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Molecular mechanisms of fragile X syndrome
Shunsuke B. Inoue, Mikiko C. Siomi and Haruhiko Siomi
Institute for Genome Research, University of Tokushima, Tokushima, Japan
Abstract : Fragile X syndrome is the most common form of inherited mental retardation.
Mutations which abolish expression of an X-linked gene, FMR1, result in pathogenesisof the disease. FMR1 encodes a cytoplasmic RNA-binding protein which interacts with two
autosomal homologs, FXR1 and FXR2. These proteins are highly expressed in neurons. In
addition, the FMR1/FXR proteins are associated with ribosomes. Given their RNA-binding
activity and association with ribosomes, these proteins are hypothesized to bind to
specific RNAs and regulate their expression at translational levels in a manner criticalfor correct development of neurons. Much progress has been made in FMR1 researchover the past several years, but little light has yet to be shed on the physiological function of these proteins. It will be critical to define the biochemical properties of these
proteins, and identify potential downstream targets to clarify the molecular mechanisms
underlying the potential roles of these proteins in translation. A basic understanding of
the function of this new family of RNA-binding proteins should then allow us to begin toaddress the question of how the lack of FMR1 expression leads to symptoms in fragile X
syndrome. J. Med. Invest. 47 : 101-107, 2000
Key words : fragile X syndrome, RNA-binding protein, translation

Fragile X Syndrome
Fragile X syndrome is the single most common
form of inherited mental retardation, and after Down’s
syndrome, the most common identified form of mental retardation (1-3). It occurs in all ethnic groups.
Estimates of the frequency of the disorder, basedon cytogenetic testing, suggested a prevalence of one
in 1,250 males and one in 2,000 females, but more
recent studies based on direct detection of the mutation indicate a more conservative figure of one in
5,000. Nevertheless, it is the most frequent cause
of inherited mental retardation. Affected individuals
suffer from moderate to severe mental retardationand various degrees of autistic behavior. The clinical features also include an elongated facial appearance, wide prominent ears and enlarged testicles (macroorchidism) in adult male patients (4).
Received for publication June 6, 2000 ; accepted July 3,
2000.
Address correspondence and reprint requests to Haruhiko
Siomi, Ph.D., Institute for Genome Research, University ofTokushima Kuramoto-cho, Tokushima 770-8503, Japan andFax : +81-88-633-9451.

