American Journal of Medical Genetics 84:268–271 (1999)
Reduced mRNA For G3BP in Fragile X Cells:
Evidence of FMR1 Gene Regulation
Nan Zhong,1* Weina Ju,1 David Nelson,2 Carl Dobkin,1 and W. Ted Brown1
1
Department of Human Genetics, New York State Institute for Basic Research, Staten Island, New York
2
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas
Although fragile X syndrome is caused by
the absence of fragile X gene expression,
little is known about the pathogenic pro-
cesses underlying the mental retardation.
Recent findings that the fragile X protein,
FMRP, contains RNA binding motifs and
nuclear transport signals and associates
with ribosomes suggest that FMRP may be
involved in either mRNA processing, trans-
port, or translation. To test the hypothesis
that absence of FMRP may affect the pro-
cessing of specific transcripts, we have used
an RNA differential display assay (RDDA) to
identify differentially expressed transcripts
in lymphoblast lines derived from fragile X
syndrome patients. A 0.9-kb cDNA fragment
that showed reduced expression in a fragile
X lymphoblast cell line was found to be iden-
tical to G3BP (Ras-GTPase-Activating pro-
tein SH3-domain-binding protein). Quanti-
tative reverse transcriptase-polymerase
chain reaction showed that the expressed
levels of G3BP mRNA in fragile X lympho-
blast cell lines were significantly less than
controls. Our results indicate that G3BP
mRNA may be regulated by FMRP and sup-
ports the hypothesis that FMRP may modu-
late the transcription of specific transcripts.
Am. J. Med. Genet. 84:268–271, 1999.
© 1999 Wiley-Liss, Inc.
KEY WORDS: mental retardation; fragile X
syndrome; G3BP; RNA differ-
ential display; RT-PCR
INTRODUCTION
Fragile X syndrome is the most common inherited
mental retardation disorder. The vast majority of frag-
*Correspondence to: Dr. Nan Zhong, Department of Human
Genetics, New York State Institute for Basic Research, 1050 For-
est Hill Road, Staten Island, NY 10314. E-mail: omrddzhong@aol.
com
Received 22 August 1997; Accepted 25 November 1998
© 1999 Wiley-Liss, Inc.
ile X cases are due to a CGG triplet repeat expansion in
the first exon of the fragile X gene, FMR1 [Fu et al.,
1991; Verkerk et al., 1991]. Usually, the expanded re-
peat results in methylation of the CGG repeat and the
FMR1 promoter region. This results in absent FMR1
expression. Lack of the FMR1-encoded protein, FMRP,
correlates with the presence of the fragile X phenotype
[Devys et al., 1993; Verheij et al., 1993; Willemsen et
al., 1995].
FMRP binds to 4–5% of brain mRNAs, including
FMR1 mRNA, through three RNA binding domains,
two KH motifs, and one RGG box region [Ashley et al.,
1993; Siomi et al., 1993], and it associates with the 60s
ribosomal subunit via RNA [Corbin et al., 1997;
Khandjian et al., 1996; Siomi et al., 1996; Tamanini et
al., 1996]. A point mutation, designated I367N [De
Boulle et al., 1993], is located within the second KH
domain of FMRP, and it affects the FMRP binding to
RNA demonstrated in a fragile X syndrome patient
with a normal triplet CGG repeat size and a normal
level of FMR1 mRNA and FMRP [Siomi et al., 1994].
Although these findings indicate that the pathogenesis
of the fragile X syndrome may result from lack of an
RNA binding function, little is known about the mecha-
nism of how the absence of FMRP results in the fea-
tures of the fragile X syndrome, including mental re-
tardation.
To study the pathogenesis of the fragile X syndrome,
we have used RNA differential display to analyze cel-
lular transcripts in affected versus unaffected lympho-
blasts. The level of mRNA for G3BP, an RNA binding
protein, was found to have lower levels in fragile X cells
compared with normal cells.
