The fragile X mental retardation protein is a ribonucleoprotein containing both nuclear localization and nuclear export signals
The fragile X mental retardation protein is a ribonucleoprotein containing both nuclear localization and nuclear export signalsDerek E. Eberhart, Henry E. Malter, Yue Feng and Stephen T. Warren*
Howard Hughes Medical Institute and Departments of Biochemistry and Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322, USA
Received May 1, 1996;Revised and Accepted May 28, 1996
Fragile X syndrome is a frequent cause of mental retardation resulting from the absence of FMRP, the protein encoded by the FMR1 gene. FMRP is an RNA-binding protein of unknown function which is associated with ribosomes. To gain insight into FMRP function, we performed immunolocalization analysis of FMRP truncation and fusion constructs which revealed a nuclear localization signal (NLS) in the amino terminus of FMRP as well as a nuclear export signal (NES) encoded by exon 14. A 17 amino acid peptide containing the FMRP NES, which closely resembles the NES motifs recently described for HIV-1 Rev and PKI, is sufficient to direct nuclear export of a microinjected protein conjugate. Sucrose gradient analysis shows that FMRP ribosome association is RNA-dependent and FMRP is found in ribonucleoprotein (RNP) particles following EDTA treatment. These data are consistent with nascent FMRP entering the nucleus to assemble into mRNP particles prior to export back into the cytoplasm and suggests that fragile X syndrome may result from altered translation of transcripts which normally bind to FMRP.
Fragile X syndrome is a common form of inherited mental retardation that is due to the functional absence of the fragile X mental retardation protein (FMRP), encoded by the FMR1 gene (1 ,2 ). The FMR1 gene is transcriptionally silent in nearly all fragile X syndrome patients due to the almost complete methylation of the gene following massive expansion of the CGG-repeat located in the 5'-untranslated region (3 -7 ). Since several FMR1 deletions result in a similar phenotype, it is accepted that the absence of FMRP is responsible for the mental retardation and various somatic signs characteristic of fragile X syndrome (8 -10 ).
FMRP is highly conserved among vertebrates (11 -13 ) and contains two hnRNP K-protein homology (KH) domains and an RGG box, which are motifs characteristic of RNA-binding proteins (14 ,15 ). FMRP demonstrates preferential binding to RNA homopolymers (15 ,16 ) as well as selective in vitro binding to a subset of brain transcripts, including FMR1 mRNA (14 ). RNA binding studies using truncated FMRP suggest that the carboxyl terminus of FMRP, which contains the RGG box, is essential for FMRP-RNA interaction (11 ,15 ). The KH domains also may influence RNA binding since a missense mutation (Ile304Asn) within the second KH domain results in severe fragile X syndrome (17 ) and impaired in vitro RNA homopolymer binding under high salt conditions (16 ,18 ).
Extensive alternative splicing near the 3' end of the FMR1 transcript results in multiple FMRP isoforms with distinct carboxyl termini (13 ,19 ). Since no FMRP isoform-specific antibodies have been reported, whether certain isoforms exhibit distinct cellular or subcellular localizations has not been established. However, immunoblot analysis suggests that those isoforms with distinct molecular weights are found in all tissues examined although sometimes at quantitatively different levels (16 ,20 ).
FMR1 expression is widespread but not ubiquitous, with abundant neuronal expression in the brain, particularly in the hippocampus and granular layer of the cerebellum (21 -23 ). FMRP is predominantly localized to the cytoplasm in a variety of cell types (16 ,24 -26 ), yet nuclear localization has occasionally been observed (25 ). These indirect immunofluorescent studies demonstrate a granular cytoplasmic appearance and Khandjian et al. recently reported that the majority of cellular FMRP is associated with ribosomes (27 ).
Since the FMR1 gene was isolated by positional cloning strategies, no prior knowledge existed regarding either the function of FMRP or the consequences of its absence, except for the clinical description of fragile X syndrome. Despite recent progress identifying FMRP as an RNA-binding protein (14 ,15 ) which is associated with ribosomes (27 ), as well as characterization of FMRP expression patterns and isoforms, the function of FMRP remains obscure, as does the mechanism by which the absence of FMRP results in mental retardation and the associated phenotype. Here we present studies indicating that FMRP contains both a nuclear localization signal (NLS) and a nuclear export signal (NES) and associates with ribosomes in a RNA-dependent manner. These data consolidate previous information on FMRP and suggest that FMRP enters the nucleus to assemble into ribonucleoprotein (RNP) particles followed by export into the cytoplasm and association with ribosomes.
