Published online 9 January 2009Nucleic Acids Research, 2009, Vol. 37, No. 41269–1279
FRAXE-associated mental retardation protein
(FMR2) is an RNA-binding protein with high
affinity for G-quartet RNA forming structure
Mounia Bensaid1,2, Mireille Melko1,2, Elias G. Bechara1,2, Laetitia Davidovic1,2,
Antonio Berretta3,4, Maria Vincenza Catania3,5, Jozef Gecz6, Enzo Lalli1,2and
1CNRS UMR 6097-Institut de Pharmacologie Mole ´culaire et Cellulaire, Valbonne,2Universite ´ de Nice
Sophia-Antipolis, Nice, France,3Institute of Neurological Sciences National Research Council (CNR), Catania,
4Department of Chemical Sciences, Section of Biochemistry and Molecular Biology, University of Catania,
5Oasi Maria SS Institute for Research on Mental Retardation and Brain Aging, Troina, Italy and6Department of
Genetic Medicine Women’s and Children’s Hospital and School of Pediatric and Reproductive Health, University of
Adelaide, Adelaide, Australia
Received October 26, 2008; Revised December 14, 2008; Accepted December 17, 2008
FRAXE is a form of mild to moderate mental retar-
dation due to the silencing of the FMR2 gene.
The cellular function of FMR2 protein is presently
unknown. By analogy with its homologue AF4,
FMR2 was supposed to have a role in transcriptional
regulation, but robust evidences supporting this
hypothesis are lacking. We observed that FMR2
co-localizes with the splicing factor SC35 in nuclear
speckles, the nuclear regions where splicing fac-
tors are concentrated, assembled and modified.
Similarly to what was reported for splicing factors,
blocking splicing or transcription leads to the accu-
mulation of FMR2 in enlarged, rounded speckles.
FMR2 is also localized in the nucleolus when spli-
cing is blocked. We show here that FMR2 is able
to specifically bind the G-quartet-forming RNA
structure with high affinity. Remarkably, in vivo,
in the presence of FMR2, the ESE action of the
G-quartet situated in mRNA of an alternatively
spliced exon of a minigene or of the putative
target FMR1 appears reduced. Interestingly, FMR1
is silenced in the fragile X syndrome, another form
of mental retardation. All together, our findings
strongly suggest that FMR2 is an RNA-binding pro-
tein, which might be involved in alternative splicing
regulation through an interaction with G-quartet
Fragile X E (FRAXE) mental retardation (OMIM
309548) is associated to a fragile site localized in Xq28
and is the cause of a non-syndromic X-linked mental
retardation affecting 1/50 000 newborn males. The disor-
der is due to the silencing of the Fragile Mental Retarda-
tion 2 (FMR2) gene, as a consequence of a CCG expansion
located upstream to this gene. In the normal population,
the number of this CCG repeat is variable between 6
and 35, while it is increased to more than 200 hyper-
methylated copies in FRAXE mentally retarded patients.
The CCG repeat of FRAXE can either expand or contract
and is equally unstable when transmitted through the male
or the female germ line (1–3).
The FRAXE mental retardation is a form of mild
to moderate mental retardation associated to learning dif-
ficulties, communication deficits, attention problems,
hyperactivity and autistic behaviour (4). Fmr2 inactivation
generated mice displaying a delay-dependent conditioned
fear impairment and a hippocampal increased long-term
potentiation (LTP) (5). FMR2 is a large gene with a major
8.75-kb transcript in placenta, fibroblasts adult and brain
and a longer 13.7-kb FMR2 isoform in fetal brain (6).
The FMR2 gene is organized in 22 exons, showing several
possibilities of alternative splicing for exons 2, 3, 5, 7 and
21. The longest of the FMR2 isoforms is composed of
1272 amino acids and contains two nuclear localization
signal (NLS) sequences that are both able to direct
GFP into the nucleus (6). The nuclear localization
of the endogenous FMR2 protein was also shown by
*To whom correspondence should be addressed. Tel: +33 4 93957755; Fax: +33 4 93957708; Email: email@example.com
? 2009 The Author(s)
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immunohistochemistry in mouse brain (7). FMR2 belongs
to a gene family including AF4 (8), LAF4 (9) and AF5q31
(10). These genes were originally cloned due to their fusion
with MLL (mixed lineage leukaemia) in different chromo-
somal translocations causing acute lymphoblastic leukae-
mia (ALL). AF4 is the best characterized member of this
family, appears to play a role in transcription, since it
interacts with Polymerase II (Pol II) and with the chroma-
tin remodelling machinery (11). AF4 deficient mice have
an altered lymphoid development. Indeed in mouse, AF4
affects early events in lymphopoiesis, such as precursor
proliferation or recruitment, but it is not required for
the terminal stages of lymphocyte differentiation (12).
The FMR2 gene family has a common ancestor in
Drosophila: the Lilliputian (lilli) gene, whose inactivation
generates flies of reduced size. In addition, lilli mutant flies
show reduced expression of some early zygotic genes such
as serendipity, fushi tarazu and huckebein, that are essential
for cellularization and embryonic patterning (13).
By analogy with AF4, FMR2 has been considered to be
a putative transcription activator. However, it is impor-
tant to underline that the ability of FMR2 to activate
transcription was proven only for some domains, but
not for the full-length protein (14). The purpose of this
study was to unravel the function of FMR2 that is cur-
rently unknown. We show here that FMR2 is localized
in SC35-containing nuclear speckles, being implicated in
splicing. This role in splicing is carried out through its
specific interaction with G-quartet RNA, a structure
known to be able to act as an exonic splicing enhancer
(ESE) (15). We also show here that FMR2 influences
in vivo the alternative splicing pattern of the mRNA of
Fragile X Mental Retardation 1 (FMR1), which contains
a G-quartet nearby an acceptor site of the alternatively
spliced exon 14 (16).
