microRNA在脆性X综合征发病机制中的研究进展

脆性X综合征(fragile X syndrome,FXS)是最常见的遗传性认知障碍疾病,也是一种与自闭症谱系障碍(autism spectrum disorder,ASD)相关的严重的基因疾病.它主要是由于脆性X智力低下基因1(fragile X mental retardation 1,FMR1)的异常扩增及其上游Cp G岛的异常甲基化,导致其编码的脆性X智力低下蛋白(fragile X mental retardation protein,FMRP)表达减少或缺失引起的.FMRP与miRNA(micro RNA)均具有翻译抑制活性,而且FMRP在生物化学和遗传学上均与miRNA调控通路有相互作用.此外,越来越多的研究发现miRNA调控通路在FXS的发病和治疗中发挥作用.因此,本文对miRNA的功能及其与脆性X蛋白家族成员间的相互作用进行阐述,为在miRNA水平了解FXS的发病机制奠定基础.     

脆性X相关蛋白Ⅰ的研究进展

脆性X相关基因I(FXR1)发现于1995年,位于3号染色体3q28。其产物脆性X相关蛋白1(FXR1P)与脆性X智障蛋白1 (FMR1P)、脆性X相关蛋白2(FXR2P)形成一个RNA结合蛋白家族。目前认为这三种蛋白能输送mRNA分子并调控其翻译过程。FXR1的翻译产物(FXR1P)分子中存在两个KH结构域和一个RGG结构域,这两种结构域与FXR1P分子的RNA结合作用有关。FXR1P的表达具有较高的组织特意性,在横纹肌中表达最高。合适的动物模型对于一种蛋白的功能研究具有十分重要的意义,目前,FXR1敲除的各种动物模型如小鼠、斑马鱼、非洲爪蟾的近亲Xenopus tropicalis已经在不同的实验室建立。本文主要介绍了FXR1P的结构特点、功能及其实验动物模型。     

FXR1, an autosomal homolog of the fragile X mental retardation gene.

Fragile X mental retardation syndrome, the most common cause of hereditary mental retardation, is directly associated with the FMR1 gene at Xq27.3. FMR1 encodes an RNA binding protein and the syndrome results from lack of expression of FMR1 or expression of a mutant protein that is impaired in RNA binding. We found a novel gene, FXR1, that is highly homologous to FMR1 and located on chromosome 12 at 12q13. FXR1 encodes a protein which, like FMR1, contains two KH domains and is highly conserved in vertebrates. The 3' untranslated regions (3'UTRs) of the human and Xenopus laevis FXR1 mRNAs are strikingly conserved (approximately 90% identity), suggesting conservation of an important function. The KH domains of FXR1 and FMR1 are almost identical, and the two proteins have similar RNA binding properties in vitro. However, FXR1 and FMR1 have very different carboxy-termini. FXR1 and FMR1 are expressed in many tissues, and both proteins, which are cytoplasmic, can be expressed in the same cells. Interestingly, cells from a fragile X patient that do not have any detectable FMR1 express normal levels of FXR1. These findings demonstrate that FMR1 and FXR1 are members of a gene family and suggest a biological role for FXR1 that is related to that of FMR1.     

Fragile X protein family member FXR1P is regulated by microRNAs

FXR1P is one of two autosomal paralogs of the fragile X mental retardation protein FMRP. The absence of FMRP causes fragile X syndrome, the leading cause of hereditary mental retardation. FXR1P plays an important role in normal muscle development and has been implicated in facioscapulohumeral muscular dystrophy (FSHD). Its absence also causes cardiac abnormalities in both mice and zebrafish. To examine miRNA-mediated regulation of FMRP and FXR1P, we studied their expression in a conditional Dicer knockdown cell line, DT40. We found that FXR1P, but not FMRP, is significantly increased upon Dicer knockdown and the consequent reduction of miRNAs, suggesting that FXR1P is regulated by miRNAs while FMRP is not in DT40 cells. Expression of a luciferase reporter bearing the 3′ untranslated region (3′UTR) of FXR1 was significantly increased in the absence of miRNAs, confirming miRNA-mediated regulation of FXR1P, while a luciferase reporter bearing the FMR1 3′UTR was not. We identified one of the regulatory regions in the 3′UTR of FXR1 by removing a conserved, 8-nucleotide miRNA seed sequence common to miRNAs 25, 32, 92, 363, and 367 and demonstrated loss of miRNA-mediated suppression. Treatment with specific miRNA hairpin inhibitors to each of the miRNAs in the seed sequence showed that miRs 92b, 363, and 367 regulated FXR1P expression. Accordingly, overexpression of the miRNA 367 mimic significantly decreased endogenous FXR1P expression in human cell lines HEK-293T and HeLa. We report for the first time that FXR1P is regulated through miRNA binding, with one site being the miR-25/32/92/363/367 seed sequence.     