Immunocytofluorescence analysis of COS-7 cells which were transiently transfected with a SV40 promoter-driven full-length FMRP construct containing the FLAG-epitope revealed the expected diffuse cytoplasmic pattern (24 -26 ), along with intense perinuclear staining (Fig. 1 A and B). The granular cytoplasmic pattern likely reflects FMRP associated with ribosomes (27 ). In contrast to this result, FMRP isoform 4 (Iso4), in which exon 14 is normally spliced out leaving a distinct carboxyl terminus (13 ), localizes to the nucleus with diffuse staining in the nucleoplasm and no visible staining in the nucleolus (Fig. 1 A and B).
Since [Delta]277 and [Delta]167 may be small enough to enter the nucleus by diffusion, fusion constructs between chicken muscle pyruvate kinase (CMPK) and the amino terminus of FMRP were generated and analyzed for subcellular localization to directly test for the presence of a functional NLS in FMRP (Fig. 2 A). CMPK normally exhibits diffuse cytoplasmic staining and has previously been utilized to define protein targeting signals (28 ). Fusion of the first 16 (FMR.PK1) or the first 117 (FMR.PK6) residues of FMRP resulted in cytoplasmic localization indistinguishable from CMPK alone (Fig. 2 A and B). However, addition of the first 184 residues of FMRP resulted in nuclear localization of the fusion protein (FMR.PK2; Fig. 2 A and B), indicating that FMRP contains an NLS sufficient to direct nuclear localization.
Although FMRP contains a functional NLS, it is predominantly localized in the cytoplasm, suggesting that the NLS is normally overridden by another domain. Since residues encoded by exon 14 appear to be necessary for cytoplasmic localization, further truncation constructs were examined to delineate this domain (Fig. 3 A). Removal of the carboxyl 25 ([Delta]464) and 41 ([Delta]448) residues encoded by exon 14 still resulted in FMRP being found in the cytoplasm (Fig. 3 A and B). Indeed, examination of [Delta]441 reveals that only the initial 17 residues of exon 14 were necessary to localize FMRP to the cytoplasm (Fig. 3 A and B). However, [Delta]432, containing the first eight residues of exon 14, partially loses this ability (Fig. 3 A). Truncation into exon 13 encoded residues ([Delta]419) abolished the predominant cytoplasmic localization (Fig. 3 A and B). A FMRP construct ([Delta]17) in which the first 17 residues encoded by exon 14 (425-441) are removed, leaving the remainder of FMRP intact, showed nuclear localization in 98% of cells (Fig. 3 A and B). Thus, the initial 17 amino acids encoded by exon 14 contain information necessary for the cytoplasmic localization of FMRP.
To determine whether the exon 14-encoded domain was sufficient to override the NLS of FMRP in a chimeric protein, we fused portions of exon 14 to the carboxyl terminus of FMR.PK2 (Fig. 4 A). Remarkably, when the 65 residues encoded by exon 14 are fused to FMR.PK2, localization is shifted to the cytoplasm (FMR.PK3; Fig. 4 A and B). Truncation of the carboxyl terminus of FMR.PK3 to the initial 40 residues of exon 14 similarly results in cytoplasmic localization of the resulting fusion protein (FMR.PK4; Fig. 4 A). Indeed, fusion of the first 17 residues encoded by exon 14 to FMR.PK2 still results in nearly 70% of the transfected cells exhibiting cytoplasmic localization (FMR.PK5, Fig. 4 A and B). Therefore, 17 amino acids (425-441) are necessary and sufficient to direct FMRP or a heterologous nuclear-targeted protein to the cytoplasm.
There are at least two mechanisms by which this 17-residue domain could direct cytoplasmic localization of FMRP. The domain could function either as a nuclear export signal (NES) or as a cytoplasmic retention region which prevents FMRP from going into the nucleus. Sittler et al. (32 ) demonstrated that FMRP isoforms lacking exon 14 localize to the nucleus and suggested that a cytoplasmic retention signal within exon 14 is responsible for the predominant cytoplasmic localization of FMRP. However, the first description of NESs recently appeared for the HIV-1 Rev protein (33 ) and protein kinase inhibitor [alpha] (PKI) (34 ). These signals appear to share many attributes of NLSs in mediating dynamic movement across the nuclear pore, such as ATP- and temperature-dependence as well as being saturable (35 ). Examination of the NES sequences of PKI and Rev reveals strong sequence similarity to residues within the 17 amino acid region responsible for cytoplasmic localization of FMRP (Fig. 4 C), especially at the leucine residues that appear to be crucial for NES function in Rev (33 ) and PKI (34 ).
