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.     

The fragile X-related gene affects the crawling behavior of Drosophila larvae by regulating the mRNA level of the DEG/ENaC protein pickpocket1

BACKGROUND: Fragile X syndrome is caused by loss-of-function mutations in the fragile X mental retardation 1 (FMR1) gene. How FMR1 affects the function of the central and peripheral nervous systems is still unclear. FMR1 is an RNA binding protein that associates with a small percentage of total mRNAs in vivo. It remains largely unknown what proteins encoded by mRNAs in the FMR1-messenger ribonuclear protein (mRNP) complex are most relevant to the affected physiological processes. RESULTS: Loss-of-function mutations in the Drosophila fragile X-related (dfmr1) gene, which is highly homologous to the human fmr1 gene, decrease the duration and percentage of time that crawling larvae spend on linear locomotion. Overexpression of DFMR1 in multiple dendritic (MD) sensory neurons increases the time percentage and duration of linear locomotion; this phenotype is similar to that caused by reduced expression of the MD neuron subtype-specific degenerin/epithelial sodium channel (DEG/ENaC) family protein Pickpocket1 (PPK1). Genetic analyses indicate that PPK1 is a key component downstream of DFMR1 in controlling the crawling behavior of Drosophila larvae. DFMR1 and ppk1 mRNA are present in the same mRNP complex in vivo and can directly bind to each other in vitro. DFMR1 downregulates the level of ppk1 mRNA in vivo, and this regulatory process also involves Argonaute2 (Ago2), a key component in the RNA interference pathway. CONCLUSIONS: These studies identify ppk1 mRNA as a physiologically relevant in vivo target of DFMR1. Our finding that the level of ppk1 mRNA is regulated by DFMR1 and Ago2 reveals a genetic pathway that controls sensory input-modulated locomotion behavior.     

Spatial code recognition in neuronal RNA targeting: role of RNA-hnRNP A2 interactions

In neurons, regulation of gene expression occurs in part through translational control at the synapse. A fundamental requirement for such local control is the targeted delivery of select neuronal mRNAs and regulatory RNAs to distal dendritic sites. The nature of spatial RNA destination codes, and the mechanism by which they are interpreted for dendritic delivery, remain poorly understood. We find here that in a key dendritic RNA transport pathway (exemplified by BC1 RNA, a dendritic regulatory RNA, and protein kinase M ζ [PKMζ] mRNA, a dendritic mRNA), noncanonical purine•purine nucleotide interactions are functional determinants of RNA targeting motifs. These motifs are specifically recognized by heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2), a trans-acting factor required for dendritic delivery. Binding to hnRNP A2 and ensuing dendritic delivery are effectively competed by RNAs with CGG triplet repeat expansions. CGG repeats, when expanded in the 5′ untranslated region of fragile X mental retardation 1 (FMR1) mRNA, cause fragile X–associated tremor/ataxia syndrome. The data suggest that cellular dysregulation observed in the presence of CGG repeat RNA may result from molecular competition in neuronal RNA transport pathways.     

Comparative Genomic Sequence Analysis of the FXR Gene Family: FMR1, FXR1, and FXR2

Mutations in the X-linked gene FMR1 cause fragile X syndrome, the leading cause of inherited mental retardation. Two autosomal paralogs of FMR1 have been identified, and are known as FXR1 and FXR2. Here we describe and compare the genomic structures of the mouse and human genes FMR1, FXR1, and FXR2. All three genes are very well conserved from mouse to human, with identical exon sizes for all but two FXR2 exons. In addition, the three genes share a conserved gene structure, suggesting they are derived from a common ancestral gene. As a first step towards exploring this hypothesis, we reexamined the Drosophila melanogaster gene Fmr1, and found it to have several of the same intron/exon junctions as the mammalian FXRs. Finally, we noted several regions of mouse/human homology in the noncoding portions of FMR1 and FXR1. Knowledge of the genomic structure and sequence of the FXR family of genes will facilitate further studies into the function of these proteins.     

