Skip Navigation


Human Molecular Genetics Advance Access originally published online on October 7, 2003
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
12/23/3087    most recent
ddg335v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (20)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Mazroui, R.
Right arrow Articles by Khandjian, E. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mazroui, R.
Right arrow Articles by Khandjian, E. W.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics, 2003, Vol. 12, No. 23 3087-3096
DOI: 10.1093/hmg/ddg335
© 2003 Oxford University Press

Fragile X Mental Retardation protein determinants required for its association with polyribosomal mRNPs

Rachid Mazroui{dagger}, Marc-Etienne Huot, Sandra Tremblay, Nathalie Boilard, Yves Labelle and Edouard W. Khandjian*

Unité de Recherche en Génétique Humaine et Moléculaire, Centre de Recherche Hôpital Saint-François d'Assise, Le CHUQ, Québec, (Qc) G1L 3L5 and Département de Biologie Médicale, Faculté de Médecine, Université Laval, Québec, Canada

Received July 17, 2003; Accepted September 29, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fragile X Mental Retardation protein (FMRP) is an RNA-binding protein that contains multiple domains with apparently differential affinity to mRNA and to the ribonucleotide homopolymer poly(G). Attempts have been made to map the RNA-binding sites along the protein sequence with a view to determining which of the KH1, KH2 and RGG domains are required to recognize and bind to RNA. While these studies have greatly contributed to the delineation of domains that bind homopolymers or mRNA in vitro, little is known concerning their implications in FMRP function(s) in vivo. To address this question, we have prepared a series of FMRP versions, in which each known in vitro functional domain has been individually deleted, leaving the rest of the protein intact. Constructs with deletions in the protein–protein interaction and RNA-binding as well as in the phosphorylation domains were expressed in STEK-KO cells lacking FMRP and their recruitment into polyribosomal mRNPs and their intra-cellular localization were determined. Our results indicate that the KH RNA-binding domains and the Protein–Protein Interacting domain are essential for FMRP to associate with polyribosomal mRNPs, while the RGG box and the phosphorylated domains are dispensable.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The RNA-binding protein Fragile X Mental Retardation (FMRP) (1,2) is widely expressed in human and mouse tissues and is particularly abundant in the brain (3) due to its high expression in neurons (4). The sole absence of this protein is responsible for the Fragile X syndrome, an X-linked disease (reviewed in 5–8). Owing to the presence of a non-canonical nuclear localization and a conserved nuclear export signal (NLS and NES, respectively) (9–11), FMRP is considered to be a putative nucleocytoplasmic shuttling protein (12). However, it is predominantly localized in the cytoplasm in association with poly(A) mRNP complexes derived from polyribosomes (13,14).

FMRP contains three potential RNA-binding domains: an RGG motif and two KH modules (10). The KH domain was originally identified in the human heterogeneous ribonucleoprotein K (15) and is a motif that is thought to make direct protein–RNA contact with a three-dimensional ß{alpha}{alpha}ßß{alpha} fold (16), although it can also mediate protein–protein interactions (17). The second RNA binding motif is the RGG box. This motif which is found in many RNA binding proteins can be arginine methylated and has been implicated in RNA interactions (18). It has been shown that the RGG motif of FMRP binds to sequences that have the potential to form an intramolecular G-quartet structure present in FMR1 mRNA and in several cellular mRNAs (19–21), suggesting that this motif may mediate specific FMRP–RNA interactions.

In vitro studies as well as immunoprecipitation experiments indicate that FMRP is able to self-associate and to heterodimerize with the two homologous proteins FXR1P and FXR2P (10,22). In addition, several FMRP interacting proteins such as NUFIP1 (23), CYFIP1 and CYFIP2 (24) and 82-FIP (25) have been described recently. All these interactor proteins bind directly to FMRP in the N-terminus region, predominantly in domains lying between the NLS and the first KH motif (26). Other proteins such as Nucleolin, p50, both RNA-binding proteins (27,28) and Pur alpha, mStauffen, Myosin Va (29) have been shown to be components of FMRP-containing complexes. In Drosophila, the dFMRP, the common ancestor and ortholog of FMRP, FXR1P and FXR2P, is associated with the RISC complex together with AGO2 and DICER (30,31). However, evidence for direct interactions of FMRP with these proteins is lacking. A new domain in human FMRP as well as in Drosophila dFMRP has been shown to be phosphorylated. This domain is situated between the RGG box and the NES and it has been proposed that phosphorylation of FMRP could play a role in regulating its physiological functions (32).

There is increasing evidence that FMRP is involved in translational control since it inhibits translation of reporter mRNAs in the rabbit reticulocyte lysate system (21,33,34) as well as in vivo following transient transfection assays (35). However, despite the identification of the domains of FMRP that are involved in protein–protein or RNA–protein interactions, little is known concerning the requirement of these domains in translational regulation activities of FMRP in vivo. The unique known case is the missense mutation I304N occurring in the second KH (KH2) domain (36) that partially impairs RNA binding in vitro (15) while in vivo it abolishes FMRP association with polyribosomal mRNP (14).