To directly test for nuclear export activity of the putative NES region, a peptide corresponding to the first 17 residues encoded by exon 14 was conjugated to bovine serum albumin (BSA) and then labeled with fluorescein isothiocyanate (FITC). FITC-labeled BSA-NES was microinjected into the nuclei of COS-7 cells along with immunoglobulin G (IgG) labeled with lissamine rhodamine (LRSC). IgG, which is too large to cross the nuclear envelope by diffusion, was coinjected as a control for injection site and leakage. BSA-NES was efficiently exported with virtually all signal in the cytoplasm after 1 h at 37oC, while coinjected IgG remained in the nucleus (Fig. 4 E). BSA-NES export is an active, temperature-dependent process as there was virtually no export when the cells were incubated at 4oC instead of 37oC (Fig. 4 E). The observed nuclear export is also sequence-dependent since BSA-M1, in which the first three leucine residues of the FMRP NES were mutated to alanine, could no longer mediate export (Fig. 4 D and E). Therefore, FMRP contains a functional NES within the first 17 residues encoded by exon 14.
It has recently been demonstrated that the hrp36 protein of Chironomus tentans, which shows similarity to the NES-containing RNA-binding protein hnRNP A1 (36 ), is incorporated into Balbiani ring mRNP particles in the nucleus and remains associated with these particles during nuclear export and throughout translation (37 ). Since hrp36 and FMRP share the features of RNA-binding, nuclear export, association with polyribosomes, and predominant cytoplasmic localization, it is possible that FMRP is also involved in the export of mRNP particles from the nucleus. To determine if FMRP is associated with RNP particles, postnuclear supernatant from cells treated with cycloheximide to arrest ribosome elongation was subjected to a 20-47% linear sucrose gradient. As shown in Figure 5 , FMRP was detected in fractions containing ribosome subunits and monosomes, but preferentially cofractionated with actively translating polysomes which is in agreement with the findings of Khandjian and coworkers (27 ). When cell lysate was treated with RNase (300 [mu]g/ml), to remove the mRNA connecting translating polyribosomes without disturbing individual ribosomes (38 ), FMRP was significantly dissociated from ribosomes into free protein and RNP fractions (Fig. 5 ). Therefore, FMRP-ribosome association appears to be RNA dependent. When cells were lysed in the presence of 10 mM EDTA, which has been shown to cause polyribosomes to dissociate into subunits (39 ), ribosome dissociation was observed and FMRP was shifted to fractions containing particles with molecular masses of ~30S to ~100S (Fig. 5 ). This behavior is consistent with previously reported EDTA-resistant mRNP particles (39 ,40 ). Furthermore, FMRP is detected in oligo dT-captured mRNPs when the cell lysate is exposed to UV-crosslinking prior to mRNP capture (data not shown). Taken as a whole, these results suggest that FMRP is a component of mRNP particles that can associate with the translation machinery.
The finding that FMRP contains a functional NLS and that Iso4 localizes to the nucleus is consistent with previously unexplained observations. Verheij et al. (25 ) detected intense nuclear staining with anti-FMRP antibodies in esophageal epithelium and the occasional isolated cell in other tissues, though the vast majority of tissues examined displayed clear cytoplasmic localization, as do cells in culture. In addition, Devys and coworkers (26 ) showed that a FMRP construct containing only the first 284 residues localized to the nucleus as did [Delta]277 and [Delta]167. A fusion construct placing the amino terminal 184 amino acids of FMRP upstream of the cytoplasmic CMPK protein (FMR.PK2) was utilized to confirm that this region of FMRP is sufficient to direct nuclear localization. Since this fusion protein is ~72 kDa and nuclear import of proteins with a molecular mass greater than ~40-60 kDa requires facilitated nuclear transport (41 ,42 ), these data strongly argue for the existence of a functional NLS in FMRP.
Despite the presence of an NLS, FMRP is found predominantly within the cytoplasm. The first 17 amino acids encoded by exon 14, 425-441, were shown above to be both necessary and sufficient for cytoplasmic localization of FMRP. This 17 amino acid region was shown by analysis of FMRP-CMPK fusion proteins and by direct nuclear microinjection to contain an NES. Thus, FMRP is one of a newly recognized class of proteins, including Rev (33 ) and PKI[alpha] (34 ), which contain NES domains. Like Rev and PKI[alpha], the FMRP NES shows no similarity to the recently described NES of hnRNP A1, termed M9, which can mediate both nuclear import and export (36 ). As reported for the other NES-containing proteins, FMRP NES activity is temperature- and sequence-dependent. Nuclear export is blocked at 4oC and mutation of the first three leucine residues in the NES to alanine results in the loss of NES activity thus further contributing to definition of the consensus sequence for leucine-rich NESs. It will be interesting to determine if FMRP interacts with the cellular cofactors hRIP/Rab from human (45 ,46 ) and/or Rip1p from yeast (47 ), which interact with the Rev NES and are considered putative NES receptors.
