- Email: [email protected]
RNA Targets of the Fragile X Protein
Cell, Vol. 107, 555–557, November 30, 2001, Copyright 2001 by Cell Press
RNA Targets of the Fragile X Protein Michael D. Kaytor and Harry T. Orr1 Institute of Human Genetics Department of Laboratory Medicine and Pathology University of Minnesota Box 206, University of Minnesota Health Center Minneapolis, Minnesota 55455
Three papers published recently in Cell bring the power of human genetics, Drosophila genetics, and genomics to bear on the understanding of fragile X syndrome. They provide further support for the importance of local protein synthesis within a neuron as a determinant of proper synaptogenesis and the development of cognitive abilities. Fragile X syndrome (FRAXA) is a common form of inherited mental retardation that occurs with a frequency of 1 in 4,000 males and 1 in 8,000 females. FRAXA is inherited as an X-linked dominant disease with reduced penetrance; 80% of males and 30% of females carrying a mutant allele of the fragile X mental retardation-1 (FMR1) gene exhibit an abnormal phenotype. Fragile X patients typically present with mental retardation, with an IQ between 20 and 60. Neurological symptoms can also consist of hyperactivity, attention deficit disorder, and autistic-like behaviors. In addition, the clinical picture of fragile X includes the abnormal facial features of a prominent jaw and large ears. Post-pubescent males have enlarged testicles. Cloning of FMR1 revealed a novel mutational mechanism—the expansion of an unstable trinucleotide repeat. The FMR1 unstable repeat is a CGG trinucleotide located within the 5⬘ untranslated region. Expansion of the CGG repeats into the mutant range of greater than 230 repeats results in hypermethylation and the transcriptional silencing of FMR1. Identification of fragile X patients with other mutations in FMR1, deletions and a point mutation, demonstrate that the absence of a single gene product, FMRP, is the cause of fragile X syndrome and its associated mental retardation. For this discussion, it is important to keep in mind that fragile X syndrome is not associated with neurodegeneration. Rather, pathology in the brain of both FRAXA patients and Fmr1 knockout mice appears to be limited to the presence of abnormal dendritic spines reminiscent of a delay in their maturation (Hinton et al.,1991; Comery et al., 1997). FMRP Has RNA Binding Activity How does the absence of FMRP cause the clinical phenotype seen in fragile X patients? The first insight came from an analysis of the FMRP sequence that revealed two types of RNA binding motifs, two ribonucleoprotein K homology domains, KH domains, and a cluster of arginine and glycine amino acids, an RGG box (Ashley et al., 1993; Siomi et al., 1993). The one point mutation allele of FMR1, found in a male with an exceptionally 1
Correspondence: [email protected]
Minireview
severe form of mental retardation, consisted of an isoleucine to asparagine at position 304 in the second KH domain of FMRP (DeBoulle et al., 1993; Siomi et al., 1994). This mutation further highlighted the importance of the putative RNA binding activity of FMRP and its role in mental function. FMRP was found to have RNA binding activity in vitro by virtue of its ability to bind homopolymers of poly(rG) and poly(rU). However, the in vitro RNA binding activity of FMRP is likely to be complex. Deletion analyses indicate that only the first KH domain binds RNA, and the I304N mutation in the second KH domain does not alter the RNA binding activity of FMRP. The N-terminal region of FMRP, with no known RNA binding motif, also binds poly(rG). It has been demonstrated that FMRP can bind to mRNAs from brain, including its own mRNA (Brown et al., 1998). Importantly, the in vivo RNA targets of FMRP remain to be determined. FMRP and the Ribosome The subcellular distribution of FMRP provided further clues regarding its function. FMRP is found predominantly in the cytoplasm. However, this protein has both a functional nuclear localization signal (NLS) within its N terminus and a nuclear export signal (NES) at the C terminus (Eberhart et al., 1996). The presence of these two motifs raised the possibility that FMRP shuttles between the cytoplasm and the nucleus, perhaps in the transport of RNA from the nucleus to the cytoplasm. In the cytoplasm, FMRP associates with polyribosomes that are actively translating protein. This association is RNA-dependent via mRNP particles. Other proteins found within the FMRP-mRNP complex include the fragile-X-related FXR1P and FXR2P, each of which also has potential RNA binding