On the aggregation properties of FMRP--a link with the FXTAS syndrome?

Fragile X mental retardation protein (FMRP) is an RNA binding protein necessary for correct spatiotemporal control of neuronal gene expression in humans. Lack of functional FMRP causes fragile X mental retardation, which is the most common inherited neurodevelopmental disorder in humans. In a previous study, we described the biochemical and biophysical aggregation properties of constructs spanning the conserved region of FMRP and of two other human fragile X related (FXR) proteins, FXR1P and FXR2P. Here, we show that the same regions have an intrinsic tendency to aggregate and spontaneously misfold towards β-rich structures, also under non-destabilizing conditions. These findings pave the way to future studies of the mechanism of formation of FXR-containing ribonucleoprotein granules and suggest a possible link with the as yet poorly understood FXR proteins' associated pathologies.     

Skeletal muscle proteome expression differentiates severity of cancer cachexia in mice and identifies loss of fragile X mental retardation syndrome-related protein 1

Tandem mass tag (TMT)-based quantitative proteomics was used to examine protein expression in skeletal muscle from mice with moderate and severe cancer cachexia to study mechanisms underlying varied cachexia severity. Weight loss of 10% (moderate) and 20% (severe) was induced by injection of colon-26 cancer cells in 10-week old Balb/c mice. In moderate cachexia, enriched pathways reflected fibrin formation, integrin/mitogen-activated protein kinase (MAPK) signaling, and innate immune system, suggesting an acute phase response and fibrosis. These pathways remained enriched in severe cachexia; however, energy-yielding pathways housed in mitochondria were prominent additions to the severe state. These enrichments suggest distinct muscle proteome expression patterns that differentiate cachexia severity. When analyzed with two other mouse models, eight differentially expressed targets were shared including serine protease inhibitor A3N (Serpina3n), synaptophysin-like protein 2 (Sypl2), Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial (Idh3a), peroxisomal acyl-coenzyme A oxidase 1 (Acox1), collagen alpha-1(VI) chain (Col6a1), myozenin 3 (Myoz3), UDP-glucose pyrophosphorylase (Ugp2), and solute carrier family 41 member 3 (Slc41a3). Acox1 and Idh3a control lipid oxidation and NADH generation in the TCA cycle, respectively, and Col6a1 comprises part of type VI collagen with reported profibrotic functions, suggesting influential roles in cachexia. A potential target was identified in fragile X mental retardation syndrome-related protein 1 (FXR1), an RNA-binding protein not previously implicated in cancer cachexia. FXR1 decreased in cachexia and related linearly with weight change and myofiber size. These findings suggest distinct mechanisms associated with cachexia severity and potential biomarkers and therapeutic targets.     

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.     

The fragile X syndrome: exploring its molecular basis and seeking a treatment

Fragile X syndrome (FXS) - the leading cause of inherited mental retardation - is an X-linked disease caused by loss of expression of the FMR1 (fragile X mental retardation 1) gene. In addition to impairment of higher-cognitive functions, FXS patients show a variety of physical and other mental abnormalities. FMRP, the protein encoded by the FMR1 gene, is thought to play a key role in translation, trafficking and targeting of mRNA in neurons. To better understand FMRP's functions, the protein partners and mRNA targets that interact with FMRP have been sought. These and functional studies have revealed links with processes such as cytoskeleton remodelling via the RhoGTPase pathway and mRNA processing via the RNA interference pathway. In this review, we focus on recent insights into the function of FMRP and speculate on how the absence of FMRP might cause the clinical phenotypes seen in FXS patients. Finally, we explore potential therapies for FXS.     