The process of nuclear export is poorly understood, but the discovery of NESs has provided a great deal of insight into RNA export (reviewed in 35 ,48 ). It has been suggested that protein-based NESs may be a common feature of nuclear RNA export (35 ). Rev binds and directs export of viral mRNAs containing the Rev response element binding site (33 ). TFIIIA, which has been implicated in the export of 5S rRNA, contains a putative leucine-rich NES which can substitute for the Rev NES (33 ,48 ). hnRNP A1, which is associated with poly(A) RNA in both the nucleus and cytoplasm, contains the M9 domain which allows shuttling between the two compartments (36 ). Other proteins which resemble hnRNP A1, such as the hrp36 protein of C. tentans (37 ), have also been implicated in RNA export (48 ). While further analysis is necessary to determine the export characteristics of FMRP, the identification of domains which mediate nucleocytoplasmic shuttling suggests that FMRP is involved in RNA export (see below). It is noteworthy that two autosomal homologs of FMR1 have recently been described. FXR1 and FXR2 show significant homology with FMRP, especially the NLS, NES, and KH domains (12 ,49 ,50 ).
It was recently reported that cytoplasmic FMRP is associated with ribosomes (27 ). Our data confirm that FMRP is preferentially associated with actively translating polysomes. In addition, we have shown that RNase treatment removes FMRP from ribosomes and that following EDTA treatment FMRP is found in mRNP particles. These data suggest that FMRP associates with ribosomes as a component of mRNP particles. Since FMRP can selectively bind mRNA in vitro (14 ), it seems possible that FMRP may bind specific transcripts within the mRNP particle and possibly influence their translation.
FMRP contains both a functional NLS and NES and appears to associate with ribosomes in a RNA-dependent fashion as a component of mRNP particles. Given the fact that many RNP particles form within the nucleus (51 ), it is reasonable to consider that nascent FMRP enters the nucleus. Immunoelectron microscopy analysis of neurons revealed FMRP in the nucleus, passing through the nuclear pore, and associated with ribosomes (S. Hersch, S. T. Warren et al., manuscript in preparation). Therefore, it seems that FMRP enters the nucleus and there assembles into a mRNP particle, interacting with specific RNA transcripts, as does the NES-containing Rev protein (33 ) (Fig. 6 ). In addition to binding to mRNAs, FMRP may interact with other proteins, such as the FMRP homolog, FXR2, which can interact with FMRP in vitro (50 ). Like other NES-containing proteins (33 ,37 ), once FMRP is assembled into a mRNP particle, it can direct export of the FMRP-mRNP particle to the cytoplasm where it could associate with translating ribosomes. It is also possible that FMRP may play some role in modulating the translation of the mRNA(s) with which it interacts. Thus, fragile X syndrome may be due to the anomalous nuclear export and ribosome presentation of specific transcripts resulting in their inappropriate translation.
pFMRP and pIso4 were constructed by subcloning the EcoRI-MscI fragment of pFLAG-Mc2.17 or the EcoRI-NaeI fragment from pFLAG-Iso4, respectively, into the EcoRI-SnaBI sites of pSV-Sport1 (Gibco/BRL). pFLAG-Mc2.17 and pFLAG-Iso4 have the 8 residue FLAG-epitope tag inserted between amino acids 2 and 3 (Small, Lakkis, and Warren, in preparation) and were derived from the full-length murine fmr1 cDNA clone, Mc2.17 (13 ), and the alternatively spliced murine fmr1 cDNA clone, Mc2.14 (13 ). p[Delta]585, p[Delta]491, and p[Delta]167 were generated from pFLAG-Mc2.17 by digesting with EcoRI followed by NaeI, XmnI, or PvuII, respectively and subcloning these fragments into the EcoRI-SnaBI site of pSV-Sport1. The EcoRI-HindIII fragment of pFLAG-Mc2.17 was subcloned into pSV-Sport1 to generate p[Delta]277.
FMRP constructs with deletions in exon 14 were constructed as follows: p[Delta]448 was constructed by subcloning the EcoRI-XbaI fragment of pFLAG-Mc2.17 into pcDNA3 (Invitrogen). p[Delta]464, p[Delta]441, p[Delta]432, and p[Delta]419 were amplified by polymerase chain reaction (PCR) from pFLAG-Mc2.17 using primer 5'BluS which annealed just 5' of the pBluescript (Stratagene) multiple cloning site allowing EcoRI digestion to release the 5' end of all the clones along with primer 1510r-N, 1440r-N, 1415r-N, or 1375r-N, respectively which contain a NotI site. The PCR protocol for all constructs consisted of one cycle at 95oC (2 min); 30 cycles of 95oC (10 s), 58-60oC (45 s), 72oC (2 min); and one cycle at 72oC (7 min). PCR products were digested with EcoRI-NotI and cloned into pcDNA3. [Delta]17 was generated from pFLAG-Mc2.17, by removing the initial 17 residues encoded by exon 14, using the Ex-Site PCR mutagenesis kit (Stratagene) with primers 1388 and 1447.
