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The RNA binding protein FMRP: new connections and missing links
Biology of the Cell 95 (2003) 221–228 www.elsevier.com/locate/bicell
Review
The RNA binding protein FMRP: new connections and missing links Céline Schaeffer, Mélanie Beaulande, Chantal Ehresmann, Bernard Ehresmann, Hervé Moine * UPR 9002, Institut de Biologie Moléculaire et Cellulaire, 15, rue René-Descartes, 67084 Strasbourg cedex, France Received 18 February 2003; accepted 5 March 2003
Abstract The loss of the fragile X mental retardation protein (FMRP) is responsible for the most common cause of inherited mental retardation called the fragile X syndrome. FMRP is suspected to participate in the synaptic plasticity of neurons by acting on posttranscriptional control of gene expression. FMRP is an RNA binding protein that associates with mRNAs together with other proteins to form large ribonucleoprotein complexes. These complexes are proposed to participate in the transport, localization and translation of target mRNAs. Progress has been made recently in the identification of the mRNAs and the proteins present in these complexes and a possible connection with the micro-RNA dependent regulatory pathway has been established. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Fragile X syndrome; FMRP; RNA binding; G–quartet; Non–coding RNA
1. The absence of FMRP causes fragile X syndrome One of the most frequent genetic diseases, the fragile X syndrome, is caused by the lack (or more rarely by a defect in the function) of an RNA binding protein: the fragile X mental retardation protein (FMRP). Mental retardation is the main symptom of the disease but behavioral disorders are also associated with it. In addition to mental retardation, other clinical features can be present, particularly in males, like macroorchidism in postpubertal males, typical face with large ears and high forehead or hyper extensible joints. Because fragile X has a prevalence of about 1 in 4000 males and 1 in 8000 females, it is considered as the most frequent form of inherited mental retardation (Crawford et al., 2001). The name of this syndrome comes from the presence of a cytogenetically visible fragile site on chromosome X. This fragile site is revealed in particular cytological growth conditions, and it was used for the positional cloning of the gene involved in the disease (for reviews, see Imbert et al., 1998; Jin and Warren, 2000). Fragile X results from the amplification of a CGG trinucleotide repeat located in the 5' untranslated region (5' UTR) of the FMR1 (fragile X mental retardation 1) gene. The number of CGG repeats is polymorphic in the normal population, ranging between 6 and 50 with an average of * Corresponding author. Tel.: +33-3-88-41-70-51; fax: +33-3-88-60-22-18. E-mail address: [email protected] (H. Moine). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 10.1016/S0248-4900(03)00037-6
about 30. Two classes of mutations have been described: premutations and full mutations. Premutations, on one hand, are characterized by moderate expansions (50–200) and are not associated with mental retardation although they have some clinical impacts (Hagerman and Hagerman, 2002). Full mutations, on the other hand, are present in affected people and are characterized by large expansions (200 to more than 1000 repeats) and by an abnormal methylation of the repeats themselves as well as of the surrounding regions. This methylation leads to the transcriptional inhibition of FMR1 and thus to the absence of its protein product, FMRP. The expansion occurs only through maternal transmission with an efficiency of amplification directly linked to the size of the premutation (Bardoni and Mandel, 2002; Imbert et al., 1998), but its mechanism is not yet understood. Although the expansion of the CGG repeat is found in the majority of affected people, some rare patients are known without amplification but bearing instead deletions or a missense mutation within the FMR1 coding sequence. These data, together with the fact that the FMR1 KO mouse reproduces a subtle phenotype reminiscent of the human phenotype (The Dutch–Belgian Fragile X Consortium, 1994), support the idea that FMR1 is the only gene implicated in this syndrome. The FMR1 gene is composed of 17 exons, spans about 38 kb and is subjected to alternative splicing. In vivo six different isoforms, ranging from 70 to 80 kDa, can be detected, with the major form being ±80 kDa (Khandjian, 1999). The
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FMRP protein is highly conserved in vertebrates and belongs to a family that contains two other proteins: FXR1 and FXR2 (fragile X related protein 1 and 2). In Drosophila melanogaster, there is only one homolog for these three proteins: dFXR (Wan et al., 2000). Localization studies have shown that FMRP is broadly expressed in most tissues, but it is predominantly found in the central nervous system and testes. In the cell, FMRP is mostly cytoplasmic but about 5% of the total FMRP is localized in the nucleus. It contains both a nuclear localization signal (NLS) and a nuclear export signal (NES) allowing it to shuttle between nucleus and cytoplasm (Bardoni et al., 1997; Eberhart et al., 1996; Fridell et al., 1996). One major issue in understanding the biology of fragile X syndrome is to define the role of FMRP. This role has been very elusive up to now. But the fact that FMRP is present mostly in cytoplasm, associated with mRNAs as part of large ribonucleoproteic complexes (mRNP), together with the polyribosomes, suggested that FMRP is somehow involved in posttranscriptional control of gene expression. Thus, FMRP could control the transport, the localization and/or the translation of specific mRNAs in the cells where it is mostly expressed: the neurons. As FMRP seems to exert its function through RNA binding, it was essential to define its RNA binding properties and identify its targets. Also, the characterization of the mRNP complex(es), in which it is involved, is crucial for understanding its function. Significant progress has been made recently in the understanding of the RNA binding properties of FMRP and in the identification of mRNAs and proteins associated with it. This review will focus on these new findings and their meaning regarding the possible role of FMRP in neuronal function. 2. The RNA binding properties of FMRP FMRP contains two K homology domains (KH) and one arginine and glycine rich region (RGG box) known to be RNA binding motifs. Indeed, FMRP binds to RNA homopolymers in vitro with a preference for poly(G) and poly(U), whereas a very weak binding to poly(A) and poly(C) is seen (Siomi et al., 1993). FMRP was shown to bind to a small fraction of mRNAs present in a pool of total human fetal brain mRNAs (approximately 4%). FMRP was also shown to be able to bind to its own mRNA with high affinity, but also to its antisense sequence (Ashley et al., 1993). Taken together these data were suggesting that FMRP could have some RNA binding selectivity, yet with a high propensity to bind RNA in general. Since FMRP is not essential for viability, a possibility is that FMRP could regulate the expression of specific mRNAs, in particular in the nervous system where FMRP is mostly expressed. In fragile X patients, the loss of FMRP would cause defects in the proper translation of many mRNAs, thus explaining the pleiotropic phenotype associated with fragile X syndrome. Moreover, the identification of a point mutant in the second KH domain in a patient with a severe fragile X phenotype
Fig. 1. Structure of G–quartets. (A) Schematic model for the G–quartets and (B) its hydrogen bonding scheme. In G–quartets, four guanines form hydrogen bonds with each other in a symmetrical square planar arrangement. The overall structure of G–quartets is stabilized by the coordination of cations, such as K+, within the central cavity of the quadruplex, and by stacking several G–quartet layers on top of each other. (C) Sequence of the FMRP binding site present in FMR1 mRNA (Schaeffer et al., 2001). In this sequence, the guanines are interspersed with adenines, which suggests that simple G tetrads (B) could also be associated with some G4A2 hexads. (D) Hydrogen bonding scheme of a G4A2 hexad (as found in GGAGG RNA by Liu et al., 2002 using NMR).