MATERIALS AND METHODS
Full-length FMR2 cDNA was amplified by RT–PCR from
NG108 RNA using LA Taq (TaKaRa) and primers whose
sequences are reported in Table 1 containing the XhoI and
BamHI restriction sites. PCR fragments were digested
with restriction enzymes and cloned into the Flag-pTL1
plasmid (17). Three regions of FMR2 (N-ter, C-ter, C1)
were amplified from full-length gene and subcloned into
Flag-pTL1 vector (17) using the same restriction enzymes.
The full-length FMR2, N-ter and C-ter domains cloned in
Flag-pTL1 were amplified by PCR using the primers
whose sequences are reported in Table 2 and cloned in a
pET-151D Topo plasmid (Invitrogen).
The FBS and FBS?35 fragments were amplified from
pTL1-N19 and pTL1-N19 ?35 plasmids, respectively (18)
using the following primers: 50-GGGTCGACGAAGAG
AGGGAGAGCTTC-30(forward) and 50-GGGGATCCG
TTTCCTTTGAAGCCTCCTC-30(reverse). The primers
contain the SalI and BamHI restriction sites, respectively.
The PCR fragments generated using these primers were
digested and subcloned either in the SXN 13 minigene
(19) or in the pGEM-T easy vector (Promega). All plas-
mids were verified by sequencing.
Antibodies, immunofluorescence and immunoblot
To generate the polyclonal anti-FMR2 antibody, a syn-
sponding to amino acids 116–133 of mouse FMR2
was coupled to ovalbumin and used for immunization of
rabbits by standard protocols (Eurogentec). The charac-
terization of this antibody is described in Supplementary
Material. The antiserum was affinity purified as described
Transfection and immunofluorescence were carried out
as previously described (20) using the following antibo-
dies: polyclonal anti-FMR2 at 1:1000 (this study), mono-
clonal anti-Flag M2 (Sigma) at 1:1000, polyclonal anti-
Flag (Sigma) at 1:1000, monoclonal anti-SC35 (Abcam)
at 1:2000, and the following secondary antibodies: Alexa
fluor 488 anti-rabbit IgG or Alexa fluor 594 goat anti-
mouse IgG (Molecular Probes). Immunoblot was carried
out as described (20) using polyoclonal anti-FMR2
(1:1000) (this study), monoclonal anti-Flag M2 (1:3000)
HeLa and NG108 cells were cultured in DMEM supple-
mented with 10% fetal bovine serum and 100mg/ml peni-
cillin/streptomycin. Human fibroblasts were grown in
RPMI 1640 supplemented with 10% fetal bovine serum
and 100mg/ml penicillin/streptomycin.
Actinomycin D treatment, microinjection
NG108 and HeLa cells were incubated with Actinomycin
D (ActD) (Sigma) at the final concentration of 5mg/ml.
Table 1. Primers used to clone full-length FMR2 and its deletion
constructs in Flag-pTL1
ConstructsForward and reverse primers
Table 2. Primers used to clone FMR2 and its deletion constructs in
Constructs Forward and reverse primers
Nucleic Acids Research, 2009, Vol. 37,No. 4
Cytoplasmic microinjection of an antisense U6 oligonu-
cleotide in SK-N-SH cells was performed as described
(21) using lysine-fixable Cascade blue-conjugated dextran
(Invitrogen) as a marker to identify injected cells. Cells
were fixed 2h after injection or 2–4h after ActD
Production and purification of recombinant proteins
The FMR2 full length, N-ter and C-ter regions of FMR2
were amplified by PCR from its cDNA using the primers
listed in Table 2. After purification, the PCR fragments
were cloned in a pET-151D Topo plasmid (Invitrogen) as
fusion proteins with a six histidine tag. The clones were
expressed in Escherichia coli BL21 star (DE3) (Invitrogen).
The cells were grown in LB medium with ampicillin
(100mg/ml), induced for 4h by addition of 1mM isopro-
pyl-b-D-thiogalacto-pyranoside (IPTG) when the cultures
reached an OD of 0.4at 600nm. The cells were harvested
and resuspended in lysis buffer [25mM Tris–HCl pH 7.6,
300mM KCl, 1mM EDTA, 1mM DTT, 20% glycerol,
5% NP-40, 0.5M urea, complete protease inhibitor cock-
tail (Roche), 1mM PMSF] sonicated for 5min and cen-
trifuged at 15000 r.p.m. for 30min at 48C. The
(QIAGEN) overnight at 48C by agitation. Beads were
washed four times with washing buffer (25mM Tris–
HCl, pH 7.6, 300mM KCl, 1mM DTT, 0.5M urea,
20% glycerol, 20mM imidazole). Fusion proteins were
eluted from the beads with elution buffer (25mM Tris–
HCl pH 7.6, 50mM KCl, 1mM EDTA, 1mM DTT,
10% glycerol, 0.1% Triton, 0.5M urea, 200mM imida-
zole) for 30min at 48C.
with Ni-NTA Agarose
RNA binding assays
RNA homopolymer binding assay were performed as
described (20). Filter binding assay and gel retardation
assay were performed as previously described (18,22).
Reverse transcriptase–polymerase chain reaction
Semiquantitative PCR was performed to evaluate the
mRNA produced by minigene constructs, using the pri-
mers SXN13 alternative exon reported in Table 3.
We evaluated the inclusion of exon 2 of the SXN13
minigene and of exon 14 of FMR1 gene by quantitative
RT–PCR as we have recently described (23). The
sequences of primers and PCR conditions used in this
set of experiments are reported in Table 3. RNA was pur-
ified from HeLa cells, normal and FRAXE fibroblasts
using the RNeasy kit (Qiagen) and retro-transcribed
by the Thermoscript RT-PCR System (Invitrogen). Real-
time PCR was performed using a 7000 Sequence Detection
System (Applied Biosystem) using the cDNA, qPCR Core
kit for SYBR Green (Eurogentec) according to the man-
ufacturer’s instructions and 200 nM of each primer. All
experiments were performed in triplicate. The relative
expression of transcripts was quantified using 2?iiCT
method (24). Human GAPDH was used as endogenous
PCR amplification for real-time PCR was performed
with 40 cycles of denaturation at 958C for 15s and
annealing/extension at 608C for 60s. Detection of PCR
product was monitored by measuring the increase in flu-
orescence caused by the binding of SYBR Green to
Localization and functional domainsof FMR2
To get insight into FMR2 function, we performed a
detailed study of its intracellular localization by generat-
ing a polyclonal anti-FMR2 antibody using a synthetic
peptide corresponding to amino acids 116–133 of mouse
FMR2 (see Supplementary Material). We then transfected
HeLa cells (which do not express endogenous FMR2,
Figure1 and Supplementary Figure 1) with a construct
encoding full-length FMR2 and we revealed its nuclear
localization in a dot-like pattern (Figure 1A). This same
result was obtained with a Flag-tagged-FMR2 (not
shown). Our anti-FMR2 antibody recognizes endogenous
FMR2 protein in neuroblastoma NG108 cells (Figure 1B)
and in primary hippocampal neurons (Figure 1C).