To generate FMRP-CMPK fusion constructs, fmr1 fragments were PCR amplified as described above from pFLAG-Mc2.17 with forward primer 72f-H, which contains a HindIII site, and reverse primers 164r-B (pFMR.PK1), 467r-B (pFMR.PK6), or 669r-B (pFMR.PK2), which contain a BglII site. The PCR products were digested with HindIII/BglII and cloned into the 5' end of the p3PK cassette (28 ) generating an in-frame fusion with CMPK (amino acids 17-476). The entire FMRP/CMPK fusion constructs were then liberated by HindIII/EcoRI digestion and cloned into pcDNA3 creating pFMR.PK1, pFMR.PK6, and pFMR.PK2. PCR amplification of exon 14 fragments from pFLAG-Mc2.17 was performed using primer 14f-B, which contains a BamHI site, with primer 1586r-E (pFMR.PK3), 1510r-E (pFMR.PK4), or 1440r-E (pFMR.PK5), which contain an EcoRI site. The PCR products were digested with BamHI/EcoRI and ligated to the 3' end of pFMR.PK2. Constructs were verified by sequencing and produced the expected size protein in SDS-PAGE immunoblot analysis of transfected COS-7 cell lysate and/or when in vitro transcribed and translated using the TnT kit (Promega). Sequence of PCR primers with the restriction sites underlined:
Approximately 1 * 105 COS-7 cells were seeded onto chamber slides, cultured overnight, and then transfected using 1-3 [mu]g DNA and 15 [mu]g lipofectin (Gibco/BRL) per manufacturer's instructions. Forty-eight hours post-transfection, cells were fixed with 3% paraformaldehyde (PF) and permeabilized with 0.1% Triton X-100. The fixed and permeabilized cells were incubated at room temperature (RT) with 2 [mu]g/ml of anti-FLAG M2 antibody (IBI) in PBS with 3% BSA in a humidified chamber for 2 h. Slides were washed in blocking solution (0.01 M PB, 0.25 M NaCl, 5% nonfat milk), incubated with 2.5 [mu]g/ml biotinylated rabbit anti-mouse IgG1 antibody (Zymed) in blocking solution for 30 min at RT, washed again, and then incubated with 3 [mu]g/ml fluorescein isothiocyanate (FITC)-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc.) in blocking solution for 20 min at RT. Coverslips were mounted with antifade solution (Oncor). Cells were visualized via epifluorescence using a 100* Zeiss oil immersion objective and Zeiss filter cube 09. Fluorescent micrographs were taken with Kodak EL 400 film using identical exposure times.
NES and M1 peptides synthesized and HPLC purified by the Emory University Microchemical facility were crosslinked to BSA (Boehringer Mannheim) via the cysteine residue using sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1- carboxylate (sulfo-SMCC; Pierce) essentially as described (33 ) and labeled with the QuickTag FITC conjugation kit (Boehringer Mannheim). FITC-labeled BSA-NES or BSA-M1 were injected into COS-7 cell nuclei at ~0.5 mg/ml in PBS (pH 7.4) along with IgG-lissamine rhodamine (LRSC, Jackson ImmunoResearch) which was coinjected to insure clean nuclear injection without leakage. Cells were either injected and then incubated at 37oC for 1 h or were equilibrated to 4oC for 15 min, injected, and then incubated at 4oC for 1 h. Following injection/incubation, cells were fixed with 3% PF at RT for 5 min and visualized via epifluorescence using a 60* Zeiss oil immersion objective with Zeiss filter cube 09 (FITC) or 15 (LRSC).
Lymphoblastoid cells were incubated for 15 min with cycloheximide (100 [mu]g/ml) prior to vacuum cavitation lysis (200 psi for 10 min) in a buffer containing 0.25 M sucrose, 50 mM Tris (pH 7.5), 25 mM KCl, 5 mM MgCl2, 1 [mu]g/ml each Aprotinin, Pepstatin and Leupeptin, and 1 mM phenyl methane-sufonyl fluoride. The lysate was subjected to 10 000 * g centrifugation for 10 min to yield the postnuclear supernatant. 500 ml of postnuclear supernatant was loaded onto a 20-47% (w/v) sucrose gradient containing 80 mM NaCl, 20 mM Tris (pH 7.5), and 5 mM MgCl2 followed by centrifugation in a Beckman SW41 rotor at 225 000 * g for 2.25 h at 4oC. The entire gradient profile was monitored at 254 nm and fractionated into twenty-one ~500 [mu]l fractions using a gradient fractionator. The fractionation profile was confirmed by distribution of 18S rRNA (52 ) and the 60S ribosomal protein P0 (53 ). For RNase treatment, RNase A was added to a final concentration of 300 [mu]g/ml and incubated at RT for 5 min. For EDTA treatment, lysis was carried out as described above except 10 mM EDTA was substituted for MgCl2 in the lysis buffer and the sucrose gradient. For slot immunoblot assays, 150 [mu]l of each gradient fraction was applied to PBS pre-wetted nitrocellulose membrane by vacuum filtration using the Bio-Dot SF assembly (Bio-Rad). Immunostaining and ECL detection were performed at RT according to the manufacturer's protocol (Amersham) using FMRP monoclonal antibody mAb1a (26 ).