sites. Nucleolin, a known component of mRNPs, is also found associated with FMRPcontaining mRNPs. These data led to the suggestion that FMRP might target mRNAs to translating ribosomes. It appears that the I304N mutation, the severe diseasecausing point mutation, alters the structure of mRNPs and their association with actively translating polyribosomes. The failure of FMRP with the I304N mutation to associate with the polyribosome has been suggested to be linked to the inability of this form of FMRP to dimerize (Feng et al., 1997). From these data, it was hypothesized that FMRP-containing mRNPs bind a subset of mRNAs whose translation is localized to the dendrite and is particularly critical for proper synaptic function. While there is some evidence in vitro that FMRP may function as a repressor of translation (Li et al., 2001; Laggerbauer et al., 2001), to what extent and how it might effect translation in vivo is unknown. Regardless, in the absence of FMRP, translation of its target mRNAs would be misregulated (Jin and Warren, 2000). Drosophila FMRP and the Synapse The Drosophilia genome contains a single gene that is homologous to FMR1, Drosophila fragile X related (dfxr), also designated dfmr1 (Wan et al., 2000). The dfxrencoded protein (dFXR) contains all of the functional motifs that have been delineated for FMRP: two KH domains, one RGG box, an NLS, and an NES. In this
Cell 556
Table 1. FMRP Target RNAs Gene
G quartet
Darnell et al.
Brown et al.
Semaphorin 3F Potassium channel Kv3.1 Arginine vasopressin receptor, V1a Srm tyrosine kinase Histone H4 Transcription differentiation inhibitor, ID3 Guanine nucleotide exchange factor
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
MAP1B
⫹
⫹
⫹
Msx2-interacting nuclear protein (Mint) GT334/TMEM1 Munc13 NAP-22 KIAA 1091, Rab6 interacting protein KIAA 0964, PSD-95 associated SAPAP4 Similar to adenylate cyclase (Hs. 9572) Similar to MKP-dusPTPase (Hs. 29106) TP63 Casein kinase 1, gamma 2 Similar to I38022 hypothetical protein KIAA 0317 protein Arg/Abl-interacting protein, ArgBP2 FMR1
⫹ ⫹ ⫹* ⫹* ⫹* ⫺ ? ⫺ ⫹ ⫺ ? ⫹ ⫺ **
⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
* G quartet reported by Brown et al. (2001). ** Schaeffer et al. (2001).
issue of Cell, Zhang and colleagues (2001) use Drosophila genetics to demonstrate that dFXR has a role in the regulation of synapse growth and function, primarily through its regulation of the expression of the microtubule-associated protein Futsch. An interesting aspect of this work is the demonstration that dFXR associates with futsch mRNA and that dFXR inversely regulates the expression of Futsch, likely as a translational repressor. While the effects of altered dFXR expression were examined at several sites in the fly nervous system, the most intriguing results were seen at the neuromuscular junction (NMJ). Null dfxr mutants had a synaptic overgrowth, an enhanced number of synaptic terminals characterized by a 50% increase in the number of synaptic boutons. In contrast, presynaptic overexpression of dFXR resulted in an almost 40% reduction in the synaptic boutons at the NMJ. Interestingly, postsynaptic overexpression of dFXR caused a similar but less severe reduction in the number of synaptic boutons. Thus, in the fly, it seems that dFXR functions to regulate growth on both sides of the synaptic cleft. Yet, at the physiological level, dFXR modulated neurotransmission by a presynaptic mechanism. Loss of dfxr resulted in an elevated neurotransmission and elevated presynaptic overexpression of dFXR enhanced spontaneous synaptic vesicle release as measured by a 5-fold increase in the miniature excitatory junctional currents. Taking a cue from the considerable similarity in phenotype between the dfxr mutants and futsch hypomorph mutants, Zhang et al. (2001) examined whether dFXR might interact with futsch mRNA. They found that futsch mRNA could be immunoprecipitated by anti-dFXR antibodies. Moreover, Futsch protein levels in the nervous system correlated inversely with the level of dfxr expression. Thus, dFXR seems to function as a repressor of Futsch translation in the nervous system. Returning
