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.     

Identification of small molecules rescuing fragile X syndrome phenotypes in Drosophila

Fragile X syndrome is caused by the functional loss of the fragile X mental retardation 1 (FMR1) gene. Deletion of the FMR1 ortholog in Drosophila melanogaster (Fmr1) recapitulates many phenotypes associated with fragile X syndrome. We have discovered that Fmr1 mutant Drosophila die during development when reared on food containing increased levels of glutamate, which is consistent with the theory that FMR1 loss results in excess glutamate signaling. Using this lethal phenotype, we screened a chemical library of 2,000 compounds and identified nine molecules that rescued the lethality, including three that implicate the GABAergic inhibitory pathway. Indeed, GABA treatment rescued several known Fmr1 mutant phenotypes in flies, including mushroom bodies defects, excess Futsch translation and abnormal male courtship behavior. These data are consistent with GABAergic inhibition of the enhanced excitatory pathway in fragile X syndrome. In addition, our screen reveals that the muscarinic cholinergic receptors may have a role in fragile X syndrome in parallel to the GABAergic pathway. These results point to potential therapeutic approaches for treating fragile X syndrome.     

Drosophila fragile X mental retardation protein developmentally regulates activity-dependent axon pruning

Fragile X Syndrome (FraX) is a broad-spectrum neurological disorder with symptoms ranging from hyperexcitability to mental retardation and autism. Loss of the fragile X mental retardation 1 (fmr1) gene product, the mRNA-binding translational regulator FMRP, causes structural over-elaboration of dendritic and axonal processes, as well as functional alterations in synaptic plasticity at maturity. It is unclear, however, whether FraX is primarily a disease of development, a disease of plasticity or both: a distinction that is vital for engineering intervention strategies. To address this crucial issue, we have used the Drosophila FraX model to investigate the developmental function of Drosophila FMRP (dFMRP). dFMRP expression and regulation of chickadee/profilin coincides with a transient window of late brain development. During this time, dFMRP is positively regulated by sensory input activity, and is required to limit axon growth and for efficient activity-dependent pruning of axon branches in the Mushroom Body learning/memory center. These results demonstrate that dFMRP has a primary role in activity-dependent neural circuit refinement during late brain development.     

Distinct contributions of KH domains to substrate binding affinity of Drosophila P-element somatic inhibitor protein

Drosophila P-element somatic inhibitor protein (PSI) regulates splicing of the P-element transposase pre-mRNA by binding a pseudo-splice site upstream of the authentic splice site using four tandem KH-type RNA binding motifs. While the binding domains and specificity of PSI have been established, little is known about the contributions of each PSI KH domain to overall protein stability and RNA binding affinity. Using a construct containing only the RNA binding domain of PSI (PSI-KH03), we introduced a physiologically relevant point mutation into each KH domain of PSI individually and measured stability and RNA binding affinity of the resulting mutant proteins. Although secondary structure, as measured by circular dichroism spectroscopy, is only subtly changed for each mutant protein relative to wild type, RNA binding affinity is reduced in each case. Mutations in the second or third KH domains of the protein are significantly more deleterious to substrate recognition than mutation of the outer (first and fourth) domains. These results show that despite the ability of a single KH domain to bind RNA in some systems, PSI requires multiple tandem KH domains for specific and high-affinity recognition of substrate RNA.     

The Drosophila fragile X protein functions as a negative regulator in the orb autoregulatory pathway

Translational regulation of maternal mRNAs in distinct temporal and spatial patterns underlies many key decisions in developing eggs and embryos. In Drosophila, Orb is responsible for mediating the translational activation of mRNAs localized within the developing oocyte. Orb is a germline-specific RNA binding protein and is one of the founding members of the CPEB family of translational regulators. Here we show that Orb associates with the Drosophila Fragile X Mental Retardation (dFMR1) protein as part of a ribonucleoprotein complex that controls the localized translation of mRNAs in developing egg chambers. One of the key orb regulatory targets is orb mRNA, and this autoregulatory activity is critical for ensuring that Orb protein is expressed at high levels in the oocyte. We show that dFMR1 functions as a negative regulator in the orb autoregulatory circuit, downregulating orb mRNA translation.     