To determine which region(s) or domain(s) of FMRP are required for its function, referred here to its ability to interact and associate with polyribosomal mRNPs, we have produced a series of different versions of FMRP. Domains known to interact with RNA and with proteins were individually deleted from the full-length FMRP. In addition, the phosphorylation domain of FMRP was also removed. We show here that, although the different FMRP versions retain some characteristics of functionality based on in vitro criteria, they fail to be integrated into functional mRNP when reintroduced in a cell line lacking FMRP. The results allow us to delineate FMRP regions and domains that are required for in vivo FMRP function.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In vitro RNA-binding properties of the His-hFMRP versions
An accepted criterion to test whether a protein has affinity for RNA is its in vitro ability to bind to homoribopolymers. In most studies carried out to determine the RNA-binding domains of FMRP, full-length as well as truncated proteins or short single domains of the protein were used in binding assays with polyribonucleotides (2,37,38). Similarly, FMRP domains that are required for protein–protein interactions either for self-association and heteromerization or binding to interactors have been determined through in vitro approaches (10,24,39). Although these studies have greatly contributed to delineate domains involved in RNA-binding activities and in protein–protein interactions in vitro, little is known concerning their implications in FMRP function(s) in vivo, such as its assembly into polyribosomal-mRNPs. To address this question, we have constructed several FMRP versions in which each described in vitro functional domain has been deleted individually leaving the rest of the protein intact (Fig. 1A). The constructs encoding the recombinant histidine-human FMRP (His-hFMRP) and its derivatives were inserted in pET21a. {Delta}KHT lacks both KH domains in tandem corresponding to positions 207–425, {Delta}KH1 and {Delta}KH2 lack the first KH1 and second KH2 domain positions 207–283 and 282–425, respectively. {Delta}RGG refers to FMRP lacking the RGG box at position 526–555, and {Delta}PPId to a construct lacking the Protein–Protein Interaction domain from 116 to 208. Finally, {Delta}Phd is a construct from which the putative phosphorylation domain 443–527 was removed. Recombinant proteins were expressed in E. coli and purified by Ni-NTA column chromatography. FMRP containing the isoleucine/asparagine missense mutation (I304N) present in one fragile X patient was similarly expressed. All recombinant proteins, except {Delta}PPId, had an apparent migration at their expected size corresponding to 80, 80, 78, 64, 53, 67, 63 and 68 kDa, respectively, as determined by SDS–PAGE (Fig. 1B). The migration of {Delta}PPId in which 92 amino acids were deleted was slightly reduced compared with that of {Delta}Phd or {Delta}KH1 in which 82 and 74 amino acids were removed, respectively. This discrepancy is most likely due to the high positive net charge of the {Delta}PPId protein.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 1. (A) Schematic diagram of the constructs used. The overall structure of full-length iso7 FMRP and its truncated versions are presented with the three RNA-binding domains (KH1, KH2 and RGG) and the nuclear localization signal (NLS) and nuclear export signal (NES) in hatched boxes. The protein–protein interaction domain (PPId), and the phosphorylation domain (Phd) are also indicated in italics. The position of the I304N mutation is represented with an asterisk. Each deleted domain is indicated by a gap. The numbers refer to the amino acid sequence of the Swiss-Prot Q06787 entry. (B) His-tagged hFMRP and its truncated variant proteins were produced in bacteria, purified, analyzed by SDS–PAGE and revealed after Coomassie Blue staining.

 
To determine which domain(s) are required for FMRP to bind RNA, the different FMRP versions were tested for their ability to be retained on poly(G) homopolymers–agarose beads. Previously, it has been shown (37) that the N-terminal part containing the protein–protein interaction domain, the central region of FMRP containing both KH domains as well as the C-terminal part containing the RGG box, all bind efficiently to poly(G). In addition, recent data showed that FMRP binds to a G-quartet structure containing mRNAs. This interaction required only the RGG motif and did not involve the KH domains. The results presented in Figure 2 show that the {Delta}RGG version did not bind to the poly(G) homopolymers while all other FMRP versions, including the I304N mutant, were retained. These results indicate that the RGG box is indeed required for FMRP to bind poly(G) homopolymers in this in vitro assay, whereas the others domains are dispensable.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. The RGG box is required for FMRP to bind in vitro to poly(G) homopolymers. His-tagged FMRP and its truncated version proteins were subjected to RNA-binding assay using poly(G) homopolymers immobilized on Agarose beads. Bound proteins were eluted, separated by SDS gel and stained with Coomassie Blue.

 
In vitro protein-binding properties of the His-hFMRP versions
We then determined whether subtracting the protein–protein interaction domain or other key domains would influence the binding of protein interactors to the different FMRP versions. The self-association and heterodimerization domain of FMRP consists of a coiled-coil N-terminal region that is highly conserved during evolution. This domain is also found in FXR1P and FXR2P, suggesting that it may play an important role in the formation of mRNP complexes in which FMRP and its partner proteins have been localized. Since the bacterial His-hFMRP versions previously used in the RNA-binding assay (see above) are not post-translationally modified, we used FMRP versions expressed in the STEK Fmr1 KO cell line (35) after transient transfection. For this purpose, FMR1 cDNA versions were removed from the pET21a bacterial vector and transferred into the eukaryotic vector pTL1. After transient transfection of the STEK cells with these new vectors, the different expressed FMRP versions were analyzed by immunoblot analyses and showed identical apparent relative mobility as seen for the purified bacterial His-hFMRP (Fig. 3).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 3. Iso7 full-length FMRP and its derivative versions were expressed in STEK Fmr1 KO cells lacking Fmrp. Immunoblot analysis was performed using mAb1C3 that slightly cross-reacts with endogenous FXR1P as indicated by the stars.

 
In vitro studies as well as immunoprecipitation experiments have shown that the protein–protein interaction domain of FMRP is required for its incorporation into complexes carrying FXR1P and FXR2P (10). We therefore determined by far-western blot analyses whether this domain is the only one required for these interactions. Cell extracts were prepared from STEK cells transiently transfected with the different constructs and the proteins were separated by SDS–PAGE, transferred to a membrane and incubated with 35S-labeled super long FXR1P isoform of 84 kDa that has been produced by in vitro transcription–translation using a rabbit reticulocyte lysate system. Autoradiography of the membrane showed as expected that deletion of the PPI domain prevented FXR1P to bind to FMRP (Fig. 4). Also, 35S-labeled FXR1P did not bind either to {Delta}KHT or to {Delta}KH1, while a positive signal was observed at the level of {Delta}KH2. One possible explanation for these results is that the protein-protein interaction domain overlaps the KH1 domain (16,17). Additional bands were also observed in all lanes at 94 kDa corresponding most probably to FXR1P/FXR2P interaction, while self-association could be revealed as faint bands at 78 kDa. The nature of the high molecular weight FXR1P targets is not known.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 4. Interaction of FXR1P with iso7 FMRP and its derivative versions. STEK cells were transfected with pTL1-Iso7 and its derivative vectors, total protein extracted, separated by SDS–PAGE and transferred to nitrocellulose membranes and proceeded for far-western analyses using in vitro synthesized [35S]-FXR1P in the rabbit reticulocyte lysate system. FXR1P refers to the [35S]-FXR1P 84 kDa used as a probe, and BSA to the control analysis.