Intriguingly, both endogenous and transfected FMR2
are present in the nucleus in large dots colocalized with
nuclear speckle domains, as revealed by the anti-SC35
Figure S2). These nuclear domains represent sites where
splicing factors are concentrated, assembled and modified
(25) (Supplementary Figure 2).
To define the functional properties of FMR2 domains,
we divided FMR2 into three regions and subcloned them
fused to Flag-tag: N-ter (residues 1–640), C-ter (residues
633–1272), C1 (residues 633–966). We used these con-
structs to transfect HeLa cells and their protein products
were revealed by immunofluorescence using the anti-Flag
antibody. The N-ter construct is localized in the cytoplasm
(Figure 2), while C-ter exhibits the same nuclear distribu-
tion as the full-length protein, co-localizing with SC35 and
C1 is nucleolar (Figure 2) (in Supplementary Figure 2B
the co-localization of C1 with fibrillarin as a nucleolar
marker is shown). We conclude that the C-ter domain
is determinant for the speckle localization of FMR2.
Interestingly, this domain also contains a nucleolar local-
ization signal in its C1 subdomain.
Table 3. Primers used to quantify relative expression of minigene
SXN13 and FMR1 mRNA
GeneForward and reverse primers
SXN13 alternative exon50-GACCATTCACCACATTGGTG-30
Primers junction SXN13
FMR1 alternative exon
FMR1 constituve exon
Nucleic Acids Research,2009, Vol.37, No. 41271
Figure 2. FMR2 determinants for its intracellular localization. (A) Co-localization of C-ter with SC35 in nuclear speckles in Hela cells.
(B) Localization of N-ter in the cytoplasm of HeLa cells. (C) Nucleolar localization of C1 in HeLa. All FMR2 domains have been detected
using the polyclonal anti-Flag antibody, and SC35 was detected by the anti SC35 monoclonal antibody. The 40? magnification, scale bar
10 mm. Twenty 40? fields were analyzed, showing a comparable result.
FMR2 SC35 Overlay
Figure 1. Endogenous and overexpressed FMR2 localizes to nuclear speckles. Co-localization of FMR2 with SC35 in nuclear speckles as detected
by polyclonal anti-FMR2 antibody in Hela cells transfected with full-length FMR2 (A) and in NG108 cells (B) and in primary hippocampal neurons
(C) expressing endogenous FMR2. FMR2 was detected with polyclonal anti-FMR2 antibody and SC35 was detected by monoclonal anti-SC35
antibody. (A) 63? magnification, scale bar 5 mm. (B and C) 40? magnification, scale bar 10 mm. Twenty-five 40? fields were analyzed, showing
a comparable result.
Nucleic Acids Research, 2009, Vol. 37,No. 4
Dynamic behaviour ofFMR2
Nuclear speckles are dynamic structures whose size and
shape vary according to the level of RNA polymerase II-
dependent transcription and splicing activity (25). For this
reason, we investigated whether inhibition of splicing or
transcription have an impact on FMR2 intracellular local-
ization (21,26). First, we studied the subcellular distribu-
tion of SC35 and FMR2 in NG108 cells after blocking
transcription by ActD. As expected, SC35 appears redis-
tributed into enlarged, rounded speckles (Figure 3A),
similar to other splicing factors (26). Interestingly, we
observed that a portion of FMR2 protein is co-localized
with SC35. We also blocked splicing in living cells
by injecting the antisense U6 oligonucleotide in the
SK-N-SH neuroblastoma cell line. This led to the
accumulation of SC35 in enlarged speckle domains
(Figure 3B). In these conditions FMR2 is partially co-
localized with SC35 in these speckles-like structures and
also concentrated in nucleoli (Figure 3B). Taken together,
these data strongly suggest that FMR2 is involved in spli-
cing and may have RNA-binding properties.
FMR2isan RNA-binding protein
To validate our hypothesis, we tested the ability of FMR2
to bind synthetic RNA homopolymers. Indeed, FMR2
selectively binds to poly (G), but not poly (A), poly (C)
or poly (U) (Figure 4A) while the control FMRP (Fragile
X Mental Retardation Protein), as expected, is able to
bind to poly (G) and poly (U) (20). The binding activity
to poly (G) was retained by the FMR2C-ter domain, the
DAPI FMR2SC35 Overlay
inj. markerFMR2 SC35Overlay
Figure 3. Blocking of transcription and splicing affects nuclear speckles localization of FMR2. (A) Blocking of transcription in NG108 neuroblas-
toma cells by ActD. The treatment affects the morphology and number of nuclear speckles, as revealed by detection of SC35 with anti-SC35
antibody. FMR2 is co-localized with SC35 in control cells and after treatment, as detected by the anti-FMR2 antibody. In ActD-treated cells, FMR2
is also localized in the cytoplasm. The 40? magnification, scale bar 10mm. Twenty-five 40? fields were analysed, showing a comparable results. (B)
Blocking of splicing in SK-N-SH neuroblastoma cells. In U6 antisense-microinjected cells, SC35 is accumulated in enlarged nuclear speckles, as
detected by monoclonal anti-SC35 antibody. In these cells, FMR2 is co-localized with SC35 and is also concentrated in the nucleoli, as detected by
polyclonal anti-FMR2 antibody. Upper panels, 20? magnification, scale bar 10mm; lower panels, 63? magnification, scale bar 5mm. Ten 20? fields
were analysed, showing a comparable result.