Drosophila fragile X protein,DFXR, regulates neuronal morphology and function in the brain

Mental retardation is a pervasive societal problem, 25 times more common than blindness for example. Fragile X syndrome, the most common form of inherited mental retardation, is caused by mutations in the FMR1 gene. Fragile X patients display neurite morphology defects in the brain, suggesting that this may be the basis of their mental retardation. Drosophila contains a single homolog of FMR1, dfxr (also called dfmr1). We analyzed the role of dfxr in neurite development in three distinct neuronal classes. We find that DFXR is required for normal neurite extension, guidance, and branching. dfxr mutants also display strong eclosion failure and circadian rhythm defects. Interestingly, distinct neuronal cell types show different phenotypes, suggesting that dfxr differentially regulates diverse targets in the brain.     

Circadian phenotypes of Drosophila fragile x mutants in alternative genetic backgrounds

Drosophila FMR1 mutants are models of human fragile X syndrome. They show a loss of locomotor activity rhythm and severe degradation of eclosion timing. We analyzed the circadian behavior of FMR1 mutants (dfmr1(B55)) in two genetic backgrounds, yellow white (yw) and Canton S (CS). The arrhythmic phenotype of circadian locomotor activity in constant darkness (DD) did not significantly change in either genetic background. Surprisingly, eclosion timing was completely restored by backcrossing dfmr1(B55) with yw or CS flies. Morphological analysis of the small ventrally located lateral neurons of FMR1 mutants revealed that the dorsal-projection area was significantly larger in arrhythmic than rhythmic flies. In addition, dfmr1(B55) mutants in both genetic backgrounds had a significantly lower evening peak in the light-dark (LD) cycle. These results indicate that lack of FMR1 does not affect eclosion timing, but alters locomotor activity patterns in both LD and DD conditions by affecting the arborization of small ventrally located lateral neurons. Thus, the FMR1 gene may regulate the circadian-related locomotor activity of Drosophila.     

Circadian rhythm-dependent alterations of gene expression in Drosophila brain lacking fragile X mental retardation protein

Fragile X syndrome is caused by the loss of the FMR1 gene product, fragile X mental retardation protein (FMRP). The loss of FMRP leads to altered circadian rhythm behaviors in both mouse and Drosophila; however, the molecular mechanism behind this phenomenon remains elusive. Here we performed a series of gene expression analyses, including of both mRNAs and microRNAs (miRNAs), and identified a number of mRNAs and miRNAs (miRNA-1 and miRNA-281) with circadian rhythm-dependent altered expression in dfmr1 mutant flies. Identification of these RNAs lays the foundation for future investigations of the molecular pathway(s) underlying the altered circadian rhythms associated with loss of dFmr1.     

Characterization of dFMR1, a Drosophila melanogaster homolog of the fragile X mental retardation protein

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by loss of FMR1 gene activity due to either lack of expression or expression of a mutant form of the protein. In mammals, FMR1 is a member of a small protein family that consists of FMR1, FXR1, and FXR2. All three members bind RNA and contain sequence motifs that are commonly found in RNA-binding proteins, including two KH domains and an RGG box. The FMR1/FXR proteins also contain a 60S ribosomal subunit interaction domain and a protein-protein interaction domain which mediates homomer and heteromer formation with each family member. Nevertheless, the specific molecular functions of FMR1/FXR proteins are unknown. Here we report the cloning and characterization of a Drosophila melanogaster homolog of the mammalian FMR1/FXR gene family. This first invertebrate homolog, termed dfmr1, has a high degree of amino acid sequence identity/similarity with the defined functional domains of the FMR1/FXR proteins. The dfmr1 product binds RNA and is similar in subcellular localization and embryonic expression pattern to the mammalian FMR1/FXR proteins. Overexpression of dfmr1 driven by the UAS-GAL4 system leads to apoptotic cell loss in all adult Drosophila tissues examined. This phenotype is dependent on the activity of the KH domains. The ability to induce a dominant phenotype by overexpressing dfmr1 opens the possibility of using genetic approaches in Drosophila to identify the pathways in which the FMR1/FXR proteins function.     

