Advances in understanding of fragile X pathogenesis and FMRP function,and in identification of X linked mental retardation genes

The fragile X mental retardation syndrome is caused by large methylated expansions of a CGG repeat in the FMR1 gene that lead to the loss of expression of FMRP, an RNA-binding protein. FMRP is proposed to act as a regulator of mRNA transport or translation that plays a role in synaptic maturation and function. The recent observations of unexpected phenotypes in some carriers of fragile X premutations suggest a pathological role, in these individuals, of an abnormal FMR1 mRNA. FMRP was recently shown to interact preferentially with mRNAs containing a G quartet structure. Mouse and Drosophila models are used to decipher the function of FMRP, which was found to inhibit translation of some mRNA targets, but may be stimulatory in other cases. Proteins interacting with FMRP have been identified, and suggest a link with the Rac1 GTPase pathway that is important in neuronal maturation. Recent advances also include identification of other genes implicated in X-linked mental retardation.     

G-quartet-dependent recognition between the FMRP RGG box and RNA

Fragile-X syndrome, the most common monogenic form of mental retardation, is caused by down-regulation of the expression of Fragile X Mental Retardation Protein (FMRP). FMRP is a multifunctional, multidomain RNA-binding protein that acts as a translational repressor in neuronal cells. Interaction between FMRP and mRNA targets involves an RGG box, a protein motif commonly thought to mediate unspecific interactions with nucleic acids. Instead, FMRP RGG box has been shown to recognize RNA G-quartet structures specifically and to be necessary in neurons for RNP particle formation and dendritic mRNA localization. In the present study, we have characterized structurally three representative RNA targets of FMRP in their unbound form and in complex with the RGG box. We observe a large heterogeneity in the conformation of the RNA targets and in their RGG binding mode, which could be the basis of recognition specificity. We also found that G-quartet formation occurs not only intramolecularly but can also be mediated by RNA dimerization. These findings suggest a potential role of RNA:RNA interactions in protein:RNA complexes and in RNP particle assembly.     

A Novel Function for Fragile X Mental Retardation Protein in Translational Activation

Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in several steps of RNA metabolism. To date, two RNA motifs have been found to mediate FMRP/RNA interaction, the G-quartet and the “kissing complex,” which both induce translational repression in the presence of FMRP. We show here a new role for FMRP as a positive modulator of translation. FMRP specifically binds Superoxide Dismutase 1 (Sod1) mRNA with high affinity through a novel RNA motif, SoSLIP (Sod1 mRNA Stem Loops Interacting with FMRP), which is folded as three independent stem-loop structures. FMRP induces a structural modification of the SoSLIP motif upon its interaction with it. SoSLIP also behaves as a translational activator whose action is potentiated by the interaction with FMRP. The absence of FMRP results in decreased expression of Sod1. Because it has been observed that brain metabolism of FMR1 null mice is more sensitive to oxidative stress, we propose that the deregulation of Sod1 expression may be at the basis of several traits of the physiopathology of the Fragile X syndrome, such as anxiety, sleep troubles, and autism.     

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.     

Translational Regulation of NeuroD1 Expression by FMRP: Involvement in Glutamatergic Neuronal Differentiation of Cultured Rat Primary Neural Progenitor Cells

Fragile X mental retardation protein (FMRP) is encoded by Fmr1 gene in which mutation is known to cause fragile X syndrome characterized by mental impairment and other psychiatric symptoms similar to autism spectrum disorders. FMRP plays important roles in cellular mRNA biology such as transport, stability, and translation as an RNA-binding protein. In the present study, we identified potential role of FMRP in the neural differentiation, using cortical neural progenitor cells from Sprague–Dawley rat. We newly found NeuroD1, an essential regulator of glutamatergic neuronal differentiation, as a new mRNA target interacting with FMRP in co-immunoprecipitation experiments. We also identified FMRP as a regulator of neuronal differentiation by modulating NeuroD1 expression. Down-regulation of FMRP by siRNA also increased NeuroD1 expression along with increased pre- and post-synaptic development of glutamatergic neuron, as evidenced by Western blot and immunocytochemistry. On the contrary, cells harboring FMRP over-expression construct showed decreased NeuroD1 expression. Treatment of cultured neural precursor cells with a histone deacetylase inhibitor, valproic acid known as an inducer of hyper-glutamatergic neuronal differentiation, down-regulated the expression of FMRP, and induced NeuroD1 expression. Our study suggests that modulation of FMRP expression regulates neuronal differentiation by interaction with its binding target mRNA, and provides an example of the gene and environmental interaction regulating glutamatergic neuronal differentiation.     

Timing of the absence of <Emphasis Type="Italic">FMR1</Emphasis> expression in full mutation chorionic villi

