Biochemical evidence for the association of fragile X
mental retardation protein with brain
Edouard W. Khandjian*†‡, Marc-Etienne Huot*†, Sandra Tremblay*†, Laetitia Davidovic*†, Rachid Mazroui*†§,
and Barbara Bardoni¶
*Unite ´ de Recherche en Ge ´ne ´tique Humaine et Mole ´culaire, Centre de Recherche Ho ˆpital Saint-Franc ¸ois d’Assise, Centre Hospitalier Universitaire de
Que ´bec, Que ´bec, QC, Canada G1L 3L5;†De ´partement de Biologie Me ´dicale, Faculte ´ de Me ´decine, Universite ´ Laval, Que ´bec, QC, Canada G1K 7P4; and
¶Institut de Ge ´ne ´tique et de Biologie Mole ´culaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Sante ´ et de la
Recherche Me ´dicale, Universite ´ Louis Pasteur, BP 10142, 67404 Illkirch Cedex, France
Communicated by William T. Greenough, University of Illinois at Urbana–Champaign, Urbana, IL, July 27, 2004 (received for review December 17, 2003)
Fragile X syndrome is caused by the absence of the fragile X mental
retardation protein (FMRP). This RNA-binding protein is widely
expressed in human and mouse tissues, and it is particularly
abundant in the brain because of its high expression in neurons,
where it localizes in the cell body and in granules throughout
dendrites. Although FMRP is thought to regulate trafficking of
in synapses, it is not known whether it has additional functions in
the control of translation in the cell body. Here, we have used
recently developed approaches to investigate whether FMRP is
the brain, FMRP is present in actively translating polyribosomes,
and we show that this association is acutely sensitive to the type
of detergent required to release polyribosomes from membranous
structures. In addition, proteomic analyses of purified brain polyri-
bosomes reveal the presence of several RNA-binding proteins that,
similarly to FMRP, have been previously localized in neuronal
granules. Our findings highlight the complex roles of FMRP both in
actively translating polyribosomes and in repressed trafficking
involved in all subsequent steps in RNA function, from matu-
ration and nucleocytoplasmic transport to subcellular localiza-
RNA contain regions or domains essential for RNA recognition
and binding. In neurons, in addition to being present in the cell
body, a small fraction of mRNA is found in dendrites and axons
at considerable distances from the nucleus (4–7). This differ-
ential distribution implies mechanisms of sorting, targeting,
transport, and delivering of specific mRNA to these particular
distal subcellular domains, where local protein synthesis is
The fragile X mental retardation protein (FMRP) is thought
to be a key player in the control of mRNA transfer to distal
locations such as dendrites (12). This protein is widely expressed
in human and mouse tissues and is particularly abundant in
neurons (13). The absence of FMRP causes fragile X syndrome,
the most common monogenic form of mental retardation (14,
15). Studies on brain of fragile X patients and Fmr1 knockout
mice strongly suggest that FMRP is involved in the proper
development of neuronal spines (16–20). These abnormalities
have been postulated to be at the basis of the mental retardation
that results from defects in the process of neurite extension,
guidance, and branching.
FMRP is an RNA-binding protein present in messenger
ribonucleoparticle (mRNP) complexes associated with the trans-
lation machinery (21–24); however, the exact role of FMRP in
translation remains unclear. High levels of FMRP act as a
negative regulator of translation in vitro and in vivo (25–28). We
he RNA-binding proteins play pivotal roles in posttranscrip-
tional regulation of gene expression. These proteins are
have proposed that, in nonneural cells, FMRP is dispensable,
whereas in neurons, a small fraction of FMRP acts as a repressor
dendrites (28). Indeed, although the great majority of FMRP has
been observed in the neuron cell body (13, 29), a small fraction
was detected either by immunofluorescent staining or immuno-
electron microcopy at distal locations such as neurites, dendrites,
and synaptosomes (22, 29–32). A series of neuronal mRNAs has
been isolated either by immunoprecipitation approaches (33) or
using antibody positioned RNA amplification (34). Although
initial cell fractionation studies have shown that proteins from
the FXR family are associated with polyribosomal mRNPs
derived from rodent brain (29, 35), recent results have proposed
that, in mouse brain, FMRP behavior is unique because it was
not detected at the level of polyribosomes. Instead, FMRP was
found exclusively associated with other classes of slow sediment-
ing ribonucleoparticles (RNPs) (36). These RNPs have been
inferred to correspond to repressed mRNPs that are distinct
from those present in the translation machinery.