 
To confirm these results we next performed immunoprecipitation analyses to determine whether the different FMRP versions are present in FXR1P-containing complexes. For this purpose, the STEK Fmr1 KO cells lacking FMRP were transfected with the respective vectors and cytoplasmic extracts prepared and subjected to immunoprecipitation using #ML-13, an anti-FXR1P mono-specific serum. The immunoprecipitated proteins were collected and separated by SDS–PAGE, transferred to membranes and analyzed to determine the presence of FMRP variants that interact with FXR1P. The first membrane was reacted with mAb2FX specific to the 80–78 kDa FXR1P major isoforms (40) and the results showed the presence of these proteins in all immunoprecipitated samples (Fig. 5). To test for the presence of FMRP, identical membranes were reacted with mAb1C3. Owing to the presence of very high concentrations of the immunoprecipitated FXR1P, the mAb1C3 also reacted with the latter as expected (40), including in the control lane loaded with the STEK KO extract. These results could not be interpreted; however, using mAb7B8 a clearer picture was obtained since newly expressed iso7 FMRP and its derivatives I304N, {Delta}RGG, {Delta}Phd and {Delta}KH2 were shown to co-immunoprecipitate with FXR1P. It is worth mentioning that RNase A treatment of the immunoprecipitates had no effect on the co-precipitation of FMRP with FXR1P, indicating that the association of the two proteins is likely to be mediated via protein–protein and not RNA–protein interactions (data not shown). On the other hand, {Delta}PPId was not detected along with FXR1P. Also, {Delta}KHT and {Delta}KH1 proteins failed to coimmunoprecipitate with FXR1P, confirming the results obtained by the far-western analyses (Fig. 4). Taken together, these results strongly suggest that the protein–protein interaction domain of FMRP is necessary for its association with FXR1P and provide further evidence that this domain overlaps in the first KH1 domain.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 5. Immunoprecipitation studies demonstrating that the protein–protein interaction (PPId) as well as the KH1 domains are both essential for FMRP to associate with FXR1P. Control untransfected STEK and HeLa cells and STEK cells transfected with plasmids encoding FMRP and its derivatives were lysed and one-tenth of the cytosolic extracts (corresponding to 5x105 cells) were immunoprecipitated with anti-FXR1P #ML13 serum. Immunoprecipitated proteins were separated on SDS–10% polyacrylamide gels, transferred to nitrocellulose, and immunoblotted with anti-FXR1P mAb2FX antibodies to dectect FXR1P and with mAb1C3 and mAb7B8 to detect FMRP. Note that mAb1C3 cross reacted with the high amounts of FXR1P present on the membrane.

 
It has recently been shown that hFMRP is post-translationally modified and can be phosphorylated in vitro by the casein kinase II (CKII) at Ser 500, while in Drosophila the corresponding dCKII phosphorylates dFMR1 at Ser 406 (32). However, hFMRP contains two additional putative phosphorylation sites at positions Thr 455 and Thr 518. To delineate the phosphorylation domains of FMRP, we deleted the region containing the putative phosphorylation sites Thr 455, Thr 518 and Ser 500 to yield the {Delta}Phd version. We tested the possibility to phosphorylate in vitro the different purified bacterial His-hFMRP versions devoid of post-translational modifications. Each FMRP variant was incubated in the presence of a HeLa S100 extract and recovered by pull-down using Ni-NTA beads. The complexes bound to the beads were incubated under phosphorylation conditions in the presence of [{gamma}-32P]-ATP. After the reaction, proteins eluted from the beads were separated by SDS–PAGE, stained with Coomassie blue and 32P-labeled proteins revealed after autoradiography. Staining with Coomassie showed that all FMR protein variants were recovered at their expected position with similar intensity (data not shown). As seen in Figure 6, all His-hFMR proteins tested except {Delta}Phd were phosphorylated in vitro. In addition, 32P-labeled bands were detected in all lanes, including the control that contained only HeLa extract, suggesting that proteins from the S100 lysate were adsorbed to the beads and became phosphorylated under the conditions used. The same experiments were also repeated using STEK S100 extracts and identical results were obtained (data not shown). These results indicate that a kinase(s) present in both HeLa and STEK extracts can phosphorylate FMRP in vitro within the predicted phosphorylation domain in agreement with Siomi et al. (32).



View larger version (59K):
[in this window]
[in a new window]
  		Figure 6. Pull-down kinase assay delineating the phosphorylation domain of FMRP. Recombinant His-tagged FMRP and its derivatives were incubated with HeLa S100 extracts. After incubation, the proteins were recovered by binding to the His-Bind resin beads, and the immobilized complexes were incubated with the phosphorylation mixture in the presence of [{gamma}-32P]ATP (see Material and Methods). Proteins were eluted in SDS sample solution, separated by SDS–PAGE followed by autoradiography to reveal phosphorylated proteins. C: control lane without His-hFMRP.

 
In vivo characterization of FMRP active domains
The results presented above clearly indicate that domains in FMRP are required for in vitro recognition of poly(G) homopolymer, for protein–protein interactions and for its phosphorylation. However, these analyses cannot answer the crucial question of whether these domains are important for a functional FMRP in vivo. We have previously shown that FMRP reintroduced in STEK cells can be recognized as a normal cellular partner that can be incorporated in pre-existing mRNPs associated with polyribosomes (35). To determine the domains of FMRP required for its integration into polyribosomal mRNPs, STEK Fmr1 KO cells were transiently transfected with the different FMRP versions. Twenty hours after transfection the cells were fixed and the cellular localization of the FMRP variants were first determined by immunofluorencence staining (Fig. 7). In cells expressing the full-length iso7 FMRP, prominent cytoplasmic granules were detected as previously described (35). These granules are induced by high levels of FMRP and contain repressed mRNPs that are trapped in cytoplasmic foci. In contrast, FMRP versions lacking the RNA-binding domains KH1, KH2, RGG and the protein–protein interaction domains were distributed in the cytoplasm indicating that the formation of the granule-like structures requires a full-length protein. Interestingly, FMRP lacking the phosphorylation domain still formed granules, and the same distribution was confirmed using an FMRP mutant in which the phosphorylated Serine 500 site was substituted by an alanine residue (Fig. 7).



View larger version (47K):
[in this window]
[in a new window]
  		Figure 7. Intracellular distribution of iso7 FMRP and its derivative versions expressed in STEK cells. After transfection with the respective vectors, the STEK cells grown on coverslips were fixed and indirect immunofluorescence staining was performed using mAb1C3.

