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.     

Fragile X mental retardation protein interactions with the microtubule associated protein 1B RNA

Fragile X mental retardation syndrome, the most common form of inherited mental retardation, is caused by the absence of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine (RGG) box to bind to a subset of RNA targets that form a G quadruplex structure. We performed a detailed analysis of the interactions between the FMRP RGG box and the microtubule associated protein 1B (MAP1B) mRNA, a relevant in vivo FMRP target. We show that MAP1B RNA forms an intramolecular G quadruplex structure, which is bound with high affinity and specificity by the FMRP RGG box. We determined that hydrophobic interactions are important in the FMRP RGG box-MAP1B RNA association, with minor contributions from electrostatic interactions. Our findings that at low protein:RNA ratios the RNA G quadruplex structure is slightly stabilized, whereas at high ratios is unfolded, suggest a mechanism by which the FMRP concentration variation in response to a neurotransmitter stimulation event could act as a regulatory switch for the protein function, from translation repressor to translation activator.     

脆性X智力低下蛋白参与非编码RNA通路的研究进展

脆性X综合征(Fragile X syndrome)是一种最常见的遗传性智力低下疾病,并且伴有语言和行为障碍等。该疾病是由脆性X智力低下基因(Fragile X mental retardation 1, FMR1)突变而导致脆性X智力低下蛋白(Fragile X mental retardation protein, FMRP)表达异常造成的。近年来,研究发现FMRP参与非编码RNA通路,并发挥多种重要生物学功能,这对理解脆性X综合征发病机理具有重要的推动作用。首先发现FMRP与siRNA和miRNA通路中Dicer酶、Ago1和Ago2蛋白相互作用,参与神经活动及生殖干细胞命运决定等重要过程。随后又发现FMRP与piRNA通路中Aub、Ago1和Piwi蛋白相互作用,维持了染色体正常结构和基因组稳定性。最新研究结果发现FMRP与lncRNA相互作用,其功能和价值正引起关注。本文从FMRP与非编码RNA通路的关系展开,着重介绍了FMRP与piRNA之间的相互作用,以期为深入理解非编码RNA通路在脆性X综合征的发病过程中作用提供参考,同时期望与临床医学领域尽快形成交叉研究,早日促进理论成果转化为临床应用。     

Methylation of the arginine-glycine-rich region in the fragile X mental retardation protein FMRP differentially affects RNA binding

The C-terminal end of the fragile X mental retardation protein contains a stretch of amino acid residues that are enriched in arginine and glycine. Recent studies using recombinant FMRPs have demonstrated that this region participates in RNA binding in vitro, with calculated Kds ranging from 1-10 nM depending on the RNA. It is known that other arginine-glycine-rich proteins are subject to site-specific methylation by protein arginine methyltransferases (PRMTs) that are particularly abundant in most cells. We have demonstrated that the interaction of homoribopolymer mimetic RNAs with human FMRP (hFMRP) made in PRMT-containing cell-free lysates is more sensitive to increasing salt concentrations than recombinant hFMRP expressed in bacteria. We have also shown that blocking methylation with adenosine-2', 3'-dialdehyde (AdOx) alters homoribopolymer binding and hFMRP target mRNA binding; both increases and decreases are observed as a function of methylation. These data suggest that changes in PRMT activity that occur during development, or arise via signal transduction may be a means of regulating the binding of hFMRP to mRNA in vivo.     

RNA and microRNAs in fragile X mental retardation

Fragile X syndrome is caused by the loss of an RNA-binding protein called FMRP (for fragile X mental retardation protein). FMRP seems to influence synaptic plasticity through its role in mRNA transport and translational regulation. Recent advances include the identification of mRNA ligands, FMRP-mediated mRNA transport and the neuronal consequence of FMRP deficiency. FMRP was also recently linked to the microRNA pathway. These advances provide mechanistic insight into this disorder, and into learning and memory in general.     

Regulatory BC1 RNA and the fragile X mental retardation protein: convergent functionality in brain

Background

BC RNAs and the fragile X mental retardation protein (FMRP) are translational repressors that have been implicated in the control of local protein synthesis at the synapse. Work with BC1 and Fmr1 animal models has revealed that phenotypical consequences resulting from the absence of either BC1 RNA or FMRP are remarkably similar. To establish functional interactions between BC1 RNA and FMRP is important for our understanding of how local protein synthesis regulates neuronal excitability.

