, F., Meijer, N., Verheij, C., Willems, P.J., Galjaard, H., Oostra, Med. Genet., 41, 289–294.
B.A. and Hoogeveen, A.T. (1996) FMRP is associated to the ribosomes via 80. Weiler, I.J., Irwin, S.A., Klintsova, A.Y., Spencer, C.M., Brazelton, A.D.,
RNA. Hum. Mol. Genet., 5, 809–813. Miyashiro, K., Comery, T.A., Patel, B., Eberwine, J. and Greenough, W.T.
67. Eberhart, D.E., Malter, H.E., Feng, Y. and Warren, S.T. (1996) The fragile (1997) Fragile X mental retardation protein is translated near synapses in
X mental retardation protein is a ribonucleoprotein containing both nuclear response to neurotransmitter activation. Proc. Natl Acad. Sci. USA, 94,
localization and nuclear export signals. Hum. Mol. Genet., 5, 1083–1091. 5395–5400.
68. Feng, Y., Absher, D., Eberhart, D.E., Brown, V., Malter, H.E. and Warren, 81. Paradee, W., Melikian, H.E., Rasmussen, D.L., Kenneson, A., Conn, P.J.
S.T. (1997) FMRP associates with polyribosomes as an mRNP and the and Warren, S.T. (1999) Fragile X mouse: strain effects of knockout
I304N mutation of severe fragile X syndrome abolishes this association. phenotype and evidence suggesting deficient amygdala function.
Mol. Cell, 1, 109–118. Neuroscience, 94, 185–192.