SMART MODE:

NORMAL GENOMIC

• Schenck A, Bardoni B, Moro A, Bagni C, Mandel JL
• A highly conserved protein family interacting with the fragile X mental retardation protein (FMRP) and displaying selective interactions with FMRP-related proteins FXR1P and FXR2P.
• Proc Natl Acad Sci U S A. 2001; 98: 8844-9
• Display abstract

• The absence of the fragile X mental retardation protein (FMRP), encoded by the FMR1 gene, is responsible for pathologic manifestations in the Fragile X Syndrome, the most frequent cause of inherited mental retardation. FMRP is an RNA-binding protein associated with polysomes as part of a messenger ribonucleoprotein (mRNP) complex. Although its function is poorly understood, various observations suggest a role in local protein translation at neuronal dendrites and in dendritic spine maturation. We present here the identification of CYFIP1/2 (Cytoplasmic FMRP Interacting Proteins) as FMRP interactors. CYFIP1/2 share 88% amino acid sequence identity and represent the two members in humans of a highly conserved protein family. Remarkably, whereas CYFIP2 also interacts with the FMRP-related proteins FXR1P/2P, CYFIP1 interacts exclusively with FMRP. FMRP--CYFIP interaction involves the domain of FMRP also mediating homo- and heteromerization, thus suggesting a competition between interaction among the FXR proteins and interaction with CYFIP. CYFIP1/2 are proteins of unknown function, but CYFIP1 has recently been shown to interact with the small GTPase Rac1, which is implicated in development and maintenance of neuronal structures. Consistent with FMRP and Rac1 localization in dendritic fine structures, CYFIP1/2 are present in synaptosomal extracts.

• Laggerbauer B, Ostareck D, Keidel EM, Ostareck-Lederer A, Fischer U
• Evidence that fragile X mental retardation protein is a negative regulator of translation.
• Hum Mol Genet. 2001; 10: 329-38
• Display abstract

• Fragile X syndrome is a common form of inherited mental retardation. Most fragile X patients exhibit mutations in the fragile X mental retardation gene 1 (FMR1) that lead to transcriptional silencing and hence to the absence of the fragile X mental retardation protein (FMRP). Since FMRP is an RNA-binding protein which associates with polyribosomes, it had been proposed to function as a regulator of gene expression at the post-transcriptional level. In the present study, we show that FMRP strongly inhibits translation of various mRNAs at nanomolar concentrations in both rabbit reticulocyte lysate and microinjected Xenopus laevis oocytes. This effect is specific for FMRP, since other proteins with similar RNA-binding domains, including the autosomal homologues of FMRP, FXR1 and FXR2, failed to suppress translation in the same concentration range. Strikingly, a disease-causing Ile-->Asn substitution at amino acid position 304 (I304N) renders FMRP incapable of interfering with translation in both test systems. Initial studies addressing the underlying mechanism of inhibition suggest that FMRP inhibits the assembly of 80S ribosomes on the target mRNAs. The failure of FMRP I304N to suppress translation is not due to its reduced affinity for mRNA or its interacting proteins FXR1 and FXR2. Instead, the I304N point mutation severely impairs homo-oligomerization of FMRP. Our data support the notion that inhibition of translation may be a function of FMRP in vivo. We further suggest that the failure of FMRP to oligomerize, caused by the I304N mutation, may contribute to the pathophysiological events leading to fragile X syndrome.

• Wang X, Tanaka Hall TM
• Structural basis for recognition of AU-rich element RNA by the HuD protein.
• Nat Struct Biol. 2001; 8: 141-5
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• Hu proteins bind to adenosine-uridine (AU)-rich elements (AREs) in the 3' untranslated regions of many short-lived mRNAs, thereby stabilizing them. Here we report the crystal structures of the first two RNA recognition motif (RRM) domains of the HuD protein in complex with an 11-nucleotide fragment of a class I ARE (the c-fos ARE; to 1.8 A), and with an 11-nucleotide fragment of a class II ARE (the tumor necrosis factor alpha ARE; to 2.3 A). These structures reveal a consensus RNA recognition sequence that suggests a preference for pyrimidine-rich sequences and a requirement for a central uracil residue in the clustered AUUUA repeats found in class II AREs. Comparison to structures of other RRM domain-nucleic acid complexes reveals two base recognition pockets in all the structures that interact with bases using residues in conserved ribonucleoprotein motifs and at the C-terminal ends of RRM domains. Different conformations of nucleic acid can be bound by RRM domains by using different combinations of base recognition pockets and multiple RRM domains.

• Mila M et al.
• Rare variants in the promoter of the fragile X syndrome gene (FMR1).
• Mol Cell Probes. 2000; 14: 115-9
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• Fragile X syndrome, the most common form of familial mental retardation, is mainly caused by the expansion of an unstable region of CGG repeats in the 5' untranslated region of the FMR1 (Fragile X Mental Retardation-1) gene. Molecular tools to detect an abnormal CGG expansion in FMR1 include Southern blot hybridization and PCR amplification. Southern blotting with the StB12.3 probe and Eco RI/Eag I double digestion is widely used as a routine test for fragile X syndrome diagnosis in laboratories around the world. A patient with mental retardation of unknown origin showed absence of digestion for Eag I due to a -149C-->G substitution in the CpG island of the FMR1 gene, which destroys that restriction enzyme site. Screening for other changes around that region also detected a -154insGGC in a patient with a phenotype highly suggestive of fragile X syndrome but without CGG expansion. Expression studies did not show any abnormal changes in FMR1 function. In summary, we have identified two different changes (a C to G substitution at -149 and a GGC insertion at -154) in the promoter of the FMR1 gene. These are the first variants described in the promoter of the FMR1 gene.

• Kooy RF, Willemsen R, Oostra BA
• Fragile X syndrome at the turn of the century.
• Mol Med Today. 2000; 6: 193-8
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• Fragile X syndrome is not only the most common form of inherited cognitive impairment, it is also one of the most frequent single gene disorders. It is caused by a stretch of CGG-repeats within the fragile X gene, which increases in length as it is transmitted from generation to generation. Once the repeat exceeds a threshold length, no fragile X protein is produced and disease results. Since the mutation was discovered, nearly a decade of research has revealed a wealth of information regarding the fragile X gene and its possible function within the cell. The fragile X story also provides a sobering example of how much time and effort might be necessary to develop beneficial treatment through understanding gene function.

• Lisbin MJ, Gordon M, Yannoni YM, White K
• Function of RRM domains of Drosophila melanogaster ELAV: Rnp1 mutations and rrm domain replacements with ELAV family proteins and SXL.
• Genetics. 2000; 155: 1789-98
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• Members of the ELAV family of proteins contain three RNA recognition motifs (RRMs), which are highly conserved. ELAV, a Drosophila melanogaster member of this family, provides a vital function and exhibits a predominantly nuclear localization. To investigate if the RNA-binding property of each of the ELAV RRMs is required for ELAV's in vivo function, amino acid residues critical in RNA binding for each RRM were individually mutated. A stringent genetic complementation test revealed that when the mutant protein was the sole source of ELAV, RNA-binding ability of each RRM was essential to ELAV function. To assess the degree to which each domain was specific for ELAV function and which domains perhaps performed a function common to related ELAV proteins, we substituted an ELAV RRM with the corresponding RRM from RBP9, the D. melanogaster protein most homologous to ELAV; HuD, a human ELAV family protein; and SXL, which, although evolutionarily related, is not an ELAV family member. This analysis revealed that RRM3 replacements were fully functional, but RRM1 and RRM2 replacements were largely nonfunctional. Under less stringent conditions RRM1 and RRM2 replacements from SXL and RRM1 replacement from RBP9 were able to provide supplemental function in the presence of a mutant hypomorphic ELAV protein.

• Limprasert P, Jaruratanasirikul S, Vasiknanonte P
• Unilateral macroorchidism in fragile X syndrome.
• Am J Med Genet. 2000; 95: 516-7

• Wan L, Dockendorff TC, Jongens TA, Dreyfuss G
• Characterization of dFMR1, a Drosophila melanogaster homolog of the fragile X mental retardation protein.
• Mol Cell Biol. 2000; 20: 8536-47
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• 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.