back to genetics, the authors tested further the interaction between dfxr and futsch by creating double mutants. The double-mutant flies with a null mutation in dfxr and a hypomorphic one in futsch had normal synaptic structure at the NMJ. This strongly suggests dFXR functions to repress futsch translation and that altered regulation of Futsch is sufficient to explain the effects seen in the dfxr mutants. To what extent do these results in Drosophila extend to fragile X syndrome as it manifests in humans? FMRP’s Fondness for G Quartets In the previous issue of Cell (November 16), papers by Brown et al. (2001) and Darnell et al. (2001) examined FMRP-associated mRNAs from mammalian brain. Using strategies that combined biochemistry with genomics, both studies found that FMRP-associated RNAs contained an intramolecular G quartet structure. Previously, Schaeffer et al. (2001) reported that FMRP binds to its own mRNA via a purine quartet. Using a sequential RNA selection strategy, Darnell et al. found that FMRP bound to sequences having the potential to form a G quartet, a structure in which four guanine residues are positioned in a planar conformation. These investigators went on to test whether the formation of a G quartet was critical for FMRP binding. By examining binding in the presence of Li⫹, an inhibitor of G quartet stacking, they found the binding of the test clone to FMRP was abolished. It was further shown that the RGG box of FMRP was required for binding to the G quartet in the RNA. Brown and colleagues used mRNA coimmunoprecipitated with murine Fmrp RNPs to probe micorarrays. This resulted in the identification of 432 mRNAs that were enriched in the Fmrp RNPs. This group also probed micorarrays using mRNAs isolated from the polyribosome fraction of a human fragile X cell line. This latter probing yielded 251 human mRNAs that had an altered polyribosome
Minireview 557
profile in the absence of FMRP. Of these human mRNAs that had a murine ortholog assayed in their first analysis, 50% were associated with the Fmrp RNPs. In Darnell et al., the authors demonstrate that 70% of these overlapping mRNAs contained a G quartet bound directly by FMRP. Darnell et al. searched the UniGene database for G-quartet-containing sequences, identifying several potential FMRP target mRNAs in the human genome. After assessing these sequences for their ability to fold in a manner consistent with formation of a G quartet, 31 RNAs were selected. Twelve of these were assayed for FMRP binding. Six RNAs bound to FMRP with affinities having a Kd ranging from 75 nM to 467 nM and 6 did not interact with FMRP. Similarly, they examined RNAs identified in the Brown et al. paper as altered in polysome distribution for G quartets and direct FMRP interaction. This collaborative effort yielded a list of 8 RNA targets—RNAs with G quartets, that directly bind to FMRP, and that are altered in polysome distribution in human fragile X cells (Table 1). This work thus identified several mRNAs containing G quartets whose misregulation might be the basis of the mental retardation in fragile X syndrome. Of Flies and Mice and Men Both in the human and Drosophila, the fragile X protein has been found to have a role in synaptic growth. In the fly, the data from Zhang et al. strongly indicate that the ability of dFXR to bind to and regulate the translation of Futsch underlies its role in synaptic development and function. At the very least, these data provide compelling evidence that dFXR does bind to and regulate the expression of a specific RNA. How about in mammals? The identification by Brown et al. and Darnell et al. that a specific structure, a G quartet, is critical for FMRP to bind to an mRNA indicates a high level of selectivity. Thus, in both flies and mammals, FMRP binds preferentially to a subset of RNAs. Since only one RNA, futsch, was examined for binding to dFXR, the number of fly mRNAs that are regulated by dFXR remains unknown. It is important to note that the mammalian homolog of Futsch, the microtubule associated protein MAP1B, was identified as a target of murine Fmrp and human FMRP in Darnell et al. and Brown et al. Thus, it is highly likely, given the results in Drosophila, that a substantial portion of the synaptic alterations seen in fragile X patients results from the misregulation of MAP1B expression, presumably locally at the synapse. However, the extent to which alterations in MAP1B expression also underlie the mental retardation in fragile X syndrome are less clear. Through a collaborative effort, several mammalian FMRP RNA targets have been identified (Table 1). One would certainly like to think that more than one of these contributes to human cognitive function. It is quite probable that Drosophila will prove useful in their prioritization. Selected Reading Ashley, C.T., Wilkinson, K.D., Reines, D., and Warren, S.T. (1993). Science 262, 563–566. Brown, V., Jin, P., Ceman, S., Darnell, J.C., O’Donnell, W.T., Tenenbaum, S.A., Jin, X., Feng, Y., Wilkinson, K.D., Keene, J.D., et al. (2001). Cell 107, 477–487.