MATERIALS AND METHODS
Isolation of DNA and RNA From
Fragile X Lymphoblasts
Fragile X lymphoblast cell lines were derived from
fragile X syndrome patients whose diagnosis was con-
firmed by molecular testing [Brown et al., 1993; Gu
et al., 1994]. Genomic DNA was isolated from the
cultured cells using a Puregene kit (Gentra, Research
Triangle Park, NC). Total RNA was isolated with an
RNeasy kit (Qiagen, Valencia, CA).

RNA Fingerprinting To Detect
Cellular Transcription
An RNA differential display assay (RDDA) [Liang
and Pardee, 1992, 1995; Linskens et al., 1995] using
arbitrary primers was carried out using an RNA fin-
gerprint kit (Clontech Laboratories, Inc., Palo Alto,
CA). The first strand cDNA was synthesized with oligo-
d(T) from 2.5 ␮g of total RNA in a 10-␮l reaction. The
synthesized cDNA was diluted to 50 ng/␮l and used for
PCR amplification. Different combinations of arbitrary
primers (T1/P4, T1/P6, T1/T7, T2/P8, T2/P9, T2/T10,
T8/P8, T8/P9, T8/P10, T9/P8, T9/P9, T9/P10) supplied
within the fingerprinting kit were tested individually
in 10 ␮l of PCR reaction that consists of 50 ng of 1st
strand cDNA, 1.5 mM MgCl2, 2.5 mM dNTPs, 1 ␮Ci
␣-32P-dCTP at 3,000 Ci/mM (Amersham, Des Plaines,
IL), 0.5 ␮l of each forward or reverse arbitrary primers,
1× Taq buffer II, and 0.25 units of AmpliTaq (Perkin-
Elmer, Norwalk, CT). PCR amplification was carried
out in an MJ PTC-100 thermal cycler (MJ Research,
MA) using 0.2-ml thin wall PCR tubes. The thermal
cycle conditions followed the RNA fingerprint kit in-
structions, which were one cycle of 94°C for 5 min, 40°C
for 5 min, 68°C for 5 min, two cycles of 94°C for 30 sec,
40°C for 30 sec, 68°C for 5 min, and 23 cycles of 94°C for
20 sec, 60°C for 30 sec, 68°C for 2 min. The last exten-
sion was performed at 68°C for 7 min. The PCR prod-
ucts were resolved by electrophoresis on Sequagel-6
(National Diagnostics, Atlanta, GA), dried, and auto-
radiographed with Blue Sensitive X-film (Molecular
Technologies, St. Louis, MO). The PCR reaction was
repeated twice if there were differentially displayed
patterns detected.
Subcloning and Sequencing Differentially
Expressed Transcripts
The differentially expressed PCR product signals on
X-film were matched back to the dried-gel. The gel po-
sition where the band of interest was located was ex-
cised from control lanes. To recover DNA, the piece of
dried-gel was soaked in 40–80 ␮l (depending upon the
size of gel-piece) of Tricine-EDTA buffer (10 mM Tri-
cine, pH 9.5, 0.2 mM EDTA) and covered by a drop of
mineral oil preventing evaporation in a 1.5-ml micro-
centrifuge tube. This tube was heated in boiling water
for 5 min and the eluted DNA was transferred into a
fresh tube. A 5-␮l aliquot of the eluted DNA was used
for reamplification in a 25-␮l reaction with the same
primers used in the original RDDA reaction. After a
10-␮l aliquot was reanalyzed for confirmation on a
0.8% LE-agarose (FMC, ME) gel, 3 ␮l (out of 25 ␮l total)
of the PCR product was directly cloned into pCRIII (In-
vitrogen, Carlsbad, CA). DNA sequencing was con-
ducted with a dsCycle sequence kit (Gibco-BRL, Gai-
thersburg, MD).
Database Searches
Differentially expressed transcript sequences were
used as quote sequences to search the GenBank DNA
database using the program BLAST through NCBI
(National Center of Biomedical Information), or by
G3BP in the Fragile X 269
FASTA in GCG (Wisconsin sequence analysis package,
Version 8).