We thank J. Frangioni and B. Neel for vector p3PK and anti-CMPK antibody, J-L Mandel and K.B. Elkon for antibodies, I. Gonzalez for rRNA probes, J. Hershey for advice on gradient preparation, and D. McCormick, D. Nelson, D. Reines, and K. Wilkinson for advice and critical review of the manuscript. S. T. W. is an investigator of the Howard Hughes Medical Institute. This work was partially funded by NIH grant HD 20521.
1 Warren, S.T. and Ashley, C.T. (1995) Triplet repeat expansion mutations: the example of fragile X syndrome. Annu. Rev. Neurosci.18, 77-99.MEDLINE Abstract
2 Warren, S.T. and Nelson, D.L. (1994) Advances in molecular analysis of fragile X syndrome. JAMA271, 536-542.MEDLINE Abstract
3 Hornstra, I.K., Nelson, D.L., Warren, S.T. and Yang, T.P. (1993) High resolution methylation analysis of the FMR1 gene trinucleotide repeat region in fragile X syndrome. Hum. Mol. Genet.2, 1659-1665.MEDLINE Abstract
4 Sutcliffe, J.S., Nelson, D.L., Zhang, F., Pieretti, M., Caskey, C.T., Saxe, D. and Warren, S.T. (1992) DNA methylation represses FMR-1 transcription in fragile X syndrome. Hum. Mol. Genet.1, 397-400.MEDLINE Abstract
5 Oberlé, I., Rousseau, F., Heitz, D., Kretz, C., Devys, D., Hanauer, A., Boué, J., Bertheas, M.F. and Mandel, J-L. (1991) Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science252, 1097-1102.MEDLINE Abstract
6 Verkerk, A.J.M.H., Pieretti, M., Sutcliffe, J.S., Fu, Y.-H., Kuhl, D.P.A., Pizutti, A., Reiner, O., Richards, S., Victoria, M.F., Zhang, F., Eussen, B.E., van Ommen, G.J.B., Blonden, L.A.J., Riggins, G.J., Chastain, J.L., Kunst, C.B., Galjaard, H., Caskey, C.T., Nelson, D.L., Oostra, B.A. and Warren, S.T. (1991) Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell65, 905-914.
7 Fu, Y.-H., Kuhl, D.P., Pizzuti, A., Pieretti, M., Sutcliffe, J.S., Richards, S., Verkerk, A.J.M.H., Holden, J.J.A., Fenwick, R.G., Jr., Warren, S.T., Oostra, B.A., Nelson, D.L. and Caskey, C.T. (1991) Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell67, 1047-1058.MEDLINE Abstract
8 Lugenbeel, K.A., Peier, A.M., Carson, N.L., Chudley, A.E. and Nelson, D.L. (1995) Intragenic loss of function mutations demonstrate the primary role of FMR1 in fragile X syndrome. Nature Genet.10, 483-485.MEDLINE Abstract
9 Hirst, M., Grewal, P., Flannery, A., Slatter, R., Maher, E., Barton, D., Fryns, J.-P. and Davies, K. (1995) Two new cases of FMR1 deletions associated with mental impairment. Am. J. Hum. Genet.56, 67-74.MEDLINE Abstract
10 Meijer, H., de Graaff, E., Merckx, D.M.L., Jongbloed, R.J.E., de Die-Smulders, C.E.M., Engelen, J.J.M., Fryns, J.-P., Curfs, P.M.G. and Oostra, B.A. (1994) A deletion of 1.6 kb proximal to the CGG repeat of the FMR1 gene causes the clinical phenotype of the fragile X syndrome. Hum. Mol. Genet.3, 615-620.MEDLINE Abstract
11 Price, D.K., Zhang, F., Ashley, C.T. and Warren, S.T. (1996) The chicken FMR1 gene is highly conserved with a CCT 5' untranslated repeat and encodes an RNA-binding protein. Genomics31, 3-12.MEDLINE Abstract
12 Siomi, M., Siomi, H., Sauer, W.H., Srinivasan, S., Nussbaum, R.L. and Dreyfuss, G. (1995) FXR1, an autosomal homolog of the fragile X mental retardation gene. EMBO J.14, 2401-2408.MEDLINE Abstract
13 Ashley, C.T., Sutcliffe, J.S., Kunst, C.B., Leiner, H.A., Eichler, E.E., Nelson, D.L. and Warren, S.T. (1993) Human and murine FMR-1: alternative splicing and translational initiation downstream of the CGG repeat. Nature Genet.4, 244-251.MEDLINE Abstract
14 Ashley, C.T., Wilkinson, K.D., Reines, D. and Warren, S.T. (1993) FMR1 protein: conserved RNP family domains and selective RNA binding. Science262, 563-566.MEDLINE Abstract
15 Siomi, H., Siomi, M.C., Nussbaum, R.L. and Dreyfuss, G. (1993) The protein product of the fragile X gene, FMR1, has characteristics of an RNA binding protein. Cell74, 291-298.MEDLINE Abstract