Individuals with the syndrome show neuropathologicalabnormalities such as thin, elongated dendritic spinesand enlargement of the hippocampus, caudate nucleus, lateral ventricle, and thalamus (5). Even height,
occipitofrontal circumference, and sometimes weightare increased, especially before puberty. Therefore,fragile X syndrome is sometimes considered to bean ‘overgrowth syndrome’.
As the name implies, this syndrome is an X-linked
genetic disorder and is frequently associated with theexpression of a folate sensitive fragile site, or a gap
observed in metaphase stains of the X chromosomes of affected individuals. The fragile site has
been mapped to region Xq27.3 on the long arm of
the X chromosome (1-3). Using a positional cloning
strategy, the FMR1 gene was isolated and the mutational basis of the syndrome was determined tobe the expansion of a trinucleotide repeat (CGG)npresent in the 5’ untranslated region of the identified gene (6-8). FMR 1 is the first cloned gene, which
has been liked to human intelligence. The inheritance of fragile X syndrome had puzzled geneticists for some years for two unusual inheritancepatterns, which fail to conform to the laws of Mendel
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S B. Inoue et al. Molecular mechanisms of fragile X syndrome(1-3). First, the risk of having the disease increases
in successive generations of a family (the so-called
Sherman paradox or genetic anticipation). Second,
phenotypically normal males produce descendants
with the fragile X syndrome. We now know that theunusual inheritance patterns are due to the trinucleotide
repeat expansion. While in normal individuals the
CGG repeat length is polymorphic, ranging from 5
to 50 copies with an average number of 30, unaffectedmale carriers possess between 50 and 200 repeats(premutation) : in nearly all cases of fragile X patients, there is a large expansion in the number of
the CGG repeats, to more than 200 copies, and sometimes to more than a thousand copies. Transmission of premutation alleles from males always results
in premutation-sized alleles in their daughters,
accounting for the lack of affected daughters of transmitting males. When females contribute premutation
alleles to their offspring, increases in size prevailover decreases. The tendency for premutation-sized
alleles to increase in length, coupled with the escalatingrisk of transmission to full mutation, provides amolecular explanation for the Sherman paradox ofincreasing penetrance. This finding has led to thediscovery of a new dynamic mutation mechanism,
the so called trinucleotide repeat expansion, whichis responsible for a number of human genetic
neurological disorders including Huntington’s disease
and spinocerebellar ataxia (9, 10). This CGG repeat
expansion is associated with abnormal DNA methylationof both a nearby CpG island and the repeat itself(1-3). As a result, the FMR1 locus becomes silent
at the transcriptional level and, thus, no translation
occurs (11-13) (Figure 1). Although the timing andmolecular mechanisms of the CGG repeat expansion and methylation on expanded CGG repeat areunder extensive study, we do not know much about
when and how CGG repeat instability and DNA
methylation occur.
It has become clear that fragile X syndrome resultsfrom the lack of FMR1 expression. In other words,
the cause of fragile X syndrome is a loss-of-functionof FMR1. This has been confirmed by the Dutch-Belgian
Consortium who generated transgenic mice models
by targeted disruption (knockout) of the mouse
Fmr1 gene (14, 15). The Fmr1 knockout mice show
enlarged testes, abnormal dendritic spine morphology, impaired cognitive function and abnormal
behavior, similar to humans with fragile X. The development of the Fmr1 knockout mice was a criticaladvance in elucidating the pathological mechanismsthat lead to mental retardation and behavioral ab-

Fig. 1. Fragile X syndrome is caused by the lack of transcription
due to the expansion of the CGG repeat located in the 5’ UTRof the FMR1 gene. Fragile X syndrome results from a massive
trinucleotide repeat expansion. As a result of the repeat expansion,
abnormal methylation of both a nearby CpG island and the repeat
itself takes place, causing transcriptional silencing of FMR1.

normalities in fragile X syndrome. This animal model
might also be useful for developing therapeuticstrategies. However, as yet, very little is known about
the physiological function of FMR1.

The FMR1 Gene
Although a strong sequence conservation between
fruitfly (16), Xenopus (17), chicken (18), mouse(19) and human (7) FMR1 cDNAs suggest that it
may have a “housekeeping” role, its physiological
function remains a mystery. One clue as to thefunction of the FMR1 protein comes from the identification in the protein sequence of motifs characteristic of RNA-binding proteins. We and the othergroup found that the protein contains an RGG box
and two KH domains (13, 20) (Figure 2a). The RGG
box is a region of 20 to 25 amino acids containing
several Arg-Gly-Gly tripeptide repeats and this motif
has been found in numerous RNA-binding proteins (21).
The KH domain was discovered on the basis of primary
sequence similarity between the pre-mRNA-binding
protein hnRNP K and other RNA associated proteinssuch as ribosomal protein S3, bacterial antiterminatorprotein NusA, and a yeast meiosis-specific alternative splicing factor Mer-1 and, therefore, it was named
K homology domain or KH domain (22, 23). The
hallmark of the KH domain is a strongly conserved
tetrapeptide Gly-x-x-Gly (x is often a positively charged

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Identification of the RNA targets of FMR1 protein
will be an important step toward understanding how
the absence of a single protein results in the pleiotropic
phenotype observed in fragile X syndrome.