Here, we report that, by using recently developed approaches,
we reproducibly found FMRP to be associated with polyribo-
somal mRNPs in brain, as is the case in nonneuronal cells grown
in culture. Also, we show that the association of FMRP as well
as of several populations of RNA-binding proteins with these
structures is acutely sensitive to the type of detergent used to
release polyribosomes from membranous structures. These ap-
proaches enabled us to characterize a series of RNA-binding
proteins that are associated with the translation apparatus.
Materials and Methods
Preparation of Homogenates. Adult (2–4 months old) and young
(10–12 days old) CD1 mice were used throughout this study.
Anesthetized animals were killed by cervical dislocation, and
their brains and livers were quickly removed, immediately chilled
in ice-cold PBS containing 50 ?g?ml cycloheximide, and finely
minced with scissors. The fragments from two brains were
pooled and transferred to 7 ml of a buffer containing 20 mM
Tris?HCl (pH 7.4), 100 mM KCl, 1.25 mM MgCl2, 1 mM DTT,
10 units?ml RNasine (Amersham Pharmacia), and protease
inhibitors (Mini Complete, Roche Biochemicals). Tissues were
slowly homogenized by hand (7–10 strokes) in a Kontes glass
homogenizer (Vineland, NJ) fitted with the loose-B pestle. A
postmitochondrial supernatant was prepared by centrifuging the
homogenate at 9,000 ? g for 15 min.
Abbreviations: FMRP, fragile X mental retardation protein; mRNP, messenger ribonucleo-
particle; DOC, deoxycholate; RNP, ribonucleoparticle.
‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
§Present address: Department of Biochemistry, McGill University, Montre ´al, QC, Canada
© 2004 by The National Academy of Sciences of the USA
September 7, 2004 ?
vol. 101 ?
no. 36 ?
Polyribosomes Studies. To concentrate polyribosomes, 1% Non-
idet P-40 was added to the postmitochondrial supernatant, and
7 ml of the solution was layered over a 3-ml pad made of 45%
(wt?wt) sucrose in an 11-ml tube and centrifuged in a Sorval
TH-641 rotor at 34,000 rpm (105,000 ? g) for 3 h. The
ribosomal pellets were then resuspended in a buffer (20 mM
Tris?HCl, pH 7.4?100 mM KCl?1.25 mM MgCl2) containing
the appropriate anionic or nonionic detergents as described in
Results. Resuspended polyribosomes were analyzed by 15–
45% (wt?wt) isokinetic sucrose gradients composed of 25 mM
Tris?HCl, pH 7.4?100 mM KCl?5 mM MgCl2. After centrifu-
gation in a Sorvall TH-641 rotor for 2 h at 34,000 rpm and 4°C,
gradients were fractionated by upward displacement using an
ISCO UA-5 flow-through spectrophotometer set at 254 nm
and connected to a gradient collector. Each fraction was
precipitated overnight at ?20°C after addition of 2 volumes of
ethanol. The precipitated material was collected by centrifu-
gation at 12,000 rpm for 20 min and solubilized in SDS sample
buffer before immunoblot analyses.
gradients were pooled, and the polyribosomes were recovered
after ultracentrifugation. Polyribosomes were resuspended in a
buffer containing 20 mM Tris?HCl (pH 7.4), 100 mM KCl, 1.25
mM MgCl2, 1 mM DTT, 10 units?ml Rnasine, and protease
inhibitors, treated either with 1% Nonidet P-40 or 1% deoxy-
cholate (DOC) for 30 min at 4°C and recentrifuged at 105,000 ?
g for 2 h. The supernatants containing the Nonidet P-40 or DOC
denatured in SDS sample buffer and analyzed by immunoblot-
ting or by SDS?PAGE, and the protein was revealed after
Coomassie blue staining.
Immunoblot analyses were carried out as described (23).
FMRP was detected with mAb1C3 (13) and mAb7B8 (obtained
from A. Tartakoff, Case Western Reserve University, Cleve-
land), FXR1P was detected with mAb2FX specific for the 78- to
80-kDa isoforms (35), FXR2P was detected with mAbA42 and
PABP1 was detected with mAb10E10 (both obtained from G.