Quantitative Reverse Transcriptase-Polymerase
Chain Reaction (RT-PCR) Detecting
mRNA Expression
Total RNA (2.5 ␮g) was reverse transcribed with a
StrataScript RT-PCR kit (Stratagene, La Jolla, CA).
Following instructions supplied with the kit, oligo-d(T)
and random primers were used to synthesize a first
strand cDNA. PCR was carried out in a 10-␮l reaction
containing 50 ng of synthesized first strand cDNA, 50
pmol each of two sets of specific primers to amplify 220
bp (nucleotide (nt) 973-1193) of G3BP [Parker et al.,
1996], and 400 bp of ␣-actin (the actin primers were
supplied from the kit and used as an internal control).
Thermal cycle conditions followed the kit instructions.
Briefly, 5 min of denaturation at 95°C was followed by
25 cycles of 95°C for 30 sec, 50°C for 30 sec, and 72°C
for 1 min. The final extension was 10 min. at 72°C. The
PCR products were separated on Sequagel-6 (National
Diagnostics). The gel was dried and autoradiographed
at −70°C for two days. The density of the PCR band on
the X-film was scanned with a Shimadzu (CS-9000)
densitometer.
RESULTS
Twelve different combinations of arbitrary primers
were applied to screen for differentially displayed tran-
scripts. Six differentially displayed PCR bands ampli-
fied with primer combination of T1/P4, T1/P6, T2/P9,
T8/P8, T8/P9, T8/P10 were detected. Five of six differ-
entially displayed bands showed either no apparent
difference in the mRNA expression level or the PCR
fragment was too small (<150 bp, amplified by T1/P6)
to reveal a specific signal due to nonspecific homologies
to 3⬘ untranslated regions of various transcripts by a
Northern blot hybridization analysis. A 0.9-kb band
generated with primers T8 and P10 was strongly am-
plified in controls (Fig. 1, lane 5 and 6) but only weakly
in fragile X (Fig. 1, lane 4). Seven colonies from the
cloned 0.9-kb band were sequenced with flanking
primer T7. This showed that the 0.9-kb band was iden-
tical in sequence (Fig. 2) to a G3BP (Ras-GTPase-
activating protein SH3-domain-binding protein) cDNA
sequence from nt 973 to 1269. This G3BP sequence
includes two heterogeneous nuclear ribonucleo-protein
(hnRNP) domains and one RGG box [Parker et al.,
1996]. We noted four bases were missing in our query
sequence compared with the G3BP sequence (under-
lined in Fig. 2), which was likely the result of sequenc-
ing or reading errors.
To verify that G3BP gene expression levels in fragile
X cells were lower, Northern blot hybridization was
performed using the cloned 0.9-kb fragment as a probe.
However, no detectable signal was obtained in either
normal control or fragile X lymphoblasts. This was
probably due to the low abundance of the expressed
G3BP mRNA [Parker et al., 1996]. To detect G3BP
mRNA expression, four fragile X lymphocyte cell lines
(Fig. 3, lanes 2, 3, and 4), including one (lane 3) with a
large intragenic deletion of FMR1 [Gu et al., 1994] and
270 Zhong et al.
Fig. 1. RDDA gel showing a 0.9-kb band with a reduced expression in
fragile X lymphoblasts (lane 4) compared with an unaffected individual
(lane 5) and from a person with Down syndrome (non-fragile X mental
retardation) (lane 6). In lanes 3–6, PCR amplification was done on the
same amounts of total RNA that had been subjected to RDDA analysis but
that had not been reverse-transcribed. The absence of bands in lanes 1–3
serves as a control to indicate that the patterns in lanes 4–6 were not
derived from genomic DNA that was contaminating the RNA preparations.
one that was used in RDDA (Fig. 3, lane 6), were tested
by quantitative RT-PCR. The band corresponding to
G3BP was much weaker than the ␣-actin internal con-
trol in the fragile X cell lines but heavier than ␣-actin
in the normal controls (lanes 1 and 5). The relative
amount of G3BP mRNA determined by densitometry
Fig. 2. Partial sequence analysis demonstrating that a 0.9-kb fragment
isolated from normal controls on RDDA gel is 99% identical to the human
G3BP gene. The five bases that are absent in the query sequence (under-
lined) may be a result of sequencing error.