provided additional support for a connection between the syndrome and the RNA binding activity of FMRP. In fact, it was shown that this mutation (Ile304Asn) affected homopolymers RNA binding in vitro (Siomi et al., 1994). However, as the initial studies had been performed with nonpurified in vitro translated FMRP and as it was shown that FMRP associates with mRNP particles, it was not clear whether the RNA binding activity could be attributed to FMRP itself or whether it required other proteins. Subsequent studies performed with purified FMRP produced in baculovirus definitely confirmed that RNA binding was really an intrinsic property of FMRP (Brown et al., 1998). As the RNA binding property of FMRP appeared to be essential for its function, the key issues were then to determine whether FMRP binds to specific sets of mRNAs and whether its absence affects the distribution of proteins encoded by these mRNAs in cells from fragile X patients. Progress in the identification of the RNA binding properties of FMRP has been performed recently by using different approaches. We provided the first evidence that purified FMRP has a very high affinity for peculiar RNA sequences able to adopt a structure forming G–quartets (Schaeffer et al., 2001). We detected this specific and high affinity binding site in the mRNA encoding FMRP, which was suspected to interact with FMRP in vitro and in vivo (Ashley et al., 1993; Ceman et al., 1999). G–quartet structure consists in the superposition of layers formed by four guanines hydrogen bonded via Hoogsteen base pairs in a square planar symmetric array (Fig. 1). G–quartets form very stable structures stabilized by the coordination of monovalent ions lying between or within the plane of the guanine tetrads. Because of their requirement for cation coordination, G–quartets exhibit unique structural properties: they are preferentially stabilized
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by K+ whereas smaller size Na+ and Li+, supposedly fitting less well in the central cavity, cause their destabilization. We took advantage of these biochemical peculiarities to demonstrate the involvement of G–quartets in FMRP binding. Furthermore, the exchange of the homologous FMRP binding site by another sequence known to be able to adopt such G–quartet, or its insertion within a heterologous mRNA, is able to promote FMRP binding. Also, the biological relevance of this interaction was supported by the fact that binding of FMRP to such a structure inserted in the 5' UTR of a reporter gene inhibited its translation in rabbit reticulocyte lysate. Thus, G–quartets appeared as good potential targets for FMRP binding on mRNAs. Using the SELEX method, Darnell et al. (2001) came to the same conclusion concerning the binding of FMRP to G–quartets. Furthermore, they showed that the RGG box of FMRP was responsible for the interaction and not the KH domain. In a combined study, Brown et al. (2001) examined the mRNAs associated with FMRP–mRNP in mouse brain and human lymphoblastoid cell lines. They used a microarray strategy to compare the results from normal and FMRP– minus cells. By comparing the input RNA used for the immunoprecipitation experiments and the RNA coimmunoprecipitated with FMRP, they identified one pool of 432 mRNAs associated with FMRP in the mouse brain. By comparing the mRNA polysomal profile from normal and fragile X patient lymphoblast cells, they found another pool of 251 mRNAs, which had variations in their polysome content while their cytoplasmic abundance remained unchanged. Between the two RNA pools, 14 mRNAs were found in common. The weak overlap between the two pools was explained by the differences in gene representation on the murine and human microarray, and in gene expression between lymphoblastoid cell and whole brain extracts. Among these mRNAs, about 50% were decreased in their polysome association, whereas the other half was increased. This apparent opposite effect, caused by the absence of FMRP on its potential mRNA targets, is very difficult to rationalize at this moment. Most interestingly, the analysis of these mRNAs revealed that most of them contain a potential G–quartet structure (whereas only a few percent of mRNAs taken randomly would be predicted to contain such structures). Again, a rationalization with respect to the role of FMRP is hard to make as the G–quartet structure can be located in the coding region as well as in the 5' or 3' UTR, independently of the variations in mRNA polysome associations. Many of these mRNAs encode proteins important for neuronal functions (microtubule associated protein 1B (MAP1B), potassium channel kv3.1, neuronal axonal protein –22 (NAP22), Rab6 interacting protein 1, semaphorin 3F, etc.). These data support the hypothesis that misregulation of certain mRNAs is the molecular basis of the fragile X syndrome. Further evidence is now required to demonstrate the impact of FMRP at the protein level of these interesting genes. In fact, a first indication in favor of a functional link between FMRP and one of these genes was already found in
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Drosophila. Remarkably, the absence of dFXR, the fly homolog of FMRP, causes a synaptic structural defect similar to an over-expression of Futsch, the fly homolog of MAP1B. Following this initial observation, Zhang et al. (2001) were able to correct the synaptic anomalies elicited by dFXR deficiency by combining the dFXR and Futsch mutations. This morphological opposing effect of the two genes enabled the authors to propose that dFXR is a translational repressor of Futsch. However, at the moment, it seems that the G–quartet region proposed to be used in MAP1B for FMRP recognition is not conserved in Futsch mRNA. Increasing the already long list of potential FMRP targets, another recent study using an original approach revealed 81 additional mRNAs. By using an antibody linked to a priming oligonucleotide, Miyashiro et al. (2003) performed in situ cDNA synthesis to identify mRNAs present in the close vicinity of FMRP in neurons. At first glance, it is disappointing to notice that none of the members of this list are present in that established by Brown et al. (2001). This result may be explained by a weak overlap between the lists of genes present on both arrays. In fact, from the top 80 candidates retained by Brown et al., only 17 were present in the 5000– membered cDNAs macroarray used by Miyashiro et al. (2003). Among those 17 genes, eight (GRIN1, IP3 receptor, Rab6–associated GDI, Tesk1, PI4 kinase, a–latroxin receptor, mRNA similar to Tensin, mRNA similar to acetylglucosaminyl transferase) could be detected with the antibody positioned amplification approach. Surprisingly, these top candidates were not retained in the final list presented by the authors. Also, among the noticeable absent genes of the list is MAP1B, but as the authors explain, this target may have been easily missed since their macroarray contains a 1 kb fragment of the MAP1B sequence while full length mRNA is 9 kb long. A number of other reasons can account for the differences found between the arrays: the choice of the initial screen (co-immunoprecipitation vs. in situ RNA amplification), the cell type (lymphoblast vs. whole brain vs. neuron), the array technology (microarray vs. macroarray), the threshold chosen to keep or discard a candidate, the additional validation tests (polysome association vs. in vitro binding experiments), etc. each with their own limitations and causes of false positive or negative. While the study by Brown et al. (2001) did not provide evidence for the protein expression of their gene candidates, Miyashiro et al. (2003) managed to detect subtle differences within neurons in the localization and amount of a few proteins when comparing brain tissues from wild type and FMRP KO mice. Among these proteins are GR (the glucocorticoid receptor a) and GRK2 (G protein-coupled receptor kinase 2, a modulator involved in cell signaling). A possible functional relationship between GR and fragile X would be that corticosteroid feedback is often affected in fragile X patients. One main conclusion from this study is that the effects caused by the absence of FMRP are essentially visible in dendrites, particularly at the postsynaptic compartments. This further supports the involvement of FMRP in mRNA
