Function Of Fragile X Mental Retardation Protein Research Articles

DrosophilaFragile X Mental Retardation Protein and Metabotropic Glutamate Receptor A Convergently Regulate the Synaptic Ratio of Ionotropic Glutamate Receptor Subclasses

A current hypothesis proposes that fragile X mental retardation protein (FMRP), an RNA-binding translational regulator, acts downstream of glutamatergic transmission, via metabotropic glutamate receptor (mGluR) G(q)-dependent signaling, to modulate protein synthesis critical for trafficking ionotropic glutamate receptors (iGluRs) at synapses. However, direct evidence linking FMRP and mGluR function with iGluR synaptic expression is limited. In this study, we use the Drosophila fragile X model to test this hypothesis at the well characterized glutamatergic neuromuscular junction (NMJ). Two iGluR classes reside at this synapse, each containing common GluRIIC (III), IID and IIE subunits, and variable GluRIIA (A-class) or GluRIIB (B-class) subunits. In Drosophila fragile X mental retardation 1 (dfmr1) null mutants, A-class GluRs accumulate and B-class GluRs are lost, whereas total GluR levels do not change, resulting in a striking change in GluR subclass ratio at individual synapses. The sole Drosophila mGluR, DmGluRA, is also expressed at the NMJ. In dmGluRA null mutants, both iGluR classes increase, resulting in an increase in total synaptic GluR content at individual synapses. Targeted postsynaptic dmGluRA overexpression causes the exact opposite GluR phenotype to the dfmr1 null, confirming postsynaptic GluR subtype-specific regulation. In dfmr1; dmGluRA double null mutants, there is an additive increase in A-class GluRs, and a similar additive impact on B-class GluRs, toward normal levels in the double mutants. These results show that both dFMRP and DmGluRA differentially regulate the abundance of different GluR subclasses in a convergent mechanism within individual postsynaptic domains.

Fragile X Mental Retardation Syndrome: Structure of the KH1-KH2 Domains of Fragile X Mental Retardation Protein

Fragile X syndrome is the most common form of inherited mental retardation in humans, with an estimated prevalence of about 1 in 4000 males. Although several observations indicate that the absence of functional Fragile X Mental Retardation Protein (FMRP) is the underlying basis of Fragile X syndrome, the structure and function of FMRP are currently unknown. Here, we present an X-ray crystal structure of the tandem KH domains of human FMRP, which reveals the relative orientation of the KH1 and KH2 domains and the location of residue Ile304, whose mutation to Asn is associated with a particularly severe incidence of Fragile X syndrome. We show that the Ile304Asn mutation both perturbs the structure and destabilizes the protein.

Role of microRNA Pathway in Mental Retardation

Deficits in cognitive functions lead to mental retardation (MR). Understanding the genetic basis of inherited MR has provided insights into the pathogenesis of MR. Fragile X syndrome is one of the most common forms of inherited MR, caused by the loss of functional Fragile X Mental Retardation Protein (FMRP).MicroRNAs (miRNAs) are endogenous, single-stranded RNAs between 18 and 25 nucleotides in length, which have been implicated in diversified biological pathways. Recent studies have linked the miRNA pathway to fragile X syndrome. Here we review the role of the miRNA pathway in fragile X syndrome and discuss its implication in MR in general.

Fragile X related protein 1 isoforms differentially modulate the affinity of fragile X mental retardation protein for G-quartet RNA structure.

Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of expression of the Fragile X Mental Retardation Protein (FMRP), an RNA binding protein with high specificity for G-quartet RNA structure. FMRP is involved in several steps of mRNA metabolism: nucleocytoplasmic trafficking, translational control and transport along dendrites in neurons. Fragile X Related Protein 1 (FXR1P), a homologue and interactor of FMRP, has been postulated to have a function similar to FMRP, leading to the hypothesis that it can compensate for the absence of FMRP in Fragile X patients. Here we analyze the ability of three isoforms of FXR1P, expressed in different tissues, to bind G-quartet RNA structure specifically. Only the longest FXR1P isoform was found to be able to bind specifically the G-quartet RNA, albeit with a lower affinity as compared to FMRP, whereas the other two isoforms negatively regulate the affinity of FMRP for G-quartet RNA. This result is important to decipher the molecular basis of fragile X syndrome, through the understanding of FMRP action in the context of its multimolecular complex in different tissues. In addition, we show that the action of FXR1P is synergistic rather than compensatory for FMRP function.