Brown, V., Small, K., Lakkis, L., Feng, Y., Gunter, C., Wilkinson, K.D., and Warren, S.T. (1998). J. Biol. Chem. 273, 15521–15527. Comery, T.A., Harris, J.B., Willems, P.J., Oostra, B.A., Irwin, S.A., Weiler, I.J., and Greenough, W.T. (1997). Proc. Natl. Acad. Sci. USA 94, 5401–5404. Darnell, J.C., Jensen, K.B., Jin, P., Brown, V., Warren, S.T., and Darnell, R.B. (2001). Cell 107, 489–499. De Boulle, K., Verkerk, A.J., Reyniers, E., Vits, L., Hendricks, J., Van Roy, B., Van den Bois, F. deGraaff, E., Oostra, B.A., and Willems, P. (1993). Nat. Genet. 3, 31–35. Eberhart, D.E., Malter, H.E., Feng, Y., and Warren, S.T. (1996). Hum. Mol. Genet. 5, 1083–1091. Feng, Y., Absher, D., Eberhart, D.E., Brown, V., Malter, H.E., and Warren, S.T. (1997). Mol. Cell 1, 109–118. Hinton, V.J., Brown, W.T., Wisniewski, K., and Rudelli, R.D. (1991). J. Med. Genet. 41, 289–294. Jin, P., and Warren, S.T. (2000). Hum. Mol. Genet. 9, 901–908. Li, Z., Zhang, Y., Ku, L., Wilkinson, K.D., Warren, S.T., and Feng, Y. (2001). Nucleic Acids Res. 29, 2276–2283. Laggerbauer, B., Ostareck, D., Keidel, E.M., Ostareck-Lederer, A., and Fischer, U. (2001). Hum. Mol. Genet. 10, 329–338. Schaeffer, C., Bardoni, B., Mandel, J.L., Ehresmann, B., Ehresmann, C., and Moine, H. (2001). EMBO J. 20, 4803–4813. Siomi, H., Siomi, M.C., Nussbaum, R.L., and Dreyfuss, G. (1993). Cell 74, 291–298. Siomi, H., Choi, M., Siomi, M.C., Nussbaum, R.L., and Dreyfuss, G. (1994). Cell 77, 33–39. Wan, L., Dockendorff, T.C., Jongens, T.A., and Dreyfuss, G. (2000). Mol. Cell. Biol. 20, 8536–8547. Zhang, Y.Q., Bailey, A.M., Matthies, H.J.G., Renden, R.B., Smith, M.A., Speese, S.D., Rubin, G.M., and Broadie, K. (2001). Cell 107, this issue, 591–603.