FMR1 Interacting ProteinsFig. 2. The FMR1 family. A, Graphical alignment of peptide
sequences for FMR1 and homologues FXR1 and FXR2. This
family of RNA-binding proteins contains two types of RNA-binding
motifs, namely RGG box and KH domain. Note the high similarity through the KH domains. B, A model summarizing theobserved interactions among the FMR1, FXR1 and FXR2.

amino acid), which is flanked with regularly spacedhydrophobic residues within a region of 60-80 aminoacids (23 and 24). Many other proteins that contain KH domains, such as Gld-1, Hex3 and quaking
have been shown to have extremely importantroles in development. Two KH domains are locatedin the middle of the FMR1 protein and an RGG box
lies downstream of the KH domains. The RNA-binding
domains in FMR1 appear to be functional because
FMR1 was demonstrated to directly bind RNA in
vitro with some degree of sequence specificity. We
demonstrated that the in vitro translated FMR1
protein can bind to poly(G) and poly(U), synthetic RNA homopolymers in a salt resistancemanner (13). Ashley et al. provided evidence that
the FMR1 protein selectively binds to a subset ofbrain-derived RNA, with approximately 4% by massof human fetal brain message specifically binding
FMR1 protein (20). These findings suggested that
FMR1 post-transcriptionally regulates expressionof downstream genes that are critical for normal
development of intelligence. Since fragile X syndrome
is often associated with overgrowth phenotypes, potential
target genes may also encode growth-regulatory proteins, and regulation of these genes by FMR1 may
affect the control of cell proliferation. However, cellular
RNA targets of FMR1 have not yet been identified.

It appears clear that a better understanding of
the function of FMR1 also requires identificationof proteins with which FMR1 protein may interact.
To identify such interacting proteins, we used theyeast two hybrid system and isolated two novel
cDNAs for proteins which specifically interact with
FMR1 (25). Surprisingly, these FMR1 interacting
genes are very similar in overall structure to FMR1;
they share ˜ 60% sequence homology with FMR1
(Figure 2A). Therefore, the two genes were named
FXR1 and FXR2 (FXR for fragile X related genes).
The importance of these genes is reflected in theconservation of gene sequence from Xenopus tohuman. Given the high degree of sequence similarity
among these proteins, it is not surprising that both
FXR1 and FXR2 proteins are also capable of binding to poly(G) and poly(U) in vitro. We have also
demonstrated, by means of a co-immunoprecipitation
assay, that FMR1 protein can form heterodimers
with FXR1 and FXR2 proteins in living cells (25and data not shown). Further characterization of
the interactions among the family members using
glutathione-S-transferase (GST) fusion proteins reveals that every family member can form dimers withevery other family member and with itself (25)
(Figure 2B). These findings clearly indicate thatthe three proteins form a gene family of proteins
related by structure and most likely by function, as
they interact with each other. Whatever the outcome of this interaction, FMR1 can no longer beconsidered to function alone but likely has, in FXR1and FXR2, partners to perform its normal function, which is presumably critical for the development of intelligence. This interaction could be amechanism of functional autoregulation of this new
family of RNA-binding proteins. For example, FXR1and FXR2 could modulate RNA binding specificity
and/or affinity through a direct interaction with FMR1.
It is also possible that the free FMR1 protein may
be poorly configured for binding to RNA in the absence of FXR proteins. There is precedence fromtwo other RNA-binding protein systems for a decisive
influence of the protein-protein interaction on the
RNA binding of the proteins. The signal recognition
particle (SRP) is made up of six proteins and one

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S B. Inoue et al. Molecular mechanisms of fragile X syndrome