Dreyfuss, University of Pennsylvania, Philadelphia), NUFIP1
was detected with rabbit Ab1375 (37), p50 was detected with
rabbit ?YB-1 (from N. Sonenberg, McGill University, Mon-
tre ´al), 82-FIP was detected with rabbit Ab1666 (38), Stauffen1
and -2 were detected with rabbit ?St1 and ?St2, respectively
(from L. DesGroseillers, University of Montre ´al), Sam68 was
detected with rabbit SC-333 (Santa Cruz Biotechnology), and
the ribosomal S6 and L7 proteins were detected with their
respective antisera (from A. Ziemiecki, University of Bern,
Proteomics Analyses. Analyses were performed at the McGill
University?Genome Que ´bec Innovation Centre facility (Mon-
tre ´al). Samples were run on SDS?PAGE (10% acrylamide), and
the gels were stained with Coomassie blue, scanned, and ana-
lyzed by using IMAGEMASTER software (Amersham Pharmacia).
Gel slices were excised, proteins were digested with trypsin, and
the resulting tryptic peptides were analyzed on a liquid chroma-
tography quadrupole time-of-flight (Micromass, Manchester,
U.K.) mass spectrometer. Peptides were electrosprayed as they
exited the column, and double or triple charged ions were
selected for passage into a collision cell. Fragmentation was
induced by collision with argon gas, and data were collected in
1-s scans for up to 5 s. Peak lists of tandem MS data were
prepared by using MASSLYNX software (Micromass) and submit-
ted to Mascot (Matrix Science, Boston) for identification by
analysis against the National Center for Biotechnology Infor-
mation nonredundant human and mouse databases.
Isolation of Brain Polyribosomes from Adult Mouse. To prepare
FMRP-containing mRNPs, we first isolated membranous struc-
tures by differential centrifugation of total brain lysate using
buffers without detergents as reported (22, 29). We consistently
observed that substantial amounts of FMRP could be recovered
in a fraction corresponding to the rough endoplasmic reticulum
(data not shown). In view of these results, we examined whether
FMRP was associated with polyribosomal mRNPs or was part of
different RNP complexes in brain. Because ?40% of the polyri-
bosomes are bound to membranes in rodent brain (39), the
choice of the detergent was critical to release polyribosomes
from these structures. Cytoplasmic extracts prepared in the
presence of Nonidet P-40, a nonionic detergent, were analyzed
by velocity sedimentation through linear sucrose density gradi-
ents. In repeated analyses, we were unable to obtain a UV
absorption profile corresponding to polyribosomes because of
contaminating light scattering, masking the distribution of sub-
cellular components that may have penetrated the gradients. To
eliminate or to reduce most of these unwanted contaminating
UV-absorbing materials, the cytoplasmic fractions were first
layered over a 45% (wt?wt) sucrose pad, and the polyribosomes
were concentrated by ultracentrifugation as described for rat
liver (40). The resulting opalescent pellets were resuspended in
the extraction buffer containing MgCl2and Nonidet P-40, and
further analyzed by velocity sedimentation through sucrose
density gradients. Unexpectedly, we constantly observed that
only trace amounts of the 80S ribosomes could be detected as
collected fractions were analyzed for the presence of the L7
ribosomal protein. In addition, we observed the presence of an
opalescent pellet in the bottom of the centrifuge tubes where
high levels of L7 and FMRP were detected (Fig. 1a). In contrast,
liver polyribosomes that were used as control were clearly
separated as shown in Fig. 1a, and FMRP was detected in
fractions corresponding to polyribosomes containing L7 pro-
indicates that unknown materials that sediment together with
brain polyribosomes have been compacted at the high g values
used trapping these structures. We have not been able to
resuspend intact polyribosomes in the presence of Nonidet P-40,
Triton X-100, or Igepal CA-630, which are all related nonionic
ever, when the pellets were treated with buffers containing 1%
DOC, an anionic detergent, the polyribosomes were released,
and typical UV profiles with peaks corresponding to the 80S
monomere and high levels of polyribosomes were obtained after
experimental conditions, FMRP was restricted to the first frac-
tions of the gradients, indicating that the protein was no longer
associated with polyribosomes. The same distribution was ob-
served also with liver polyribosomes treated in the same way, as
FMRP remained at the top of the gradients (Fig. 1b).
We also tested a different procedure for the preparation of
free and bound polyribosomes in the presence of a combination
of nonionic and ionic detergents (1% Triton X-100 plus 1%
approach allowed us to successfully isolate polyribosomes; how-
ever, under these conditions, FMRP could not be detected at the
level of polyribosomes, but was instead present in the upper part
of the sucrose gradients (data not shown).