• Tassone F, Hagerman RJ, Taylor AK, Gane LW, Godfrey TE, Hagerman PJ
• Elevated levels of FMR1 mRNA in carrier males: a new mechanism of involvement in the fragile-X syndrome.
• Am J Hum Genet. 2000; 66: 6-15
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• Fragile-X syndrome is a trinucleotide-repeat-expansion disorder in which the clinical phenotype is believed to result from transcriptional silencing of the fragile-X mental retardation 1 (FMR1) gene as the number of CGG repeats exceeds approximately 200. For premutation alleles ( approximately 55-200 repeats), no abnormalities in FMR1-gene expression have been described, despite growing evidence of clinical involvement in premutation carriers. To address this (apparent) paradox, we have determined, for 16 carrier males (55-192 repeats), the relative levels of leukocyte FMR1 mRNA, by use of automated fluorescence-detection reverse transcriptase-PCR, and the percent of lymphocytes that are immunoreactive for FMR1 protein (FMRP). For some alleles with>100 repeats, there was a reduction in the number of FMRP-positive cells. Unexpectedly, FMR1 mRNA levels were elevated at least fivefold within this same range. No significant increase in FMR1 mRNA stability was observed in a lymphoblastoid cell line (160 repeats) derived from one of the carrier males, suggesting that the increased message levels are due to an increased rate of transcription. Current results support a mechanism of involvement in premutation carriers, in which reduced translational efficiency is at least partially compensated through increased transcriptional activity. Thus, diminished translational efficiency may be important throughout much of the premutation range, with a mechanistic switch occurring in the full-mutation range as the FMR1 gene is silenced.

• Mazzocco MM
• Advances in research on the fragile X syndrome.
• Ment Retard Dev Disabil Res Rev. 2000; 6: 96-106
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• Fragile X syndrome is a neurodevelopmental disorder that results from a single gene mutation on the X chromosome. The purpose of this review is to summarize key advances made in understanding the fragile X premutation gene seen in carriers and the full mutation gene seen in persons with the syndrome. DNA testing has replaced cytogenetic testing as the primary method for identification of fragile X, although the efficacy of protein level screening is being explored. The premutation is associated with no effects, although there is evidence of physical effects-primarily premature menopause and mild outward features of the fragile X syndrome-among premutation carriers. There is much controversy regarding premutation effects on psychological development. The few experimental studies carried out to date do not suggest noticeable or significant effects. One challenge in addressing this controversy is the sometimes ambiguous differentiation between premutation and full mutation genes. There is a well-established yet highly variable phenotype of the full mutation. Research from this decade has helped to address specific aspects of this phenotype, including the early course of its development in males, the influence of home and family environments, the nature of social difficulties and autistic features seen in boys and girls with fragile X, and the potential role of hyperarousal or hyper-reactivity. Studies in these areas, and on the role of FMR protein, will contribute towards ongoing advances in our understanding of fragile X syndrome and its mechanisms. The variability in physical, social, and cognitive features, as described in this review, is one that prohibits clear-cut screening guidelines designed to avoid high rates of both false positives and false negatives. Results from recent studies indicate the need to consider behavioral features in selecting candidates for fragile X screening. MRDD Research Reviews 2000;6:96-106.

• Todd PK, Mack KJ
• Sensory stimulation increases cortical expression of the fragile X mental retardation protein in vivo.
• Brain Res Mol Brain Res. 2000; 80: 17-25
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• Fragile X syndrome is a common cause of mental retardation that results from the absence of the fragile X mental retardation protein (FMRP), an RNA binding protein whose function remains unclear. Recent in vitro work has demonstrated that the protein is translated near the synapse in an activity dependent manner [33]. We therefore asked whether expression of FMRP might be altered by neuronal activity in vivo. Using immunoblots of different sub-cellular fractions of the rat somatosensory cortex, we show that the levels of FMRP increase significantly following unilateral whisker stimulation, a model of experience dependent plasticity. This increase is greatest between 2 and 8 h after the stimulus and is seen in both a synaptosomal fraction as well as a sub-cellular fraction enriched for polyribosomal complexes. In contrast, detectable levels of FMRP within the somatosensory cortex show either a decrease or no change after a kainic acid induced seizure compared to water treated controls. Our findings demonstrate that FMRP expression levels are modulated in vivo in response to neuronal activity and suggest a role for FMRP in activity dependent plasticity.

• Barinaga M
• Translational roots for mental retardation?
• Science. 2000; 290: 737-737

• Castellvi-Bel S et al.
• Detection of the fragile X syndrome protein for the evaluation of FMR1 intermediate alleles.
• Hum Genet. 2000; 107: 195-6
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• Molecular screening programs in mentally retarded individuals have been performed in several populations worldwide. One finding has been an excess of FMR1 intermediate alleles in a population with learning difficulties. However, other published reports with similar characteristics did not corroborate those previous results. In order to contribute additional data from our population, we studied 563 patients affected with nonspecific mental retardation (MRX) that did not present a CGG expansion in the FMR1 gene and 208 individuals as a control population. Forty MRX patients presented alleles within the intermediate range. Among them, one case showed a pattern of expression of the FMR1 protein (FMRP) concordant with a fragile X syndrome case with an intermediate allele/full mutation mosaicism, although it was not detected by Southern blot analysis. Statistical analysis was performed again showing no statistically significant difference regarding the intermediate allele frequency in the MRX and control populations. This finding is in agreement with the hypothesis that the incidence of intermediate FMR1 alleles in MRX populations does not seem to be higher than in control populations, and it emphasizes the importance of FMRP detection as a diagnostic tool for fragile X syndrome.

• Tamanini F et al.
• The fragile X-related proteins FXR1P and FXR2P contain a functional nucleolar-targeting signal equivalent to the HIV-1 regulatory proteins.
• Hum Mol Genet. 2000; 9: 1487-93
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• Fragile X syndrome is caused by the absence of the fragile X mental-retardation protein (FMRP). FMRP and the fragile X-related proteins 1 and 2 (FXR1P and FXR2P) form a gene family with functional similarities, such as RNA binding, polyribosomal association and nucleocytoplasmic shuttling. In a previous study, we found that FMRP and FXR1P shuttle between cytoplasm and nucleoplasm, while FXR2P shuttles between cytoplasm and nucleolus. The nuclear and nucleolar-targeting properties of these proteins were investigated further. Here, we show that FXR2P contains in its C-terminal part, a stretch of basic amino acids 'RPQRRNRSRRRRFR' that resemble the nucleolar-targeting signal (NoS) of the viral protein Rev. This particular sequence is also present within exon 15 of the FXR1 gene. This exon undergoes alternative splicing and is therefore only present in some of the FXR1P isoforms. We investigated the intracellular distribution of various FXR1P isoforms with (iso-e and iso-f) and without (iso-d) the potential NoS in transfected COS cells treated with the nuclear export inhibitor leptomycin-B. Both iso-e and iso-f showed a nucleolar localization, as observed for FXR2P; iso-d was detected in the nucleo-plasm outside the nucleoli. Further, when a labelled 16-residue synthetic peptide corresponding to the NoS of FXR1P was added to human fibroblast cultures a clear nucleolar signal was observed. Based on these data we argue that the intranuclear distribution of FXR2P and FXR1P isoforms is very likely to be mediated by a similar NoS localized in their C-terminal region. This domain is absent in some FXR1P isoforms as well as in all FMRP isoforms, suggesting functional differences for this family of proteins, possibly related to RNA metabolism in different tissues.

• Sung YJ, Conti J, Currie JR, Brown WT, Denman RB
• RNAs that interact with the fragile X syndrome RNA binding protein FMRP.
• Biochem Biophys Res Commun. 2000; 275: 973-80
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• The Fragile X protein FMRP is an RNA binding protein whose targets are not well known; yet, these RNAs may play an integral role in the disease's etiology. Using a biotinylated-FMRP affinity resin, we isolated RNAs from the parietal cortex of a normal adult that bound FMRP. These RNAs were amplified by differential display (DDRT-PCR) and cloned and their identities determined. Nine candidate RNAs were isolated; five RNAs, including FMR1 mRNA, encoded known proteins. Four others were novel. The specificity of binding was demonstrated for each candidate RNA. The domains required for binding a subset of the RNAs were delineated using FMRP truncation mutant proteins and it was shown that only the KH2 domain was required for binding. Binding occurred independently of homoribopolymer binding to the C-terminal arginine-glycine-rich region (RGG box), suggesting that FMRP may bind multiple RNAs simultaneously.

• Brown WT, Nolin SL
• Apparent FMR1 allele instability in non-fragile X males.
• Genet Test. 2000; 4: 241-2

• Tzountzouris J et al.
• Apparently unstable normal FMR1 alleles in nine developmentally delayed patients: implications for molecular diagnosis of the fragile X syndrome.
• Genet Test. 2000; 4: 235-9
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• The Fragile X syndrome is a common form of X-linked mental retardation, affecting approximately 1 in 4,000 males. Since the discovery of the FMR1 gene responsible for the syndrome, molecular, rather than cytogenetic, diagnosis of Fragile X syndrome has become the gold standard. Numerous molecular diagnostic centers worldwide use PCR and Southern blotting to characterize the size of the CGG repeats within the gene, expansion of which has been shown to be associated with the vast majority of cases of Fragile X syndrome. Instability of this repeat through successive generations has been demonstrated in many patients and has been associated with numerous factors, including repeat length and molecular structure of the repeat. Nine males with normal-size alleles that exhibit repeat length instability by the presence of a second normal length distinct band by repeated PCR analysis from peripheral lymphocytes are reported. Many hypotheses addressing the reason for this apparent instability were tested without elucidating the underlying molecular causes, including cytogenetic analysis, sequence analysis of the repeat locus, and analysis of flanking dinucleotide repeat loci. All patients exhibited a normal complement of sex chromosomes by cytogenetic and molecular analysis. These results from the widely used PCR analysis illustrate an interesting molecular phenomenon and raise many questions relating to the factors and mechanisms involved in trinucleotide instability as well as having implications for the diagnostic testing of the Fragile X syndrome.