Methodology/Principal Findings

We generated BC1−/− Fmr1−/− double knockout (dKO) mice. We examined such animals, lacking both BC1 RNA and FMRP, in comparison with single knockout (sKO) animals lacking either one repressor. Analysis of neural phenotypical output revealed that at least three attributes of brain functionality are subject to control by both BC1 RNA and FMRP: neuronal network excitability, epileptogenesis, and place learning. The severity of CA3 pyramidal cell hyperexcitability was significantly higher in BC1−/− Fmr1−/− dKO preparations than in the respective sKO preparations, as was seizure susceptibility of BC1−/− Fmr1−/− dKO animals in response to auditory stimulation. In place learning, BC1−/− Fmr1−/− dKO animals were severely impaired, in contrast to BC1−/− or Fmr1−/− sKO animals which exhibited only mild deficits.

Conclusions/Significance

Our data indicate that BC1 RNA and FMRP operate in sequential-independent fashion. They suggest that the molecular interplay between two translational repressors directly impacts brain functionality.     

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).     

Fragile X mental retardation protein in learning-related synaptic plasticity

Fragile X syndrome (FXS) is caused by a lack of the fragile X mental retardation protein (FMRP) due to silencing of the Fmr1 gene. As an RNA binding protein, FMRP is thought to contribute to synaptic plasticity by regulating plasticity-related protein synthesis and other signaling pathways. Previous studies have mostly focused on the roles of FMRP within the hippocampus - a key structure for spatial memory. However, recent studies indicate that FMRP may have a more general contribution to brain functions, including synaptic plasticity and modulation within the prefrontal cortex. In this brief review, we will focus on recent studies reported in the prefrontal cortex, including the anterior cingulate cortex (ACC). We hypothesize that alterations in ACC-related plasticity and synaptic modulation may contribute to various forms of cognitive deficits associated with FXS.     

Temporal requirements of the fragile X mental retardation protein in the regulation of synaptic structure

Fragile X syndrome (FraX), caused by the loss-of-function of one gene (FMR1), is the most common inherited form of both mental retardation and autism spectrum disorders. The FMR1 product (FMRP) is an mRNA-binding translation regulator that mediates activity-dependent control of synaptic structure and function. To develop any FraX intervention strategy, it is essential to define when and where FMRP loss causes the manifestation of synaptic defects, and whether the reintroduction of FMRP can restore normal synapse properties. In the Drosophila FraX model, dFMRP loss causes neuromuscular junction (NMJ) synapse over-elaboration (overgrowth, overbranching, excess synaptic boutons), accumulation of development-arrested satellite boutons, and altered neurotransmission. We used the Gene-Switch method to conditionally drive dFMRP expression to define the spatiotemporal requirements in synaptic mechanisms. Constitutive induction of targeted neuronal dFMRP at wild-type levels rescues all synaptic architectural defects in Drosophila Fmr1 (dfmr1)-null mutants, demonstrating a presynaptic requirement for synapse structuring. By contrast, presynaptic dFMRP expression does not ameliorate functional neurotransmission defects, indicating a postsynaptic dFMRP requirement. Strikingly, targeted early induction of dFMRP effects nearly complete rescue of synaptic structure defects, showing a primarily early-development role. In addition, acute dFMRP expression at maturity partially alleviates dfmr1-null defects, although rescue is not as complete as either early or constitutive dFMRP expression, showing a modest capacity for late-stage structural plasticity. We conclude that dFMRP predominantly acts early in synaptogenesis to modulate architecture, but that late dFMRP introduction at maturity can weakly compensate for early absence of dFMRP function.     

Biophysical characterization of G-quadruplex forming FMR1 mRNA and of its interactions with different fragile X mental retardation protein isoforms

Fragile X syndrome, the most common form of inherited mental impairment in humans, is caused by the absence of the fragile X mental retardation protein (FMRP) due to a CGG trinucleotide repeat expansion in the 5′-untranslated region (UTR) and subsequent translational silencing of the fragile x mental retardation-1 (FMR1) gene. FMRP, which is proposed to be involved in the translational regulation of specific neuronal messenger RNA (mRNA) targets, contains an arginine-glycine-glycine (RGG) box RNA binding domain that has been shown to bind with high affinity to G-quadruplex forming mRNA structures. FMRP undergoes alternative splicing, and the binding of FMRP to a proposed G-quadruplex structure in the coding region of its mRNA (named FBS) has been proposed to affect the mRNA splicing events at exon 15. In this study, we used biophysical methods to directly demonstrate the folding of FMR1 FBS into a secondary structure that contains two specific G-quadruplexes and analyze its interactions with several FMRP isoforms. Our results show that minor splice isoforms, ISO2 and ISO3, created by the usage of the second and third acceptor sites at exon 15, bind with higher affinity to FBS than FMRP ISO1, which is created by the usage of the first acceptor site. FMRP ISO2 and ISO3 cannot undergo phosphorylation, an FMRP post-translational modification shown to modulate the protein translation regulation. Thus, their expression has to be tightly regulated, and this might be accomplished by a feedback mechanism involving the FMRP interactions with the G-quadruplex structures formed within FMR1 mRNA.     