 
To answer the question whether FMRP incorporated in polyribosomal mRNPs requires a specific or all of the domains described above, cytoplasmic extracts were prepared from the STEK Fmr1 KO transfected cells and analyzed by velocity sedimentation through sucrose density gradients. No major differences in the UV profiles of sedimenting material in the gradients and in the distribution of the ribosomes were observed between the different extracts prepared in the presence of Mg2+ (Fig. 8). In repeated experiments, we constantly observed that iso7 FMRP was mainly detected in the polysomal fractions as judged by immunoblot analyses with a distribution similar to that observed for endogenous FMRP in different cell types (13,14,41). We also constantly observed that low levels (~30%) of the {Delta}RGG version remained associated with the polyribosomes although it has lost its ability to bind RNA based to the poly(G) binding assay as described above. Interestingly, the sedimentation distribution of the {Delta}Phd variant was similar, if not identical to that of full-length iso7 FMRP, and the same distribution was also observed for FMRP carrying the S500A substitution. These results strongly suggest that phosphorylation of FMRP is not required for its incorporation into mRNPs. All {Delta}PPId, {Delta}KHT, {Delta}KH1, {Delta}KH2 versions were absent from polyribosomes and the majority of these proteins were detected in the top fractions of the sucrose gradients (Fig. 8). A clearer picture of the incorporation of the FMRP variants into mRNPs was obtained when polyribosomes, first concentrated by ultracentrifugation, were treated with EDTA that causes the dissociation of the polyribosomes into the large and small ribosomal sub-units and the release of the mRNPs. Consistent with the results described above, iso7 FMRP and its {Delta}Phd and S500A derivatives were detected sedimenting with the released mRNPs and reduced amounts of {Delta}RGG were also detected along with mRNPs. Concerning {Delta}KHT, {Delta}KH1, {Delta}KH2 and {Delta}PPId, all these variants were absent from the gradients, indicating that these variants could not associate with mRNPs. Finally, to confirm these results, the distribution of FMRP variants into different sub-cellular fractions obtained after differential centrifugations was examined. Fractions containing predominantly nuclei, ribosomes and the soluble final supernatant were prepared and the FMR proteins determined in each fraction. A substantial amount (~40–60%) for each protein was recovered in the crude nuclear pellet (Fig. 9). These high amounts of FMRP associated with the insoluble fraction are due to the non-physiological overexpression of the proteins, which were found to be tightly associated with the cytoskeleton framework (unpublished data). Iso7 FMRP, {Delta}Phd and S500A were detected in the ribosomal fraction while trace amounts were present in the supernatant fractions. On the other hand, {Delta}RGG was distributed in both the ribosomal and supernatant fractions. All other FMRP variants were recovered strictly in the supernatant fractions. These results are consistent with those obtained by sedimentation analyses in sucrose density gradients (Fig. 8) and taken together they clearly show that the protein–protein interaction domain and both KH domains of FMRP are required for its incorporation into translationally competent mRNPs. The RGG box is not essential for the recruitment of FMRP into polyribosomes, however its absence renders the process less efficient. Finally, the phosphorylation domain of FMRP appears entirely dispensable for the recruitment of FMRP into polyribosomal RNPs.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 8. FMRP domains required for its polyribosomal mRNPs association. STEK cells lacking FMRP were transfected with expression vectors encoding FMRP or its derivatives. Left panels: cytoplasmic extracts were sedimented through 15–45% (w/w) sucrose density gradient in the presence of MgCl2. Right panels: sedimentation of polyribosomal fractions treated with EDTA onto 5–20% (w/w) sucrose density gradient. (A) Representative absorption profiles of 15–45% (left) and 5–20% (right) sucrose density gradients and distribution of the ribosomal L7 protein as revealed by immunoblot analyses using anti-L7 serum. (B) The distribution of FMRP and its derivative proteins that were revealed with mAb1C3.

 


View larger version (35K):
[in this window]
[in a new window]
  		Figure 9. Subcellular distribution of Iso7 full-length FMRP and its derivative versions expressed in STEK cells as compared to the normal distribution of endogenous FMRP in HeLa cells. Total (T), nuclear (N), ribosomal (R), and post-ribosomal supernatant (S) fractions were obtained after differential centrifugation at increasing g forces as described (13). All fractions were resuspended in a final volume of 1 ml of SDS sample buffer equivalent to the starting volume used to lyze the cells, and equal volumes (40 µl) of each fraction were analyzed by immunoblotting.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Here we investigated the requirement of each described domain of FMRP for its polyribosomal association in vivo. We found that both KH domains and the protein–protein interaction domain of FMRP that is known to mediate self-association and heterodimerization with FXR1P and FXR2P and other interactors such as the CYFIPs are essential for FMRP to assemble into functional polyribosomal mRNPs. Also we demonstrated that the RGG box of FMRP is not essential for its recruitment into polyribosomes, suggesting that a subpopulation of FMRP without RGG activity is able to join the mRNPs. This speculation is supported by the fact that an FMR1 spliced mRNA variant resulting from the loss of the 5' end of exon 15 containing the RGG box has been detected (42). Thus our results suggest that protein–protein and RNA–protein interactions are both required for FMRP to be recruited into mRNPs. Interestingly, while phosphorylation of FMRP is not required for its recruitment into polyribosomes, this post-translational modification might still modulate its biological activity as proposed by Siomi et al. (32).

The RNA-binding properties of FMRP have been defined on the basis that recombinant and in vitro translated protein can bind in vitro with high affinity to poly(G) and poly(U) synthetic homoribopolymers, whereas only trace binding was observed with poly(A) or poly(C) (2). While binding to poly(G) that mimics the G-quartet structure (43) anticipated the discovery of FMRP affinity to the G-quartet structural elements (20,21), its binding to poly(U) has generated far less excitement. However, recent genetic assays showed that FMRP interacts with U-rich elements in the 3'-UTR (44), and a novel series of mRNAs containing U-rich target sequences has been identified (45). Clearly, this suggests that the G-quartet structures are not the common denominators of all putative mRNA targets. To determine the requirement of the two KH domains for the interaction of FMRP to polyribosomal mRNPs, we constructed and tested three versions lacking either KH1 or KH2 or both. All versions failed to be incorporated into polyribosomal mRNPs in vivo. The results obtained with {Delta}KH2 may reflect the situation observed with the natural I304N mutant which fails to be integrated into polyribosomes in vivo (14, and our unpublished data). These results differ from those described by Tamanini et al. (46), who showed that I304N can be captured by ribosomes in reconstitution experiments using the reticulocyte lysate. The results obtained with the {Delta}KH1 showed that this FMRP version does not associate with polyribosomes and, contrary to the {Delta}KH2, it does not bind to FXR1P. These results raise the possibility that either the KH1 domain would be required to stabilize protein–protein interactions, as is the case for Sam68, a KH containing RNA-binding protein (17), or that it overlaps the protein–protein interaction domain. It is worth mentioning that a new protein interaction domain called NDF has been recently detected in the N-terminal domain of FMRP (47) which might play a central role in the integration of FMRP into RNPs.

It has been proposed that an intra-molecular RNA G-quartet structure is essential for FMRP–RNA interaction and that the RGG box, but not the KH domains, is responsible for the specific mRNAs target recognition using in vitro binding assays (20,21). In addition, a series of neuronal mRNAs have been isolated either by immunoprecipitation approaches (19) or using a new technique called ‘APRA’ for Antibody Positioned RNA Amplification (48). In both studies, some brain FMRP-associated mRNAs were shown to contain G-quartet structures, the majority of which interestingly are located in the 3'-UTR. However, mRNAs that do not contain the G-quartet structures were also identified and might be putative candidates to search for specific U-rich sequences recognized perhaps by the KH domains. Supporting this speculation, mutations in each of the KH domains, including the I304N mutant, abolish poly(U) binding in vitro while binding to poly(G) remained unaffected (15).