• Allain FH, Gilbert DE, Bouvet P, Feigon J
• Solution structure of the two N-terminal RNA-binding domains of nucleolin and NMR study of the interaction with its RNA target.
• J Mol Biol. 2000; 303: 227-41
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• Nucleolin is an abundant 70 kDa nucleolar protein involved in many aspects of ribosomal RNA biogenesis. The central region of nucleolin contains four tandem consensus RNA-binding domains (RBD). The two most N-terminal domains (RBD12) bind with nanomolar affinity to an RNA stem-loop containing the consensus sequence UCCCGA in the loop. We have determined the solution structure of nucleolin RBD12 in its free form and have studied its interaction with a 22 nt RNA stem-loop using multidimensional NMR spectroscopy. The two RBDs adopt the expected beta alpha beta beta alpha beta fold, but the position of the beta 2 strand in both domains differs from what was predicted from sequence alignments. RBD1 and RBD2 are significantly different from each others and this is likely important in their sequence specific recognition of the RNA. RBD1 has a longer alpha-helix 1 and a shorter beta 2-beta 3 loop than RBD2, and differs from most other RBDs in these respects. The two RBDs are separated by a 12 amino acid flexible linker and do not interact with one another in the free protein. This linker becomes ordered when RBD12 binds to the RNA. Analysis of the observed NOEs between the protein and the RNA indicates that both RBDs interact with the RNA loop via their beta-sheet. Each domain binds residues on one side of the loop; specifically, RBD2 contacts the 5' side and RBD1 contacts the 3'.

• Tuncbilek E et al.
• Screening for the fragile X syndrome among mentally retarded males by hair root analysis.
• Am J Med Genet. 2000; 95: 105-7
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• A noninvasive antibody test was used to identify male fragile X patients in special education schools, on the basis of the lack of FMRP in hair roots. We studied 300 males with mental retardation of unknown cause attending special schools. Patients were divided into two groups, based on the scores according to a fragile X check list (Group 1 /= 10 points). Group 2 consists of 51 males and only 5 males in this group showed no FMRP expression in hair roots within the abnormal range (91%). Fragile X diagnosis in these cases was confirmed by DNA analysis. None of the males scoring more than 10 on the check list was diagnosed positive for the fragile X syndrome using DNA analysis. With our antibody test on hair roots we did not detect a fragile X patient in Group 1. The FMRP antibody test on hair roots is suitable in a screening program for the fragile X syndrome among mentally retarded males attending special education schools.

• Conklin DC, Rixon MW, Kuestner RE, Maurer MF, Whitmore TE, Millar RP
• Cloning and gene expression of a novel human ribonucleoprotein.
• Biochim Biophys Acta. 2000; 1492: 465-9
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• This paper reports on the cloning and characterization of a novel human ribonucleoprotein, RBM8, containing a single RNA binding domain comprising the two RNP-CS and RNP-2 consensus motifs. The protein has 55% identity to a segment of a C. elegans ribonucleoprotein of unknown function. The RBM8 gene shows ubiquitous tissue expression, predominantly as a 0.9 kb transcript. An interesting feature of the RBM8 transcript is an homology of 42% in the 3' untranslated region, in the antisense orientation, to the human gonadotropin-releasing hormone receptor polypeptide. RBM8 maps to human chromosome 14 in the 14q21-q23 region.

• Tan BS, Law HY, Zhao Y, Yoon CS, Ng IS
• DNA testing for fragile X syndrome in 255 males from special schools in Singapore.
• Ann Acad Med Singapore. 2000; 29: 207-12
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• INTRODUCTION: Fragile X syndrome, the most common cause of inherited mental retardation, results from unstable expansion of a trinucleotide (CGG)n repeat in the FMR1 gene. Phenotypic expression is variable making clinical diagnosis difficult, while diagnosis by Southern blotting is relatively expensive and labour intensive. The prevalence in Singapore has not been studied. MATERIALS AND METHODS: We developed a rapid screening test using a PCR analysis. We studied 255 males with unexplained cause for learning difficulties from 8 special schools. A clinical scoring system based on characteristic features described was devised. RESULTS: PCR analysis showed absence of the band for the normal allele in 11 samples, 6 of which were confirmed by Southern blotting to be positive for FMR1 expansion, giving a 2% false-positive rate with PCR. Sensitivity of the PCR test was evaluated by performing Southern blotting in all PCR-normal samples; all of which were confirmed to be normal. This PCR test was shown to be highly reproducible. Clinical criteria were not predictive. CONCLUSIONS: Six (2.4%) new cases of fragile X syndrome were detected. There is a need to incorporate fragile X testing in routine screening of patients with developmental delay and learning difficulties. The use of PCR could eliminate the need for Southern blotting in up to 95% of cases. PCR analysis provides a simple, reliable and rapid tool for screening.

• Inoue SB, Siomi MC, Siomi H
• Molecular mechanisms of fragile X syndrome.
• J Med Invest. 2000; 47: 101-7
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• Fragile X syndrome is the most common form of inherited mental retardation Mutations which abolish expression of an X-linked gene, FMR1, result in pathogenesis of the disease. FMR1 encodes a cytoplasmic RNA-binding protein which interacts with two autosomal homologs, FXR1 and FXR2. These proteins are highly expressed in neurons. In addition, the FMR1/FXR proteins are associated with ribosomes. Given their RNA-binding activity and association with ribosomes, these proteins are hypothesized to bind to specific RNAs and regulate their expression at translational levels in a manner critical for correct development of neurons. Much progress has been made in FMR1 research over the past several years, but little light has yet to be shed on the physiological function of these proteins. It will be critical to define the biochemical properties of these proteins, and identify potential downstream targets to clarify the molecular mechanisms underlying the potential roles of these proteins in translation. A basic understanding of the function of this new family of RNA-binding proteins should then allow us to begin to address the question of how the lack of FMR1 expression leads to symptoms in fragile X syndrome.

• Eliez S, Reiss AL
• Genetics of childhood disorders: XI. Fragile X syndrome.
• J Am Acad Child Adolesc Psychiatry. 2000; 39: 264-6

• Jeong EJ, Hwang GS, Kim KH, Kim MJ, Kim S, Kim KS
• Structural analysis of multifunctional peptide motifs in human bifunctional tRNA synthetase: identification of RNA-binding residues and functional implications for tandem repeats.
• Biochemistry. 2000; 39: 15775-82
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• Human bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) contains three tandem repeats linking the two catalytic domains. These repeated motifs have been shown to be involved in protein-protein and protein-nucleic acid interactions. The single copy of the homologous motifs has also been found in several different aminoacyl-tRNA synthetases. The solution structure of repeat 1 (EPRS-R1) and the secondary structure of the whole appended domain containing three repeated motifs in EPRS (EPRS-R123) was determined by nuclear magnetic resonance (NMR) spectroscopy. EPRS-R1 consists of two helices (residues 679-699 and 702-721) arranged in a helix-turn-helix, which is similar to other RNA binding proteins and the j-domain of DnaJ, and EPRS-R123 is composed of three helix-turn-helix motifs linked by an unstructured loop. When tRNA is bound to the appended domain, chemical shifts of several residues in each repeat are perturbed. However, the perturbed residues in each repeat are not the same although they are in the same binding surface, suggesting that each repeat in the appended domain is dynamically arranged to maximize contacts with tRNA. The affinity of tRNA to the three-repeated motif was much higher than to the single motif. These results indicate that each of the repeated motifs has a weak intrinsic affinity for tRNA, but the repetition of the motifs may be required to enhance binding affinity. Thus, the results of this work gave information on the RNA-binding mode of the multifunctional peptide motif attached to different ARSs and the functional reason for the repetition of this motif.