Biology of the fragile X mental retardation protein, an RNA-binding protein.

The fragile X syndrome, an X-linked disease, is the most frequent cause of inherited mental retardation. The syndrome results from the absence of expression of the FMR1 gene (fragile mental retardation 1) owing to the expansion of a CGG trinucleotide repeat located in the 5' untranslated region of the gene and the subsequent methylation of its CpG island. The FMR1 gene product (FMRP) is a cytoplasmic protein that contains two KH domains and one RGG box, characteristics of RNA-binding proteins. FMRP is associated with mRNP complexes containing poly(A)+mRNA within actively translating polyribosomes and contains nuclear localization and export signals making it a putative transporter (chaperone) of mRNA from the nucleus to the cytoplasm. FMRP is the archetype of a novel family of cytoplasmic RNA-binding proteins that includes FXR1P and FXR2P. Both of these proteins are very similar in overall structure to FMRP and are also associated with cytoplasmic mRNPs. Members of the FMR family are widely expressed in mouse and human tissues, albeit at various levels, and seem to play a subtle choreography of expression. FMRP is most abundant in neurons and is absent in muscle. FXR1P is strongly expressed in muscle and low levels are detected in neurons. The complex expression patterns of the FMR1 gene family in different cells and tissues suggest that independent, however similar, functions for each of the three FMR-related proteins might be expected in the selection and metabolism of tissue-specific classes of mRNA. The molecular mechanisms altered in cells lacking FMRP still remain to be elucidated as well as the putative role(s) of FXR1P and FXR2P as compensatory molecules.     

Analysis of the Fragile X mental retardation protein isoforms 1, 2 and 3 interactions with the G-quadruplex forming semaphorin 3F mRNA

Fragile X syndrome, the most prevalent inheritable mental retardation, is caused by the loss of fragile X mental retardation protein (FMRP) expression. FMRP is an RNA-binding protein with nucleo-cytoplasmic shuttle activity, proposed to act as a translation regulator of specific mRNAs in the brain. It has been shown that FMRP uses its arginine-glycine-glycine (RGG) box domain to bind a subset of mRNA targets that form a G-quadruplex structure. FMRP has also been shown to undergo the post-translational modifications of arginine methylation and phosphorylation, as well as alternative splicing, resulting in multiple isoforms. The alternative splice isoforms investigated in this study, isoform 1 (ISO1), isoform 2 (ISO2), and isoform 3 (ISO3), are created by the alternative splicing acceptor site at exon 15. FMRP ISO2 and ISO3 are truncated by 12 and 13 residues, respectively, relative to the longest FMRP isoform ISO1. These truncations, which are in the close proximity of the RGG box domain, preserve the integrity of the RGG box in all three isoforms, but eliminate the in vivo phosphorylation sites, present only on FMRP ISO1. We have expressed and purified recombinant FMRP ISO1, ISO2 and ISO3 in Escherichia coli, free of post-translational modifications, and by using fluorescence spectroscopy, we show that each FMRP isoform binds G-quadruplex RNA, albeit with different binding affinities, suggesting that naturally occurring sequence modifications in the proximity of the RGG box modulate its G-quadruplex RNA binding ability.     

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.     

The structure of the N-terminal domain of the fragile X mental retardation protein: a platform for protein-protein interaction

FMRP, whose lack of expression causes the X-linked fragile X syndrome, is a modular RNA binding protein thought to be involved in posttranslational regulation. We have solved the structure in solution of the N-terminal domain of FMRP (NDF), a functionally important region involved in multiple interactions. The structure consists of a composite fold comprising two repeats of a Tudor motif followed by a short alpha helix. The interactions between the three structural elements are essential for the stability of the NDF fold. Although structurally similar, the two repeats have different dynamic and functional properties. The second, more flexible repeat is responsible for interacting both with methylated lysine and with 82-FIP, one of the FMRP nuclear partners. NDF contains a 3D nucleolar localization signal, since destabilization of its fold leads to altered nucleolar localization of FMRP. We suggest that the NDF composite fold determines an allosteric mechanism that regulates the FMRP functions.