RNA Targets of the Fragile X Protein Michael D. Kaytor and Harry T. Orr1 Institute of Human Genetics Department of Laboratory Medicine and Pathology University of Minnesota Box 206, University of Minnesota Health Center Minneapolis, Minnesota 55455
Three papers published recently in Cell bring the power of human genetics, Drosophila genetics, and genomics to bear on the understanding of fragile X syndrome. They provide further support for the importance of local protein synthesis within a neuron as a determinant of proper synaptogenesis and the development of cognitive abilities. Fragile X syndrome (FRAXA) is a common form of inherited mental retardation that occurs with a frequency of 1 in 4,000 males and 1 in 8,000 females. FRAXA is inherited as an X-linked dominant disease with reduced penetrance; 80% of males and 30% of females carrying a mutant allele of the fragile X mental retardation-1 (FMR1) gene exhibit an abnormal phenotype. Fragile X patients typically present with mental retardation, with an IQ between 20 and 60. Neurological symptoms can also consist of hyperactivity, attention deficit disorder, and autistic-like behaviors. In addition, the clinical picture of fragile X includes the abnormal facial features of a prominent jaw and large ears. Post-pubescent males have enlarged testicles. Cloning of FMR1 revealed a novel mutational mechanism—the expansion of an unstable trinucleotide repeat. The FMR1 unstable repeat is a CGG trinucleotide located within the 5⬘ untranslated region. Expansion of the CGG repeats into the mutant range of greater than 230 repeats results in hypermethylation and the transcriptional silencing of FMR1. Identification of fragile X patients with other mutations in FMR1, deletions and a point mutation, demonstrate that the absence of a single gene product, FMRP, is the cause of fragile X syndrome and its associated mental retardation. For this discussion, it is important to keep in mind that fragile X syndrome is not associated with neurodegeneration. Rather, pathology in the brain of both FRAXA patients and Fmr1 knockout mice appears to be limited to the presence of abnormal dendritic spines reminiscent of a delay in their maturation (Hinton et al.,1991; Comery et al., 1997). FMRP Has RNA Binding Activity How does the absence of FMRP cause the clinical phenotype seen in fragile X patients? The first insight came from an analysis of the FMRP sequence that revealed two types of RNA binding motifs, two ribonucleoprotein K homology domains, KH domains, and a cluster of arginine and glycine amino acids, an RGG box (Ashley et al., 1993; Siomi et al., 1993). The one point mutation allele of FMR1, found in a male with an exceptionally 1
Correspondence: [email protected]
Minireview
severe form of mental retardation, consisted of an isoleucine to asparagine at position 304 in the second KH domain of FMRP (DeBoulle et al., 1993; Siomi et al., 1994). This mutation further highlighted the importance of the putative RNA binding activity of FMRP and its role in mental function. FMRP was found to have RNA binding activity in vitro by virtue of its ability to bind homopolymers of poly(rG) and poly(rU). However, the in vitro RNA binding activity of FMRP is likely to be complex. Deletion analyses indicate that only the first KH domain binds RNA, and the I304N mutation in the second KH domain does not alter the RNA binding activity of FMRP. The N-terminal region of FMRP, with no known RNA binding motif, also binds poly(rG). It has been demonstrated that FMRP can bind to mRNAs from brain, including its own mRNA (Brown et al., 1998). Importantly, the in vivo RNA targets of FMRP remain to be determined. FMRP and the Ribosome The subcellular distribution of FMRP provided further clues regarding its function. FMRP is found predominantly in the cytoplasm. However, this protein has both a functional nuclear localization signal (NLS) within its N terminus and a nuclear export signal (NES) at the C terminus (Eberhart et al., 1996). The presence of these two motifs raised the possibility that FMRP shuttles between the cytoplasm and the nucleus, perhaps in the transport of RNA from the nucleus to the cytoplasm. In the cytoplasm, FMRP associates with polyribosomes that are actively translating protein. This association is RNA-dependent via mRNP particles. Other proteins found within the FMRP-mRNP complex include the fragile-X-related FXR1P and FXR2P, each of which also has potential RNA binding sites. Nucleolin, a known component of mRNPs, is also found associated with FMRPcontaining mRNPs. These data led to the suggestion that FMRP might target mRNAs to translating ribosomes. It appears that the I304N mutation, the severe diseasecausing point mutation, alters the structure of mRNPs and their association with actively translating polyribosomes. The failure of FMRP with the I304N mutation to associate with the polyribosome has been suggested to be linked to the inability of this form of FMRP to dimerize (Feng et al., 1997). From these data, it was hypothesized that FMRP-containing mRNPs bind a subset of mRNAs whose translation is localized to the dendrite and is particularly critical for proper synaptic function. While there is some evidence in vitro that FMRP may function as a repressor of translation (Li et al., 2001; Laggerbauer et al., 2001), to what extent and how it might effect translation in vivo is unknown. Regardless, in the absence of FMRP, translation of its target mRNAs would be misregulated (Jin and Warren, 2000). Drosophila FMRP and the Synapse The Drosophilia genome contains a single gene that is homologous to FMR1, Drosophila fragile X related (dfxr), also designated dfmr1 (Wan et al., 2000). The dfxrencoded protein (dFXR) contains all of the functional motifs that have been delineated for FMRP: two KH domains, one RGG box, an NLS, and an NES. In this
Cell 556
Table 1. FMRP Target RNAs Gene
G quartet
Darnell et al.