• Nagy E, Henics T, Eckert M, Miseta A, Lightowlers RN, Kellermayer M
• Identification of the NAD(+)-binding fold of glyceraldehyde-3-phosphate dehydrogenase as a novel RNA-binding domain.
• Biochem Biophys Res Commun. 2000; 275: 253-60
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• There is growing evidence that metabolic enzymes may act as multifunctional proteins performing diverse roles in cellular metabolism. Among these functions are the RNA-binding activities of NAD(+)-dependent dehydrogenases. Previously, we have characterized the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an RNA-binding protein with preference to adenine-uracil-rich sequences. In this study, we used GST-GAPDH fusion proteins generated by deletion mutagenesis to search for the RNA binding domain. We established that the N-terminal 43 amino acid residues of GAPDH, which correspond to the first mononucleotide-binding domain of the NAD(+)-binding fold is sufficient to confer RNA-binding. We also provide evidence that this single domain, although it retains most of the RNA-binding activity, loses sequence specificity. Our results suggest a molecular basis for RNA-recognition by NAD(+)-dependent dehydrogenases and (di)nucleotide-binding metabolic enzymes that had been reported to have RNA-binding activity with different specificity. To support this prediction we also identified other members of the family of NAD(+)-dependent dehydrogenases with no previous history of nucleic acid binding as RNA binding proteins in vitro. Based on our findings we propose the addition of the NAD(+)-binding domain to the list of RNA binding domains/motifs.

• Beresford RG et al.
• Absence of fragile X syndrome in Nova Scotia.
• J Med Genet. 2000; 37: 77-9

• Uliel L, Weisman-Shomer P, Oren-Jazan H, Newcomb T, Loeb LA, Fry M
• Human Ku antigen tightly binds and stabilizes a tetrahelical form of the Fragile X syndrome d(CGG)n expanded sequence.
• J Biol Chem. 2000; 275: 33134-41
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• Hairpin and tetrahelical structures of a d(CGG)(n) sequence in the FMR1 gene have been implicated in its expansion in fragile X syndrome. The identification of tetraplex d(CGG)(n) destabilizing proteins (Fry, M., and Loeb, L. A.(1999) J. Biol. Chem. 274, 12797-12803; Weisman-Shomer, P., Naot, Y., and Fry, M. (2000) J. Biol. Chem. 275, 2231-2238) suggested that proteins might modulate d(CGG)(n) folding and aggregation. We assayed human TK-6 lymphoblastoid cell extracts for d(CGG)(8) oligomer binding proteins. The principal binding protein was identified as Ku antigen by its partial amino acid sequence and antigenicity. The purified 88/75-kDa heterodimeric Ku bound with similar affinities (K(d) approximately 1. 8-10.2 x 10(-9) mol/liter) to double-stranded d(CGG)(8).d(CCG)(8), hairpin d(CGG)(8), single-stranded d(CII)(8), or tetraplex structures of telomeric or IgG switch region sequences. However, Ku associated more tightly with bimolecular G'2 tetraplex d(CGG)(8) (K(d) approximately 0.35 x 10(-9) mol/liter). Binding to Ku protected G'2 d(CGG)(8) against nuclease digestion and impeded its unwinding by the tetraplex destabilizing protein qTBP42. Stabilization of d(CGG)(n) tetraplex domains in FMR1 by Ku or other proteins might promote d(CGG) expansion and FMR1 silencing.

• Bakker CE et al.
• Immunocytochemical and biochemical characterization of FMRP, FXR1P, and FXR2P in the mouse.
• Exp Cell Res. 2000; 258: 162-70
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• Fragile X syndrome is caused by the absence of expression of the FMR1 gene. Both FXR1 and FXR2 are autosomal gene homologues of FMR1. The products of the three genes are belonging to a family of RNA-binding proteins, called FMRP, FXR1P, and FXR2P, respectively, and are associated with polyribosomes as cytoplasmic mRNP particles. The aim of the present study is to obtain more knowledge about the cellular function of the three proteins (Fxr proteins) and their interrelationships in vivo. We have utilized monospecific antibodies raised against each of these proteins and performed Western blotting and immunolabeling at the light-microscopic level on tissues of wild-type and Fmr1 knockout adult mice. In addition, we have performed immunoelectron microscopy on hippocampal neurons of wild-type mice to study the subcellular distribution of the Fxr proteins. A high expression was found in brain and gonads for all three proteins. Skeletal muscle tissue showed only a high expression for Fxr1p. In the brain the three proteins were colocalized in the cytoplasm of the neurons; however, in specific neurons Fxr1p was also found in the nucleolus. Immunoelectronmicrsocopy on hippocampal neurons demonstrated the majority of the three proteins in association with ribosomes and a minority in the nucleus. The colocalization of the Fxr proteins in neurons is consistent with similar cellular functions in those specific cells. The presence of the three proteins in the nucleus of hippocampal neurons suggests a nucleocytoplasmic shuttling for the Fxr proteins. In maturing and adult testis a differential expression was observed for the three proteins in the spermatogenic cells. The similarities and differences between the distribution of the Fxr proteins have implications with respect to their normal function and the pathogenesis of the fragile X syndrome.

• Markovtsov V, Nikolic JM, Goldman JA, Turck CW, Chou MY, Black DL
• Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein.
• Mol Cell Biol. 2000; 20: 7463-79
• Display abstract

• Splicing of the c-src N1 exon in neuronal cells depends in part on an intronic cluster of RNA regulatory elements called the downstream control sequence (DCS). Using site-specific cross-linking, RNA gel shift, and DCS RNA affinity chromatography assays, we characterized the binding of several proteins to specific sites along the DCS RNA. Heterogeneous nuclear ribonucleoprotein (hnRNP) H, polypyrimidine tract binding protein (PTB), and KH-type splicing-regulatory protein (KSRP) each bind to distinct elements within this sequence. We also identified a new 60-kDa tissue-specific protein that binds to the CUCUCU splicing repressor element of the DCS RNA. This protein was purified, partially sequenced, and cloned. The new protein (neurally enriched homolog of PTB [nPTB]) is highly homologous to PTB. Unlike PTB, nPTB is enriched in the brain and in some neural cell lines. Although similar in sequence, nPTB and PTB show significant differences in their properties. nPTB binds more stably to the DCS RNA than PTB does but is a weaker repressor of splicing in vitro. nPTB also greatly enhances the binding of two other proteins, hnRNP H and KSRP, to the DCS RNA. These experiments identify specific cooperative interactions between the proteins that assemble onto an intricate splicing-regulatory sequence and show how this hnRNP assembly is altered in different cell types by incorporating different but highly related proteins.

• Ceman S, Nelson R, Warren ST
• Identification of mouse YB1/p50 as a component of the FMRP-associated mRNP particle.
• Biochem Biophys Res Commun. 2000; 279: 904-8
• Display abstract

• Fragile X mental retardation is caused by the absence of FMRP, an RNA-binding protein found in a large mRNP complex. Although there is evidence that FMRP exists as a homo-multimer, additional proteins have been identified that associate with FMRP in the mRNP. The autosomal paralogs of FMRP, FXR1P, and FXR2P, associate with FMRP, as do nucleolin and NUFIP1, all RNA binding proteins. Using cell lines that were stably transfected with Flag-Fmr1, we identified an additional protein that coimmunoprecipitates with FMRP. The approximately 50 kDa protein was identified by mass spectrometry as mouse Y box-binding protein 1 (YB1), which is 97% identical to the core mRNP protein p50, an RNA-binding protein. An anti-p50 antiserum recognized the 50 kDa protein, confirming the identification. The association of the FMRP-mRNP with a Y box protein, the latter commonly found in mRNPs, further suggests the involvement of FMRP in translation modulation.

• Jin P, Warren ST
• Understanding the molecular basis of fragile X syndrome.
• Hum Mol Genet. 2000; 9: 901-8
• Display abstract

• Fragile X syndrome, a common form of inherited mental retardation, is mainly caused by massive expansion of CGG triplet repeats located in the 5'-untranslated region of the fragile X mental retardation-1 ( FMR1 ) gene. In patients with fragile X syndrome, the expanded CGG triplet repeats are hypermethylated and the expression of the FMR1 gene is repressed, which leads to the absence of FMR1 protein (FMRP) and subsequent mental retardation. FMRP is an RNA-binding protein that shuttles between the nucleus and cytoplasm. This protein has been implicated in protein translation as it is found associated with polyribosomes and the rough endoplasmic reticulum. We discuss here the recent progress made towards understanding the molecular mechanism of CGG repeat expansion and physiological function(s) of FMRP. These studies will not only help to illuminate the molecular basis of the general class of human diseases with trinucleotide repeat expansion but also provide an avenue to understand aspects of human cognition and intelligence.

• Beaulieu MA
• A distinct FMRP polysomal population at an advanced stage of mammalian erythropoiesis.
• Biochem Biophys Res Commun. 2000; 275: 608-10
• Display abstract

• The fragile-X syndrome is a mental disorder caused by the absence of FMRP (the Fragile-X Mental Retardation Protein). While FMRP is found to be associated with the ribosomal components, its precise translational function remains to be defined. Here we report that FMRP is not found with the abundant free polysomes of the reticulocyte lysate, but rather with a heavy ribonucleoprotein complex sedimenting over 400S. This unusual distribution of FMRP at an advanced stage of mammalian erythropoiesis may unveil the discrete role of FMRP in translation.