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localization and/or local translation in dendrites. Another conclusion is that G–quartets could be postulated in a number of these RNA targets (23%), but as far as they can be predicted, these structures do not seem to be the main common denominator of this latter list of mRNA targets. In fact, a recent report proposes that the mRNA selectivity of FMRP could be mediated also via an “adaptor” RNA: the RNA BC1 in rodents and its counterpart BC200 in primates (Zalfa et al., 2003). Indeed, these noncoding RNAs, abundant in neurons, are able both to bind directly to FMRP and to base pair with sequences present in mRNAs regulated by FMRP. The mRNAs proposed to be under the control of FMRP via BC1/BC200 are Arc, a–CaMKII, and MAP1B, which had been previously demonstrated to be localized in dendrites or play some important neuronal function. Moreover, BC1 RNA was recently shown to be involved in translational repression at the level of initiation (Wang et al., 2002). Taken together, these data suggest that FMRP could use at least two signals to target an mRNA: the G–quartet motif that was found in an important number of potential mRNA targets, and a small RNA hybridized with the mRNA, explaining the targeting of the non–G–quartet mRNAs. In the case of MAP1B mRNA, both signals would be present. 3. The FMRP mRNP particle(s) The capture of cytoplasmic FMRP by affinity chromatography using oligo(dT) suggested its association in vivo with mRNAs (Corbin et al., 1997; Feng et al., 1997), thus qualifying FMRP as part of mRNP complexes. These mRNP are associated with the translating ribosomes (polyribosomes) as demonstrated by the use of translation initiation inhibitors (Corbin et al., 1997; Feng et al., 1997). Still, the question of a functional interaction between FMRP and the ribosome seems unsolved. After an initial conclusion that FMRP was intimately associated with the ribosome, further studies suggested that FMRP does not interact directly with the ribosomes themselves but rather through the interaction with mRNA. Indeed, ribosomes can be depleted from mRNPs by EDTA treatment, while FMRP remains associated to a complex of large size, with a sedimentation peak centered at 100S, clearly distinct from heaviest 60S ribosomal subunits (Feng et al., 1997; Ohashi et al., 2002). However, the recent report of the isolation of ribosomal proteins directly associated with the Drosophila FMRP homolog suggests that a direct interaction between FMRP and the ribosome is likely to occur at some point (Ishizuka et al., 2002). As already mentioned, FMRP is mostly cytoplasmic, and microscopic studies have shown that it overlaps with polyribosomes in the rough endoplasmic reticulum (RER) of the perikaryon. Also, in neurons, where FMRP is mostly found, a part is present in the dendritic synapses. Roughly, FMRP is where the mRNA engaged in translation is. Moreover, FMRP associated with mRNAs and ribosomes as granules was visualized “en route” while trafficking between perikaryon and
neurites (DeDiego Otero et al., 2002). Furthermore, the FMRP mRNP particle seems to use the “long distance” transportation system, which is microtubule and kinesin dependent, rather than the “short distance” transportation, which uses the actin microfilaments. From their physical appearance, these granules seem to be reminiscent of stress granules (Mazroui et al., 2002). 4. The proteins that associate with FMRP The identification of the members of the FMRP mRNP particles has already revealed several key players but the list is still open. FXR1 and FXR2, two close homologs of FMRP, have been identified as interactors of FMRP by yeast two hybrid studies (Zhang et al., 1995). Clearly, these proteins seem to have some functional redundancy with FMRP, as they share the same protein domains (KH1, KH2, RGG), have the same RNA polymer binding properties, associate with mRNPs in polysomes, are capable to heterodimerize with FMRP and have some shuttling activity. Furthermore, they share some similar distribution in certain tissues (e.g. adult brain). However, FXR1 and FXR2 seem to have acquired also distinct functions and/or tissue specificity. In particular, they show some differences in their expression patterns in many organs (e.g. in human fetal brain, in fetal and adult testis, in muscles) (Tamanini et al., 1997), and their shuttling activity (for FXR2 and some alternatively spliced isoforms of FXR1) is between nucleolus and cytoplasm unlike FMRP. Also, the FXR proteins preferentially homodimerize rather than heterodimerize (Tamanini et al., 2000) and do not interact with all the other interactors of FMRP (see below), suggesting that they might belong to distinct mRNPs. NUFIP1 has been also detected by two-hybrid screening (Bardoni et al., 1999). NUFIP1 mostly locates in nucleus in association with interchromatin fibrils, and it can shuttle between nucleus and cytoplasm. In the brain, its expression pattern is similar to that of FMRP. Interestingly, NUFIP1 does not interact with FXR1 and FXR2, thus underlining the possibility that the FXR proteins have acquired distinct specificities. Despite the presence of two Zinc-finger motifs, suggesting nucleic acid binding properties, the function of NUFIP is still unknown. Also, its association with FMRP as an integral part of FMRP–mRNPs is not certain. The identification of CYFIP1 and CYFIP2 as interactors of FMRP (Schenck et al., 2001), two homologous proteins that contain no known motifs, is of great interest. CYFIP1 had been found previously to be involved in the interaction with the factor Rac1 (Kobayashi et al., 1998). Rac1 is a member of the wide family of the Rho–GTPases, which are specific molecular switches controlling a plethora of cellular pathways, from membrane trafficking to cell growth and division. Most interestingly, Rho–GTPases, through their control of the actin cytoskeleton, play a critical role in the maturation and maintenance of the dentritic spines (the structures found affected in FMRP–minus neurons). This pathway
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was previously found to be involved in other known forms of mental retardation (Ramakers, 2002). Remarkably, CYFIP2 (also known as P53 Induced mRNA, PIR121) was recently found to be involved in the complex repressing the WAVE1 protein. WAVE1 triggers the actin polymerization process (Eden et al., 2002), its repression is alleviated by Rac1, probably by its interaction with CYFIP2. Thus, because FMRP is also an interactor of CYFIP, one could hypothesize that in fragile X patient, the absence of FMRP would cause an increase in the amount of CYFIP available; this change is likely to affect the actin polymerization process and thereby the dendritic spine maturation. Epitope tag immunoprecipitation experiments have revealed that nucleolin and YB1/P50 are coimmunoprecipitated with FMRP in an RNase treatment independent manner, suggesting that they are part of the same particle as FMRP (Ceman et al., 1999, 2000). It is still not known whether they are able to interact directly with FMRP. FMRP was also found associated by coimmunoprecipitation with the other RNA binding proteins Pura, mStaufen, and Myosin Va, which have been proposed to regulate the transport of polyribosomes along microtubules and actin filaments via RER structures (Ohashi et al., 2002). The association of FMRP with these proteins is sensitive to RNase treatment, suggesting that they belong to distinct particles associated on a same mRNA. The mRNAs bound by FMRP would thus benefit from the cargo/kinesin motor associated with the RER, in this case, FMRP per se would not be responsible for the transport. More recently, two groups reported at the same time that the dFXR protein (the Drosophila homolog of the FMRP and FXR1/2 human family of proteins) interacts directly in vivo with a core component of the RNA–induced silencing complex (RISC): the protein Argonaute 2 (AGO2) (Caudy et al., 2002). Together with AGO2, Ishizuka et al. (2002) also found the two ribosomal protein L5 and L11, 5S rRNA, an RNA helicase (Dmp68) and some yet unidentified micro-RNAs (miRNAs). Dicer, the RNase III like enzyme responsible for both the formation of the short interfering RNAs (siRNA) and the maturation of miRNAs, was also detected in the complex. Thus, these findings suggest that, in Drosophila, dFXR could play a role in the gene silencing pathways of siRNAs and/or miRNAs. miRNAs and siRNAs are present in similar RNP complexes, but their function differs. While siRNAs are 21–23 double stranded nucleotide fragments produced from long dsRNA duplex (in particular those encoded by viruses or transposons), miRNAs instead are small RNAs excised from 60– to 80–nt hairpin RNAs, themselves matured from longer sequences transcribed from endogenous genes. The siRNAs and the miRNAs are present in similar RNP complexes but their function seems to depend on their matching with their target mRNA. Thus, while the siRNAs trigger the degradation of the target RNA, miRNAs that form bulged structures are believed to inhibit translation after the initiation stage (Olsen and Ambros, 1999; Zeng et al., 2002).