Nucleic Acids Research,2009, Vol.37, No. 41273
same region directing its localization in nuclear speckles
(Figure 2), but not by the N-ter domain (Figure 2). Even if
no homology with other RNA-binding domains is present
in FMR2, two putative RNA-binding motifs were identi-
fied within this region (the first between residues 787 and
815 and the second between residues 890 and 917) using
the RNABindR program (27). Since FMR2 has affinity
for poly (G) RNA, we questioned whether it can bind
any RNA containing a stretch of G nucleotides or if it is
able to recognize and bind a specific G-rich RNA struc-
ture. For this reason, we tested the ability of N-ter and
C-ter recombinant FMR2 proteins to bind a RNA probe
containing a G-quartet forming structure, the N-19 RNA
in a gel retardation assay. Previously, we have shown that
FMR1 mRNA contains a G-quartet forming structure, the
N19 sequence, localized between nucleotides 1470 and
1896 of FMR1, which is bound by FMRP itself (18,28).
Indeed, as for the poly G-sequence increasing amounts of
C-ter bind the N19 probe while of N-ter does not interact
with the RNA (Figure 4B), suggesting a specificity of
FMR2 for G-quartet structure via its C-terminal domain.
FMR2has highaffinity fortheG-quartet RNA structure
G-quartets or quadruplexes are four stranded nucleic acid
structures formed by stacking of planar layers of guanine
tetrad units. In the tetrads, four guanines interact two by
two in a cyclic Hoogsteen hydrogen bonding arrangement.
G-quartet RNA folds in a 3D structure preferentially in
the presence of K+ ions in comparison with Li+ and
Na+ ions (18,29), as it was also shown for G-quartet
RNA structure localized in FMR1 mRNA (18,22).
To test our hypothesis, we generated recombinant N-ter,
C-ter and full-length His-tagged FMR2 proteins and we
performed a filter-binding assay with the N19 probe, using
FMRP as a positive control. Surprisingly, in the presence
of K+, full-length FMR2 and C-ter proteins were able to
bind N19 as tightly as FMRP, whereas the N-terminal
domain, in agreement with the results shown in Figure
4A and 4B, was not (Figure 5B). Additionally, in the pres-
ence of Na+, that partially destabilizes the G-quartet for-
mation, FMR2 and C-ter bound to N19 to a much lower
extent (Figure 5C), while in the presence of Li+the bind-
ing of FMR2 proteins to N19 RNA was abolished
(Figure 5D). To measure binding affinity we used compe-
tition assays that are more sensitive and allow for allevi-
ation of the contribution of the non-specific binding
properties of FMR2 (18,22). Therefore a constant concen-
tration of labeled N19 RNA was incubated with a fixed
amount of His-FMR2 and C-ter in the presence of
increasing concentrations of unlabeled N19 RNA as a
competitor. Binding of FMR2 and C-ter to the labeled
N19 RNA was efficiently competed by the cold probe
(Figure 5E). As little as 1 nM of competitor RNA is
able to displace 50% of FMR2 from the G-quartet labeled
probe, an affinity comparable to that of FMRP for G-
quartet RNA structure (17,18,22). When we used the unla-
belled N8 competitor RNA (corresponding to the 30UTR
of PP2Ac mRNA and not containing G-quartet RNA)
(17) as a negative control, no displacement of the binding
was observed for FMR2 (Figure 5E). Then we tested the
ability of FMR2 and C-ter to bind the FBS (FMRP bind-
ing site) RNA, encompassing only the G-quartet forming
structure inside the N19 RNA (Figure 5A) (nt 1557–1658
of the FMR1 mRNA coding region), FMRP, FMR2 and
C-ter proteins were able to bind specifically to FBS
(Figure 5F). Conversely, when we deleted the G-quartet
structure generating the ?BR ?35 sequence [described
in Figure 5A and in ref. (18)] full-length FMR2 and its
C-terminal domain were unable to bind (Figure 5G). We
can conclude that FMR2 binds G-quartet RNA specifi-
cally and with high affinity.
FMR2/G-quartet RNA interaction in vivo
The G-quartet being a purine-rich element, it has features
of an exonic splicing enhancer (ESE), a group of discrete
sequences within exons that promote both constitutive
and regulated splicing (30). In particular, the G-quartet
present in the FMR1 mRNA has been recently shown to
be a potent ESE (15). To confirm these data, we intro-
duced the FBS sequence into the middle exon of the
SXN13 minigene (19), generating SXN/FBS and we trans-
fected HeLa cells with one or the other minigene. The
presence of a G-quartet determines the complete inclusion
of exon 2 in the SXN/FBS mRNA, which was detected by
semiquantitative RT–PCR using primers localized in exon
Figure 4. FMR2 is an RNA-binding protein.(A) In vitro translated [35S]
methionine labelled full-length FMR2, full-length FMRP, N-ter and
C-ter proteins were incubated with each RNA homopolymer linked
to agarose in the presence of 0.25M KCl. The same volume (10ml)
of each eluate was analysed by SDS–PAGE followed by fluorography.
(B) Labelled N19 probe was incubated in the presence of increasing
amounts of recombinant N-ter (lanes 2–4: 0.2, 0.4 and 0.6 pmol, respec-
tively) and C-ter (lanes 5–7: 0.2, 0.4 and 0.6 pmol, respectively) pro-
teins. As a control, the labelled N19 probe was shown in lane 1.