Isolation of Brain Polyribosomes from Young Mouse. The results
presented above clearly showed that under the conditions used,
we were not able to prepare brain polyribosomes that carry
FMRP from adult mice. Attempts to homogenize the brains
more vigorously to fragment the endoplasmic reticulum resulted
www.pnas.org?cgi?doi?10.1073?pnas.0405398101Khandjian et al.
in breakage of the high molecular weight polyribosomes, which
were shifted to the upper parts of the gradients. Because we have
reported that brains from young animals contain 2- to 3-fold
more FMRP as compared with adult (41) and that these tissues
are easier to homogenize, we reasoned that using brains from
younger animals would facilitate polyribosome extraction and
reduce mechanical stress. A representative analysis of polyribo-
some preparations from young animals is presented in Fig. 2 and
shows high levels of heavy sedimenting structures containing
FMRP. These structures corresponded to polyribosomes be-
of the polyribosomes into the large and small ribosomal subunits
and the release of the mRNPs. Polyribosomes were also de-
stroyed after RNase treatment, and FMRP was found free
floating in the top fractions of the gradient (data not shown).
When polyribosome preparations were treated with 1% of DOC
instead of Nonidet P-40, the displacement of FMRP was evident
as it was detected in the upper fractions of the gradient, whereas
the UV profile remained unaffected. Similar results were ob-
tained for 3T3 and HeLa cells grown in culture (data not shown).
To estimate the yield of polyribosomes recovered, different
subcellular fractions were prepared by using sequential extrac-
tions with nonionic followed by ionic detergents (schematically
illustrated in Fig. 3) as adapted from the classic procedure of
Blobel and Potter (42) for total liver polyribosomes preparation.
Fig. 4 illustrates the results obtained after sedimentation anal-
yses of the resuspended pellets and depicts the distribution of
polyribosomes and FMRP. Densitometric and planometric anal-
yses of the polyribosomal absorption profiles of three indepen-
dent experiments indicated that ?98% of FMRP was detected
in the postmitochondrial supernatants treated with Nonidet
P-40, whereas only trace amounts were detected after subse-
quent washing of the pellets with DOC. Finally, when the
postribosomal fraction was centrifuged at 105,000 ? g for 18 h,
no enrichment of free RNPs was seen (Fig. 4). These results
clearly show that the vast majority of FMRP was detected in
association with polyribosomes, whereas only minute amounts of
nonsedimentable FMRP were present in the upper fractions of
the gradients loaded with materials that had been collected after
a long ultracentrifugation for 18 h. The free FMRP likely
correspond to molecules that had been released by the long and
severe treatments imposed to the polyribosomes that are rela-
tively fragile structures.
RNA-Binding Proteins Are Released from Polyribosomes After DOC
Treatment. In view of the altered sedimentation properties of
FMRP stripped off from the polyribosomes, we wondered
Postmitochondrial fraction was treated with 1% nonionic detergent Nonidet
P-40, and total polyribosomes were first concentrated by ultracentrifugation,
resuspended, and analyzed by sedimentation velocity throughout sucrose
of the centrifuge tube, whereas the distribution of liver polyribosomes is
distribute throughout the gradients according to their sedimentation values,
whereas FMRP is detected at the top of the gradients. The integrity and
distribution of polyribosomes were based on the UV profile as well as the
presence of L7, a core protein of the large ribosomal subunit. Distribution of
FMRP in different fractions was revealed by immunoblotting with mAb1C3.
Analyses of brain polyribosomes prepared from adult mice. (a)
between the two polyribosomal profiles, the distribution of FMRP is clearly
affected after treatment with DOC.
the postmitochondrial supernatant and the residual fractions derived from
homogenates of young mouse brain.
Schematic diagram of the steps used to prepare polyribosomes from
Khandjian et al.
September 7, 2004 ?
vol. 101 ?
no. 36 ?
whether this observation was unique to FMRP or was a gener-
alized phenomenon that affected other RNA-binding proteins.