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5. The possible function(s) of FMRP There is strong evidence to support the notion that FMRP is important in regulating mRNA translation. A general inhibitory effect on translation is observed when FMRP purified from Escherichia coli is preincubated with mRNAs in cell-free systems and in frog oocytes (Laggerbauer et al., 2001). On the opposite, a specific inhibitory effect with respect to the nature of the mRNAs is obtained when the protein is produced in baculovirus (Li et al., 2001; Schaeffer et al., 2001). Indeed, posttranslational modifications are likely to affect RNA binding properties of FMRP, in particular phosphorylation (Siomi et al., 2002). The translation inhibition effect is specific for FMRP, because FXR1P and FXR2P do not block mRNA translation. Moreover, FMRP carrying the missense mutation (I304N) in KH2 domain that prevents this protein from homodimerizing causes the loss of translational repression (Laggerbauer et al., 2001). In neurons, FMRP is found at postsynaptic sites enriched in ribosomes where some mRNAs (including FMR1 mRNA) are translated in response to synaptic activation. This response appears impaired in mice that lack FMRP (Huber et al., 2002; Weiler and Greenough, 1999; Weiler et al., 1997). The local protein synthesis in neurons has crucial roles in providing the synaptic plasticity, the process permitting to a synapse to constantly modify its transmission efficiency depending on received signals (see Jiang and Schuman, 2002 for a review). Synaptic plasticity is probably at the cellular basis of learning and long-term memory. In the brain of FMRP–deficient mice and of fragile X patients examined, there are subtle anomalies in the number and morphology of dendritic spines, the small protuberances on dendrites that form synapses with other neurons (Greenough et al., 2001; Nimchinsky et al., 2001). Synaptic defects were also observed in the Drosophila fragile X model (Zhang et al., 2001). Taken together, these observations suggest that by modulating protein synthesis, FMRP is important for the formation and pruning of synapses during development, and for synaptic activity in the adult. Clear progress has been made in the identification of potential mRNA targets and of new protein interactors of FMRP. According to the accumulated data, FMRP is able to interact with mRNAs that contain a G–quartet structure and with others that apparently do not. The finding that mRNAs can be co-immunoprecipitated with FMRP in a BC1–dependent manner is suggesting that FMRP could also selectively associate with mRNAs by binding to a noncoding RNA that base pairs specifically to these mRNAs. Thus, one could propose the hypothesis that FMRP would be able to participate to the formation of different mRNP subsets depending on the use of the G–quartet USER code (a term standing for untranslated sequence element for regulation proposed by Keene and Tenenbaum, 2002) or on the use of an “adaptor” noncoding RNA (i.e. BC1/BC200, Zalfa et al., 2003) (Fig. 2). FMRP would thus bind a finite number of mRNAs (probably already in the nucleus) thus defining mRNA subsets. These
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Fig. 2. An FMRP working model. (1) In the cellular body, FMRP assembles on mRNAs subsets according to the G–quartet USER code or via noncoding adaptor RNA (i.e. BC1/BC200). The process is likely to start in the nucleus and proceed in the cytoplasm. The known interactors of FMRP are listed. In the assembled mRNP, translation is repressed. Whether the same particle will form on each mRNA species and whether the different species will undergo the same fate is unknown. (2) mRNPs are transported into the dendrites via microtubules using motor proteins (e.g. kinesin). (3) Upon receiving a signal (e.g. synaptic stimulation through the metabotropic glutamate receptor activation), mRNA translation resumes and protein is locally produced thereby influencing synapse plasticity.
subsets could represent FMRP posttranslational operons (Keene and Tenenbaum, 2002). One cannot completely exclude that FMRP could also bind other sites with lower affinity, in particular if its molar amount exceeds the number of high affinity binding targets. In this case, FMRP may well be bound nonspecifically to other RNA sites, as this seems to be the case for hnRNP proteins for instance (with which FMRP seems to share many similarities). Finally, how FMRP might exert its translational inhibitory effect? The new connections discovered between dFXR and the RNA silencing pathway on one hand, and between FMRP and the noncoding RNAs BC1/BC200 on the other hand, seem to shed some light on this question. The role of dFXR in RNA interference efficiency seems to be marginal as the absence of dFXR has only a minor effect of on the RNAi activity (i.e. by triggering the degradation of the target RNA) (Caudy et al., 2002; Ishizuka et al., 2002). More work might be required to completely exclude a role for dFXR in RNAi. However, there is another very attractive possibility: dFXR could play a role in the pathway of the miRNA-dependent
translation control. Translation inhibition through this process has been clearly established for only a few cases (Schwarz and Zamore, 2002). But, as the list of miRNAs is growing exponentially (hundreds have been identified to date), the role of miRNAs in the posttranscriptional regulation of gene expression may appear to be very important. In this respect, the presence of BC1/BC200 in the mammal mRNP complex and the association of dFXR with the RISC factor in Drosophila are probably suggestive. However, the evidence for a functional link between RISC and human FMRP mRNP complexes remains to be established. In these mRNPs, the mRNAs would be maintained translationaly silenced through the formation of a complex with FMRP and noncoding RNAs. Consistent with this view, BC1 RNA has been demonstrated to be a specific repressor of translation (Wang et al., 2002). These complexes are likely to be maintained silent throughout their transport until the protein translation resumes when a particular signal is delivered (e.g. through synaptic activation of the metabotropic glutamate receptors, Huber et al., 2002).
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It remains now to be established whether the different mRNAs targeted by FMRP undergo the same fate. To start answering this question, it will be important to determine if FMRP recognizes similar features on a G–quartet structure and on the adaptor RNAs. Also, it will be necessary to know whether the same interactors associate with FMRP on the different mRNAs; the assembly of different FMRP mRNP complexes will probably be related to different functions (e.g. translation and transport). A careful study at the level of the individual mRNA targets is now necessary. This issue will certainly be complicated by the fact that each mRNA species is decorated with a unique combinatorial arrangement of other mRNP proteins that are also likely to influence (positively, neutrally or negatively) the action of FMRP on the fate of mRNAs. The recent data accumulated, if they do not yet answer all the questions about FMRP’s function, are certainly providing very rich material for future experiments. Moreover, animal models (FMR1 KO mouse and fly model) are now available and will be essential to validate the proposed function of FMRP. Research in the fragile X field is certainly promising new exciting results to come.
Acknowledgements We thank Pascale Romby for critical reading of the manuscript, and Jean-Louis Mandel, Barbara Bardoni, Anette Schenck, Marie Castets and Angela Giangrande for exemplary collaboration.