Brown et al.
Semaphorin 3F Potassium channel Kv3.1 Arginine vasopressin receptor, V1a Srm tyrosine kinase Histone H4 Transcription differentiation inhibitor, ID3 Guanine nucleotide exchange factor
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
MAP1B
⫹
⫹
⫹
Msx2-interacting nuclear protein (Mint) GT334/TMEM1 Munc13 NAP-22 KIAA 1091, Rab6 interacting protein KIAA 0964, PSD-95 associated SAPAP4 Similar to adenylate cyclase (Hs. 9572) Similar to MKP-dusPTPase (Hs. 29106) TP63 Casein kinase 1, gamma 2 Similar to I38022 hypothetical protein KIAA 0317 protein Arg/Abl-interacting protein, ArgBP2 FMR1
⫹ ⫹ ⫹* ⫹* ⫹* ⫺ ? ⫺ ⫹ ⫺ ? ⫹ ⫺ **
⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
* G quartet reported by Brown et al. (2001). ** Schaeffer et al. (2001).
issue of Cell, Zhang and colleagues (2001) use Drosophila genetics to demonstrate that dFXR has a role in the regulation of synapse growth and function, primarily through its regulation of the expression of the microtubule-associated protein Futsch. An interesting aspect of this work is the demonstration that dFXR associates with futsch mRNA and that dFXR inversely regulates the expression of Futsch, likely as a translational repressor. While the effects of altered dFXR expression were examined at several sites in the fly nervous system, the most intriguing results were seen at the neuromuscular junction (NMJ). Null dfxr mutants had a synaptic overgrowth, an enhanced number of synaptic terminals characterized by a 50% increase in the number of synaptic boutons. In contrast, presynaptic overexpression of dFXR resulted in an almost 40% reduction in the synaptic boutons at the NMJ. Interestingly, postsynaptic overexpression of dFXR caused a similar but less severe reduction in the number of synaptic boutons. Thus, in the fly, it seems that dFXR functions to regulate growth on both sides of the synaptic cleft. Yet, at the physiological level, dFXR modulated neurotransmission by a presynaptic mechanism. Loss of dfxr resulted in an elevated neurotransmission and elevated presynaptic overexpression of dFXR enhanced spontaneous synaptic vesicle release as measured by a 5-fold increase in the miniature excitatory junctional currents. Taking a cue from the considerable similarity in phenotype between the dfxr mutants and futsch hypomorph mutants, Zhang et al. (2001) examined whether dFXR might interact with futsch mRNA. They found that futsch mRNA could be immunoprecipitated by anti-dFXR antibodies. Moreover, Futsch protein levels in the nervous system correlated inversely with the level of dfxr expression. Thus, dFXR seems to function as a repressor of Futsch translation in the nervous system. Returning
back to genetics, the authors tested further the interaction between dfxr and futsch by creating double mutants. The double-mutant flies with a null mutation in dfxr and a hypomorphic one in futsch had normal synaptic structure at the NMJ. This strongly suggests dFXR functions to repress futsch translation and that altered regulation of Futsch is sufficient to explain the effects seen in the dfxr mutants. To what extent do these results in Drosophila extend to fragile X syndrome as it manifests in humans? FMRP’s Fondness for G Quartets In the previous issue of Cell (November 16), papers by Brown et al. (2001) and Darnell et al. (2001) examined FMRP-associated mRNAs from mammalian brain. Using strategies that combined biochemistry with genomics, both studies found that FMRP-associated RNAs contained an intramolecular G quartet structure. Previously, Schaeffer et al. (2001) reported that FMRP binds to its own mRNA via a purine quartet. Using a sequential RNA selection strategy, Darnell et al. found that FMRP bound to sequences having the potential to form a G quartet, a structure in which four guanine residues are positioned in a planar conformation. These investigators went on to test whether the formation