Specific sequences in the fragile X syndrome protein FMR1 and the FXR proteins mediate their binding to 60S ribosomal subunits and the interactions among them.

Fragile X syndrome, the most common form of hereditary mental retardation, usually results from lack of expression of the FMR1 gene. The FMR1 protein is a cytoplasmic RNA-binding protein. The RNA-binding activity of FMR1 is an essential feature of FMR1, as fragile X syndrome can also result from the expression of mutant FMR1 protein that is impaired in RNA binding. Recently, we described two novel cytoplasmic proteins, FXR1 and FXR2, which are both very similar in amino acid sequence to FMR1 and which also interact strongly with FMR1 and with each other. To understand the function of FMR1 and the FXR proteins, we carried out cell fractionation and sedimentation experiments with monoclonal antibodies to these proteins to characterize the complexes they form. Here, we report that the FMR1 and FXR proteins are associated with ribosomes, predominantly with 60S large ribosomal subunits. The FXR proteins are associated with 60S ribosomal subunits even in cells that lack FMR1 and that are derived from a fragile X syndrome patient, indicating that FMR1 is not required for this association. We delineated the regions of FMR1 that mediate its binding to 60S ribosomal subunits and the interactions among the FMR1-FXR family members. Both regions contain sequences predicted to have a high propensity to form coiled coil interactions, and the sequences are highly evolutionarily conserved in this protein family. The association of the FMR1, FXR1, and FXR2 proteins with ribosomes suggests they have functions in translation or mRNA stability.     

FMRP蛋白6种异构体与FXR1蛋白间的相互作用

脆性Ｘ综合征是最常见的遗传性智力低下疾病,其致病基因ＦＭＲ１存在复杂的选择剪接．ＦＭＲ１基因的功能及其选择剪接的生物学意义尚未阐明．ＦＭＲ１蛋白（ＦＭＲＰ）与脆性Ｘ相关蛋白１（ＦＸＲ１）可形成异源二聚体．采用酵母双杂交体系研究了由ＦＭＲ１第１２、１４、１５外显子不同选择剪接方式产生的６种ＦＭＲＰ异构体与ＦＸＲ１蛋白的相互作用,以期从蛋白质相互作用的角度探讨ＦＭＲ１基因选择剪接表达的生物学意义．结果表明各种异构体与ＦＸＲ１相互作用的强度随异构体蛋白肽链长度的增长而减弱．外显子１２、１４、１５的选择剪接虽然不能开关式控制ＦＭＲＰ与ＦＸＲ１的相互作用,但其Ｃ端亲水区在一定程度上影响相互作用的强弱．提示选择剪接对ＦＭＲＰ与ＦＸＲ１异源二聚体的稳定性产生影响．     

Cellular stress-induced up-regulation of FMRP promotes cell survival by modulating PI3K-Akt phosphorylation cascades

Background  

Fragile X syndrome (FXS), the most commonly inherited mental retardation and single gene cause of autistic spectrum disorder, occurs when the Fmr1 gene is mutated. The product of Fmr1, fragile X linked mental retardation protein (FMRP) is widely expressed in HeLa cells, however the roles of FMRP within HeLa cells were not elucidated, yet. Interacting with a diverse range of mRNAs related to cellular survival regulatory signals, understanding the functions of FMRP in cellular context would provide better insights into the role of this interesting protein in FXS. Using HeLa cells treated with etoposide as a model, we tried to determine whether FMRP could play a role in cell survival.     

Altered mTOR signaling and enhanced CYFIP2 expression levels in subjects with fragile X syndrome