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Siomi, H., Siomi, M.C., Nussbaum, R.L., Dreyfuss, G., 1993. The protein product of the fragile X gene, FMR1, has characteristics of an RNA–binding protein. Cell 74, 291–298. Siomi, M.C., Higashijima, K., Ishizuka, A., Siomi, H., 2002. Casein kinase II phosphorylates the fragile X mental retardation protein and modulates its biological properties. Mol. Cell Biol. 22, 8438–8447. Tamanini, F., Kirkpatrick, L.L., Schonkeren, J., van Unen, L., Bontekoe, C., Bakker, C., Nelson, D.L., Galjaard, H., Oostra, B.A., Hoogeveen, A.T., 2000. The fragile X–related proteins FXR1P and FXR2P contain a functional nucleolar-targeting signal equivalent to the HIV–1 regulatory proteins. Hum. Mol. Genet. 9, 1487–1493. Tamanini, F., Willemsen, R., van Unen, L., Bontekoe, C., Galjaard, H., Oostra, B.A., Hoogeveen, A.T., 1997. Differential expression of FMR1, FXR1 and FXR2 proteins in human brain and testis. Hum. Mol. Genet. 6, 1315–1322. Wan, L., Dockendorff, T.C., Jongens, T.A., Dreyfuss, G., 2000. Characterization of dFMR1, a Drosophila melanogaster homolog of the fragile X mental retardation protein. Mol. Cell Biol. 20, 8536–8547. Wang, H., Iacoangeli, A., Popp, S., Muslimov, I.A., Imataka, H., Sonenberg, N., Lomakin, I.B., Tiedge, H., 2002. Dendritic BC1 RNA: functional role in regulation of translation initiation. J. Neurosci. 22, 10232–10241. Weiler, I.J., Greenough, W.T., 1999. Synaptic synthesis of the Fragile X protein: possible involvement in synapse maturation and elimination. Am. J. Med. Genet. 83, 248–252. Weiler, I.J., Irwin, S.A., Klintsova, A.Y., Spencer, C.M., Brazelton, A.D., Miyashiro, K., Comery, T.A., Patel, B., Eberwine, J., Greenough, W.T., 1997. Fragile X mental retardation protein is translated near synapses in response to neurotransmitter activation. Proc. Natl. Acad. Sci. USA 94, 5395–5400. Zalfa, F., Giorgi, M., Primerano, B., Moro, A., Di Penta, A., Reis, S., Oostra, B., Bagni, C., 2003. Protein FMRP associates with BC1 RNA and regulates the translation of specific mRNAs at synapses. Cell 112, 317–327. Zeng, Y., Wagner, E.J., Cullen, B.R., 2002. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9, 1327–1333. Zhang, Y., O’Connor, J.P., Siomi, M.C., Srinivasan, S., Dutra, A., Nussbaum, R.L., Dreyfuss, G., 1995. The fragile X mental retardation syndrome protein interacts with novel homologs FXR1 and FXR2. Embo J. 14, 5358–5366. Zhang, Y.Q., Bailey, A.M., Matthies, H.J., Renden, R.B., Smith, M.A., Speese, S.D., Rubin, G.M., Broadie, K., 2001. Drosophila fragile X–related gene regulates the MAP1B homolog Futsch to control synaptic structure and function. Cell 107, 591–603.
Review
The RNA binding protein FMRP: new connections and missing links Céline Schaeffer, Mélanie Beaulande, Chantal Ehresmann, Bernard Ehresmann, Hervé Moine * UPR 9002, Institut de Biologie Moléculaire et Cellulaire, 15, rue René-Descartes, 67084 Strasbourg cedex, France Received 18 February 2003; accepted 5 March 2003
Abstract The loss of the fragile X mental retardation protein (FMRP) is responsible for the most common cause of inherited mental retardation called the fragile X syndrome. FMRP is suspected to participate in the synaptic plasticity of neurons by acting on posttranscriptional control of gene expression. FMRP is an RNA binding protein that associates with mRNAs together with other proteins to form large ribonucleoprotein complexes. These complexes are proposed to participate in the transport, localization and translation of target mRNAs. Progress has been made recently in the identification of the mRNAs and the proteins present in these complexes and a possible connection with the micro-RNA dependent regulatory pathway has been established. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Fragile X syndrome; FMRP; RNA binding; G–quartet; Non–coding RNA
1. The absence of FMRP causes fragile X syndrome One of the most frequent genetic diseases, the fragile X syndrome, is caused by the lack (or more rarely by a defect in the function) of an RNA binding protein: the fragile X mental retardation protein (FMRP). Mental retardation is the main symptom of the disease but behavioral disorders are also associated with it. In addition to mental retardation, other clinical features can be present, particularly in males, like macroorchidism in postpubertal males, typical face with large ears and high forehead or hyper extensible joints. Because fragile X has a prevalence of about 1 in 4000 males and 1 in 8000 females, it is considered as the most frequent form of inherited mental retardation (Crawford et al., 2001). The name of this syndrome comes from the presence of a cytogenetically visible fragile site on chromosome X. This fragile site is revealed in particular cytological growth conditions, and it was used for the positional cloning of the gene involved in the disease (for reviews, see Imbert et al., 1998; Jin and Warren, 2000). Fragile X results from the amplification of a CGG trinucleotide repeat located in the 5' untranslated region (5' UTR) of the FMR1 (fragile X mental retardation 1) gene. The number of CGG repeats is polymorphic in the normal population, ranging between 6 and 50 with an average of * Corresponding author. Tel.: +33-3-88-41-70-51; fax: +33-3-88-60-22-18. E-mail address: [email protected] (H. Moine). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 10.1016/S0248-4900(03)00037-6
about 30. Two classes of mutations have been described: premutations and full mutations. Premutations, on one hand, are characterized by moderate expansions (50–200) and are not associated with mental retardation although they have some clinical impacts (Hagerman and Hagerman, 2002). Full mutations, on the other hand, are present in affected people and are characterized by large expansions (200 to more than 1000 repeats) and by an abnormal methylation of the repeats themselves as well as of the surrounding regions. This methylation leads to the transcriptional inhibition of FMR1 and thus to the absence of its protein product, FMRP. The expansion occurs only through maternal transmission with an efficiency of amplification directly linked to the size of the premutation (Bardoni and Mandel, 2002; Imbert et al., 1998), but its mechanism is not yet understood. Although the expansion of the CGG repeat is found in the majority of affected people, some rare patients are known without amplification but bearing instead deletions or a missense mutation within the FMR1 coding sequence. These data, together with the fact that the FMR1 KO mouse reproduces a subtle phenotype reminiscent of the human phenotype (The Dutch–Belgian Fragile X Consortium, 1994), support the idea that FMR1 is the only gene implicated in this syndrome. The FMR1 gene is composed of 17 exons, spans about 38 kb and is subjected to alternative splicing. In vivo six different isoforms, ranging from 70 to 80 kDa, can be detected, with the major form being ±80 kDa (Khandjian, 1999). The
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FMRP protein is highly conserved in vertebrates and belongs to a family that contains two other proteins: FXR1 and FXR2 (fragile X related protein 1 and 2). In Drosophila melanogaster, there is only one homolog for these three proteins: dFXR (Wan et al., 2000). Localization studies have shown that FMRP is broadly expressed in most tissues, but it is predominantly found in the central nervous system and testes. In the cell, FMRP is mostly cytoplasmic but about 5% of the total FMRP is localized in the nucleus. It contains both a nuclear localization signal (NLS) and a nuclear export signal (NES) allowing it to shuttle between nucleus and cytoplasm (Bardoni et al., 1997; Eberhart et al., 1996; Fridell et al., 1996). One major issue in understanding the biology of fragile X syndrome is to define the role of FMRP. This role has been very elusive up to now. But the fact that FMRP is present mostly in cytoplasm, associated with mRNAs as part of large ribonucleoproteic complexes (mRNP), together with the polyribosomes, suggested that FMRP is somehow involved in posttranscriptional control of gene expression. Thus, FMRP could control the transport, the localization and/or the translation of specific mRNAs in the cells where it is mostly expressed: the neurons. As FMRP seems to exert its function through RNA binding, it was essential to define its RNA binding properties and identify its targets. Also, the characterization of the mRNP complex(es), in which it is involved, is crucial for understanding its function. Significant progress has been made recently in the understanding of the RNA binding properties of FMRP and in the identification of mRNAs and proteins associated with it. This review will focus on these new findings and their meaning regarding the possible role of FMRP in neuronal function. 2. The RNA binding properties of FMRP FMRP contains two K homology domains (KH) and one arginine and glycine rich region (RGG box) known to be RNA binding motifs. Indeed, FMRP binds to RNA homopolymers in vitro with a preference for poly(G) and poly(U), whereas a very weak binding to poly(A) and poly(C) is seen (Siomi et al., 1993). FMRP was shown to bind to a small fraction of mRNAs present in a pool of total human fetal brain mRNAs (approximately 4%). FMRP was also shown to be able to bind to its own mRNA with high affinity, but also to its antisense sequence (Ashley et al., 1993). Taken together these data were suggesting that FMRP could have some RNA binding selectivity, yet with a high propensity to bind RNA in general. Since FMRP is not essential for viability, a possibility is that FMRP could regulate the expression of specific mRNAs, in particular in the nervous system where FMRP is mostly expressed. In fragile X patients, the loss of FMRP would cause defects in the proper translation of many mRNAs, thus explaining the pleiotropic phenotype associated with fragile X syndrome. Moreover, the identification of a point mutant in the second KH domain in a patient with a severe fragile X phenotype
Fig. 1. Structure of G–quartets. (A) Schematic model for the G–quartets and (B) its hydrogen bonding scheme. In G–quartets, four guanines form hydrogen bonds with each other in a symmetrical square planar arrangement. The overall structure of G–quartets is stabilized by the coordination of cations, such as K+, within the central cavity of the quadruplex, and by stacking several G–quartet layers on top of each other. (C) Sequence of the FMRP binding site present in FMR1 mRNA (Schaeffer et al., 2001). In this sequence, the guanines are interspersed with adenines, which suggests that simple G tetrads (B) could also be associated with some G4A2 hexads. (D) Hydrogen bonding scheme of a G4A2 hexad (as found in GGAGG RNA by Liu et al., 2002 using NMR).