Fragile X syndrome is caused by the expansion of the CGG repeat in the 5' untranslated region of the FMR1 gene. This expansion leads to methylation of the FMR1 promoter region thereby blocking FMR1 protein (FMRP) expression. Prenatal diagnosis can be performed on chorionic villi samples (CVS) by Southern blot analysis. Alternatively, for males, an immunohistochemical method has been introduced for CVS. In this study, we have used this immunocytochemical method for CVS in full mutation male fetuses at different times of gestational age, varying from 10.0-12.5 weeks, and in two cases of full mutation female fetuses (>13 weeks). FMRP expression studies in CVS from full mutation male fetuses (10.0-12.5 weeks) illustrate the timing of the disappearance of FMRP expression in these CVS. Until approximately 10 weeks of gestation, FMRP is expressed normally in full mutation male CVS, whereas FMRP is completely absent at 12.5 weeks of gestation. FMRP expression in full mutation female CVS (>13 weeks) is completely absent in a number of villi, whereas other villi show normal FMRP expression. Unlabelled villi can only be present in the absence of the expression of the full mutation FMR1 gene on one X-chromosome together with the X-inactivation of the normal X allele. FMRP positive villi can be explained by an active normal X allele. The presence of both positive and negative villi indicates that X-inactivation in human CVS is a random process. No villi are found with a mixture of both FMRP-expressing and non-FMRP-expressing cells. This indicates that X-inactivation occurs very early in development, before the villi start to proliferate, and that X-inactivation in villi is a clonal process. In addition, our results indicate that the timing of both X-inactivation and full mutation FMR1 allele inactivation is different, i.e. X-inactivation occurs earlier in development than inactivation of the full mutation.     

Cells Lacking the Fragile X Mental Retardation Protein (FMRP) have Normal RISC Activity but Exhibit Altered Stress Granule Assembly

The fragile X mental retardation protein (FMRP) is an RNA-binding protein involved in the mRNA metabolism. The absence of FMRP in neurons leads to alterations of the synaptic plasticity, probably as a result of translation regulation defects. The exact molecular mechanisms by which FMRP plays a role in translation regulation have remained elusive. The finding of an interaction between FMRP and the RNA interference silencing complex (RISC), a master of translation regulation, has suggested that both regulators could be functionally linked. We investigated here this link, and we show that FMRP exhibits little overlap both physically and functionally with the RISC machinery, excluding a direct impact of FMRP on RISC function. Our data indicate that FMRP and RISC are associated to distinct pools of mRNAs. FMRP, unlike RISC machinery, associates with the pool of mRNAs that eventually goes into stress granules upon cellular stress. Furthermore, we show that FMRP plays a positive role in this process as the lack of FMRP or a point mutant causing a severe fragile X alter stress granule formation. Our data support the proposal that FMRP plays a role in controlling the fate of mRNAs after translation arrest.     

The fragile X mental retardation protein interacts with U-rich RNAs in a yeast three-hybrid system

We recently identified several ESTs that bind to the fragile X mental retardation protein (FMRP) in vitro. To determine whether they interacted in vivo we performed three-hybrid screens in a Saccharomyces cerevisiae histidine auxotroph. We demonstrate that two of the ESTs support growth on histidine and transduce beta-galactosidase activity when co-expressed with FMRP under selective growth conditions. In contrast, the iron response element (IRE) RNA does not. Likewise, the ESTs do not support growth or transduce beta-galactosidase activity when co-expressed with the iron response element binding protein (IRP). Each EST is relatively small and has 40% identity with a sequence in FMR1 mRNA harboring FMRP binding determinants. Interestingly, while neither the ESTs contain a G-quartet structural motif they do contain U-rich sequences that are found in mRNA with demonstrated in vitro binding and in vivo association with FMRP. This indicates that U-rich elements comprise another motif recognized by FMRP.     

Dynamic association of the fragile X mental retardation protein as a messenger ribonucleoprotein between microtubules and polyribosomes

The fragile X mental retardation protein (FMRP) is a selective RNA-binding protein that regulates translation and plays essential roles in synaptic function. FMRP is bound to specific mRNA ligands, actively transported into neuronal processes in a microtubule-dependent manner, and associated with polyribosomes engaged in translation elongation. However, the biochemical relationship between FMRP-microtubule association and FMRP-polyribosome association remains elusive. Here, we report that although the majority of FMRP is incorporated into elongating polyribosomes in the soluble cytoplasm, microtubule-associated FMRP is predominantly retained in translationally dormant, polyribosome-free messenger ribonucleoprotein (mRNP) complexes. Interestingly, FMRP-microtubule association is increased when mRNPs are dynamically released from polyribosomes as a result of inhibiting translation initiation. Furthermore, the I304N mutant FMRP that fails to be incorporated into polyribosomes is associated with microtubules in mRNP particles and transported into neuronal dendrites in a microtubule-dependent, 3,5-dihydroxyphenylglycine-stimulated manner with similar kinetics to that of wild-type FMRP. Hence, polyribosome-free FMRP-mRNP complexes travel on microtubules and wait for activity-dependent translational derepression at the site of function. The dual participation of FMRP in dormant mRNPs and polyribosomes suggests distinct roles of FMRP in dendritic transport and translational regulation, two distinct phases that control local protein production to accommodate synaptic plasticity.     

The fragile X syndrome protein FMRP associates with BC1 RNA and regulates the translation of specific mRNAs at synapses

The Fragile X syndrome, which results from the absence of functional FMRP protein, is the most common heritable form of mental retardation. Here, we show that FMRP acts as a translational repressor of specific mRNAs at synapses. Interestingly, FMRP associates not only with these target mRNAs, but also with the dendritic, non-translatable RNA BC1. Blocking of BC1 inhibits the interaction of FMRP with its target mRNAs. Furthermore, BC1 binds directly to FMRP and can also associate, in the absence of any protein, with the mRNAs regulated by FMRP. This suggests a mechanism where BC1 could determine the specificity of FMRP function by linking the regulated mRNAs and FMRP. Thus, when FMRP is not present, loss of translational repression of specific mRNAs at synapses could result in synaptic dysfunction phenotype of Fragile X patients.