Nucleic Acids Research, 2009, Vol. 37,No. 4
Protein quantity (pmol)
Protein quantity (pmol)
Protein quantity (pmol)
log concentration competitor (M)
FMR2 - N19
C-ter - N19
FMR2 - N8
C-ter - N8
% bound N19
% bound N19
% bound N19
% bound N19
% bound FBS
Figure 5. FMR2 binds with high specificity the G-quartet RNA structure. (A) Sequence of the FBS purine-rich region encompassing the G-quartet forming structure inside the N19 RNA. The 35
nucleotides indicated in bold and underlined were deleted to generate the ?BR ?35 sequence. (B) Filter-binding assay using increasing amounts of full-length FMR2, FMRP, N-ter and C-ter
proteins in the presence of K+using the N19 RNA as labelled probe. (C) The same experience described in (B) was repeated in the presence of Na+and in (D) in the presence of Li+. (E)
Competition experiments in a nitrocellulose binding assay using the N19 unlabelled RNA as competitor and the unlabelled 30UTR of PP2Ac RNA (N8) not containing any G-quartet forming
structure as a negative control. (F) Filter-binding assay using an increasing amount of full-length FMR2, FMRP and C-ter proteins. The labelled probe is FBS RNA. (G) Filter-binding assay
using an increasing amount of full-length FMR2 and C-ter proteins. The labelled probe is the ?BR ?35 RNA. Each point shows the mean of the results obtained in three independent
experiments (see Supplementary Table 1 for details of each binding assay).
Nucleic Acids Research,2009, Vol.37, No. 41275
1 and 3 of the minigene (Figure 6A). Starting from this
observation, we tested whether FBS/FMR2 interaction
could regulate the splicing of the SXN/FBS minigene.
To this purpose, we co-transfected HeLa cells with the
SXN/FBS minigene and with full-length FMR2, C-ter,
N-ter or the empty vector. Since differences in the expres-
sion of the SXN/FBS minigene were difficult to appreciate
in non-quantitative conditions, we evaluated the relative
expression of transcripts with alternative exon 2 inclusion
by real-time quantitative PCR (primers are localized in
exon 1 and exon junction between exon 2 and 3 of mini-
gene. Their sequences are described in Table 3). As shown
in Figure 6B, inclusion of the exon containing FBS was
decreased about 2-fold in cells co-transfected with full-
length FMR2 or C-ter relative to cells co-transfected
with the empty vector or with the N-ter. In this experi-
ment, the expression level of each FMR2 protein was
tested by western blot and quantitative RT–PCR, as illu-
strated in Supplementary Figure 3. To exclude that this
result is due to a different decay of the mRNA coding for
minigene isoforms in the presence or in the absence of
FMR2, we tested the stability of SXN/FBS minigene in
HeLa cells transfected and treated with ActD for 6h. No
difference in decay of exon2-containing minigene mRNA
was observed during this treatment in cell transfected with
FMR2-pTL or the pTL empty vector (Supplementary
Exons 7-8 (constitutive)
Exon 14 (alternative)
1-6789 101112131415 1617
Exon2-FBS level relative to control
Figure 6. Effect of FMR2 on splicing of an alternative exon in minigene-transfected cells and in the endogenous FMR1 transcript. (A) Schematic
representation of SXN13 minigene and of SXN13/FBS minigene. This last one includes G-quartet in exon 2. Visualization of splicing products of the
minigene by semiquantitative RT–PCR. Black arrows indicate primers positions. (B) Relative expression of the inclusion of minigene exon 2 using
real-time quantitative PCR in the presence or in the absence of FMR2, C-ter and N-ter proteins. Results were obtained by analysis of three
independent transfection experiments. Red arrows indicate primers postitions, GAPDH was used as a standard. Error bars represent standard
deviation. (C) Schematic structure of the FMR1 full-length mRNA, showing alternative splicing of exon 14. (D) Relative expression of FMR1
constitutive exons 7–8 using real-time quantitative PCR in normal fibroblasts and in fibroblasts obtained from a patient carrying the deletion of
FMR2 gene. No difference is observed between the two samples. (E) Relative expression of exon 14 sequence-including FMR1 isoforms using real-
time quantitative PCR in normal fibroblasts and in fibroblasts obtained from a patient carrying the deletion of FMR2 gene. An increased expression
level of 50% is observed for exon 14-containing FMR1 isoforms mRNA in FRAXE fibroblasts.
Nucleic Acids Research, 2009, Vol. 37,No. 4
Figure 4A). Also in this case, the expression level of
FMR2 was tested by western blot (Supplementary
Figure 4B) using the monoclonal anti-Flag antibody.
This finding suggests that the FBS/FMR2 interaction
modulates and influences the splicing efficiency of a mini-
gene containing FBS and that this function is retained by
the C-ter, but not by the N-ter domain, which does not
bind to the FBS. These data suggest that inclusion of a
G-quartet-containing exon in a mature mRNA might be
modulated in vivo by FMR2. We asked whether the
absence of FMR2 has an impact on the alternative spli-
cing pattern of the FMR1 gene, containing a G-quartet
structure in its exon 15 near to the 30acceptor sites neces-
sary for alternative splicing of exon 14 (16). This structure
is a potent exonic splicing enhancer [(15) and this study].
For this reason, we tested the expression level of exon
14-containing FMR1 isoforms in normal fibroblasts and
in a cell line of a FRAXE patient carrying a deletion of
FMR2, that completely abolishes the expression of this
gene (1). For this quantification, we used real-time PCR
using different sets of primers: a couple of primers ampli-
fying the N-terminal region of FMR1 mRNA (exons 7
and 8) and a couple of primers located in exon 13 and
exon 14 (Figure 6C), giving the possibility to evaluate
the inclusion of the exon 14 sequence in the FMR1
mature mRNA. Interestingly, the overall expression of
FMR1 (all isoforms) is not influenced by the absence of
FMR2 (Figure 6D), while in FRAXE fibroblasts the inclu-
sion of exon 14 in FMR1 mRNA is increased of 50%
when compared with normal fibroblasts (Figure 6E).
This demonstrated altered inclusion of exon 14 in the
mature mRNA of FMR1 in the absence of FMR2.