• Mizutani A, Fukuda M, Ibata K, Shiraishi Y, Mikoshiba K
• SYNCRIP, a cytoplasmic counterpart of heterogeneous nuclear ribonucleoprotein R, interacts with ubiquitous synaptotagmin isoforms.
• J Biol Chem. 2000; 275: 9823-31
• Display abstract

• Synaptotagmins (Syts) are a large family of membrane proteins consisted of at least 12 isoforms. They are categorized in neuron-specific isoforms (I-V, X, and XI) and ubiquitous isoforms (VI-IX) based on their expression patterns. Syt-I, a neuron-specific and abundant isoform, has been well characterized and postulated to be the exocytotic Ca(2+) sensor. However, the functions of other isoforms remain obscure. Here, we report that ubiquitous isoforms of synaptotagmins, Syt-VII, Syt-VIII, and Syt-IX, interacted with a cytoplasmic RNA-binding protein, SYNCRIP (Synaptotagmin-binding, cytoplasmic RNA-interacting protein), through their C2B domains. SYNCRIP was originally found in the Syt-II C2AB domain bound fraction from the mouse brain lysate. cDNA cloning of SYNCRIP cDNA revealed that the protein was highly homologous to heterogeneous nuclear ribonucleoprotein R (hnRNP R) recently identified. SYNCRIP protein was ubiquitously and constantly expressed in various tissues of mice parallel to hnRNP R. SYNCRIP indeed bound RNA with preference to poly(A) RNA; however, in contrast to the nuclear localization of hnRNP R, SYNCRIP was distributed predominantly in the cytoplasm as judged by both biochemical fractionation and immunohistochemical studies. In vitro binding experiments showed the potential interaction of SYNCRIP with C2B domains of Syts except for those of Syt-V, -VI, and -X. Furthermore, the interaction between SYNCRIP and Syt-VII, -VIII, or -IX was revealed by co-immunoprecipitation experiments using COS cells transiently expressing each Syt isoform. These findings suggested that SYNCRIP was a target of ubiquitous type of Syts and implied the involvement of ubiquitous Syts in the regulation of dynamics of the cytoplasmic mRNA.

• Pimentel MM
• Fragile X syndrome (review).
• Int J Mol Med. 1999; 3: 639-45
• Display abstract

• Fragile X syndrome is the most common form of inherited mental retardation currently known, associated with a wide range of developmental disabilities in both males and females, caused by a large expansion of a (CGG)n repeat in the first exon of the FMR1 gene. Fragile X syndrome occurs in all racial and ethnic groups, and it is a condition of major epidemiological importance among mentally handicapped males. Therefore, this disease must be considered in the differential diagnosis of any child with developmental delay, mental retardation or learning disability. The fragile X syndrome is due to the shutdown of the FMR1 gene transcription, and the pathogenesis of this syndrome is a consequence of absence of the protein product of the FMR1 gene (FMRP). Since the great majority of fragile X patients have the same type of mutation in a specific location of the gene, molecular analysis is extremely accurate for diagnosis of the disease, and important for genetic counseling of family members. Others genetic disorders are also caused by expanded trinucleotide repeats.

• Ai LS, Lin CH, Hsieh M, Li C
• Arginine methylation of a glycine and arginine rich peptide derived from sequences of human FMRP and fibrillarin.
• Proc Natl Sci Counc Repub China B. 1999; 23: 175-80
• Display abstract

• N-methylation at the arginine residues in RNA binding proteins with the arginine and glycine rich RGG box has been identified. We show that a synthetic peptide R9 (GGRGRGGGF) with the RGG sequence present in human fibrillarin and fragile X mental retardation protein (FMRP) can be specifically methylated by rat brain extract. A control peptide K9 with all arginines replaced by lysines could not be methylated under the same conditions, indicating that the arginines in the peptide were the methylation sites. A novel missense mutation, which changes an arginine to a histidine in the RGG box region of FMRP in a typical fragile X patient, has been identified. A synthetic peptide with this Arg-->His (GGRGHGGGF) substitution was methylated by our in vitro methylation system to a much less extent. Amino acid analysis of the methylated R9 peptide identified the methylated amino acid as monomethylarginine. The R9 peptide may be useful for further studies on arginine methylation in RGG proteins.

• Bardoni B, Schenck A, Mandel JL
• A novel RNA-binding nuclear protein that interacts with the fragile X mental retardation (FMR1) protein.
• Hum Mol Genet. 1999; 8: 2557-66
• Display abstract

• Silenced expression of the FMR1 gene is responsible for the fragile X syndrome. The FMR1 gene codes for an RNA binding protein (FMRP), which can shuttle between the nucleus and the cytoplasm and is found associated to polysomes in the cytoplasm. By two-hybrid assay in yeast, we identified a novel protein interacting with FMRP: nuclear FMRP interacting protein (NUFIP). NUFIP mRNA expression is strikingly similar to that of the FMR1 gene in neurones of cortex, hippocampus and cerebellum. At the subcellular level, NUFIP colocalizes with nuclear isoforms of FMRP in a dot-like pattern. NUFIP presents a C2H2 zinc finger motif and a nuclear localization signal, but has no homology to known proteins and shows RNA binding activity in vitro. NUFIP does not interact with the FMRP homologues encoded by the FXR1 and FXR2 genes. Thus, these results indicate a specific nuclear role for FMRP.

• Ceman S, Brown V, Warren ST
• Isolation of an FMRP-associated messenger ribonucleoprotein particle and identification of nucleolin and the fragile X-related proteins as components of the complex.
• Mol Cell Biol. 1999; 19: 7925-32
• Display abstract

• The loss of FMR1 expression due to trinucleotide repeat expansion leads to fragile X syndrome, a cause of mental retardation. The encoded protein, FMRP, is a member of a gene family that also contains the fragile X-related proteins, FXR1P and FXR2P. FMRP has been shown to be a nucleocytoplasmic shuttling protein that selectively binds a subset of mRNAs, forms messenger ribonucleoprotein (mRNP) complexes, and associates with translating ribosomes. Here we describe a cell culture system from which we can isolate epitope-tagged FMRP along with mRNA, including its own message, and at least six other proteins. We identify two of these proteins as FXR1P and FXR2P by using specific antisera and identify a third protein as nucleolin by using mass spectrometry. The presence of nucleolin is confirmed by both reactivity with a specific antiserum as well as reverse coimmunoprecipitation where antinucleolin antiserum immunoprecipitates endogenous FMRP from both cultured cells and mouse brain. The identification of nucleolin, a known component of other mRNPs, adds a new dimension to the analysis of FMRP function, and the approach described should also allow the identification of the remaining unknown proteins of this FMRP-associated mRNP as well as the other bound mRNAs.

• Glaser D, Wohrle D, Salat U, Vogel W, Steinbach P
• Mitotic behavior of expanded CGG repeats studied on cultured cells: further evidence for methylation-mediated triplet repeat stability in fragile X syndrome.
• Am J Med Genet. 1999; 84: 226-8

• Petek E, Kroisel PM, Schuster M, Zierler H, Wagner K
• Mosaicism in a fragile X male including a de novo deletion in the FMR1 gene.
• Am J Med Genet. 1999; 84: 229-32
• Display abstract

• In most cases the fragile X syndrome is caused by an amplification of the CGG trinucleotide repeat in the 5' untranslated region of the FMR1 gene, in combination with the hypermethylation of the proximal CpG island. Recently, also a few cases with deletions or a mosaic of a deletion and a full mutation in the FMR1 gene, leading to the same phenotype, have been described. Here we report the molecular analysis of a patient with typical fragile X phenotype and mosaicism of the FMR1 genomic region consisting of a premutation, a full mutation of the CGG repeats, and a 215 bp deletion, diagnosed by Southern blot hybridisation and polymerase chain reaction (PCR). Sequence analysis of the deletion demonstrated that the 5' breakpoint of the deletion is located within a putative hotspot region 75-53 bp proximal to the CGG repeat.

• Taylor AK et al.
• Tissue heterogeneity of the FMR1 mutation in a high-functioning male with fragile X syndrome.
• Am J Med Genet. 1999; 84: 233-9
• Display abstract

• Few studies have been conducted comparing the FMR1 mutation in multiple tissues of individuals affected with fragile X syndrome. We report a postmortem study of the FMR1 mutation in multiple tissues from a high-functioning male with fragile X syndrome. This man was not mentally retarded and had only a few manifestations of the disorder such as learning disabilities and mild attention problems. Southern blot analysis of leukocytes demonstrated an unmethylated mutation with a wide span of sizes extending from the premutation to full mutation range. A similar pattern was seen in most regions of the brain. In contrast, a methylated full mutation of a single size was seen in the parietal lobe and in most non-brain tissues studied. Therefore, there were striking differences in both FMR1 mutation size and methylation status between tissues. Lack of mental retardation in this individual may have been due to sufficient expression of FMR1 protein (FMRP) in most areas of the brain. Immunocytochemistry showed FMRP expression in regions of the brain with the unmethylated mutation (superior temporal cortex, frontal cortex, and hippocampus) and no expression in the region with the methylated full mutation (parietal). Neuroanatomical studies showed no dendritic spine pathology in any regions of the brain analyzed.