provided additional support for a connection between the syndrome and the RNA binding activity of FMRP. In fact, it was shown that this mutation (Ile304Asn) affected homopolymers RNA binding in vitro (Siomi et al., 1994). However, as the initial studies had been performed with nonpurified in vitro translated FMRP and as it was shown that FMRP associates with mRNP particles, it was not clear whether the RNA binding activity could be attributed to FMRP itself or whether it required other proteins. Subsequent studies performed with purified FMRP produced in baculovirus definitely confirmed that RNA binding was really an intrinsic property of FMRP (Brown et al., 1998). As the RNA binding property of FMRP appeared to be essential for its function, the key issues were then to determine whether FMRP binds to specific sets of mRNAs and whether its absence affects the distribution of proteins encoded by these mRNAs in cells from fragile X patients. Progress in the identification of the RNA binding properties of FMRP has been performed recently by using different approaches. We provided the first evidence that purified FMRP has a very high affinity for peculiar RNA sequences able to adopt a structure forming G–quartets (Schaeffer et al., 2001). We detected this specific and high affinity binding site in the mRNA encoding FMRP, which was suspected to interact with FMRP in vitro and in vivo (Ashley et al., 1993; Ceman et al., 1999). G–quartet structure consists in the superposition of layers formed by four guanines hydrogen bonded via Hoogsteen base pairs in a square planar symmetric array (Fig. 1). G–quartets form very stable structures stabilized by the coordination of monovalent ions lying between or within the plane of the guanine tetrads. Because of their requirement for cation coordination, G–quartets exhibit unique structural properties: they are preferentially stabilized
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by K+ whereas smaller size Na+ and Li+, supposedly fitting less well in the central cavity, cause their destabilization. We took advantage of these biochemical peculiarities to demonstrate the involvement of G–quartets in FMRP binding. Furthermore, the exchange of the homologous FMRP binding site by another sequence known to be able to adopt such G–quartet, or its insertion within a heterologous mRNA, is able to promote FMRP binding. Also, the biological relevance of this interaction was supported by the fact that binding of FMRP to such a structure inserted in the 5' UTR of a reporter gene inhibited its translation in rabbit reticulocyte lysate. Thus, G–quartets appeared as good potential targets for FMRP binding on mRNAs. Using the SELEX method, Darnell et al. (2001) came to the same conclusion concerning the binding of FMRP to G–quartets. Furthermore, they showed that the RGG box of FMRP was responsible for the interaction and not the KH domain. In a combined study, Brown et al. (2001) examined the mRNAs associated with FMRP–mRNP in mouse brain and human lymphoblastoid cell lines. They used a microarray strategy to compare the results from normal and FMRP– minus cells. By comparing the input RNA used for the immunoprecipitation experiments and the RNA coimmunoprecipitated with FMRP, they identified one pool of 432 mRNAs associated with FMRP in the mouse brain. By comparing the mRNA polysomal profile from normal and fragile X patient lymphoblast cells, they found another pool of 251 mRNAs, which had variations in their polysome content while their cytoplasmic abundance remained unchanged. Between the two RNA pools, 14 mRNAs were found in common. The weak overlap between the two pools was explained by the differences in gene representation on the murine and human microarray, and in gene expression between lymphoblastoid cell and whole brain extracts. Among these mRNAs, about 50% were decreased in their polysome association, whereas the other half was increased. This apparent opposite effect, caused by the absence of FMRP on its potential mRNA targets, is very difficult to rationalize at this moment. Most interestingly, the analysis of these mRNAs revealed that most of them contain a potential G–quartet structure (whereas only a few percent of mRNAs taken randomly would be predicted to contain such structures). Again, a rationalization with respect to the role of FMRP is hard to make as the G–quartet structure can be located in the coding region as well as in the 5' or 3' UTR, independently of the variations in mRNA polysome associations. Many of these mRNAs encode proteins important for neuronal functions (microtubule associated protein 1B (MAP1B), potassium channel kv3.1, neuronal axonal protein –22 (NAP22), Rab6 interacting protein 1, semaphorin 3F, etc.). These data support the hypothesis that misregulation of certain mRNAs is the molecular basis of the fragile X syndrome. Further evidence is now required to demonstrate the impact of FMRP at the protein level of these interesting genes. In fact, a first indication in favor of a functional link between FMRP and one of these genes was already found in
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Drosophila. Remarkably, the absence of dFXR, the fly homolog of FMRP, causes a synaptic structural defect similar to an over-expression of Futsch, the fly homolog of MAP1B. Following this initial observation, Zhang et al. (2001) were able to correct the synaptic anomalies elicited by dFXR deficiency by combining the dFXR and Futsch mutations. This morphological opposing effect of the two genes enabled the authors to propose that dFXR is a translational repressor of Futsch. However, at the moment, it seems that the G–quartet region proposed to be used in MAP1B for FMRP recognition is not conserved in Futsch mRNA. Increasing the already long list of potential FMRP targets, another recent study using an original approach revealed 81 additional mRNAs. By using an antibody linked to a priming oligonucleotide, Miyashiro et al. (2003) performed in situ cDNA synthesis to identify mRNAs present in the close vicinity of FMRP in neurons. At first glance, it is disappointing to notice that none of the members of this list are present in that established by Brown et al. (2001). This result may be explained by a weak overlap between the lists of genes present on both arrays. In fact, from the top 80 candidates retained by Brown et al., only 17 were present in the 5000– membered cDNAs macroarray used by Miyashiro et al. (2003). Among those 17 genes, eight (GRIN1, IP3 receptor, Rab6–associated GDI, Tesk1, PI4 kinase, a–latroxin receptor, mRNA similar to Tensin, mRNA similar to acetylglucosaminyl transferase) could be detected with the antibody positioned amplification approach. Surprisingly, these top candidates were not retained in the final list presented by the authors. Also, among the noticeable absent genes of the list is MAP1B, but as the authors explain, this target may have been easily missed since their macroarray contains a 1 kb fragment of the MAP1B sequence while full length mRNA is 9 kb long. A number of other reasons can account for the differences found between the arrays: the choice of the initial screen (co-immunoprecipitation vs. in situ RNA amplification), the cell type (lymphoblast vs. whole brain vs. neuron), the array technology (microarray vs. macroarray), the threshold chosen to keep or discard a candidate, the additional validation tests (polysome association vs. in vitro binding experiments), etc. each with their own limitations and causes of false positive or negative. While the study by Brown et al. (2001) did not provide evidence for the protein expression of their gene candidates, Miyashiro et al. (2003) managed to detect subtle differences within neurons in the localization and amount of a few proteins when comparing brain tissues from wild type and FMRP KO mice. Among these proteins are GR (the glucocorticoid receptor a) and GRK2 (G protein-coupled receptor kinase 2, a modulator involved in cell signaling). A possible functional relationship between GR and fragile X would be that corticosteroid feedback is often affected in fragile X patients. One main conclusion from this study is that the effects caused by the absence of FMRP are essentially visible in dendrites, particularly at the postsynaptic compartments. This further supports the involvement of FMRP in mRNA