Fractions corresponding to 150–500 S from sucrose density
gradients were first pooled, and the polyribosomes were con-
centrated by ultracentrifugation in duplicate. One series of
purified polyribosomes was resuspended in a buffer containing
1% Nonidet P-40, whereas the other was incubated in buffer
containing 1% DOC. After a 30-min incubation period at 4°C,
both samples were recentrifuged at 33,000 rpm to pellet the
polyribosomes. The supernatants and pellets were analyzed by
immunoblotting using a series of antibodies. Fig. 5 shows the
results that can be summarized as follows. After Nonidet P-40
treatment, all RNA-binding proteins tested remained associated
with the polyribosomes as monitored by using the ribosomal
proteins S6 and L7. In contrast, although polyribosomes treated
with DOC still contained the ribosomal proteins, all RNA-
binding proteins tested were absent and, instead, were detected
in the supernatants. These results clearly prove that the effects
of DOC are not selective, because many other proteins associ-
ated with polyribosomes are stripped off similarly to FMRP.
Furthermore, SDS?PAGE analyses of resistant and labile pro-
teins detected after Coomassie brilliant blue staining showed
that ?15% of the total polyribosomal protein complement were
extracted after DOC treatment. To identify the nature of the
major polypeptide bands indicated by boxes in Fig. 6, these were
cut, subjected to trypsin digestion, and analyzed by MS. The
the majority of the released proteins that have been identified
are RNA-binding proteins.
Previous studies have shown that FMRP was absent from
polyribosomes in mouse brain and instead was exclusively asso-
ciated with light-sedimenting RNP complexes inferred to cor-
respond to repressed mRNPs (36). These unexpected localiza-
tions led the authors to propose an original mechanism of action
for FMRP in neurons. In the present study, we demonstrate that
considerable amounts of FMRP cosediment with large polyri-
bosomes isolated from young mouse brain, whereas no FMRP
was found associated with light-sedimenting RNP complexes.
These discrepancies between the association of FMRP with
heavy sedimenting structures (this report) and the presence of
FMRP in slow sedimenting RNPs (36) are likely due to the
different procedures used to prepare polyribosomes from brain
tissue. We found that the use of the anionic detergent DOC was
necessary to release polyribosomes from adult mouse brain,
presumably because of the chemical composition of the mem-
branous structures at this age, whereas this harsh treatment was
not required for other tissues such as liver. However, the
complexes prepared in the presence of DOC seem to represent
naked polyribosomes, as this detergent has been shown to
selectively remove proteins associated with mRNA (43), includ-
ing FMRP (22). Moreover, when added to the total homogenate,
DOC releases slow sedimenting nuclear RNPs that contaminate
the cytoplasmic fraction and that could be confounded with
small cytoplasmic mRNPs (44, 45). However, we showed that
can be prepared from young animals by using the nonionic
detergents Nonidet P-40 or Triton X-100 of the polyoxyethene
p-t-octylphenol family (43, 46). Under these experimental con-
ditions, FMRP that has been assigned to neuronal granules (31,
32) and that has been reported to be exclusively present in
fractions prepared by differential sedimentations, as described in Fig. 3. Note
that most of FMRP is associated with heavy sedimenting polyribosomes (P1),
whereas only trace amounts are detected at the top of the gradient in panel
FP, which stands for the final pellet obtained after centrifugation of the
postribosomal pellet for 18 h at 105,000 ? g.
Quantitative distribution of brain polyribosomes and FMRP from the
polyribosomes after treatment with DOC, whereas Nonidet P-40 has no del-
eterious effects on the purification of polyribosomes that still carry these
RNA-binding proteins. Immunoblot analyses were performed with the indi-
cated specific antibodies. Note that neither Nonidet P-40 nor DOC has an
effect on the core ribosomal proteins S6 and L7. P, polyribosomes recovered
after ultracentrifugation; S, protein soluble either in Nonidet P-40 or DOC.
FMRP and several other RNA-binding proteins are released from
www.pnas.org?cgi?doi?10.1073?pnas.0405398101Khandjian et al.
light-sedimenting structures (36) was consistently present in
polyribosomes. This was also the case for proteins that interact
directly with FMRP, such as FXR1P, FXR2P, NUFIP, and
82-FIP (14). Furthermore, proteins interacting with the trans-
lation apparatus such as p50 (47), PABP1 (48), Sam68 (49), and
Staufen 1?2 (50) were present.
Because DOC has the property to leave the polyribosome
skeleton intact, we were able to selectively extract and charac-
terize by proteomic analyses nonribosomal proteins. Two classes
of proteins extracted with DOC were identified: the RNA-
binding proteins and those that interact with the cytoskeleton
framework, the local support of translation (51, 52). In addition
to confirming the results obtained by immunoblotting, these
analyses also revealed the presence of a series of RNA-binding
proteins that directly interact with the translation machinery,
such as the different eIFs, tRNA ligases, and RNA helicases (53,
54). Also, RNA-binding proteins known to be predominantly
localized in the nucleus (3), such as the hnRNPs A?B?U?R and
the ELAV, were also detected associated with polyribosomes.