Joint morphology in the insect leg: evolutionary history inferred from Notch loss-of-function phenotypes in Drosophila

Joints permit efficient locomotion, especially among animals with a rigid skeleton. Joint morphologies vary in the body of individual animals, and the shapes of homologous joints often differ across species. The diverse locomotive behaviors of animals are based, in part, on the developmental and evolutionary history of joint morphogenesis. We showed previously that strictly coordinated cell-differentiation and cell-movement events within the epidermis sculpt the interlocking ball-and-socket joints in the adult Drosophila tarsus (distal leg). Here, we show that the tarsal joints of various insect species can be classified into three types: ball-and-socket, side-by-side and uniform. The last two probably result from joint formation without the cell-differentiation step, the cell-movement step, or both. Similar morphological variations were observed in Drosophila legs when Notch function was temporarily blocked during joint formation, implying that the independent acquisition of cell differentiation and cell movement underlay the elaboration of tarsal joint morphologies during insect evolution. These results provide a framework for understanding how the seemingly complex morphology of the interlocking joint could have developed during evolution by the addition of simple developmental modules: cell differentiation and cell movement.     

RNA targets of the fragile X protein.

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.     

The Drosophila fragile X mental retardation protein controls actin dynamics by directly regulating profilin in the brain

Loss of Fragile X mental retardation protein (FMRP) function causes the highly prevalent Fragile X syndrome [1 and 2]. Identifying targets for the RNA binding FMRP is a major challenge and an important goal of research into the pathology of the disease. Perturbations in neuronal development and circadian behavior are seen in Drosophila dfmr1 mutants. Here we show that regulation of the actin cytoskeleton is under dFMRP control. dFMRP binds the mRNA of the Drosophila profilin homolog and negatively regulates Profilin protein expression. An increase in Profilin mimics the phenotype of dfmr1 mutants. Conversely, decreasing Profilin levels suppresses dfmr1 phenotypes. These data place a new emphasis on actin misregulation as a major problem in fmr1 mutant neurons.     

An assay for social interaction in Drosophila fragile X mutants

We developed a novel assay to examine social interactions in Drosophila and, as a first attempt, apply it here at examining the behavior of Drosophila Fragile X Mental Retardation gene (dfmr1) mutants. Fragile X syndrome is the most common cause of single gene intellectual disability (ID) and is frequently associated with autism. Our results suggest that dfmr1 mutants are less active than wild-type flies and interact with each other less often. In addition, mutants for one allele of dfmr1, dfmr1^B55, are more likely to come in close contact with a wild-type fly than another dfmr1^B55 mutant. Our results raise the possibility of defective social expression with preserved receptive abilities. We further suggest that the assay may be applied in a general strategy of examining endophenoypes of complex human neurological disorders in Drosophila, and specifically in order to understand the genetic basis of social interaction defects linked with ID.     

An assay for social interaction in Drosophila fragile X mutants

We developed a novel assay to examine social interactions in Drosophila and, as a first attempt, apply it here at examining the behavior of Drosophila Fragile X Mental Retardation gene (dfmr1) mutants. Fragile X syndrome is the most common cause of single gene intellectual disability (ID) and is frequently associated with autism. Our results suggest that dfmr1 mutants are less active than wild-type flies and interact with each other less often. In addition, mutants for one allele of dfmr1, dfmr1^B55, are more likely to come in close contact with a wild-type fly than another dfmr1^B55 mutant. Our results raise the possibility of defective social expression with preserved receptive abilities. We further suggest that the assay may be applied in a general strategy of examining endophenoypes of complex human neurological disorders in Drosophila, and specifically in order to understand the genetic basis of social interaction defects linked with ID.Key words: Drosophila, Fragile X, autism, social behavior, novel assay     