Although the FMRP version lacking the protein–protein interaction domain ({Delta}PPId) still binds to synthetic RNA, it fails to associate with polyribosomes in vivo. This indicates that the self-association and heterodimerization activities mediated by this domain are important for FMRP function(s). These observations raise the possibility that interactor proteins such as NUFIP, CYFIP1, CYFIP2, 82-FIP or others yet to be discovered, modulate the affinity of FMRP to different classes of mRNAs by inducing structural changes in its conformation, thus exposing differentially the RNA binding domains. In line with this hypothesis is the fact that FMRP binds and is released from 82-FIP in a cell cycle-dependant manner (25), suggesting that FMRP might change its affinity to different mRNAs. We recently proposed (35) that FMRP might have a dual role. A housekeeping role in RNA transport in the great majority of cells where the absence of FMRP can be compensated by other homologous RNA-binding proteins such as FXR1P and/or FXR2P. The second function assigned to a minor fraction of FMRP would be a repressor for mRNA to be transported at distal locations such as synaptosomes (49,50) in large cells such as neurons. These two functions might be under the regulation of interactors proteins which would modulate conformational changes in the protein, by exposing the different KH and RGG domains, independently or in concert, resulting in the differential functional activities of these RNA-binding domains.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
DNA manipulation
Eukaryotic expression vectors.
pTL1-FMR1 Iso7 vector encoding FMRP was described previously (11). To generate the vector pTL1-FMR1 I304N, the EcoRI–BamHI insert was removed from pTL1–FMR1 Iso7 and replaced by the corresponding fragment isolated from pET21a–FMR1 I304N plasmid (33). pTL1 vectors coding for FMRP variants were generated by ligation of PCR products amplified from pTL1–iso7 FMR1. The PCR products were first digested with the corresponding restriction enzymes whose sites are present in the primers used for PCR amplification before ligation. For pTL1–{Delta}PPId, EcoRI F and PstI R1 oligos were used to amplify the first PCR fragment. Those used to amplify the second PCR fragments were PstI F1 and HindIII R oligos. Both fragments were digested and joined to pTL1–iso7 FMR1 previously digested with EcoRI and HindIII. For pTL1–{Delta}KH1, the first PCR fragment was amplified with the EcoRI F and PstI R2 primers, and the second PCR fragment amplified with the PstI F2 and BamHI R1 oligos. Amplified fragments were digested and joined together into the pTL1-iso7 FMR1 vector that has been previously digested with EcoRI and BamHI. For pTL1–{Delta}KH2 construct, we used the EcoRI F and PstI R3 oligos to amplify the first fragment and the PstI F3 with SacI R oligos to generate the second fragment. The PCR fragments were digested and ligated into the pTL1-iso7 vector that was digested with EcoRI and SacI.

For pTL1–{Delta}KHT, the first PCR fragment was identical to that of pTL1–{Delta}KH1 and the second one was identical to that of pTL1–{Delta}KH2. For pTL1–{Delta}Phd, the first PCR fragment was amplified using HindIII F and PstI R4 primers and the second PCR fragment was amplified with PstI F4 and SacI R. The PCR fragments were introduced into EcoRI and SacI sites of the pTL1–iso7 vector. For pTL1–{Delta}RGG, the first PCR fragment was amplified with HindIII F and PstI R5 primers and the second was amplified with PstI F5 and SacI R oligos. Both PCR fragments were digested and ligated with pTL1–FMR1 vector that was previously digested with EcoRI and SacI. The following oligos were used: EcoRI F, 5'-GGCGAATTCATGGAGGAGCTGGTGGT-3'; PstI R1, 5'-GGCCTGCAGATTAACAGATCTTAGACGTTC-3'; PstI F1, 5'-GGCCTGCAGCAGCAGCTGGAGAGTTCAAGG-3'; HindIII R, 5'-GGCAAGCTTCTAGCTTTTTTCACTGCATC-3'; PstI R2, 5'-GGCCTGCAGCTTACTAGCTTCTTCATTTC-3'; PstI F2, 5'-GGCCTGCAGGAAGATGTAATACAAGTTCCA-3'; BamHI R1, 5'-GGCGGATCCATCTGTTGTTCTTCCTTTAG-3'; PstI R3, 5'-GGCCTGCAGAGAGCAAATTCGAGAAAGC-3'; PstI F3, 5'-GGCCTGCAGAAGGAAGTAGACCAGTTGC-3'; SacI R, 5'-GGCGAGCTCTCAGGGTACTCCATTCAC-3'; PstI R4, 5'-GGCCTGCAGCTGTCGCAACTGCTCATC-3'; PstI F4, 5'-GGCCTGCAGCGCAGAGGAGACGGACGG-3'; PstI R5, 5'-GGCCTGCAGCAGGAAGCTCTCCCTCT-3'; PstI F5, 5'-GGCCTGCAGGATCACTCCCGAACAGATA-3'.

Prokaryotic expression vectors.
The pET21a plasmids encoding His-tagged FMRP and His-tagged FMRP I304N were kindly provided by Bernhard Laggenbauer and Utz Fischer. The pET21a plasmids encoding His-tagged FMRP versions were generated by ligation of PCR products amplified from pET21-FMRP. Before ligation, the PCR products were digested with the corresponding restriction enzymes whose sites are present in the primers used for PCR amplification. For pET–{Delta}PPId the fragment EcoRI–HindIII isolated from pET21–FMRP was replaced by the corresponding fragment isolated from pTL1–{Delta}PPId. For pET21–{Delta}KH1, the fragment EcoRI–KpnI isolated from pET21–FMRP was replaced by the corresponding fragment isolated from pTL1–{Delta}KH1. For pET21–{Delta}KH2, the first PCR fragment was amplified with PstI F3 and NotI R (5'-AGTGCGGCCGCGGGTAC-3') oligos and digested with PstI and NotI. This fragment was joined to restriction EcoRI–PstI fragments isolated from pTL1–{Delta}KH2 vector. Both fragment were ligated within the pET21–FMR1 plasmid that was prepared by digestion with EcoRI and NotI. The pET21–{Delta}KHT was made exactly as for pET21–{Delta}KH2 except that the restriction fragment was isolated from pTL1–{Delta}KHT. For pET21–{Delta}Phd, a PCR fragment was amplified with PstI F4 and NotI R primers, digested and joined into the pET21a plasmid together with the restriction EcoRI–PstI fragment isolated from pTL1–{Delta}Phd. pET21–{Delta}RGG construct was made as pET21–{Delta}Phd except that the forward primer used for PCR amplification was PstI F5 and the restriction EcoRI–PstI fragment was isolated from pTL1–{Delta}RGG.

Bacterial expression of recombinant proteins
Recombinant His6-tagged proteins were produced in E. coli strain BL21 (DE3) and purified by affinity chromatography using Ni-NTA Probond beads (Invitrogen) according to the manufacturer's protocol.