Hippocampal pyramidal cells in adult Fmr1 knockout mice exhibit an immature-appearing profile of dendritic spines

Fragile X syndrome (FXS) is a common form of mental retardation caused by the absence of functional fragile X mental retardation protein (FMRP). FXS is associated with elevated density and length of dendritic spines, as well as an immature-appearing distribution profile of spine morphologies in the neocortex. Mice that lack FMRP ( Fmr1 knockout mice) exhibit a similar phenotype in the neocortex, suggesting that FMRP is important for dendritic spine maturation and pruning. Examination of Golgi-stained pyramidal cells in hippocampal subfield CA1 of adult Fmr1 knockout mice reveals longer spines than controls and a morphology profile that, while essentially opposite of that described in the Fmr1 knockout neocortex, appears similarly immature. This finding strongly suggests that FMRP is required for the processes of spine maturation and pruning in multiple brain regions and that the specific pathology depends on the cellular context.

Behavioral and Neurochemical Alterations in Mice Lacking the RNA-Binding Protein Translin

Synapse-specific local protein synthesis is thought to be important for neurodevelopment and plasticity and involves neuronal RNA-binding proteins that regulate the transport and translation of dendritically localized transcripts. The best characterized of these RNA-binding proteins is the fragile X mental retardation protein (FMRP). Mutations affecting the expression or function of FMRP cause fragile X syndrome in humans, and targeted deletion of the gene encoding FMRP results in developmental and behavioral alterations in mice. Translin is an RNA-binding protein that regulates mRNA transport and translation in mouse male germ cells and is proposed to play a similar role in neurons. Like FMRP, translin is present in neuronal dendrites, binds dendritically localized RNA, and associates with microtubules and motor proteins. We reported previously the production of viable homozygous translin knock-out mice, which demonstrate altered expression of multiple mRNA transcripts in the brain and mild motor impairments. Here, we report that translin knock-out mice also exhibit sex-specific differences in tests of learning and memory, locomotor activity, anxiety-related behavior, and sensorimotor gating, as well as handling-induced seizures and alterations in monoamine neurotransmitter levels in several forebrain regions. Similar behavioral and neurochemical alterations have been observed in mice lacking FMRP, suggesting that both proteins may act within the same neuronal systems and signaling pathways. Our results in mice indicate that mutations in translin may contribute to fragile X-like syndromes, mental retardation, attention deficit hyperactivity disorder, epilepsy, and autism spectrum disorders in humans.

Prolonged Epileptiform Discharges Induced by Altered Group I Metabotropic Glutamate Receptor-Mediated Synaptic Responses in Hippocampal Slices of a Fragile X Mouse Model

Mutations in FMR1, which encodes the fragile X mental retardation protein (FMRP), are the cause of fragile X syndrome (FXS), an X-linked mental retardation disorder. Inactivation of the mouse gene Fmr1 confers a number of FXS-like phenotypes including an enhanced susceptibility to epileptogenesis during development. We find that in a FXS mouse model, in which the function of FMRP is suppressed, synaptically released glutamate induced prolonged epileptiform discharges resulting from enhanced group I metabotropic glutamate receptor (mGluR)-mediated responses in hippocampal slices. The induction of the group I mGluR-mediated, prolonged epileptiform discharges was inhibited in preparations that were pretreated with inhibitors of ERK1/2 (extracellular signal-regulated kinase 1/2) phosphorylation or of mRNA translation, and their maintenance was suppressed by group I mGluR antagonists. The results suggest that FMRP plays a key role in the control of signaling at the recurrent glutamatergic synapses in the hippocampus. The absence of this control causes the synaptically activated group I mGluRs to elicit translation-dependent epileptogenic activities.