Fig. 3. Quantitative RT-PCR showing that the signal intensity of the
amplified G3BP band in the control lanes (nl) was stronger than the -actin
that was used as an internal control. However, the G3BP signals were
much weaker than the -actin in the fragile X lanes (fx) and almost invisible
in the lane marked fx-del where the RNA was isolated from a fragile X cell
line with an FMR1 deletion.
was 7- to 10-fold less in the fragile X cells than in the
controls.
DISCUSSION
Unlike cellular housekeeping genes, FMRP appears
to play a role in the machinery of transcription and
translation in the transporting of RNA across the
nuclear membrane via RNA-binding and in assembling
to polyribosomes [Warren, 1997]. However, little is
known about how the absence of FMRP causes the dif-
ferent aspects of the fragile X syndrome phenotype. We
hypothesize that FMR1 is a “trigger” gene. It may ini-
tiate a series of processes regulating normal transcrip-
tion of some genes. Absence of these processes, result-
ing from lack of the FMRP, may be pathogenic and
cause the fragile X syndrome phenotype. These absent
processes could include lack of FMRP-bound RNAs
that have not been transported to their natural cellular
localizations as driven by the FMRP nuclear exporting
signal motif [Eberhart et al., 1996].
To test our hypothesis, we carried out an RDDA to
compare transcription patterns between normal and
fragile X lymphoblastoid cells. We identified a specific
gene transcript, G3BP, that shows markedly reduced
expression in fragile X cells. G3BP is an SH3 domain
binding protein [Parker et al., 1996] that includes two
ribonucleoprotein (RPN) motifs and an RGG box that
function as RNA-binding domains [Mayeda et al.,
1992]. G3BP has been characterized as a Ras-GAP SH3
domain-binding protein [Parker et al., 1996]. It may
function as an effector of downstream Ras activity
[Tocque et al., 1997] and be involved in the Ras signal
transduction pathway. This gene is widely expressed in
human fetal and adult lung, liver, kidney, and brain
[Parker et al., 1996]. In peripheral blood leukocytes,
the expressed mRNA level is almost undetectable by
Northern blot assay [Parker et al., 1996].
At present, we are not able to suggest specifically
how the G3BP could be involved in fragile X syndrome
pathogenesis. The reduced G3BP mRNA levels in frag-
ile X cells may result directly from the G3BP mRNA
not being transported to its natural cellular localiza-
tion, although it could be normally synthesized, or it
may be more prone to degradation due to the lack of

FMRP binding. In addition, the possibility that G3BP
may be transcriptionally repressed should also be con-
sidered. As indicated, G3BP has RNA binding motifs.
Therefore, a second possible mechanism to explain the
pathogenesis is that the G3BP is not directly bound by
FMRP but rather a “sandwich” complex of FMRP-RNA-
G3BP may form in which both FMRP and G3BP bind to
the same RNA molecule. In this way the G3BP levels
may be indirectly affected by the lack of FMRP in the
fragile X cells. To characterize this interaction, addi-
tional studies are needed to determine if FMRP binds
to G3BP mRNA, which may indicate whether the
G3BP is directly or indirectly regulated by FMRP.
Our finding that G3BP showed lower levels of ex-
pression in fragile X syndrome lymphoblasts supports
the hypothesis that FMRP may regulate the expression
of other functioning genes. We suggest the absence of
this gene product could be a part of the pathogenic
pathway in the fragile X syndrome. In this view, ab-
sence of FMRP in the fragile X pathogenetic pathway
acts as a “trigger” to initiate the pathological pheno-
type. There are likely to be other transcripts affected by
reduced FMRP. Variation in RNA differential display
assay may reveal these transcripts.
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