Poly(G) homopolymer binding assay
Binding assays were performed according to established procedures (51). Briefly, 0.5 µg of recombinant His-tagged FMRP versions were incubated with immobilized poly(G) polyacrylhydrazido-agarose beads (Sigma) in 0.5 ml of binding buffer containing 20 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.5% NP-40 and 2.5 mM MgCl2 supplemented with Protease Inhibitor Cocktail (Roche) for 2 h at 4°C. After incubation, the beads were washed with binding buffer and bound proteins eluted by addition of SDS sample buffer followed by heat denaturation. Control analyses were also performed with 35S-labeled FMRP variants produced in the rabbit reticulocyte lysate system (not shown). Proteins were separated by SDS–PAGE and stained with Coomassie Blue. In vitro translated proteins were detected after exposure of the gel to X-ray film.

In vitro phosphorylation assay
HeLa S100 extracts were prepared as described (32) and incubated with recombinant His-tagged FMR proteins in 0.5 ml of binding buffer containing 20 mM Tris–HCl pH 8, 100 mM NaCl, 0.5% NP-40 and complete Mini, EDTA-free Protease Inhibitor Cocktail (Roche) for 2 h at 4°C. DTT was omitted in the reaction since it interferes with the binding of His-tagged proteins to the His-Bind resin that were added. After 2 h incubation under constant rotation, the beads were washed and then resuspended in phophorylation buffer assay containing 50 mM Tris–HCl pH 8, 100 mM NaCl, 10 mM MgCl2 in the presence of 1 µC[{gamma}-32P] ATP (Amersham), and incubated for 30 min at 30°C. After extensive washing of the beads, proteins were eluted in SDS-sample buffer and analyzed in 10% acrylamide SDS–PAGE. Gels were stained with Coomassie Blue, dried, and exposed to X-ray film to visualize the phosphorylated proteins.

Immunoprecipitation, immunoblot and far-western analyses
STEK cells transfected with the pTL1-vectors expressing the different FMRP variants were lysed in 20 mM Tris–HCl, pH 8, containing 100 mM NaCl, 1.25 mM MgCl2, 0.5% NP40 and protease inhibitors. After clarification at 12 000 rpm for 20 min, the supernatant was adjusted to 20 mM EDTA and reacted with 5 µl of the rabbit #ML13 anti-FXR1P serum for 2 h at room temperature under constant agitation. The #ML13 serum was raised in rabbit using the synthetic peptide RAESQSRQRNLPRETLAKNK (40). Protein A-Sepharose beads (Amersham) were then added for an additional 2 h. After extensive washing with the immunoprecipitation buffer, bound proteins were eluted with SDS sample buffer.

Immunoblot analyses were carried out as described (3). FXR1P was detected with mAb2FX specific for the 78–80 kDa FXR1P isoforms (40) and FMRP with mAb1C3 and mAb7B8 (35). Far-western blot analyses were performed as described (52) using as a probe 35S-labeled FXR1P produced in the rabbit reticulocyte lysate system using cDNA FXR1P-7 coding for the 84 kDa protein isoform.

Transient transfection, subcellular fractionation and sucrose density ultracentrifugation
The STEK Fmr1 KO cell line (35) was transiently transfected with the differents pTL1-vectors using Effectene according to the manufacturer's recommendations (Qiagene). For subcellular fractionation, cell monolayers were washed twice with ice-cold PBS, and 3–5x106 cells were lysed in 1 ml buffer containing 20 mM Tris–HCl pH 7.4, 100 mM KCl, 1.25 mM MgCl2, 1 mM DTT, 10 U/ml RNasine (Pharmacia) and 0.5% NP-40, and subcellular fractions were obtained after differential centrifugation as described (13). All manipulations were carried out at 4°C.

For sucrose density ultracentrifugation, aliquots of 1 ml of cytoplasmic extracts, containing 15–20 OD at 260 nm, were analyzed by sedimentation velocity into 15–45% (w/w) linear sucrose density gradients made up in 25 mM Tris–HCl pH 7.4, 100 mM KCl, 5 mM MgCl2. After centrifugation 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 UA5 flow-through spectrophotometer set at 254 nm and connected to a gradient collector (41). Each fraction was precipitated overnight at -20°C after addition of 2 vols of ethanol. The precipitated material was collected by centrifugation at 12 000 rpm for 20 min and solubilized in SDS-sample buffer before immunoblot analysis. For mRNPs studies, the ribosomal pellets obtained after ultracentrifugation of cytoplasmic extracts over a 45% (w/w) sucrose cushion, were resuspended in NP-40 buffer containing 30 mM EDTA and the released particles were analyzed by sedimentation velocity in 5–20% (w/w) linear sucrose gradients as described above.

Indirect immunofluorescence studies
Cells grown on glass coverslips were fixed with acetone/methanol (7/3) for 30 min at -20°C. To detect FMRP, fixed cells were reacted with mAb1C3 followed by Alexa fluor 488 goat anti-mouse IgG (Molecular Probe). Immunofluorescence staining was viewed through a Leica DMMB microscope equiped with epifluorescence illuminator and connected to a JAI M300 CCD Camera using a 100x oil immersion objective. Images were transferred to the Adobe PhotoShop program.


   ACKNOWLEDGEMENTS
 
We are grateful to Barbara Bardoni and Jean-Louis Mandel for the pTL1-Iso7 FMR1 vector, Bernard Laggerbauer and Utz Fisher for the pET21–FMRP and pET21–I304N plasmids, Alan Tartakoff and Andrew Ziemiecki for providing the mAb7B8 and L7 antibodies. This work was supported by the Canadian Institutes of Health Research. R.M. was a recipient of a postdoctoral fellowship from the Fragile X Research Foundation of Canada/Canadian Institutes of Health Research Partnership Challenge Fund program and M.E.H. holds a scholarship from the Canadian Institutes of Health Research. Y.L. is a Scholar of the Fonds de la Recherche en Santé du Québec.