New insights into Fragile X syndrome

Lack of functional Fragile X mental retardation protein (FMRP) is the primary cause of the Fragile-mental retardation syndrome in humans. In most cases, the disease results from transcriptional silencing of fragile mental retardation gene 1, fmr1, which encodes FMRP. However, a single missense mutation (I304N) in the second KH domain of FMRP gives rise to a particularly severe case of Fragile X syndrome. A Drosophila homolog of FMRP has been identified, Drosophila Fragile X related protein (dFXRP). The corresponding missense mutation in dFXRP, the I307N, has pronounced effects on the in vivo activity of the protein. The effect of the point mutation on the structure and function of FMRP is unclear, and published data are contradictory. No in vitro structural or stability studies have been performed on dFXRP. Here we show that a construct that contains only the tandem KH1-KH2 domains is a stable, well-folded unit suitable for detailed structural and functional characterization. Using this KH1-KH2 construct we explicitly test a hypothesis that has been proposed to explain the effect of the Ile-->Asn mutation: that it causes complete unfolding of the protein. Here we show that the I307N point mutation does not completely unfold the KH domain. The KH1-KH2 construct bearing I307N substitution is stable in isolation and adopts a native-like fold. Thus our data favor alternative explanations for the in vivo observed loss of dFXRP activity associated with I307N mutation: (a) the point mutation might affect intra and/or inter-molecular interactions of dFXRP; or (b) it might impair dFXRP's interactions with its RNA target(s).

Transport kinetics of FMRP containing the I304N mutation of severe fragile X syndrome in neurites of living rat PC12 cells

Lack of fragile X mental retardation protein (FMRP) causes the fragile X syndrome, a common form of inherited mental retardation. The syndrome usually results from the expansion of a CGG repeat in the FMR1 gene with consequent transcriptional silencing of FMR1. However, one missense mutation (Ile304Asn) was reported in the second KH domain of the protein involved in RNA binding. The protein containing this mutation showed an impaired function, leading to an extremely severe phenotype. In the present report, we have studied the role of FMRP I304N in living PC12 cells to better understand the (dys) function of this mutant FMRP. We have generated an FMR1 I304N-EGFP stably transfected PC12 cell line with an inducible expression system (Tet-On) for regulated expression of the FMRP I304N-EGFP fusion protein. After Dox-induction, FMRP I304N-EGFP was localized in the neurites of PC12 cells; however, no granules were formed as has been recently demonstrated for the normal FMRP. Time-lapse microscopy in combination with bleaching technology illustrated that although FMRP I304N-EGFP does not form visible granules, the transport into the neurites is microtubule dependent. Immunoprecipitation with antibodies against GFP demonstrates that FMRP I304N-EGFP coprecipitate with both the 60S ribosomal protein P0 and FXR1P, suggesting that the mutant FMRP is still able to form complexes, however, with different characteristics compared to normal FMRP.

The fragile X syndrome: From molecular genetics to neurobiology

Since the identification of the FMR1 gene basic research has been focused on the molecular characterization of the FMR1 gene product, the fragile X mental retardation protein (FMRP). Recent developments in fragile X research have provided new insights and knowledge about the physiological function of FMRP in the cell and the nerve cell in particular. Currently, compelling evidence suggests a role for FMRP in the transport/translation of dendritically localized mRNAs. In addition, the identification of some of the target mRNAs of FMRP have led to an increased interest in the neurobiology of the syndrome. This review highlights the role of FMRP in dendritic mRNA transport/translation in relation to synaptic plasticity, a molecular mechanism implicated in learning and memory.

Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway.

Fragile X syndrome is caused by a loss of expression of the fragile X mental retardation protein (FMRP). FMRP is a selective RNA-binding protein which forms a messenger ribonucleoprotein (mRNP) complex that associates with polyribosomes. Recently, mRNA ligands associated with FMRP have been identified. However, the mechanism by which FMRP regulates the translation of its mRNA ligands remains unclear. MicroRNAs are small noncoding RNAs involved in translational control. Here we show that in vivo mammalian FMRP interacts with microRNAs and the components of the microRNA pathways including Dicer and the mammalian ortholog of Argonaute 1 (AGO1). Using two different Drosophila melanogaster models, we show that AGO1 is critical for FMRP function in neural development and synaptogenesis. Our results suggest that FMRP may regulate neuronal translation via microRNAs and links microRNAs with human disease.