16 Verheij, C., de Graaff, E., Bakker, C.E., Willemsen, R., Willems, P.J., Meijer, N., Galjaard, H., Reuser, A.J.J., Oostra, B.A. and Hoogeveen, A.T. (1995) Characterization of FMR1 proteins isolated from different tissues. Hum. Mol. Genet.4, 895-901.MEDLINE Abstract
17 De Boulle, K., Verkerk, A.J.M.H., Reyniers, E., Vits, L., Hendrickx, J., Van Roy, B., Van den Bos, F., de Graaff, E., Oostra, B.A. and Willems, P.J. (1993) A point mutation in the FMR-1 gene associated with fragile X mental retardation. Nature Genet.3, 31-35.MEDLINE Abstract
18 Siomi, H., Choi, M., Siomi, M.C., Nussbaum, R.L. and Dreyfuss, G. (1994) Essential role for KH domains in RNA binding: impaired RNA binding by a mutation in the KH domain of FMR1 that causes fragile X syndrome. Cell77, 33-39.MEDLINE Abstract
19 Verkerk, A.M.J.H., de Graaff, E., De Boulle, K., Eichler, E.E., Konecki, D.S., Reyniers, E., Manca, A., Poustka, A., Willems, P.J., Nelson, D.L. and Oostra, B.A. (1993) Alternative splicing in the fragile X gene FMR1. Hum. Mol. Genet.2, 399-404.
20 Khandjian, E.W., Fortin, A., Thibodeau, A., Tremblay, S., Cote, F., Devys, D., Mandel, J.-L. and Rousseau, F. (1995) A heterogeneous set of FMR1 proteins is widely distributed in mouse tissues and modulated in cell culture. Hum. Mol. Genet. 4, 783-789.MEDLINE Abstract
21 Hergersberg, M., Matsuo, K., Gassman, M., Schaffner, W., Luscher, B., Rulicke, T. and Aguzzi, A. (1995) Tissue-specific expression of a FMR1/[beta]-galactosidase fusion gene in transgenic mice. Hum. Mol. Genet.4, 359-366.MEDLINE Abstract
22 Hinds, H.L., Ashley, C.T., Nelson, D.L., Warren, S.T., Housman, D.E. and Schalling, M. (1993) Tissue specific expression of FMR1 provides evidence for a functional role in fragile X syndrome. Nature Genet.3, 36-43.MEDLINE Abstract
23 Abitbol, M., Menini, C., Delezoide, A.-L., Rhyner, T., Vekemans, M. and Mallet, J. (1993) Nucleus basalis magnocellularis and hippocampus are the major sites of FMR-1 expression in the human fetal brain. Nature Genet.4, 147-152.MEDLINE Abstract
24 Feng, Y., Zhang, F., Lokey, L.K., Chastain, J.L., Lakkis, L., Eberhart, D. and Warren, S.T. (1995) Translational suppression by trinucleotide repeat expansion at FMR1. Science268, 731-734.MEDLINE Abstract
25 Verheij, C., Bakker, C.E., de Graaff, E., Keulemans, J., Willemsen, R., Verkerk, A.J.M.H., Galjaard, H., Reuser, A.J.J., Hoogeveen, A.T. and Oostra, B.A. (1993) Characterization and localization of the FMR-1 gene product associated with fragile X syndrome. Nature363, 722-724.MEDLINE Abstract
26 Devys, D., Lutz, Y., Rouyer, N., Bellocq, J.-P. and Mandel, J-L. (1993) The FMR-1 protein is cytoplasmic, most abundant in neurons, and appears normal in carriers of the fragile X premutation. Nature Genet.4, 335-340.MEDLINE Abstract
27 Khandjian, E.W., Corbin, F., Woerly, S. and Rousseau, F. (1996) The fragile X mental retardation protein is associated with ribosomes. Nature Genet.12, 91-93.MEDLINE Abstract
28 Frangioni, J.V. and Neel, B.G. (1993) Use of a general purpose mammalian expression vector for studying intracellular protein targeting: identification of critical residues in the nuclear lamin A/C nuclear localization signal. J. Cell Sci.105, 481-488.MEDLINE Abstract
30 Kambach, C. and Mattaj, I.W. (1992) Intracellular distribution of the U1A protein depends on active transport and nuclear binding to U1 snRNA. J. Cell Biol.118, 11-21.MEDLINE Abstract
31 Siomi, H. and Dreyfuss, G. (1995) A nuclear localization domain in the hnRNP A1 protein. J. Cell Biol.129, 551-560.MEDLINE Abstract
32 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.MEDLINE Abstract