RNA (7SL). Two RNA-binding SRP proteins, SRP9and SRP14, bind 7SL RNA efficiently only when bothare present in a complex with each other (26). The
mammalian U2 snRNA-binding proteins, U2B” andU2A’ also form a complex in the absence of RNA(27). Neither of these proteins is capable on theirown of specific interaction with U2 RNA, although
U2B” binds RNA in a relatively non-specific mannerand U2A’ interacts weakly with double-stranded
RNA. Formation of the U2A’ : U2B” complex is thought
to alter the conformation of U2B” in a manner that
greatly enhances its specific binding to the U2 snRNA
element and also reduces its non-specific binding
to U1 snRNA, a different class of snRNA. Notably,both U2B” and U2A’ proteins are very similar in
overall structure as is the case for the FMR1/FXR
family.
Protein-protein interactions are intrinsic to virtually
every cellular process. There are a large number oftransient protein-protein interactions, which in turncontrol a large number of cellular processes. All modifications of proteins necessarily involve such transient
protein-protein interactions. These include the interactions of protein kinases, protein phosphatases,
glycosyl transferases, etc., with their substrate proteins. Such protein-modifying enzymes encompass
a large number of protein-protein interactions in
the cell and regulate all manner of fundamental
processes such as cell growth, cell cycle and signaltransduction. We have demonstrated the interactions
among the FMR1 family members. This raises thepossibility that FMR1 might function in a complex
with FXR1, FXR2 or both. It is of interest to determine whether complex formation or function isregulated by other factors, such as phosphorylation,or potential interactions with other cellular components. In particular, the question of what can abrogate
or dissociate the interactions among the FMR1 family
proteins may be as important as the originally identified interaction partners.
Given the strong overall similarity and similar
biological properties, this family of RNA-binding
proteins is likely to show partial overlap in theirfunction and these proteins might be functionally
redundant in some cellular processes. However, it
is already clear that FXR1 and FXR2 cannot fullycomplement the lack of FMR1 function in the fragile
X patients, since expression of FXR1 and FXR2 does
not appear to be effected in fragile X patients (17,25). Thus they may have both partially complementary functions and independent functions. It
can be anticipated that, as is the case for FMR1,

hitherto unknown genetic disorders caused by aberrant expression of the FXR genes exist. In contrast to FMR1, both FXR1 and FXR2 are encodedby autosomal genes (3q28 and 17p13, respectively :17, 25 and 28). To our knowledge, no human genetic
disorder genes including mental retardation genes
have been mapped in their regions of the genome.
Therefore, it can also be assumed that if FXR1 andFXR2 have independent functions which are essential during early development, most mutationsof human FXR1 and FXR2 would be early embryonic lethal. In either case, it is of great interest in
determining whether or not loss of the FXR1 or FXR2
function is linked to mental retardation and/or otherabnormalities.

Ribosomal Association of FMR1 FamilyProteins
In addition to the interaction between the family
members, we have demonstrated an association ofthis family of RNA-binding proteins with ribosomes(29). Simultaneously and independently, several
other groups also reported that FMR1 protein is
associated with ribosomes in an RNA-dependent
manner (30-33). Although initially the FMR1 protein was interpreted to be associated with the 60S
large ribosomal subunit based upon cofractionation,
the more recent findings showed that FMR1 protein
is associated with elongating polyribosomes via large
mRNP particles (31, 33). However, these findings do
not eliminate the possible interactions between FMR1
protein-mRNP particles and the 60S ribosomal subunitin vivo during various stages of translation, because
FMR1 protein has been found to co-immunoprecipitate
with the 60S subunit. Based upon the RNA-binding
activity and the ribosomal association, a hypothesis has been raised that the FMR1 and FXR proteins may have roles in translation. There are atleast two possible scenarios about how these proteinstranslationally regulate expression of their target
mRNAs (Figure 3). First, since these proteins are
RNA-binding proteins with some sequence specificity
and associate ribosomes as mRNA-protein (mRNP)
complex, FMR1 and FXR proteins act as chaperones
or selectors for recruiting specific mRNAs onto theribosomes. Second, since considerable amounts of
FMR1 and FXR proteins are found in soluble fractions, the distribution of these proteins between
free and ribosome-associated states may give rise
to structural heterogeneity of ribosomes with regard
to their complement of these proteins, which, inThe Journal of Medical Investigation

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implicated in cap-independent initiation. Therefore,
FMR1 family proteins could regulate translation ofa class of mRNAs without directly binding to themin such a way that FMR1 family proteins mask
sites in rRNA needed for preferential translation of
a subset of mRNAs, and loss of the FMR1 protein
in fragile X patients unmasks or modifies the sites
and, thus, influences translation of the mRNAs. In
either case, to understand the mechanisms underlying the potential role of FMR1 and FXR proteinsin translation, it will be important to identify biologically important genes that are translationallyregulated by the FMR1 and FXR proteins.