   FOOTNOTES
 
* To whom correspondence should be addressed at: URGHM, Centre de Recherche Hôpital St-François d'Assise, 10 rue de l'Espinay, Québec G1L 3L5, PQ, Canada. Tel: +1 4185254402; Fax: +1 4185254195; Email: edward.khandjian{at}crsfa.ulaval.ca Back

{dagger} Present address: Department of Biochemistry, McGill University, Montréal, PQ, Canada. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Ashley, C.T., Jr, Wilkinson, K.D., Reines, D. and Warren, S.T. (1993) FMR1 protein: conserved RNP family domains and selective RNA binding. Science, 262, 563–566.[Abstract/Free Full Text]

  2. 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. Cell, 74, 291–298.[CrossRef][ISI][Medline]

  3. Khandjian, E.W., Fortin, A., Thibodeau, A., Tremblay, S., Côté, F., Devys, D., Mandel, J.L. and Rousseau, F. (1995) A heterogeneous set of FMR1 proteins is widely distributed in mouse tissues and is modulated in cell culture. Hum. Mol. Genet., 4, 783–789.[Abstract/Free Full Text]

  4. 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 a fragile X premutation. Nat. Genet., 4, 335–340.[CrossRef][ISI][Medline]

  5. Imbert, G., Feng, Y., Nelson, D.L., Warren, S.T. and Mandel, J.L. (1998) FMR1 and mutations in fragile X syndrome: molecular biology, biochemistry, and genetics. In Warren, S.T. and Wells, R.D. (eds), Genetic Instabilities and Hereditary Neurological Diseases. Academic Press, New York, pp. 27–53.

  6. Khandjian, E.W. (1999) Biology of the fragile X mental retardation protein, an RNA-binding protein. Biochem. Cell. Biol., 77, 331–342.[CrossRef][ISI][Medline]

  7. Bardoni, B. and Mandel, J.L. (2002) Advances in understanding of fragile X pathogenesis and FMRP function, and in identification of X linked mental retardation genes. Curr. Opin. Genet. Dev., 12, 284–293.[CrossRef][ISI][Medline]

  8. O'Donnell, W.T. and Warren, S.T. (2002) A decade of molecular studies of fragile X syndrome. Annu. Rev. Neurosci., 25, 315–338.[Medline]

  9. Eberhart, D.E., Malter, H.E., Feng, Y. and Warren, S.T. (1996) The fragile X mental retardation protein is a ribonucleoprotein containing both nuclear localization and nuclear export signals. Hum. Mol. Genet., 5, 1083–1091.[Abstract/Free Full Text]

  10. Siomi, M.C., Zhang, Y., Siomi, H. and Dreyfuss, G. (1996) Specific sequences in the fragile X syndrome protein FMR1 and the FXR proteins mediate their binding to 60S ribosomal subunits and the interactions among them. Mol. Cell. Biol., 16, 3825–3832.[Abstract]

  11. 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.[Abstract/Free Full Text]

  12. Tamanini, F., Bontekoe, C., Bakker, C.E., van Unen, L., Anar, B., Willemsen, R., Yoshida, M., Galjaard, H., Oostra, B.A. and Hoogeveen, A.T. (1999) Different targets for the fragile X-related proteins revealed by their distinct nuclear localizations. Hum. Mol. Genet., 8, 863–869.[Abstract/Free Full Text]

  13. Corbin, F., Bouillon, M., Fortin, A., Morin, S., Rousseau, F. and Khandjian, E.W. (1997) The fragile X mental retardation protein is associated with poly(A)+ mRNA in actively translating polyribosomes. Hum. Mol. Genet., 6, 1465–1472.[Abstract/Free Full Text]

  14. Feng, Y., Absher, D., Eberhart, D.E., Brown, V., Malter, H.E. and Warren, S.T. (1997) FMRP associates with polyribosomes as an mRNP, and the I304N mutation of severe fragile X syndrome abolishes this association. Mol. Cell, 1, 109–118.[CrossRef][ISI][Medline]

  15. 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. Cell, 77, 33–39.[CrossRef][ISI][Medline]

  16. Musco, G., Stier, G., Joseph, C., Castiglione Morelli, M.A., Nilges, M., Gibson, T.J. and Pastore, A. (1996) Three-dimensional structure and stability of the KH domain: molecular insights into the fragile X syndrome. Cell, 85, 237–245.[CrossRef][ISI][Medline]

  17. Chen, T., Damaj, B.B., Herrera, C., Lasko, P. and Richard, S. (1997) Self-association of the single-KH-domain family members Sam68, GRP33, GLD-1, and Qk1: role of the KH domain. Mol. Cell. Biol., 17, 5707–5718.[Abstract]

  18. Liu, Q. and Dreyfuss, G. (1995) In vivo and in vitro arginine methylation of RNA-binding proteins. Mol. Cell. Biol., 15, 2800–2808.[Abstract]

  19. 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., Darnell, R.B. and Warren, S.T. (2001) Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell, 107, 477–487.[CrossRef][ISI][Medline]

  20. Darnell, J.C., Jensen, K.B., Jin, P., Brown, V., Warren, S.T. and Darnell, R.B. (2001) Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function. Cell, 107, 489–499.[CrossRef][ISI][Medline]

  21. Schaeffer, C., Bardoni, B., Mandel, J.L., Ehresmann, B., Ehresmann, C. and Moine, H. (2001) The fragile X mental retardation protein binds specifically to its mRNA via a purine quartet motif. EMBO J., 20, 4803–4813.[CrossRef][ISI][Medline]

  22. Tamanini, F., Van Unen, L., Bakker, C., Sacchi, N., Galjaard, H., Oostra, B.A. and Hoogeveen, A.T. (1999) Oligomerization properties of fragile-X mental-retardation protein (FMRP) and the fragile-X-related proteins FXR1P and FXR2P. Biochem. J., 343, 517–523.[Medline]

  23. 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.[Abstract/Free Full Text]

  24. Schenck, A., Bardoni, B., Moro, A., Bagni, C. and Mandel, J.L. (2001) A highly conserved protein family interacting with the fragile X mental retardation protein (FMRP) and displaying selective interactions with FMRP-related proteins FXR1P and FXR2P. Proc. Natl Acad. Sci. USA, 98, 8844–8849.[Abstract/Free Full Text]

  25. Bardoni, B., Castets, M., Huot, M.E., Schenck, A., Adinolfi, S., Corbin, F., Pastore, A., Khandjian, E.W. and Mandel, J.L. (2003) 82-FIP, a novel FMRP (Fragile X Mental Retardation Protein) interacting protein, shows a cell cycle-dependent intracellular localization. Hum. Mol. Genet., 12, 1689–1698.[Abstract/Free Full Text]

  26. Bardoni, B., Schenck, A. and Mandel, J.L. (2001) The fragile X mental retardation protein. Brain Res. Bull., 56, 375–382.[CrossRef][ISI][Medline]

  27. Ceman, S., Brown, V. and Warren, S.T. (1999) Isolation of an FMRP-associated messenger ribonucleoprotein particle and identification of nucleolin and the fragile X-related proteins as components of the complex. Mol. Cell. Biol., 19, 7925–7932.[Abstract/Free Full Text]

  28. Ceman, S., Nelson, R. and Warren, S.T. (2000) Identification of mouse YB1/p50 as a component of the FMRP-associated mRNP particle. Biochem. Biophys. Res. Commun., 279, 904–908.[CrossRef][ISI][Medline]

  29. Ohashi, S., Koike, K., Omori, A., Ichinose, S., Ohara, S., Kobayashi, S., Sato, T.A. and Anzai, K. (2002) Identification of mRNA/protein (mRNP) complexes containing Puralpha, mStaufen, fragile X protein, and myosin Va and their association with rough endoplasmic reticulum equipped with a kinesin motor. J. Biol. Chem., 277, 37804–37810.[Abstract/Free Full Text]

  30. Caudy, A.A., Myers, M., Hannon, G.J. and Hammond, S.M. (2002) Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev., 16, 2491–2496.[Abstract/Free Full Text]

  31. Ishizuka, A., Siomi, M.C. and Siomi, H. (2002) A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev., 19, 2497–2508.