FMR2 expression is silenced in FRAXE mental retarda-
tion and its precise function is not known. Previous studies
have been suggested that FMR2 function is related to
transcriptional regulation in the nucleus. This assumption
was based on the observation that FMR2 was exclusively
detected in the nuclear compartment and that short
N-terminal fragments of the protein have been reported
to act as transcriptional activators of the adenovirus E1b
minimal promoter (14). This function was also supported
by analogy and homology with AF4, a member of the
same gene family, that has been reported to interact
with PolII transcription elongation factor b (P-TEFb)
kinase and in complex with AF9, ENL and AF10 med-
iates methylation of histone H3-K79 (11). Our study
reveals an additional important function of FMR2, pro-
viding a novel insight on the molecular basis of FRAXE
mental retardation by providing evidence that FMR2 is
an RNA binding protein having a putative role in the
regulation of splicing of the G-quartet structure-contain-
ing mRNA. We show here that FMR2 has high affinity
for RNA and, in particular, we demonstrate its specificity
towards G-quartet RNA. G-quartet RNA motifs are
known to play a role in translational control (17,31) and
in controlling splicing efficiency working as an ESE [(15)
and this study]. We show that FMR2 is co-localized with
SC35 in the nuclear speckle domains, the compartments
where splicing factors are stored, assembled and modified
(25). Interestingly, blocking pre-mRNA splicing induces
the redistribution of a subpopulation of FMR2 molecules
in nuclear speckles containing the SC35 splicing factor,
while another pool of the protein accumulates in the
nucleolus. This observation suggests that FMR2 shuttles
between speckles and splicing sites, where it can pick up
cargo RNAs and transfer them to the nucleolus where
they can undergo subsequent modifications. Indeed, the
nucleolus performs additional roles beyond generating
ribosomal subunits, since about 5% of nucleolar compo-
nents are proteins involved in mRNA processing (32).
Intriguingly, the ability of the herpes virus saimiri
ORF57 protein to export of virus mRNA to the cytoplasm
is dependent on its nucleolar localization, suggesting a
critical role of the nucleolus in mRNA export process
(33). On the other hand, blocking transcription by ActD
treatment determines FMR2 localization within enlarged
nuclear speckles-like structure and also its accumulation
in the cytoplasm. Indeed, it is known that inhibition of
PolII transcription by actinomycin D induces the accumu-
lation of some RNA-binding proteins and transcription
factors in the cytoplasm (e.g. hnRNP A1 protein) where
they are confined when no exported RNA is synthetized in
nucleus (34). Taken together, our data suggest a possible
role of FMR2 in post-transcriptional modifications of
RNA and, in particular, in splicing. We validated this
hypothesis using a model minigene system which allowed
us to demonstrate that the over-expression of FMR2 has
an impact on the inclusion of the alternative exon 2 con-
taining the G-quartet structure bound by FMR2 in the
mature RNA. Indeed, we show here that the presence of
FMR2 does not influence the stability of the minigene
mRNA. Moreover, we tested the expression level of
exon-14 containing FMR1 isoforms in fibroblasts null
for FMR2. The interest to study these splicing variants
is due to the fact that FMR1 exon 14 encodes a nuclear
export signal (NES) sequence. Exon 14-containing FMR1
isoforms display cytoplasmic localization, while exon
14-lacking isoforms are localized in the nucleus (16,35).
Interestingly, the absence of FMR2 does not affect the
total expression of FMR1 mRNA but increases the expres-
sion of the isoforms containing exon 14 and encoding a
protein localized in the cytoplasm.
The role of FMR2 in splicing is not exclusive to its
role in transcriptional regulation, since we cannot exclude
that the function of FMR2 is modulated by the interaction
with transcription factors, as, shown, for example, for
SWI/SNF, a complex involved both in chromatin remo-
delling and regulationof
Remarkably, the N-terminal region of FMR2 is able to
affect transcription (14), while we show here that its
C-terminal domain modulates splicing. Furthermore, one
of the proteins belonging to the same family as FMR2,
AF4, has been shown to stimulate RNA polymerase II
transcriptional elongation and mediate coordinated chro-
matin remodeling (11). We can also add that the role of
transciption factor in post-transcription RNA metabolism
is more and more evident, as, for example, recently
reported (37). Indeed, these authors have shown the
Nucleic Acids Research,2009, Vol.37, No. 41277
molecular mechanism by which RA/RARa regulates
translation of specific mRNA in neuronal dendrites (37).
Even the orphan nuclear receptor DAX-1 was shown to
have a role in the mRNA metabolism (38).
Our data indicate a role of FMR2 in splicing of FMR1
pre-mRNA. This suggests that alteration of FMRP
expression in FRAXE patients may participate in the phy-
siopathology of this syndrome. The partial effect of
FMR2 on FMR1 exon14 inclusion may be due to partial
rescue of FMR2 function by its homologous proteins,
which are expressed in a broad range of tissues, as also
recently described for RBM5 and its homologues RBM6
and RBM10 (39). Alternatively, it is worth to underline
that probably other RNA-binding proteins control the
complex splicing pattern of FMR1 with a different
impact in different tissues, as shown for NMDA receptor
1 (NR1). The inclusion of exon 19 into mature NR1
mRNA is in fact regulated by the balanced action of sev-
eral RNA-binding proteins: hnRNP A1, hnRNP H, Nova
and NAPOR (40).
A long list of RNAs exists containing putative G-quar-
tet structures that are located in the proximity of an alter-
natively spliced site (http://bioinformatics.ramapo.edu/
grsdb/) (41). Subtle alterations of the alternative splicing
of some of these genes (e.g. FMR1, ATRX, Neuroligin 3,
etc.) in the absence of FMR2 may then be relevant to the
pathogenesis of the FRAXE syndrome. Indeed, mutations
in ATRX cause a form of mental retardation (42) and
Neuroligin 3 has been shown to be implicated in autism
(43), a phenotype also described in FRAXE patients (4).
In conclusion, here we have described a novel function
FMR2 in splicing. While further analysis will be necessary
to completely identify all the FMR2 targets mRNAs and
its precise mechanism of action, our data show that
FMR2 is a multifunctional protein linking RNA metabo-
lism and transcriptional control.
Supplementary Data are available at NAR Online.