of a G quartet was critical for FMRP binding. By examining binding in the presence of Li⫹, an inhibitor of G quartet stacking, they found the binding of the test clone to FMRP was abolished. It was further shown that the RGG box of FMRP was required for binding to the G quartet in the RNA. Brown and colleagues used mRNA coimmunoprecipitated with murine Fmrp RNPs to probe micorarrays. This resulted in the identification of 432 mRNAs that were enriched in the Fmrp RNPs. This group also probed micorarrays using mRNAs isolated from the polyribosome fraction of a human fragile X cell line. This latter probing yielded 251 human mRNAs that had an altered polyribosome
Minireview 557
profile in the absence of FMRP. Of these human mRNAs that had a murine ortholog assayed in their first analysis, 50% were associated with the Fmrp RNPs. In Darnell et al., the authors demonstrate that 70% of these overlapping mRNAs contained a G quartet bound directly by FMRP. Darnell et al. searched the UniGene database for G-quartet-containing sequences, identifying several potential FMRP target mRNAs in the human genome. After assessing these sequences for their ability to fold in a manner consistent with formation of a G quartet, 31 RNAs were selected. Twelve of these were assayed for FMRP binding. Six RNAs bound to FMRP with affinities having a Kd ranging from 75 nM to 467 nM and 6 did not interact with FMRP. Similarly, they examined RNAs identified in the Brown et al. paper as altered in polysome distribution for G quartets and direct FMRP interaction. This collaborative effort yielded a list of 8 RNA targets—RNAs with G quartets, that directly bind to FMRP, and that are altered in polysome distribution in human fragile X cells (Table 1). This work thus identified several mRNAs containing G quartets whose misregulation might be the basis of the mental retardation in fragile X syndrome. Of Flies and Mice and Men Both in the human and Drosophila, the fragile X protein has been found to have a role in synaptic growth. In the fly, the data from Zhang et al. strongly indicate that the ability of dFXR to bind to and regulate the translation of Futsch underlies its role in synaptic development and function. At the very least, these data provide compelling evidence that dFXR does bind to and regulate the expression of a specific RNA. How about in mammals? The identification by Brown et al. and Darnell et al. that a specific structure, a G quartet, is critical for FMRP to bind to an mRNA indicates a high level of selectivity. Thus, in both flies and mammals, FMRP binds preferentially to a subset of RNAs. Since only one RNA, futsch, was examined for binding to dFXR, the number of fly mRNAs that are regulated by dFXR remains unknown. It is important to note that the mammalian homolog of Futsch, the microtubule associated protein MAP1B, was identified as a target of murine Fmrp and human FMRP in Darnell et al. and Brown et al. Thus, it is highly likely, given the results in Drosophila, that a substantial portion of the synaptic alterations seen in fragile X patients results from the misregulation of MAP1B expression, presumably locally at the synapse. However, the extent to which alterations in MAP1B expression also underlie the mental retardation in fragile X syndrome are less clear. Through a collaborative effort, several mammalian FMRP RNA targets have been identified (Table 1). One would certainly like to think that more than one of these contributes to human cognitive function. It is quite probable that Drosophila will prove useful in their prioritization. Selected Reading Ashley, C.T., Wilkinson, K.D., Reines, D., and Warren, S.T. (1993). Science 262, 563–566. Brown, V., Jin, P., Ceman, S., Darnell, J.C., O’Donnell, W.T., Tenenbaum, S.A., Jin, X., Feng, Y., Wilkinson, K.D., Keene, J.D., et al. (2001). Cell 107, 477–487.
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