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localization and/or local translation in dendrites. Another conclusion is that G–quartets could be postulated in a number of these RNA targets (23%), but as far as they can be predicted, these structures do not seem to be the main common denominator of this latter list of mRNA targets. In fact, a recent report proposes that the mRNA selectivity of FMRP could be mediated also via an “adaptor” RNA: the RNA BC1 in rodents and its counterpart BC200 in primates (Zalfa et al., 2003). Indeed, these noncoding RNAs, abundant in neurons, are able both to bind directly to FMRP and to base pair with sequences present in mRNAs regulated by FMRP. The mRNAs proposed to be under the control of FMRP via BC1/BC200 are Arc, a–CaMKII, and MAP1B, which had been previously demonstrated to be localized in dendrites or play some important neuronal function. Moreover, BC1 RNA was recently shown to be involved in translational repression at the level of initiation (Wang et al., 2002). Taken together, these data suggest that FMRP could use at least two signals to target an mRNA: the G–quartet motif that was found in an important number of potential mRNA targets, and a small RNA hybridized with the mRNA, explaining the targeting of the non–G–quartet mRNAs. In the case of MAP1B mRNA, both signals would be present. 3. The FMRP mRNP particle(s) The capture of cytoplasmic FMRP by affinity chromatography using oligo(dT) suggested its association in vivo with mRNAs (Corbin et al., 1997; Feng et al., 1997), thus qualifying FMRP as part of mRNP complexes. These mRNP are associated with the translating ribosomes (polyribosomes) as demonstrated by the use of translation initiation inhibitors (Corbin et al., 1997; Feng et al., 1997). Still, the question of a functional interaction between FMRP and the ribosome seems unsolved. After an initial conclusion that FMRP was intimately associated with the ribosome, further studies suggested that FMRP does not interact directly with the ribosomes themselves but rather through the interaction with mRNA. Indeed, ribosomes can be depleted from mRNPs by EDTA treatment, while FMRP remains associated to a complex of large size, with a sedimentation peak centered at 100S, clearly distinct from heaviest 60S ribosomal subunits (Feng et al., 1997; Ohashi et al., 2002). However, the recent report of the isolation of ribosomal proteins directly associated with the Drosophila FMRP homolog suggests that a direct interaction between FMRP and the ribosome is likely to occur at some point (Ishizuka et al., 2002). As already mentioned, FMRP is mostly cytoplasmic, and microscopic studies have shown that it overlaps with polyribosomes in the rough endoplasmic reticulum (RER) of the perikaryon. Also, in neurons, where FMRP is mostly found, a part is present in the dendritic synapses. Roughly, FMRP is where the mRNA engaged in translation is. Moreover, FMRP associated with mRNAs and ribosomes as granules was visualized “en route” while trafficking between perikaryon and
neurites (DeDiego Otero et al., 2002). Furthermore, the FMRP mRNP particle seems to use the “long distance” transportation system, which is microtubule and kinesin dependent, rather than the “short distance” transportation, which uses the actin microfilaments. From their physical appearance, these granules seem to be reminiscent of stress granules (Mazroui et al., 2002). 4. The proteins that associate with FMRP The identification of the members of the FMRP mRNP particles has already revealed several key players but the list is still open. FXR1 and FXR2, two close homologs of FMRP, have been identified as interactors of FMRP by yeast two hybrid studies (Zhang et al., 1995). Clearly, these proteins seem to have some functional redundancy with FMRP, as they share the same protein domains (KH1, KH2, RGG), have the same RNA polymer binding properties, associate with mRNPs in polysomes, are capable to heterodimerize with FMRP and have some shuttling activity. Furthermore, they share some similar distribution in certain tissues (e.g. adult brain). However, FXR1 and FXR2 seem to have acquired also distinct functions and/or tissue specificity. In particular, they show some differences in their expression patterns in many organs (e.g. in human fetal brain, in fetal and adult testis, in muscles) (Tamanini et al., 1997), and their shuttling activity (for FXR2 and some alternatively spliced isoforms of FXR1) is between nucleolus and cytoplasm unlike FMRP. Also, the FXR proteins preferentially homodimerize rather than heterodimerize (Tamanini et al., 2000) and do not interact with all the other interactors of FMRP (see below), suggesting that they might belong to distinct mRNPs. NUFIP1 has been also detected by two-hybrid screening (Bardoni et al., 1999). NUFIP1 mostly locates in nucleus in association with interchromatin fibrils, and it can shuttle between nucleus and cytoplasm. In the brain, its expression pattern is similar to that of FMRP. Interestingly, NUFIP1 does not interact with FXR1 and FXR2, thus underlining the possibility that the FXR proteins have acquired distinct specificities. Despite the presence of two Zinc-finger motifs, suggesting nucleic acid binding properties, the function of NUFIP is still unknown. Also, its association with FMRP as an integral part of FMRP–mRNPs is not certain. The identification of CYFIP1 and CYFIP2 as interactors of FMRP (Schenck et al., 2001), two homologous proteins that contain no known motifs, is of great interest. CYFIP1 had been found previously to be involved in the interaction with the factor Rac1 (Kobayashi et al., 1998). Rac1 is a member of the wide family of the Rho–GTPases, which are specific molecular switches controlling a plethora of cellular pathways, from membrane trafficking to cell growth and division. Most interestingly, Rho–GTPases, through their control of the actin cytoskeleton, play a critical role in the maturation and maintenance of the dentritic spines (the structures found affected in FMRP–minus neurons). This pathway
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was previously found to be involved in other known forms of mental retardation (Ramakers, 2002). Remarkably, CYFIP2 (also known as P53 Induced mRNA, PIR121) was recently found to be involved in the complex repressing the WAVE1 protein. WAVE1 triggers the actin polymerization process (Eden et al., 2002), its repression is alleviated by Rac1, probably by its interaction with CYFIP2. Thus, because FMRP is also an interactor of CYFIP, one could hypothesize that in fragile X patient, the absence of FMRP would cause an increase in the amount of CYFIP available; this change is likely to affect the actin polymerization process and thereby the dendritic spine maturation. Epitope tag immunoprecipitation experiments have revealed that nucleolin and YB1/P50 are coimmunoprecipitated with FMRP in an RNase treatment independent manner, suggesting that they are part of the same particle as FMRP (Ceman et al., 1999, 2000). It is still not known whether they are able to interact directly with FMRP. FMRP was also found associated by coimmunoprecipitation with the other RNA binding proteins Pura, mStaufen, and Myosin Va, which have been proposed to regulate the transport of polyribosomes along microtubules and actin filaments via RER structures (Ohashi et al., 2002). The association of FMRP with these proteins is sensitive to RNase treatment, suggesting that they belong to distinct particles associated on a same mRNA. The mRNAs bound by FMRP would thus benefit from the cargo/kinesin motor associated with the RER, in this case, FMRP per se would not be responsible for the transport. More recently, two groups reported at the same time that the dFXR protein (the Drosophila homolog of the FMRP and FXR1/2 human family of proteins) interacts directly in vivo with a core component of the RNA–induced silencing complex (RISC): the protein Argonaute 2 (AGO2) (Caudy et al., 2002). Together with AGO2, Ishizuka et al. (2002) also found the two ribosomal protein L5 and L11, 5S rRNA, an RNA helicase (Dmp68) and some yet unidentified micro-RNAs (miRNAs). Dicer, the RNase III like enzyme responsible for both the formation of the short interfering RNAs (siRNA) and the maturation of miRNAs, was also detected in the complex. Thus, these findings suggest that, in Drosophila, dFXR could play a role in the gene silencing pathways of siRNAs and/or miRNAs. miRNAs and siRNAs are present in similar RNP complexes, but their function differs. While siRNAs are 21–23 double stranded nucleotide fragments produced from long dsRNA duplex (in particular those encoded by viruses or transposons), miRNAs instead are small RNAs excised from 60– to 80–nt hairpin RNAs, themselves matured from longer sequences transcribed from endogenous genes. The siRNAs and the miRNAs are present in similar RNP complexes but their function seems to depend on their matching with their target mRNA. Thus, while the siRNAs trigger the degradation of the target RNA, miRNAs that form bulged structures are believed to inhibit translation after the initiation stage (Olsen and Ambros, 1999; Zeng et al., 2002).