We thank Tom Cooper, Herve ´ Moine, David Nelson,
Franc ¸ oise Presse, Ce ´ line Verheggen and Jasper Saris for
gift of material, Josiane Grosgeorge and Fre ´ de ´ ric Brau for
excellent technical assistance.
CNRS, INSERM, GIS-Maladies Rares, ANR (to B.B.);
AFM (to B.B. and E.L.); INCa and Conseil Ge ´ ne ´ ral
Alpes-Maritimes (to E.L.); Italian Telethon (to M.V.C.);
Fondation Telecom Autisme (to M.B. partial); PhD
fellowship by ‘La Ligue Contre le Cancer’ (to M.M.);
‘Fondation pour la Recherche Me ´ dicale’ (to M.M.);
Boerhinger Ingelheim Fund (to M.M.); intraeuropean fel-
lowship from the Marie Curie 6th Framework Program
(to L.D.). Funding for open access charge: Agence
Nationale de la Recherche (ANR-06-NEURO-015-01).
Conflict of interest statement. None declared.
1. Gecz,J., Gedeon,A.K., Sutherland,G.R. and Mulley,J.C. (1996)
Identification of the gene FMR2, associated with FRAXE mental
retardation. Nat. Genet., 13, 105–108.
2. Gu,Y., Shen,Y., Gibbs,R.A. and Nelson,D.L. (1996) Identification
of FMR2, a novel gene associated with the FRAXE CCG repeat
and CpG island. Nat. Genet., 13, 109–113.
3. Knight,S.J., Flannery,A.V., Hirst,M.C., Campbell,L.,
Christodoulou,Z., Phelps,S.R., Pointon,J., Middleton-Price,H.R.,
Barnicoat,A., Pembrey,M.E. et al. (1993) Trinucleotide repeat
amplification and hypermethylation of a CpG island in FRAXE
mental retardation. Cell, 74, 127–134.
4. Abrams,M.T., Doheny,K.F., Mazzocco,M.M., Knight,S.J.,
Baumgardner,T.L., Freund,L.S., Davies,K.E. and Reiss,A.L. (1997)
Cognitive, behavioral, and neuroanatomical assessment of two
unrelated male children expressing FRAXE. Am. J. Med. Genet.,
5. Gu,Y., McIlwain,K.L., Weeber,E.J., Yamagata,T., Xu,B.,
Antalffy,B.A., Reyes,C., Yuva-Paylor,L., Armstrong,D., Zoghbi,H.
et al. (2002) Impaired conditioned fear and enhanced long-term
potentiation in Fmr2 knock-out mice. J. Neurosci., 22, 2753–2763.
6. Gecz,J., Bielby,S., Sutherland,G.R. and Mulley,J.C. (1997) Gene
structure and subcellular localization of FMR2, a member of a new
family of putative transcription activators. Genomics, 44, 201–213.
7. Miller,W.J., Skinner,J.A., Foss,G.S. and Davies,K.E. (2000)
Localization of the fragile X mental retardation 2 (FMR2) protein
in mammalian brain. Eur. J. Neurosci., 12, 381–384.
8. Morrissey,J., Tkachuk,D.C., Milatovich,A., Francke,U., Link,M.
and Cleary,M.L. (1993) A serine/proline-rich protein is fused to
HRX in t(4;11) acute leukemias. Blood, 81, 1124–1131.
9. Ma,C. and Staudt,L.M. (1996) LAF-4 encodes a lymphoid nuclear
protein with transactivation potential that is homologous to AF-4,
the gene fused to MLL in t(4;11) leukemias. Blood, 87, 734–745.
10. Taki,T., Kano,H., Taniwaki,M., Sako,M., Yanagisawa,M. and
Hayashi,Y. (1999) AF5q31, a newly identified AF4-related gene, is
fused to MLL in infant acute lymphoblastic leukemia with
ins(5;11)(q31;q13q23). Proc. Natl Acad. Sci. USA, 96, 14535–14540.
11. Bitoun,E., Oliver,P.L. and Davies,K.E. (2007) The mixed-lineage
leukemia fusion partner AF4 stimulates RNA polymerase II
transcriptional elongation and mediates coordinated chromatin
remodeling. Hum. Mol. Genet., 16, 92–106.
12. Isnard,P., Core,N., Naquet,P. and Djabali,M. (2000) Altered lym-
phoid development in mice deficient for the mAF4 proto-oncogene.
Blood, 96, 705–710.
13. Tang,A.H., Neufeld,T.P., Rubin,G.M. and Muller,H.A. (2001)
Transcriptional regulation of cytoskeletal functions and segmenta-
tion by a novel maternal pair-rule gene, lilliputian. Development,
14. Hillman,M.A. and Gecz,J. (2001) Fragile XE-associated familial
mental retardation protein 2 (FMR2) acts as a potent transcription
activator. J. Hum. Genet., 46, 251–259.
15. Didiot,M.C., Tian,Z., Schaeffer,C., Subramanian,M., Mandel,J.L.
and Moine,H. (2008) The G-quartet containing FMRP binding site
in FMR1 mRNA is a potent exonic splicing enhancer. Nucleic Acids
Res., 36, 4902–4912.
16. Sittler,A., Devys,D., Weber,C. and Mandel,J.L. (1996) Alternative
splicing of exon 14 determines nuclear or cytoplasmic localisation of
fmr1 protein isoforms. Hum. Mol. Genet., 5, 95–102.
17. Castets,M., Schaeffer,C., Bechara,E., Schenck,A., Khandjian,E.W.,
Luche,S., Moine,H., Rabilloud,T., Mandel,J.L. and Bardoni,B.
(2005) FMRP interferes with the Rac1 pathway and controls actin
cytoskeleton dynamics in murine fibroblasts. Hum. Mol. Genet., 14,
18. Schaeffer,C., Bardoni,B., Mandel,J.L., Ehresmann,B.,
Ehresmann,C. and Moine,H. (2001) The fragile X mental retarda-
tion protein binds specifically to its mRNA via a purine quartet
motif. EMBO J., 20, 4803–4813.