• Chiurazzi P et al.
• DNA panel for interlaboratory standardization of haplotype studies on the fragile X syndrome and proposal for a new allele nomenclature.
• Am J Med Genet. 1999; 83: 347-9

• Linden MG, Tassone F, Gane LW, Hills JL, Hagerman RJ, Taylor AK
• Compound heterozygous female with fragile X syndrome.
• Am J Med Genet. 1999; 83: 318-21
• Display abstract

• We report on a 15-year-old compound heterozygous young woman with fragile X syndrome who has a full mutation of 363 repeats on one X chromosome and a premutation of 103 repeats on the other X chromosome. As predicted, subsequent testing demonstrated that her father carries a premutation (98 repeats) as does her mother (146 repeats). There is only one previous report of a compound heterozygous female with fragile X syndrome. By quantitation of Southern blot signals, the activation ratio for the premutation (the proportion of the premutation on the active X chromosome) was determined to be 0.78. Immunocytochemistry of blood smears showed fragile X mental retardation-1 protein (FMRP) expression in 63.5% of lymphocytes. Cognitively, this woman is functioning in the mid-range of involvement for fragile X females. She attends regular classes and receives supplemental assistance for her learning disabilities. She experiences behavior characteristics typical of females with fragile X syndrome including severe shyness, anxiety, panic episodes, mood swings, and attention deficits. She has responded very well to appropriate treatment including fluoxetine for anxiety, methylphenidate for attentional problems, and educational therapy.

• Zhong N, Ju W, Nelson D, Dobkin C, Brown WT
• Reduced mRNA for G3BP in fragile X cells: evidence of FMR1 gene regulation.
• Am J Med Genet. 1999; 84: 268-71
• Display abstract

• Although fragile X syndrome is caused by the absence of fragile X gene expression, little is known about the pathogenic processes underlying the mental retardation. Recent findings that the fragile X protein, FMRP, contains RNA binding motifs and nuclear transport signals and associates with ribosomes suggest that FMRP may be involved in either mRNA processing, transport, or translation. To test the hypothesis that absence of FMRP may affect the processing of specific transcripts, we have used an RNA differential display assay (RDDA) to identify differentially expressed transcripts in lymphoblast lines derived from fragile X syndrome patients. A 0.9-kb cDNA fragment that showed reduced expression in a fragile X lymphoblast cell line was found to be identical to G3BP (Ras-GTPase-Activating protein SH3-domain-binding protein). Quantitative reverse transcriptase-polymerase chain reaction showed that the expressed levels of G3BP mRNA in fragile X lymphoblast cell lines were significantly less than controls. Our results indicate that G3BP mRNA may be regulated by FMRP and supports the hypothesis that FMRP may modulate the transcription of specific transcripts.

• Currie JR, Brown WT
• KH domain-containing proteins of yeast: absence of a fragile X gene homologue.
• Am J Med Genet. 1999; 84: 272-6
• Display abstract

• The KH domain is a region defined by its homology to the RNA-binding domains of the heterogeneous nuclear ribonucleoprotein K (hnRNPK). There are two such domains in the FMR1 protein which is underexpressed in the fragile X syndrome. We developed a computer method to search the S. cerevisiae protein sequences as they became available for the KH domain of the FMR1 protein. Using our motif and FINDPATTERNS of the Wisconsin Package of GCG, nine proteins were identified in the completed yeast ORF database that contain KH domains. Five proteins have known or predicted functions; four await functional analysis. Using GeneWorks and GeneJockeyII alignments, we found that the yeast protein KH domain showing the most similarity to either FMR1P KH domain was a KH domain in HX/SCP160. Its sequence is 50% identical to the second KH domain of FMR1P. However, SCP160 contains eight conserved and six degenerate KH domains. Further analysis showed that SCP160 is a better match overall to the vertebrate and C. elegans protein Vigilin, which also contains 14 KH domains. The next most similar yeast KH domain was found in YB83, a protein shorter than FMR1P and containing three KH domains, one of which shares 45% identity with the second KH domain in FMR1P. There is no significant overall sequence similarity between this yeast protein and FMR1P. Thus, while several proteins in yeast contain KH domains, no apparent yeast homologue exists for the FMR1 protein of the fragile X gene family.

• Rudenko G, Nguyen T, Chelliah Y, Sudhof TC, Deisenhofer J
• The structure of the ligand-binding domain of neurexin Ibeta: regulation of LNS domain function by alternative splicing.
• Cell. 1999; 99: 93-101
• Display abstract

• Neurexins are expressed in hundreds of isoforms on the neuronal cell surface, where they may function as cell recognition molecules. Neurexins contain LNS domains, folding units found in many proteins like the G domain of laminin A, agrin, and slit. The crystal structure of neurexin Ibeta, a single LNS domain, reveals two seven-stranded beta sheets forming a jelly roll fold with unexpected structural similarity to lectins. The LNS domains of neurexin and agrin undergo alternative splicing that modulates their affinity for protein ligands in a neuron-specific manner. These splice sites are localized within loops at one edge of the jelly roll, suggesting a distinct protein interaction surface in LNS domains that is regulated by alternative splicing.

• Hagerman RJ, Hills J, Scharfenaker S, Lewis H
• Fragile X syndrome and selective mutism.
• Am J Med Genet. 1999; 83: 313-7
• Display abstract

• This is the first report that details an association between fragile X syndrome (FXS) and selective mutism (SM). This 12-year-old girl with heterozygous full mutation at FMR1 has a long history of social anxiety and shyness in addition to SM. Her sister also has the full mutation and a history of SM that resolved in adolescence. A beneficial response to fluoxetine and psychotherapy is described. The FMR1 mutation appears to be the first gene mutation associated with SM and further studies are recommended to assess what percentage of patients with SM have the FMR1 mutation.

• Sermon K et al.
• Preimplantation diagnosis for fragile X syndrome based on the detection of the non-expanded paternal and maternal CGG.
• Prenat Diagn. 1999; 19: 1223-30
• Display abstract

• Fragile X syndrome is the most common monogenic cause of mental retardation in boys. It is always characterized clinically by moderate mental retardation and often by a long face with large everted ears and macro-orchidism. The causal mutation is an expansion of a CGG triplet repeat in a 5' exon of the FMR-1 gene in Xq27.3. We report here for the first time a method for preimplantation genetic diagnosis (PGD) for fragile X syndrome based on the amplification of the CGG triplet in the normal allele. Our candidate-patient population, as well as two clinical preimplantation genetic diagnosis (PGD) cycles which led to a pregnancy with an unaffected fetus, are presented in this paper.

• Kaufmann WE, Abrams MT, Chen W, Reiss AL
• Genotype, molecular phenotype, and cognitive phenotype: correlations in fragile X syndrome.
• Am J Med Genet. 1999; 83: 286-95
• Display abstract

• The study of the neurobehavioral consequences of mutations of FMR1, the gene responsible for fragile X syndrome (FraX), has been based largely on correlations between mutation patterns and cognitive profile. Following the characterization of FMRP, the FMR1 gene product, preliminary correlations between FMRP levels, and neurologic phenotype have been established. However, most of these investigations have focused on individuals at both ends of the genetic and cognitive spectra of FraX, subjects with normal or premutation (PM) alleles or males with the FMR1 full mutation (FM). The present study is designed to characterize FMRP expression and to correlate it with IQ, in a sample representing a wide spectrum of FMR1 mutations. For this purpose we developed a highly sensitive immunoblotting assay using peripheral leukocytes. Three distinct patterns of FMRP immunoreactivity (-ir) emerged. Individuals with normal (n = 28) and PM (n = 8) alleles as well as most females with the FM (n = 14) showed the highest levels with multiple approximately 70-80 kDa FMRP-ir bands. Males with the FM (n = 10) demonstrated only a 70 kDa FMRP-ir band, and had significantly lower levels when compared with any previous groups. Males with mosaicism and three of 14 females with FM displayed a doublet with equal amounts of the highest and lowest molecular weight FMRP-ir bands. Multiple regression models that adjust for the effect of parental IQ indicated that both activation ratio and FMRP-ir are significantly correlated to subject IQ. We conclude that FMRP-ir offers promise as an indicator of the impact of FMR1 mutations upon neurologic function. Furthermore, our unexpected finding of FMRP-ir in all males with FM suggests that most of them are not transcriptionally silent.