Our findings that FMRP is associated with actively translating
polyribosomes, in addition to its neuronal granule localization
(32), strongly suggest a dual role for FMRP reminiscent to
YB-1?p50, a nucleic acid chaperone (47, 55). Although the
stoichiometry of FMRP to mRNA is not known, it is possible to
propose that levels of FMRP above a certain threshold, yet to be
determined, induce repression of translation by remodeling in
concert with other interactor proteins that may influence FMRP
function(s) (14), the conformational status of mRNAs in RNPs
that have to be translocated to distal locations in neurons (56,
57). However, lower levels of FMRP might have complex roles
in translation control because, when absent, the steady-state
levels of many brain mRNAs are altered, some being increased
and some being decreased (33, 34, 58). Finally, recent results
suggest that FMRP may regulate neuronal translation via mi-
croRNAs (59), which in turn are found associated with polyri-
It is tempting to speculate whether the recently observed
FMRP-containing granules trafficking throughout dentrites (32)
correspond to the class of repressed granules described earlier
(61). Interestingly, these later repressed structures are many
hundreds, if not thousands, of Svedbergs (S20.w) away from the
small repressed mRNPs described by Zalfa et al. (36) because
they sediment faster and ahead of polyribosomes. One of the key
issues to unravel FMRP function(s) is to understand its RNA-
binding selectivity and protein interaction properties. It seems
that the nature of the approaches used to analyze the localiza-
tion(s) and function(s) of FMRP has direct impacts on the
interpretations of the results. On the basis of these consider-
ations, we believe that our results will provide a useful basis for
the future isolation and purification of brain FMRP-containing
We thank Enzo Lalli, Urs-Peter Roos, Timothy Rose, Annette Schenk,
Paul de Koninck, and Susan James for helpful discussions; Paul Nac-
cache and Richard Kinkead for critically reading the manuscript; Luc
DesGroseillers, Gideon Dreyfuss, Nahum Sonenberg, Alan Tartakoff,
and Andrew Ziemiecki for providing antibodies; and anonymous refer-
ees for their constructive comments and suggestions. This work was
supported by the Canadian Institutes of Health Research (E.W.K.), the
National Institutes of Health Human Frontiers Science Program, and the
Institut National de la Sante ´ et de la Recherche Me ´dicale (B.B.). L.D.
holds a postdoctoral fellowship from the FRAXA Research Foundation,
R.M. was the recipient of a postdoctoral fellowship from the Fragile X
Research Foundation of Canada?Canadian Institutes of Health Re-
search Partnership Challenge Fund program, and M.-E.H. holds a
scholarship from the Canadian Institutes of Health Research.
1. Darnell, R. B. (2002) Cell 110, 545–550.
2. Dever, T. E. (2002) Cell 108, 545–556.
3. Dreyfuss, G., Kim, V. N. & Kataoka, N. (2002) Nat. Rev. Mol. Cell Biol. 3,
4. Kuhl, D. & Skehel, P. (1998) Curr. Opin. Neurobiol. 8, 600–606.
5. Kiebler, M. A. & DesGroseillers, L. (2000) Neuron 25, 19–28.
6. Jansen, R.-P. (2001) Nat. Rev. Mol. Cell Biol. 2, 247–256.
7. Richter, J. D. & Lorenz, L. J. (2002) Curr. Opin. Neurobiol. 12, 300–304.
8. Campenot, R. B. & Eng, H. (2000) J. Neurocytol. 29, 793–798.
9. Martin, K. C., Barad, M. & Kandel, E. R. (2000) Curr. Opin. Neurobiol. 10,
10. Brittis, A. P., Lu, Q. & Flanagan, J. G. (2002) Cell 110, 223–235.
11. Steward, O. & Schuman, E. M. (2003) Neuron 40, 347–359.
12. Antar, L. N. & Bassell, G. J. (2003) Neuron 37, 555–558.
13. Devys, D., Lutz, Y., Rouyer, N., Bellocq, J.-P. & Mandel, J.-L. (1993) Nat.
Genet. 4, 335–340.
eluted and analyzed by MS. Identified proteins are classified as RNA-binding proteins and other proteins. P, polyribosomal proteins resistant to DOC treatment;
S, soluble proteins after DOC treatment.