A role for the Drosophila fragile X-related gene in circadian output

Mutations that abolish expression of an X-linked gene, FMR1, result in the pathogenesis of fragile X syndrome, the most common form of inherited mental retardation. To understand the normal function of the FMR1 protein, we have produced fly strains bearing deletions in a Drosophila homolog of FMR1 (dfmr1). Since fragile X patients show a number of abnormal behaviors including sleep problems, we investigated whether a loss-of-function mutation of dfmr1 affect circadian behavior. Here we show that under constant darkness (DD), a lack of dfmr1 expression causes arrhythmic locomotor activity, but in light:dark cycles, their behavioral rhythms appear normal. In addition, the clock-controlled eclosion rhythm is normal in DFMR1-deficient flies. These results suggest that DFMR1 plays a critical role in the circadian output pathway regulating locomotor activity in Drosophila.     

Restoring the phenotype of fragile X syndrome: insight from the mouse model

A mouse model for the fragile X syndrome, the most common form of inherited mental retardation, was generated a number of years ago. It shows characteristics compatible with the clinical symptoms of human patients. These include pathological changes such as macroorchidism, behavioral problems, and diminished visuo-spatial abilities. To investigate whether the fragile X syndrome is a potentially correctable disorder, several groups attempted to 'rescue' the knockout mutation by introduction of an intact copy of the FMR1 gene in the knockout mouse. Two different types of rescue mice have been created by injection of constructs based on FMR1 cDNA or on FMR1 genomic DNA. Several pathological, behavioral and cognitive function tests were performed on these two different rescue mouse lines to compare their characteristics with those of the knockout and control littermates. Each rescue line resembled the control in some aspects though neither of the 2 lines was a full 'rescue', e.g. resemble the control in all aspects investigated. Thus, rescue of some aspects of the phenotype has been achieved by introduction of FMR1 constructs in the fragile X knockout mice. The results implicate that, even if FMR1 production is cell type specific, the quantity of the FMRP expression is highly critical as overproduction may have a harmful effect.     

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.     

Understanding fragile X syndrome: insights from retarded flies

Fragile X syndrome, the most common form of inherited mental retardation, is caused by loss-of-function mutations in the fragile X mental retardation 1 (fmr1) gene. FMR1 is an RNA binding protein that is highly expressed in neurons of the central nervous system. Recent studies in Drosophila indicate that FMR1 plays an important role in synaptogenesis and axonal arborization, which may underlie the observed deficits in flight ability and circadian behavior of fmr1 mutant flies. The relevance of these studies to our understanding of fragile X syndrome is discussed.     

甲基化特异性PCR检测FMR1 和XIST基因甲基化实验方法的建立

建立一种快速、灵敏的检测脆性X智障基因（Fragile X mental retardation, FMR1）和X染色体失活基因（X chromosome inactivation,XIST）甲基化的方法,用亚硫酸氢钠和对苯二酚对基因组DNA进行脱氨基修饰。以修饰后的DNA为模板,用两套不同的引物对：1对甲基化特异性引物和1对非甲基化特异性引物扩增FMR1基因（CGG）n重复序列区、FMR1 和XIST 基因的启动子区。PCR产物进一步克隆、测序。以亚硫酸氢钠和对苯二酚脱氨基修饰后的DNA为模板,进行PCR扩增后的产物与预期基因目的基因片段大小相符合,无非特异性扩增产物。测序结果表明,FMR1、XIST基因中的非甲基化的C碱基转变为U碱基,而CpG岛被甲基化的C碱基不改变。成功地建立了检测FMR1、XIST甲基化的方法,为实验室诊断脆性X综合征提供了新的方法。     

Key microRNAs in the biology of breast cancer; emerging evidence in the last decade

microRNAs (miRNAs) are a family of small noncoding RNAs that play a pivotal role in the regulation of main biological and physiological processes, including cell cycle regulation, proliferation, differentiation, apoptosis, stem cell maintenance, and organ development. Dysregulation of these tiny molecules has been related to different human diseases, such as cancer. It has been estimated that more than 50% of these noncoding RNA sequences are placed on fragile sites or cancer-associated genomic regions. After the discovery of the first specific miRNA signatures in breast cancer, many studies focused on the involvement of these small RNAs in the pathophysiology of breast tumors and their possible clinical implications as reliable prognostic biomarkers or as a new therapeutic approach. Therefore, the present review will focus on the recent findings on the involvement of miRNAs in the biology of breast cancer associated with their clinical implications.     