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and autism. The protein (FMRP) encoded by the fragile X mental retardation gene (FMR1), is an RNA-binding protein linked to translational control. Recently, in the Fmr1 knockout mouse model of FXS, dysregulated translation initiation signaling was observed. To investigate whether an altered signaling was also a feature of subjects with FXS compared to typical developing controls, we isolated total RNA and translational control proteins from lymphocytes of subjects from both groups (38 FXS and 14 TD). Although we did not observe any difference in the expression level of messenger RNAs (mRNAs) for translational initiation control proteins isolated from participant with FXS, we found increased phosphorylation of the mammalian target of rapamycin (mTOR) substrate, p70 ribosomal subunit 6 kinase1 (S6K1) and of the mTOR regulator, the serine/threonine protein kinase (Akt), in their protein lysates. In addition, we observed increased phosphorylation of the cap binding protein eukaryotic initiation factor 4E (eIF4E) suggesting that protein synthesis is upregulated in FXS. Similar to the findings in lymphocytes, we observed increased phosphorylation of S6K1 in brain tissue from patients with FXS (n = 4) compared to normal age-matched controls (n = 4). Finally, we detected increased expression of the cytoplasmic FMR1-interacting protein 2 (CYFIP2), a known FMRP interactor. This data verify and extend previous findings using lymphocytes for studies of neuropsychiatric disorders and provide evidence that misregulation of mTOR signaling observed in the FXS mouse model also occurs in human FXS and may provide useful biomarkers for designing targeted treatments in FXS.     

Prevalence of fragile X syndrome among patients with mental retardation in the west of Iran

Background

Fragile X syndrome (FXS), an X-linked disorder, is the most common cause of inherited mental retardation. This is caused by a trinucleotide CGG repeat expansion (>200) on the fragile X mental retardation 1 gene (FMR1) becoming methylated leading to a deficiency or absence of the FMR1 protein. Determining FXS prevalence in the mentally retarded individuals in the west of Iran was the aim of this study.

Methods

200 patients with moderate mental retardation who were clinically suspicious to FXS were screened using cytogenetic and molecular methods. Blood samples were collected and cultured in the specific culture media. The G-Banding method was used for karyotyping and DNA sequencing performed for verifying the results of the cytogenetic tests.

Results

16 patients (8%) were found to have fragile X syndrome. The results showed that there is no significant association between the fragile X syndrome and economic status and place of residence, however, the relationship between fragile X syndrome and mental retardation in the family history is significant.

Conclusion

The frequency of FXS was similar to other reports in the preselected patients. For diagnosis of FXS, chromosome analysis must be accompanied by molecular studies.

     

From FMRP Function to Potential Therapies for Fragile X Syndrome

Fragile X syndrome (FXS) is caused by mutations in the fragile X mental retardation 1 (FMR1) gene. Most FXS cases occur due to the expansion of the CGG trinucleotide repeats in the 5′ un-translated region of FMR1, which leads to hypermethylation and in turn silences the expression of FMRP (fragile X mental retardation protein). Numerous studies have demonstrated that FMRP interacts with both coding and non-coding RNAs and represses protein synthesis at dendritic and synaptic locations. In the absence of FMRP, the basal protein translation is enhanced and not responsive to neuronal stimulation. The altered protein translation may contribute to functional abnormalities in certain aspects of synaptic plasticity and intracellular signaling triggered by Gq-coupled receptors. This review focuses on the current understanding of FMRP function and potential therapeutic strategies that are mainly based on the manipulation of FMRP targets and knowledge gained from FXS pathophysiology.     

Comprehensive Analysis of Ultrasonic Vocalizations in a Mouse Model of Fragile X Syndrome Reveals Limited, Call Type Specific Deficits

Fragile X syndrome (FXS) is a well-recognized form of inherited mental retardation, caused by a mutation in the fragile X mental retardation 1 (Fmr1) gene. The gene is located on the long arm of the X chromosome and encodes fragile X mental retardation protein (FMRP). Absence of FMRP in fragile X patients as well as in Fmr1 knockout (KO) mice results, among other changes, in abnormal dendritic spine formation and altered synaptic plasticity in the neocortex and hippocampus. Clinical features of FXS include cognitive impairment, anxiety, abnormal social interaction, mental retardation, motor coordination and speech articulation deficits. Mouse pups generate ultrasonic vocalizations (USVs) when isolated from their mothers. Whether those social ultrasonic vocalizations are deficient in mouse models of FXS is unknown. Here we compared isolation-induced USVs generated by pups of Fmr1-KO mice with those of their wild type (WT) littermates. Though the total number of calls was not significantly different between genotypes, a detailed analysis of 10 different categories of calls revealed that loss of Fmr1 expression in mice causes limited and call-type specific deficits in ultrasonic vocalization: the carrier frequency of flat calls was higher, the percentage of downward calls was lower and that the frequency range of complex calls was wider in Fmr1-KO mice compared to their WT littermates.