Identification of proteins extracted from polyribosomes after treatment with 1% DOC. The major Coomassie brilliant blue-stained bands in lane S were
Khandjian et al.
September 7, 2004 ?
vol. 101 ?
no. 36 ?
14. Bardoni, B. & Mandel, J.-L. (2002) Curr. Opin. Genet. Dev. 12, 284–293. Download full-text
15. O’Donnell, W. T. & Warren, S. T. (2002) Annu. Rev. Neurosci. 25, 315–338.
Genet. 41, 289–294.
17. Comery, T. A., Harris, J. B., Willems, P. J., Oostra, B. A., Irwin, S. A., Weiler,
I. J. & Greenough, W. T. (1997) Proc. Natl. Acad. Sci. USA 94, 5401–5404.
18. Greenough, W. T., Klintsova, A. Y., Irwin, S. A., Galvez, R., Bates, K. E. &
Weiler, I. J. (2001) Proc. Natl. Acad. Sci. USA 98, 7101–7106.
19. Irwin, S. A., Patel, B., Idupulapati, M., Harris, J. B., Crisostomo, R. A., Larsen,
Genet. 98, 161–167.
20. Nimchinsky, E. A., Oberlander, A. M. & Svoboda, K. (2001) J. Neurosci. 21,
21. Eberhart, D. E., Malter, H. E., Feng, Y. & Warren, S. T. (1996) Hum. Mol.
Genet. 5, 1083–1091.
22. Khandjian, E. W., Corbin, F., Woerly, S. & Rousseau, F. (1996) Nat. Genet. 12,
23. Corbin, F., Bouillon, M., Fortin, A., Morin, S., Rousseau, F. & Khandjian,
E. W. (1997) Hum. Mol. Genet. 6, 1465–1472.
24. Feng, Y., Absher, D., Eberhart, D. E., Brown, V., Malter, H. E. & Warren, S. T.
(1997) Mol. Cell 1, 109–118.
25. Laggerbauer, B., Ostareck, D., Keidel, E. M., Ostareck-Lederer, A. & Fischer,
U. (2001) Hum. Mol. Genet. 10, 329–338.
26. Li, Z., Zhang, Y., Ku, L., Wilkinson, K. D., Warren, S. T. & Feng, Y. (2001)
Nucleic Acids Res. 29, 2276–2283.
27. Schaeffer, C., Bardoni, B., Mandel, J.-L., Ehresmann, B., Ehresmann, C. &
Moine, H. (2001) EMBO J. 20, 4803–4813.
28. Mazroui, R., Huot, M.-E., Tremblay, S., Filion, C., Labelle, Y. & Khandjian,
E. W. (2002) Hum. Mol. Genet. 11, 3007–3017.
29. Feng, Y., Gutekunst, C. A., Eberhart, D. E., Yi, H., Warren, S. T. & Hersch,
S. M. (1997) J. Neurosci. 17, 1539–1547.
30. Weiler, I. J., Irwin, S. A., Klintsova, A. Y., Spencer, C. M., Brazelton, A. D.,
Miyashiro, K., Comery, T. A., Patel, B., Eberwine, J. & Greenough, W. T.
(1997) Proc. Natl. Acad. Sci. USA 94, 5395–5400.
31. De Diego Otero, Y., Severijnen, L.-A., van Cappellen, G., Schrier, M., Oostra,
B. & Willemsen, R. (2002) Mol. Cell. Biol. 22, 8332–8341.
32. Antar, L. N., Afroz, R., Dictenberg, J. B., Carroll, R. C. & Bassell, G. J. (2004)
J. Neurosci. 24, 2648–2655.
33. Brown, V., Jin, P., Ceman, S., Darnell, J. C., O’Donnell, W. T., Tenenbaum,
S. A., Jin, X., Feng, Y., Wilkinson, K. D., Keene, J. D., et al. (2001) Cell 107,
34. Miyashiro, K. Y., Beckel-Mitchener, A., Purk, T. P., Becker, K. G., Barret, T.,
Liu, L., Carbonetto, S., Weiler, I. J., Greenough, W. T. & Eberwine, J. (2003)
Neuron 37, 417–431.
35. Khandjian, E. W., Bardoni, B., Corbin, F., Sittler, A., Giroux, S., Heitz, D.,
Tremblay, S., Pinset, C., Montarras, D., Rousseau, F. & Mandel, J.-L. (1998)
Hum. Mol. Genet. 7, 2121–2128.