Involvement of the Spliceosomal U4 Small Nuclear RNA in Heterochromatic Gene Silencing at Fission Yeast Centromeres

prp13-1 is one of the mutants isolated in a screen for defective pre-mRNA splicing at a nonpermissive temperature in fission yeast Schizosaccharomyces pombe. We cloned the prp13⁺ gene and found that it encodes U4 small nuclear RNA (snRNA) involved in the assembly of the spliceosome. The prp13-1 mutant produced elongated cells, a phenotype similar to cell division cycle mutants, and displays a high incidence of lagging chromosomes on anaphase spindles. The mutant is hypersensitive to the microtubule-destabilizing drug thiabendazole, supporting that prp13-1 has a defect in chromosomal segregation. We found that the prp13-1 mutation resulted in expression of the ura4⁺ gene inserted in the pericentromeric heterochromatin region and reduced recruitment of the heterochromatin protein Swi6p to that region, indicating defects in the formation of pericentromeric heterochromatin, which is essential for the segregation of chromosomes, in prp13-1. The formation of centromeric heterochromatin is induced by the RNA interference (RNAi) system in S. pombe. In prp13-1, the processing of centromeric noncoding RNAs to siRNAs, which direct the heterochromatin formation, was impaired and unprocessed noncoding RNAs were accumulated. These results suggest that U4 snRNA is required for the RNAi-directed heterochromatic gene silencing at the centromeres. In relation to the linkage between the spliceosomal U4 snRNA and the RNAi-directed formation of heterochromatin, we identified a mRNA-type intron in the centromeric noncoding RNAs. We propose a model in which the assembly of the spliceosome or a sub-spliceosome complex on the intron-containing centromeric noncoding RNAs facilitates the RNAi-directed formation of heterochromatin at centromeres, through interaction with the RNA-directed RNA polymerase complex.     

A fragile gene

Fragile X syndrome is the most common cause of inherited mental retardation in humans. The fragile X gene (FMR1) has been cloned and the mutation causing the disease is known. The molecular basis of the disease is an expansion of a trinucleotide repeat sequence (CGG) present in the first exon within the 5′ untranslated region of the FMR1 gene. Affected individuals have repeat CGG sequences of above 200. As a result the gene is not producing protein. It has been shown that the FMR1 protein has RNA binding activity, but the function of this RNA binding activity is not known. The timing and mechanism of repeat amplification are not yet understood. An animal model for fragile X syndrome has been generated, which can be used to study the clinical and biochemical abnormalities caused by absence of FMR1 protein product.     

An origin of DNA replication in the promoter region of the human fragile X mental retardation (FMR1) gene

Fragile X syndrome, the most common form of inherited mental retardation in males, arises when the normally stable 5 to 50 CGG repeats in the 5' untranslated region of the fragile X mental retardation protein 1 (FMR1) gene expand to over 200, leading to DNA methylation and silencing of the FMR1 promoter. Although the events that trigger local CGG expansion remain unknown, the stability of trinucleotide repeat tracts is affected by their position relative to an origin of DNA replication in model systems. Origins of DNA replication in the FMR1 locus have not yet been described. Here, we report an origin of replication adjacent to the FMR1 promoter and CGG repeats that was identified by scanning a 35-kb region. Prereplication proteins Orc3p and Mcm4p bind to chromatin in the FMR1 initiation region in vivo. The position of the FMR1 origin relative to the CGG repeats is consistent with a role in repeat maintenance. The FMR1 origin is active in transformed cell lines, fibroblasts from healthy individuals, fibroblasts from patients with fragile X syndrome, and fetal cells as early as 8 weeks old. The potential role of the FMR1 origin in CGG tract instability is discussed.