The Functions of FMR1 and FXR ProteinsFig. 3. Possible function of the FMR1 family in translation. A,
A ‘direct selector’ model for the role of FMR1/FXR proteins intranslation. In this model, FMR1 and FXR proteins act as chaperones or selectors for recruiting specific mRNAs onto the ribosomes. B, A ‘indirect selector’ model. The distribution of
FMR1/FXR proteins between free and ribosome-associated states
may give rise to structural heterogeneity of ribosomes with regard
to their complement of these proteins, which, in turn, influences mRNA selection.

turn, influences mRNA selection. The former scenario
envisions that these proteins have specific bindingsites on a class of mRNAs and regulate their translation through their binding. This is akin to Drosophilamorphogen bicoid (bcd). bcd directly binds to thediscrete target sequences within the 3’ untranslated
regions (UTR) of the homeodomain protein caudal
mRNA and regulates, (in this case, represses) thetranslation of the mRNA (34). This class of RNA-binding
proteins includes C. elegans fem-3 binding factor(35) and mammalian hnRNP K (36). The second modelis related to the E. coli ribosomal protein S2 (EcoS2). Eco S2 is one of the few ribosomal proteinsthat exchanges on and off the ribosome, which gives
rise to structural heterogeneity of ribosomes (37,38). While ribosomes containing Eco S2 efficiently
translate mRNAs containing Shine-Dalgarno sequencesin the 5’ leader sequences probably by affecting
base-pairing between the leader sequences and the
16S rRNA, ribosomes lacking Eco S2 preferentiallytranslate such leaderless mRNAs that are normallytranslated inefficiently. In eukaryotes, base-pairing
interactions between 18S rRNA and mRNA has been

The expectation is that FMR1 and FXR proteins,
being ribosome-associated and RNA-binding proteins
with some sequence specificity, have important rolesin the translation of some specific mRNAs. However,
little is known about the specific function of this new
family of RNA-binding proteins. A major problem
in elucidating the function of FMR1 family proteinshas been the inability to identify a downstream gene(s)whose expression may be regulated by this familyof proteins. To some extent this is partly because
biochemical characterization of these proteins, suchas extensive cell fractionation, has only recently
begun, and partly because the probes for the individual proteins have only recently been obtained.
Furthermore, since FMR1 is clearly not essential
for general translation, it is possible that the effectof the lack of the FMR1 protein may be subtle in in
vitro assays that have been commonly used for studyof translation such as rabbit reticulocyte lysates. It
is also possible that there may be enough functional
redundancy between FMR1 and FXR proteins, so
that, in these in vitro assays, their function will beobserved only when two or all of the FMR1 andFXR proteins are completely removed from the test
fractions. However, the situation is very different
in vivo, where a subtle difference in protein concentration often appears sufficient to distinguishbetween on or off states of gene expression, as is
the case for morphogens in Drosophila (39). It isalso possible that FMR1 and FXR proteins havean individual function that may be either similar or
distinct, but together they promote high fidelity forsome process such as those required for neuronaldevelopment. As described above, several observations are, nevertheless, strongly suggestive of arole for FMR1 and FXR proteins in translational

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S B. Inoue et al. Molecular mechanisms of fragile X syndrome

regulation of gene expression. Identification of thetarget genes of FMR1 family proteins should help
to clarify the function of this family and, thus, themechanisms leading to fragile X syndrome, and may
also suggest new approaches to developing therapeutic strategies for the syndrome.

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