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5. The possible function(s) of FMRP There is strong evidence to support the notion that FMRP is important in regulating mRNA translation. A general inhibitory effect on translation is observed when FMRP purified from Escherichia coli is preincubated with mRNAs in cell-free systems and in frog oocytes (Laggerbauer et al., 2001). On the opposite, a specific inhibitory effect with respect to the nature of the mRNAs is obtained when the protein is produced in baculovirus (Li et al., 2001; Schaeffer et al., 2001). Indeed, posttranslational modifications are likely to affect RNA binding properties of FMRP, in particular phosphorylation (Siomi et al., 2002). The translation inhibition effect is specific for FMRP, because FXR1P and FXR2P do not block mRNA translation. Moreover, FMRP carrying the missense mutation (I304N) in KH2 domain that prevents this protein from homodimerizing causes the loss of translational repression (Laggerbauer et al., 2001). In neurons, FMRP is found at postsynaptic sites enriched in ribosomes where some mRNAs (including FMR1 mRNA) are translated in response to synaptic activation. This response appears impaired in mice that lack FMRP (Huber et al., 2002; Weiler and Greenough, 1999; Weiler et al., 1997). The local protein synthesis in neurons has crucial roles in providing the synaptic plasticity, the process permitting to a synapse to constantly modify its transmission efficiency depending on received signals (see Jiang and Schuman, 2002 for a review). Synaptic plasticity is probably at the cellular basis of learning and long-term memory. In the brain of FMRP–deficient mice and of fragile X patients examined, there are subtle anomalies in the number and morphology of dendritic spines, the small protuberances on dendrites that form synapses with other neurons (Greenough et al., 2001; Nimchinsky et al., 2001). Synaptic defects were also observed in the Drosophila fragile X model (Zhang et al., 2001). Taken together, these observations suggest that by modulating protein synthesis, FMRP is important for the formation and pruning of synapses during development, and for synaptic activity in the adult. Clear progress has been made in the identification of potential mRNA targets and of new protein interactors of FMRP. According to the accumulated data, FMRP is able to interact with mRNAs that contain a G–quartet structure and with others that apparently do not. The finding that mRNAs can be co-immunoprecipitated with FMRP in a BC1–dependent manner is suggesting that FMRP could also selectively associate with mRNAs by binding to a noncoding RNA that base pairs specifically to these mRNAs. Thus, one could propose the hypothesis that FMRP would be able to participate to the formation of different mRNP subsets depending on the use of the G–quartet USER code (a term standing for untranslated sequence element for regulation proposed by Keene and Tenenbaum, 2002) or on the use of an “adaptor” noncoding RNA (i.e. BC1/BC200, Zalfa et al., 2003) (Fig. 2). FMRP would thus bind a finite number of mRNAs (probably already in the nucleus) thus defining mRNA subsets. These
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Fig. 2. An FMRP working model. (1) In the cellular body, FMRP assembles on mRNAs subsets according to the G–quartet USER code or via noncoding adaptor RNA (i.e. BC1/BC200). The process is likely to start in the nucleus and proceed in the cytoplasm. The known interactors of FMRP are listed. In the assembled mRNP, translation is repressed. Whether the same particle will form on each mRNA species and whether the different species will undergo the same fate is unknown. (2) mRNPs are transported into the dendrites via microtubules using motor proteins (e.g. kinesin). (3) Upon receiving a signal (e.g. synaptic stimulation through the metabotropic glutamate receptor activation), mRNA translation resumes and protein is locally produced thereby influencing synapse plasticity.
subsets could represent FMRP posttranslational operons (Keene and Tenenbaum, 2002). One cannot completely exclude that FMRP could also bind other sites with lower affinity, in particular if its molar amount exceeds the number of high affinity binding targets. In this case, FMRP may well be bound nonspecifically to other RNA sites, as this seems to be the case for hnRNP proteins for instance (with which FMRP seems to share many similarities). Finally, how FMRP might exert its translational inhibitory effect? The new connections discovered between dFXR and the RNA silencing pathway on one hand, and between FMRP and the noncoding RNAs BC1/BC200 on the other hand, seem to shed some light on this question. The role of dFXR in RNA interference efficiency seems to be marginal as the absence of dFXR has only a minor effect of on the RNAi activity (i.e. by triggering the degradation of the target RNA) (Caudy et al., 2002; Ishizuka et al., 2002). More work might be required to completely exclude a role for dFXR in RNAi. However, there is another very attractive possibility: dFXR could play a role in the pathway of the miRNA-dependent
translation control. Translation inhibition through this process has been clearly established for only a few cases (Schwarz and Zamore, 2002). But, as the list of miRNAs is growing exponentially (hundreds have been identified to date), the role of miRNAs in the posttranscriptional regulation of gene expression may appear to be very important. In this respect, the presence of BC1/BC200 in the mammal mRNP complex and the association of dFXR with the RISC factor in Drosophila are probably suggestive. However, the evidence for a functional link between RISC and human FMRP mRNP complexes remains to be established. In these mRNPs, the mRNAs would be maintained translationaly silenced through the formation of a complex with FMRP and noncoding RNAs. Consistent with this view, BC1 RNA has been demonstrated to be a specific repressor of translation (Wang et al., 2002). These complexes are likely to be maintained silent throughout their transport until the protein translation resumes when a particular signal is delivered (e.g. through synaptic activation of the metabotropic glutamate receptors, Huber et al., 2002).
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It remains now to be established whether the different mRNAs targeted by FMRP undergo the same fate. To start answering this question, it will be important to determine if FMRP recognizes similar features on a G–quartet structure and on the adaptor RNAs. Also, it will be necessary to know whether the same interactors associate with FMRP on the different mRNAs; the assembly of different FMRP mRNP complexes will probably be related to different functions (e.g. translation and transport). A careful study at the level of the individual mRNA targets is now necessary. This issue will certainly be complicated by the fact that each mRNA species is decorated with a unique combinatorial arrangement of other mRNP proteins that are also likely to influence (positively, neutrally or negatively) the action of FMRP on the fate of mRNAs. The recent data accumulated, if they do not yet answer all the questions about FMRP’s function, are certainly providing very rich material for future experiments. Moreover, animal models (FMR1 KO mouse and fly model) are now available and will be essential to validate the proposed function of FMRP. Research in the fragile X field is certainly promising new exciting results to come.
Acknowledgements We thank Pascale Romby for critical reading of the manuscript, and Jean-Louis Mandel, Barbara Bardoni, Anette Schenck, Marie Castets and Angela Giangrande for exemplary collaboration.
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