  32. Siomi, M.C., Higashijima, K., Ishizuka, A. and Siomi, H. (2002) Casein kinase II phosphorylates the fragile X mental retardation protein and modulates its biological properties. Mol. Cell. Biol., 22, 8438–8447.[Abstract/Free Full Text]

  33. Laggerbauer, B., Ostareck, D., Keidel, E.-M., Ostareck-Lederer, A. and Fischer, U. (2001) Evidence that fragile X mental retardation protein is a negative regulator of translation. Hum. Mol. Genet., 10, 329–338.[Abstract/Free Full Text]

  34. Li, Z., Zhang, Y., Ku, L., Wilkinson, K.D., Warren, S.T. and Feng, Y. (2001) The fragile X mental retardation protein inhibits translation via interacting with mRNA. Nucl. Acids Res., 29, 2276–2283.[Abstract/Free Full Text]

  35. Mazroui, R., Huot, M.E., Tremblay, S., Filion, C., Labelle, Y. and Khandjian, E.W. (2002) Trapping of messenger RNA by fragile X mental retardation protein into cytoplasmic granules induces translation repression. Hum. Mol. Genet., 11, 3007–3017.[Abstract/Free Full Text]

  36. De Boulle, K., Verkerk, A.J., 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. Nat. Genet., 3, 31–35.[CrossRef][ISI][Medline]

  37. Adinolfi, S., Bagni, C., Musco, G., Gibson, T., Mazzarella, L. and Pastore, A. (1999) Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains. RNA, 5, 1248–1258.[Abstract]

  38. Denman, R.B. and Sung, Y.J. (2002) Species-specific and isoform-specific RNA binding of human and mouse fragile X mental retardation proteins. Biochem. Biophys. Res. Commun., 292, 1063–1069.[CrossRef][ISI][Medline]

  39. 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.[ISI][Medline]

  40. Khandjian, E.W., Bardoni, B., Corbin, F., Sittler, A., Giroux, S., Heitz, D., Tremblay, S., Pinset, C., Montarras, D., Rousseau, F. and Mandel, J.L. (1998) Novel isoforms of the fragile X related protein FXR1P are expressed during myogenesis. Hum. Mol. Genet., 7, 2121–2128.[Abstract/Free Full Text]

  41. Khandjian, E.W., Corbin, F., Woerly, S. and Rousseau, F. (1996) The fragile X mental retardation protein is associated with ribosomes. Nat. Genet., 12, 91–93.[CrossRef][ISI][Medline]

  42. Kirkpatrick, L.L., McIlwain, K.A. and Nelson, D.L. (1999) Alternative splicing in the murine and human FXR1 genes. Genomics, 59, 193–202.[CrossRef][ISI][Medline]

  43. Kim, J., Cheong, C. and Moore, P.B. (1991) Tetramerization of an RNA oligonucleotide containing a GGGG sequence. Nature, 351, 331–332.[CrossRef][Medline]

  44. Dolzhanskaya, N., Sung, Y.J., Conti, J., Currie, J.R. and Denman, R.B. (2003) The fragile X mental retardation protein interacts with U-rich RNAs in a yeast three-hybrid system. Biochem. Biophys. Res. Commun., 305, 434–441.[CrossRef][ISI][Medline]

  45. Chen, L., Yun, S.-W., Seto, J., Liu, W. and Toth, M. (2003) The fragile X mental retardation protein binds and regulates a novel class of mRNAs containing U rich target sequences. Neuroscience, 120, 1005–1017.[CrossRef][ISI][Medline]

  46. Tamanini, F., Meijer, N., Verheij, C., Willems, P.J., Galjaard, H., Oostra, B.A. and Hoogeven, A.T. (1996) FMRP is associated to the ribosomes via RNA. Hum. Mol. Genet., 5, 809–813.[Abstract/Free Full Text]

  47. Adinolfi, S., Ramos, A., Martin, S.R., Dal Piaz, F., Pucci, P., Bardoni, B., Mandel, J.L. and Pastore, A. (2003) The N-terminus of the fragile X mental retardation protein contains a novel domain involved in dimerization and RNA binding. Biochemistry, 42, 10437–10444.[CrossRef][Medline]

  48. Miyashiro, K.Y., Beckel-Mitchener, A., Purk, T.P., Becker, K.G., Barret, T., Liu, L., Carbonetto, S., Weiler, I.J., Greenough, W.T. and Eberwine, J. (2003) RNA cargoes associating with FMRP reveal deficits in cellular functioning in Fmr1 null mice. Neuron, 37, 417–431.[CrossRef][ISI][Medline]

  49. Weiler, I.J., Irwin, S.A., Klintsova, A.Y., Spencer, C.M., Brazelton, A.D., Miyashiro, K., Comery, T.A., Patel, B., Eberwine, J. and Greenough, W.T. (1997) Fragile X mental retardation protein is translated near synapses in response to neurotransmitter activation. Proc. Natl Acad. Sci. USA, 94, 5395–5400.[Abstract/Free Full Text]

  50. Greenough, W.T., Klintsova, A.Y., Irwin, S.A., Galvez, R., Bates, K.E. and Weiler, I.J. (2001) Synaptic regulation of protein synthesis and the fragile X protein. Proc. Natl Acad. Sci. USA, 98, 7101–7106.[Abstract/Free Full Text]

  51. Swanson, M.S. and Dreyfuss, G. (1988) Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities. Mol. Cell. Biol., 8, 2237–2241.[Abstract/Free Full Text]

  52. Schwartz, C.J., Sampson, H.M., Hlousek, D., Percival-Smith, A., Copeland, J.W., Simmonds, A.J. and Krause, H.M. (2001) FTZ-Factor1 and Fushi tarazu interact via conserved nuclear receptor and coactivator motifs. EMBO J., 20, 510–519.[CrossRef][ISI][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
N. Piazzon, F. Rage, F. Schlotter, H. Moine, C. Branlant, and S. Massenet