19. Coulter,L.R., Landree,M.A. and Cooper,T.A. (1997) Identification
of a new class of exonic splicing enhancers by in vivo selection.
Mol. Cell Biol., 17, 2143–2150.
Nucleic Acids Research, 2009, Vol. 37,No. 4
20. Bardoni,B., Schenck,A. and Mandel,J.L. (1999) A novel RNA-
binding nuclear protein that interacts with the fragile X mental
retardation (FMR1) protein. Hum. Mol. Genet., 8, 2557–2566.
21. Ohe,K., Lalli,E. and Sassone-Corsi,P. (2002) A direct role of SRY
and SOX proteins in pre-mRNA splicing. Proc. Natl Acad. Sci.
USA, 99, 1146–1151.
22. Bechara,E., Davidovic,L., Melko,M., Bensaid,M., Tremblay,S.,
Grosgeorge,J., Khandjian,E.W., Lalli,E. and Bardoni,B. (2007)
Fragile X related protein 1 isoforms differentially modulate the
affinity of fragile X mental retardation protein for G-quartet RNA
structure. Nucleic Acids Res., 35, 299–306.
23. Davidovic,L., Sacconi,S., Bechara,E.G., Delplace,S., Allegra,M.,
Desnuelle,C. and Bardoni,B. (2008) Alteration of expression of
muscle specific isoforms of the fragile X related protein 1 (FXR1P)
in facioscapulohumeral muscular dystrophy patients. J. Med.
Genet., 45, 679–685.
24. Livak,K.J. and Schmittgen,T.D. (2001) Analysis of relative gene
expression data using real-time quantitative PCR and the 2(-Delta
Delta C(T)) Method. Methods, 25, 402–408.
25. Lamond,A.I. and Spector,D.L. (2003) Nuclear speckles: a model for
nuclear organelles. Nat. Rev. Mol. Cell. Biol., 4, 605–612.
26. O’Keefe,R.T., Mayeda,A., Sadowski,C.L., Krainer,A.R. and
Spector,D.L. (1994) Disruption of pre-mRNA in vivo results in
reorganization of splicing factors. J. Cell Biol., 124, 249–260.
27. Terribilini,M., Sander,J.D., Lee,J.H., Zaback,P., Jernigan,R.L.,
Honavar,V. and Dobbs,D. (2007) RNABindR:a server for
analyzing and predicting RNA-binding sites proteins. Nucleic Acids
Res., 35, W578–W584.
28. Menon,L. and Mihailescu,M.R. (2007) Interactions of the
G-quartet forming semaphorin 3F RNA with the RGG box domain
of the fragile X protein family. Nucleic Acids Res., 35, 5379–5392.
29. Sundquist,W.I. and Klug,A. (1989) Telomeric DNA dimerizes by
formation of guanine tetrads between hairpin loops. Nature, 342,
30. Blencowe,B.J. (2000) Exonic splicing enhancers: mechanism of
action, diversity and role in human genetic diseases. Trends
Biochem. Sci., 25, 106–110.
31. Kumari,S., Bugaut,A., Huppert,J.L. and Balasubramianan,S. (2007)
An RNA G-quadruplex in the 5’ UTR of the NRAS proto-
oncogene modulates translation. Nat. Chem. Biol., 3, 18–21.
32. Lam,Y.W., Trinkle-Mulcahy,L. and Lamond,A.I. (2005) The
nucleolus. J. Cell Sci., 118, 1335–1337.
33. Boyne,J.R. and Whitehouse,A. (2006) Nucleolar trafficking is
essential for nuclear export of intronless herpesvirus mRNA.
Proc. Natl Acad. Sci. USA, 103, 15190–15195.
34. Pinol-Roma,S. and Dreyfuss,G. (1992) Shuttling of pre-mRNA
binding proteins between nucleus and cytoplasm. Nature, 355,
35. Bardoni,B., Sittler,A., Shen,Y. and Mandel,J.L. (1997) Analysis of
domains affecting intracellular localization of the FMRP protein.
Neurobiol. Dis., 4, 329–336.
36. Batsche,E., Yaniv,M. and Muchardt,C. (2006) The human SWI/
SNF subunit Brm is a regulator of alternative splicing. Nat. Struct.
Mol. Biol., 13, 22–29.
37. Poon,M.M. and Chen,L. (2008) Retinoic acid-gated sequence-
specific translational control by RARa. Proc. Natl Acad. Sci. USA,
38. Lalli,E., Ohe,K., Hindelang,C. and Sassone-Corsi,P. (2000)
Orphan receptor DAX-1 is a shuttling RNA binding protein
associated with polyribosomes via mRNA. Mol. Cell Biol., 20,
39. Bonnal,S., Martinez,C., Forch,P., Bachi,A., Wilm,M. and
Valcarcel,J. (2008) RBM5/Luca-15/H37 regulates Fas alternative
splice site pairing after exon definition. Mol. Cell, 32, 81–95.
40. Ule,J. and Darnell,R.B. (2006) RNA binding proteins and the
regulation of neuronal synaptic plasticity. Curr. Opin. Neurobiol.,
41. Kikin,O., Zappala,Z., D’Antonio,L. and Bagga,P.S. (2008)
GRSDB2 and GRS_UTRdb: databases of quadruplex formi,g
G-rich sequences in pre-mRNA and mRNAs. Nucleic Acids Res.,
42. Ion,A., Telvi,L., Chaussain,J.L., Galacteros,F., Valayer,J.,
Fellous,M. and McElreavey,K. (1996) A novel mutation in the
putative DNA helicase XH2 is responsible for male-to-female sex
reversal associated with an atypical form of the ATR-X syndrome.
Am. J. Hum. Genet., 58, 1185–1191.
43. Tabuchi,K., Blundell,J., Etherton,M.R., Hammer,R.E., Liu,X.,
Powell,C.M. and Sudhof,T.C. (2007) A neuroligin-3 mutation
implicated in autism increases inhibitory synaptic transmission in
mice. Science, 318, 71–76.
Nucleic Acids Research,2009, Vol.37, No. 41279