36. Zalfa, F., Giorgi, M., Primerano, B., Moro, A., Di Penta, A., Reis, S., Oostra,
B. & Bagni, C. (2003) Cell 112, 317–327.
37. Bardoni, B., Willemsen, R., Weiler, I. J., Schenck, A., Severijnen, L.-A.,
Hindelang, C., Lalli, E. & Mandel, J.-L. (2003) Exp. Cell Res. 289, 95–107.
38. Bardoni, B., Castets, M., Huot, M.-E., Schenck, A., Adinolfi, S., Corbin, F.,
Pastore, A., Khandjian, E. W. & Mandel, J.-L. (2003) Hum. Mol. Genet. 12,
39. Ramsey, J. C. & Steele, W.J. (1977) J. Neurochem. 28, 517–527.
40. Wettstein, F. O., Staehelin, T. & Noll, H. (1963) Nature 197, 430–435.
41. Khandjian, E. W., Fortin, A., Thibodeau, A., Tremblay, S., Co ˆte ´, F., Devys, D.,
Mandel, J.-L. & Rousseau, F. (1995) Hum. Mol. Genet. 4, 783–789.
42. Blobel, G. & Potter, V. R. (1967) J. Mol. Biol. 26, 279–292.
43. Olsnes, S. (1970) Eur. J. Biochem. 15, 464–471.
44. Moule ´, Y. & Chauveau, J. (1968) J. Mol. Biol. 33, 465–481.
45. Olsnes, S. (1970) Biochim. Biophys. Acta 213, 149–158.
46. Borun, T. W., Scharff, M. D. & Robbins, E. (1967) Biochim. Biophys. Acta 149,
47. Davydova, E. K., Evdokimona, V. M., Ovchinnikov, L. P. & Hershey, J. W. B.
(1997) Nucleic Acids Res. 25, 2911–2916.
48. Adam, S. A., Nakagawa, T., Swanson, M. S., Woodruff, T. K. & Dreyfuss, G.
(1986) Mol. Cell. Biol. 6, 2932–2943.
49. Grange, J., Boyer, V., Fabian-Fine, R., Ben Fredj, N., Sadoul, R. & Goldberg,
Y. (2004) J. Neurosci. Res. 75, 654–666.
50. Duchaı ˆne, T. F., Hemraj, I., Furic, L., Deitinghoff, A., Kiebler, M. A. &
DesGroseillers, L. (2002) J. Cell Sci. 115, 3285–3295.
51. Jansen, R.-P. (1999) FASEB J. 13, 455–466.
52. Bassell, G. & Singer, R. H. (1997) Curr. Biol. 9, 109–115.
53. Gingras, A.-C., Rasught, B. & Sonenberg, N. (1999) Annu. Rev. Biochem. 68,
54. Vala ´sek, L., Mathew, A. A., Shin, B.-S., Nielsen, K. H., Szamecz, B. &
Hinnebusch, A. G. (2003) Genes Dev. 17, 786–799.
55. Kohno, K., Izumi, H., Uchiumi, T., Ashizuka, M. & Kuwano, M. (2003)
BioEssays 25, 691–698.
56. Gabus, C., Mazroui, R., Tremblay, S., Khandjian, E. W. & Darlix, J.-L. (2004)
Nucleic Acids Res. 32, 2129–2137.
57. Mazroui, R., Huot, M.-E., Tremblay, S., Boilard, N., Labelle, Y. & Khandjian,
E. W. (2003) Hum. Mol. Genet. 12, 3087–3096.
58. D’Agata, V., Warren, S. T., Zhao, W., Torre, E. R., Alkon, D. L. & Cavallaro,
S. (2002) Neurobiol. Dis. 10, 211–218.
59. Jin, P., Zarnescu, D. C., Ceman, S., Nakamoto, M., Mowrey, J., Jongens,
T. A., Nelson, D. L., Moses, K. & Warren, S. T. (2004) Nat. Neurosci. 7,
60. Kim, J., Krichevsky, A., Grad, Y., Hayes, G. D., Kosik, K. S., Church, G. M.
& Ruvkun, G. (2004) Proc. Natl. Acad. Sci. USA 101, 360–365.
61. Krichevsky, A. M. & Kosik, K. S. (2001) Neuron 32, 683–696.
www.pnas.org?cgi?doi?10.1073?pnas.0405398101Khandjian et al.