Functional domains of the Drosophila bicaudal-D protein

The localization of oocyte-specific determinants in the form of mRNAs to the pro-oocyte is essential for the establishment of oocyte identity. Localization of the Bicaudal-D (Bic-D) protein to the presumptive oocyte is required for the accumulation of Bic-D and other mRNAs to the pro-oocyte. The Bic-D protein contains four well-defined heptad repeat domains characteristic of intermediate filament proteins, and several of the mutations in Bic-D map to these conserved domains. We have undertaken a structure-function analysis of Bic-D by testing the function of mutant Bic-D transgenes (Bic-D(H)) deleted for each of the heptad repeat domains in a Bic-D null background. Our transgenic studies indicate that only the C-terminal heptad repeat deletion results in a protein that has lost zygotic and ovarian functions. The three other deletions result in proteins with full zygotic function, but with affected ovarian function. The functional importance of each domain is well correlated with its conservation in evolution. The analysis of females heterozygous for Bic-D(H) and the existing alleles Bic-D(PA66) or Bic-D(R26) reveals that Bic-D(R26) as well as some of Bic-D(H) transgenes have antimorphic effects. The yeast two-hybrid interaction assay shows that Bic-D forms homodimers. Furthermore, we found that Bic-D exists as a multimeric protein complex consisting of Egl and at least two Bic-D monomers.     

Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains.

FMR1 is an RNA-binding protein that is either absent or mutated in patients affected by the fragile X syndrome, the most common inherited cause of mental retardation in humans. Sequence analysis of the FMR1 protein has suggested that RNA binding is related to the presence of two K-homologous (KH) modules and an RGG box. However, no attempt has been so far made to map the RNA-binding sites along the protein sequence and to identify possible differential RNA-sequence specificity. In the present article, we describe work done to dissect FMR1 into regions with structurally and functionally distinct properties. A semirational approach was followed to identify four regions: an N-terminal stretch of 200 amino acids, the two KH regions, and a C-terminal stretch. Each region was produced as a recombinant protein, purified, and probed for its state of folding by spectroscopical techniques. Circular dichroism and NMR spectra of the N-terminus show formation of secondary structure with a strong tendency to aggregate. Of the two homologous KH motifs, only the first one is folded whereas the second remains unfolded even when it is extended both N- and C-terminally. The C-terminus is, as expected from its amino acid composition, nonglobular. Binding assays were then performed using the 4-nt homopolymers. Our results show that only the first KH domain but not the second binds to RNA, and provide the first direct evidence for RNA binding of both the N-terminal and the C-terminal regions. RNA binding for the N-terminus could not be predicted from sequence analysis because no known RNA-binding motif is identifiable in this region. Different sequence specificity was observed for the fragments: both the N-terminus of the protein and KH1 bind preferentially to poly-(rG). The C-terminal region, which contains the RGG box, is nonspecific, as it recognizes the bases with comparable affinity. We therefore conclude that FMR1 is a protein with multiple sites of interaction with RNA: sequence specificity is most likely achieved by the whole block that comprises the first approximately 400 residues, whereas the C-terminus provides a nonspecific binding surface.     

Programming social behavior by the maternal fragile X protein

The developing fetus and neonate are highly sensitive to maternal environment. Besides the well‐documented effects of maternal stress, nutrition and infections, maternal mutations, by altering the fetal, perinatal and/or early postnatal environment, can impact the behavior of genetically normal offspring. Mutation/premutation in the X‐linked FMR1 (encoding the translational regulator FMRP) in females, although primarily responsible for causing fragile X syndrome (FXS) in their children, may also elicit such maternal effects. We showed that a deficit in maternal FMRP in mice results in hyperactivity in the genetically normal offspring. To test if maternal FMRP has a broader intergenerational effect, we measured social behavior, a core dimension of neurodevelopmental disorders, in offspring of FMRP‐deficient dams. We found that male offspring of Fmr1^+/? mothers, independent of their own Fmr1 genotype, exhibit increased approach and reduced avoidance toward conspecific strangers, reminiscent of ‘indiscriminate friendliness’ or the lack of stranger anxiety, diagnosed in neglected children and in patients with Asperger's and Williams syndrome. Furthermore, social interaction failed to activate mesolimbic/amygdala regions, encoding social aversion, in these mice, providing a neurobiological basis for the behavioral abnormality. This work identifies a novel role for FMRP that extends its function beyond the well‐established genetic function into intergenerational non‐genetic inheritance/programming of social behavior and the corresponding neuronal circuit. As FXS premutation and some psychiatric conditions that can be associated with reduced FMRP expression are more prevalent in mothers than full FMR1 mutation, our findings potentially broaden the significance of FMRP‐dependent programming of social behavior beyond the FXS population.