• Shtang S, Perry MD, Percy ME
• Search for a Caenorhabditis elegans FMR1 homologue: identification of a new putative RNA-binding protein (PRP-1) that hybridizes to the mouse FMR1 double K homology domain.
• Am J Med Genet. 1999; 84: 283-5
• Display abstract

• A mixed stage lambdagt10 Caenorhabditis elegans cDNA library was screened with a probe derived by polymerase chain reaction from the double K homology (KH) domain of mouse FMR1 cDNA, a region that is highly conserved in the human, mouse, chicken, and frog FMR1 proteins. Four positively hybridizing cDNAs were cloned and characterized by sequencing. The overlapping sequences map to cosmid R119 from C. elegans linkage group (chromosome) I, and encode a novel proline-, polyglutamine-, and RGG box-rich putative RNA-binding protein. While the cDNA has two regions with similarity to the mouse double KH domain probe at the nucleotide level, there is no significant similarity of the amino acid sequence with human FMR1, FXR1 or FXR2, nor with KH amino acid motifs. The R119 protein, therefore, does not represent an FMR1 homologue.

• Weiler IJ, Greenough WT
• Synaptic synthesis of the Fragile X protein: possible involvement in synapse maturation and elimination.
• Am J Med Genet. 1999; 83: 248-52
• Display abstract

• Fragile X mental retardation syndrome results from the absence of or a defect in the protein (FMRP) encoded by the FMR1 gene. FMRP is found in dendrites and synapses as well as in the neuronal cell soma and nucleus, and although it is known to bind to RNA, the function of the protein in neurons is not known. We have studied activity-dependent changes in postsynaptically localized protein translation in central nervous system neurons. We find that FMRP is one of the proteins produced at synapses following stimulation of metabotropic glutamate receptors. We have also observed that Fragile X knockout mice, like human Fragile X patients, have excess numbers of long, thin, immature-appearing dendritic processes. Together, these findings suggest that FMRP plays a role in the process whereby synaptic activity during development results in structural and functional maturation of the synapse. We hypothesize that synaptic synthesis of FMRP may be essential for activity-based synapse maturation and elimination, a key process in normal brain development.

• Mathisen PM, Johnson JM, Kawczak JA, Tuohy VK
• Visinin-like protein (VILIP) is a neuron-specific calcium-dependent double-stranded RNA-binding protein.
• J Biol Chem. 1999; 274: 31571-6
• Display abstract

• Double-stranded RNA-binding proteins function in regulating the stability, translation, and localization of specific mRNAs. In this study, we have demonstrated that the neuron-specific, calcium-binding protein, visinin-like protein (VILIP) contains one double-stranded RNA-binding domain, a protein motif conserved among many double-stranded RNA-binding proteins. We showed that VILIP can specifically bind double-stranded RNA, and this interaction specifically requires the presence of calcium. Mobility shift studies indicated that VILIP binds double-stranded RNA as a single protein-RNA complex with an apparent equilibrium dissociation constant of 9.0 x 10(-6) M. To our knowledge, VILIP is the first double-stranded RNA-binding protein shown to be calcium-dependent. Furthermore, VILIP specifically binds the 3'-untranslated region of the neurotrophin receptor, trkB, an mRNA localized to hippocampal dendrites in an activity-dependent manner. Given that VILIP is also expressed in the hippocampus, these data suggest that VILIP may employ a novel, calcium-dependent mechanism to regulate its binding to important localized mRNAs in the central nervous system.

• Millan JM et al.
• Screening for FMR1 mutations among the mentally retarded: prevalence of the fragile X syndrome in Spain.
• Clin Genet. 1999; 56: 98-9

• Tamanini F et al.
• Different targets for the fragile X-related proteins revealed by their distinct nuclear localizations.
• Hum Mol Genet. 1999; 8: 863-9
• Display abstract

• Fragile X syndrome is caused by the absence of the fragile X mental retardation protein (FMRP). FMRP and its structural homologues FXR1P and FXR2P form a family of RNA-binding proteins (FXR proteins). The three proteins associate with polyribosomes as cytoplasmic mRNP particles. Here we show that small amounts of FMRP, FXR1P and FXR2P shuttle between cytoplasm and nucleus. Mutant FMRP of a severely affected fragile X patient (FMRPI304N) does not associate with polyribosomes and shuttles more frequently than normal FMRP, indicating that the association with polyribosomes regulates the shuttling process. Using leptomycin B we demonstrate that transport of the FXR proteins out of the nucleus is mediated by the export receptor exportin1. Finally, inactivation of the nuclear export signal in two FXR proteins shows that FMRP shuttles between cytoplasm and nucleoplasm, while FXR2P shuttles between cytoplasm and nucleolus. Therefore, molecular dissection of the shuttling routes used by the FXR proteins suggests that they transport different RNAs.

• Moore SJ, Strain L, Cole GF, Miedzybrodzka Z, Kelly KF, Dean JC
• Fragile X syndrome with FMR1 and FMR2 deletion.
• J Med Genet. 1999; 36: 565-6
• Display abstract

• We report a 13 year old boy with fragile X syndrome resulting from a de novo deletion of the FMR1 and FMR2 genes extending from (and including) DXS7536 proximally to FMR2 distally. The patient has severe developmental delay, epilepsy, and behavioural difficulties, including autistic features. He has epicanthic folds, in addition to facial features typical of fragile X syndrome, and marked joint hypermobility. We compare our patient to the three other cases reported in which both FMR1 and FMR2 are deleted. This case has the smallest deletion reported to date. All four patients have epilepsy and a more severe degree of mental retardation than is usual in fragile X syndrome resulting from FMR1 triplet repeat expansion. Three of the patients have joint laxity and two have epicanthic folds. We suggest that these features, in particular severe developmental delay and epilepsy, may form part of the characteristic phenotype resulting from deletion of both FMR1 and FMR2 genes. The diagnosis in this case was delayed because routine cytogenetics showed no abnormality and standard molecular tests for FMR1 triplet repeat expansion (PCR and Southern blotting) failed. Further DNA studies should be undertaken to investigate for a deletion where clinical suspicion of fragile X syndrome is strong and routine laboratory tests fail.

• Adinolfi S, Bagni C, Castiglione Morelli MA, Fraternali F, Musco G, Pastore A
• Novel RNA-binding motif: the KH module.
• Biopolymers. 1999; 51: 153-64
• Display abstract

• The KH motif has recently been identified in single or multiple copies in a number of RNA associated proteins. Here we review the current knowledge accumulated about the sequence, structure, and functions of the KH. The multidomain architecture of most of the KH-containing proteins inspired an approach based on the production of peptides spanning the sequence of an isolated KH motif. Correct identification of the minimal length necessary for producing a folded peptide has had a number of important consequences for interpreting functional data. The presence of the KH motifs in fmr1, the protein responsible for the fragile X syndrome, and their possible role in the fmr1 functions are also discussed.

• Sakai K, Kitagawa Y, Hirose G
• Analysis of the RNA recognition motifs of human neuronal ELAV-like proteins in binding to a cytokine mRNA.
• Biochem Biophys Res Commun. 1999; 256: 263-8
• Display abstract

• Human neuronal Elav-like proteins contain three RNP-type RNA recognition motifs (RRMs). Previous reports demonstrated that a single RRM of the proteins is not sufficient to bind to the uridine-rich stretch in the 3' untranslated region of mRNAs and that the bi-RRM peptide consisting of the first two RRMs is necessary for the binding. The present study was designed to examine the potential contributions of the first two RRMs when binding to a cytokine mRNA. Deletions of the internal or terminal amino acid residues of the first RRM (RRM1) of the HuC/ple21 ELAV-like protein completely abolished RNA binding. However, removal of any region of the second RRM (RRM2) except for the eight amino acid residues, which correspond to the potent fourth beta-sheet structure of RRM2, did not affect RNA binding. Conjugation of the eight amino acid residues to RRM1 enhanced the RNA binding as well as the entire RRM2, indicating that the octapeptide of RRM2 can be compensated for by the binding function of RRM2. The present study also showed that the substitutions of glutamic acid at 42 for aspartic acid and leucine at 44 for phenylalanine in the first potent alpha-helix structure of RRM1, as were seen in another ELAV-like protein Hel-N1, markedly affected the RNA binding.