33 Fischer, U., Huber, J., Boelens, W.C., Mattaj, I.W. and Luhrmann, R. (1995) The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell82, 475-483.MEDLINE Abstract
34 Wen, W., Meinkoth, J.L., Tsien, R.Y. and Taylor, S.S. (1995) Identification of a signal for rapid export of proteins from the nucleus. Cell82, 463-473.MEDLINE Abstract
35 Gerace, L. (1995) Nuclear export signals and the fast track to the cytoplasm. Cell 82, 341-344.MEDLINE Abstract
36 Michael, W.M., Choi, M. and Dreyfuss, G. (1995) A nuclear export signal in hnRNP A1: a signal-mediated, temperature-dependent nuclear protein export pathway. Cell83, 415-422.MEDLINE Abstract
37 Visa, N., Alzhanova-Ericsson, A.T., Sun, X., Kiseleva, E., Bjorkroth, B., Wurtz, T. and Daneholt, B. (1996) A pre-mRNA-binding protein accompanies the RNA from the gene through the nuclear pores and into polysomes. Cell84, 253-264.MEDLINE Abstract
38 Nelson, R.J., Ziegelhoffer, T., Nicolet, C., Werner-Washburne, M. and Craig, E.A. (1992) The translation machinery and 70 kd heat shock protein cooperate in protein synthesis. Cell71, 97-105.MEDLINE Abstract
39 Nielsen, F., Ostergaard, L., Nielsen, J. and Christiansen, J. (1995) Growth-dependent translation of IGF-II mRNA by a rapamycin-sensitive pathway. Nature377, 358-362.MEDLINE Abstract
40 Nielsen, F., Gammeltoft, S. and Christiansen, J. (1990) Translational discrimination of mRNAs coding for human insulin-like growth factor II. J. Biol. Chem.265, 13431-13434.MEDLINE Abstract
41 Silver, P.A. (1991) How proteins enter the nucleus. Cell64, 489-497.MEDLINE Abstract
42 Peters, R. (1986) Fluorescence microphotolysis to measure nucleocytoplasmic transport and intracellular mobility. Biochim. Biophys. Acta864, 305-359.MEDLINE Abstract
43 Roberts, B.L., Richardson, W.D. and Smith, A.E. (1987) The effect of protein context on nuclear location signal function. Cell50, 465-475.MEDLINE Abstract
44 Nelson, M. and Silver, P. (1989) Context affects nuclear protein localization in Saccharomyces cerevisiae. Mol. Cell. Biol.9, 384-389.MEDLINE Abstract
45 Fritz, C.C., Zapp, M.L. and Green, M.R. (1995) A human nucleoporin-like protein that specifically interacts with HIV Rev. Nature376, 530-533.MEDLINE Abstract
46 Bogerd, H.B., Fridell, R.A., Madore, S. and Cullen, B. (1995) Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins. Cell82, 485-494.MEDLINE Abstract
47 Stutz, F., Neville, M. and Rosbash, M. (1995) Identification of a novel nuclear pore-associated protein as a functional target of the HIV-1 Rev protein in yeast. Cell82, 495-506.MEDLINE Abstract
48 Gorlich, D. and Mattaj, I. (1996) Nucleocytoplasmic transport. Science271, 1513-1518.MEDLINE Abstract
49 Coy, J.F., Sedlacek, Z-S., Bachner. D., Hameister, H., Joos, S., Lichter, P., Delius, H., and Poustka, A. (1995) Highly conserved 3' UTR and expression pattern of FXR1 points to a divergent gene regulation of FXR1 and FMR1. Hum. Mol. Genet.4, 2209-2218.MEDLINE Abstract
50 Zhang, Y., O'Connor, J.P., Siomi, M.C., Srinivasan, S., Dutra, A., Nussbaum, R.L. and Dreyfuss, G. (1995) The fragile X mental retardation syndrome protein interacts with novel homologs FXR1 and FXR2. EMBO J. 14, 5358-5366.MEDLINE Abstract
51 Dreyfuss, G. (1986) Structure and function of nuclear and cytoplasmic ribonucleoprotein particles. Annu. Rev. Cell Biol.2, 459-498.MEDLINE Abstract
52 Gonzalez, I. and Schmickel, R. (1986) The human 18S ribosomal RNA gene: evolution and stability. Am. J. Hum. Genet.38, 419-427.MEDLINE Abstract
53 Bonfa, E., Parnassa, A.P., Rhoads, D.D., Roufa, D.J., Wool, I.G. and Elkon, K.B. (1989) Antiribosomal S10 antibodies in humans and MRL/lpr mice with systemic lupus erythematosus. Arthritis Rheum.32, 1252-1261.MEDLINE Abstract
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