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

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

Trapping of messenger RNA by Fragile X Mental Retardation protein into cytoplasmic granules induces translation repression.

Absence of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein, is responsible for the Fragile X syndrome, the most common form of inherited mental retardation. FMRP is a cytoplasmic protein associated with mRNP complexes containing poly(A)+mRNA. As a step towards understanding FMRP function(s), we have established the immortal STEK Fmr1 KO cell line and showed by transfection assays with FMR1-expressing vectors that newly synthesized FMRP accumulates into cytoplasmic granules. These structures contain mRNAs and several other RNA-binding proteins. The formation of these cytoplasmic granules is dependent on determinants located in the RGG domain. We also provide evidence that FMRP acts as a translation repressor following co-transfection with reporter genes. The FMRP-containing mRNPs are dynamic structures that oscillate between polyribosomes and cytoplasmic granules reminiscent of the Stress Granules that contain repressed mRNAs. We speculate that, in neurons, FMRP plays a role as a mRNA repressor in incompetent mRNP granules that have to be translocated from the cell body to distal locations such as dendritic spines and synaptosomes.

Fragile X mental retardation protein in plasticity and disease.

Fragile X syndrome is the most common cause of mental retardation known to be inherited. The syndrome results from the suppressed expression of a single protein, the fragile X mental retardation protein (FMRP). Understanding the function and regulation of FMRP can, therefore, offer insights into both the pathophysiology of fragile X syndrome and the molecular mechanisms of learning and memory. We provide an overview of current concepts of how FMRP functions in the nervous system, with special emphasis on recent evidence that FMRP has a role in metabotropic glutamate receptor-activated protein translation and synaptic plasticity.

A nuclear role for the Fragile X mental retardation protein.

Fragile X syndrome results from lack of expression of a functional form of Fragile X mental retardation protein (FMRP), a cytoplasmic RNA-binding protein of uncertain function. Here, we report that FMRP contains a nuclear export signal (NES) that is similar to the NES recently identified in the Rev regulatory protein of human immunodeficiency virus type 1 (HIV-1). Mutation of this FMRP NES results in mis-localization of FMRP to the cell nucleus. The FMRP NES is encoded within exon 14 of the FMR1 gene, thus explaining the aberrant nuclear localization of a natural isoform of FMRP that lacks this exon. The NES of FMRP can substitute fully for the Rev NES in mediating Rev-dependent nuclear RNA export and specifically binds a nucleoporin-like cellular cofactor that has been shown to mediate Rev NES function. Together, these findings demonstrate that the normal function of FMRP involves entry into the nucleus followed by export via a pathway that is identical to the one utilized by HIV-1 Rev. In addition, these data raise the possibility that FMRP could play a role in mediating the nuclear export of its currently undefined cellular RNA target(s).

The ChickenFMR1Gene Is Highly Conserved with a CCT 5′-Untranslated Repeat and Encodes an RNA-Binding Protein

The transcriptional silencing of the human gene, fragile X mental retardation 1 (FMR1), is due to abnormal methylation in response to an expanded 5′-untranslated CGG trinucleotide repeat and accounts for most cases of fragile X syndrome, a frequent inherited form of mental retardation. Although the encoded fragile X mental retardation protein (FMRP) is known to have properties of a RNA-binding protein, the precise function of FMRP remains to be elucidated. We report the cloning of the chicken homolog ofFMR1and show strong evolutionary conservation, with nucleotide and amino acid identities of 85 and 92%, respectively, between chicken and human. In place of the mammalian CGG trinucleotide repeat, a 99-nt tripartite repetitive element containing a CCT trinucleotide repeat flanked on both sides by dinucleotide repeats was identified. Blocks of highly conserved 3′-untranslated sequence were also found. Within the coding region, two copies each of the highly conserved K homology motif and the Arg-Gly-Gly (RGG) box motif, both ribonucleotide particle family domains implicated in RNA binding, were identified. Chicken FMRP was found to bind RNAin vitro,and this activity correlated with the presence of the carboxy-terminal portion of the protein that includes the RGG motifs.