• Kaufmann WE, Reiss AL
• Molecular and cellular genetics of fragile X syndrome.
• Am J Med Genet. 1999; 88: 11-24

• Agulhon C et al.
• Expression of FMR1, FXR1, and FXR2 genes in human prenatal tissues.
• J Neuropathol Exp Neurol. 1999; 58: 867-80
• Display abstract

• We analyzed the distribution of FMR1, FXR1, FXR2 mRNA, and FMRP in whole normal human embryos and in the brains of normal and fragile X fetuses. The distributions of mRNA for the 3 genes in normal whole embryos and in the brains of normal male and female carrier fetuses were similar, with large amounts of mRNA in the nervous system and in several non-nervous system tissues. No FMR1 (mRNA and protein) was detected and no evident neuropathologic abnormalities found in the brains of male carrier fetuses, suggesting that the FMR1 product (FMRP) may have no crucial function in early stages of nervous system development. FXR1 and FXR2 mRNA had the same distribution and similar intensity in the brains of normal and pathologic fetuses (female and male carriers). The coexpression in the same tissues of FMR1, FXR1, and FXR2, associated with the normal expression of FXR1 and FXR2 and the absence of obvious neuropathological abnormalities in pathological brains, supports the notion that the FXR1 and FXR2 proteins partially compensate for FMRP function. However, the absence of significant overexpression of FXR1 and FXR2 in pathological brains suggests that these genes do not compensate for the lack of FMR1 expression. Alternatively, FMR1, FXR1, and FXR2 proteins may not have compensatory functions, but instead may regulate functions by hetero or homo oligomerization, as suggested by other studies. Thus, a dominant negative effect of abnormal multimeric protein complexes lacking FMRP (e.g. by modification of FXR1 and FXR2 protein functions) may result in the fragile X syndrome phenotype.

• Khandjian EW
• Biology of the fragile X mental retardation protein, an RNA-binding protein.
• Biochem Cell Biol. 1999; 77: 331-42
• Display abstract

• 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.

• de Vries BB, van den Boer-van den Berg HM, Niermeijer MF, Tibben A
• Dilemmas in counselling females with the fragile X syndrome.
• J Med Genet. 1999; 36: 167-70
• Display abstract

• The dilemmas in counselling a mildly retarded female with the fragile X syndrome and her retarded partner are presented. The fragile X syndrome is an X linked mental retardation disorder that affects males and, often less severely, females. Affected females have an increased risk of having affected offspring. The counselling of this couple was complicated by their impaired comprehension which subsequently impaired their thinking on the different options. The woman became pregnant and underwent CVS, which showed an affected male fetus. The pregnancy was terminated. Whether nondirective counselling for this couple was the appropriate method is discussed and the importance of a system oriented approach, through involving relatives, is stressed.

• Goldman A, Jenkins T, Krause A
• Molecular evidence that fragile X syndrome occurs in the South African black population.
• J Med Genet. 1998; 35: 878-878

• Baskaran S et al.
• Triplet repeat polymorphism & fragile X syndrome in the Indian context.
• Indian J Med Res. 1998; 107: 29-36
• Display abstract

• Mental retardation due to fragile X syndrome is one of the genetic disorders caused by triplet repeat expansion. CGG repeat involved in this disease is known to exhibit polymorphism even among normal individuals. Here we describe the development of suitable probes for detection of polymorphism in CGG repeat at FMR1 locus as well as the diagnosis of fragile X syndrome. Using these methods polymorphism at the FMR1 locus has been examined in 161 individuals. Ninety eight patients with unclassified mental retardation were examined, of whom 7 were found to have the expanded (CGG) allele at the FMR1 locus. The hybridization pattern for two patients has been presented as representative data.

• Silva JA, Ferrari MM, Leong GB
• Erotomania in a case of fragile-X syndrome.
• Gen Hosp Psychiatry. 1998; 20: 126-7

• Chiurazzi P, Pomponi MG, Willemsen R, Oostra BA, Neri G
• In vitro reactivation of the FMR1 gene involved in fragile X syndrome.
• Hum Mol Genet. 1998; 7: 109-13
• Display abstract

• Fragile X syndrome is the most frequent cause of heritable mental retardation. Most patients have a mutation in the 5' untranslated region of the FMR1 gene, consisting of the amplification of a polymorphic (CGG)nrepeat sequence, and cytogenetically express the folate-sensitive fragile site FRAXA in Xq27.3. Fragile X patients harbour an expanded sequence with >200 CGG repeats (full mutation), accompanied by methylation of most cytosines of the sequence itself and of the upstream CpG island. This abnormal hypermethylation of the promoter suppresses gene transcription, resulting in the absence of the FMR1 protein. Rare individuals of normal intelligence were shown to carry a completely or partially unmethylated full mutation and to express the FMR1 protein. Given this observation and knowing that the open reading frame of the mutated FMR1 gene is intact, we decided to investigate whether its activity could be restored in vitro by inducing DNA demethylation with 5-azadeoxycytidine (5-azadC) in fragile X patients' lymphoblastoid cells. We report that treatment with 5-azadC causes reactivation of fully mutated FMR1 genes with 300-800 repeats, as shown by the restoration of specific mRNA and protein production. This effect correlates with the extent of promoter demethylation, determined by restriction analysis with methylation-sensitive enzymes. These results confirm the critical role of FMR1 promoter hypermethylation in the pathogenesis of the fragile X syndrome, provide an additional explanation for the normal IQ of the rare males with unmethylated full mutations and pave the way to future attempts at pharmacologically restoring mutant FMR1 gene activity in vivo.

• Accardo PJ, Shapiro LR
• FRAXA and FRAXE: to test or not to test?
• J Pediatr. 1998; 132: 762-4

• Fuerstenberg S, Peng CY, Alvarez-Ortiz P, Hor T, Doe CQ
• Identification of Miranda protein domains regulating asymmetric cortical localization, cargo binding, and cortical release.
• Mol Cell Neurosci. 1998; 12: 325-39
• Display abstract

• An important question in cellular and developmental biology is how a cell divides to produce daughter cells with different fates. Drosophila neuroblasts are a model system for studying asymmetric cell division: at each division, neuroblasts retain stem cell-like features, whereas their sibling ganglion mother cell (GMC) has a more restricted fate. Establishing neuroblast/GMC differences involves the asymmetric localization of proteins (Inscuteable, Miranda, Prospero, and Staufen) and RNA (prospero). All of these factors are apically localized during interphase, and all except Inscuteable move to the basal cortex at mitosis prior to being partitioned solely into the GMC. In this study, we show that Miranda is colocalized with Staufen and Prospero in neuroblasts, and is required for the asymmetric cortical localization of both proteins. Analysis of miranda mutants reveals three functional domains within the Miranda protein: (1) an N-terminal domain (1-290 aa) sufficient for association of Miranda with the cell cortex and basal localization in mitotic neuroblasts; (2) a central domain (446-727 aa) necessary for apical localization in interphase neuroblasts as well as for "cargo binding" of Prospero, Staufen, and prospero mRNA; and (3) a C-terminal domain (727-830 aa) necessary for the timely degradation of Miranda and release of its cargo from the cortex of the newborn GMC. In addition, Miranda is asymmetrically localized in epithelial cells that lack Inscuteable and divide symmetrically; thus the mechanism regulating Miranda localization is common to epithelial cells and neuroblasts, and Inscuteable is not an obligate component. Finally, we define a C-terminal domain of Staufen sufficient for Miranda-dependent cortical localization in neuroblasts.

• Brown V et al.
• Purified recombinant Fmrp exhibits selective RNA binding as an intrinsic property of the fragile X mental retardation protein.
• J Biol Chem. 1998; 273: 15521-7
• Display abstract

• Fragile X syndrome is caused by the transcriptional silencing of the FMR1 gene due to a trinucleotide repeat expansion. The encoded protein, Fmrp, has been found to be a nucleocytoplasmic RNA-binding protein containing both KH domains and RGG boxes that associates with polyribosomes as a ribonucleoprotein particle. RNA binding has previously been demonstrated with in vitro-translated Fmrp; however, it remained uncertain whether the selective RNA binding observed was an intrinsic property of Fmrp or required an associated protein(s). Here, baculovirus-expressed and affinity-purified FLAG-tagged murine Fmrp was shown to bind directly to both ribonucleotide homopolymers and human brain mRNA. FLAG-Fmrp exhibited selectivity for binding poly(G) > poly(U) >> poly(C) or poly(A). Moreover, purified FLAG-Fmrp bound to only a subset of brain mRNA, including the 3' untranslated regions of myelin basic protein message and its own message. Recombinant isoform 4, lacking the RGG boxes but maintaining both KH domains, was also purified and was found to only weakly interact with RNA. FLAG-purified I304N Fmrp, harboring the mutation of severe fragile X syndrome, demonstrated RNA binding, in contrast to previous suggestions. These data demonstrate the intrinsic property of Fmrp to selectively bind RNA and show FLAG-Fmrp as a suitable reagent for structural characterization and identification of cognate RNA ligands.

• Joseph DR
• The rat androgen-binding protein (ABP/SHBG) gene contains triplet repeats similar to unstable triplets: evidence that the ABP/SHBG and the fragile X-related 2 genes overlap.
• Steroids. 1998; 63: 2-4
• Display abstract

• The rat androgen-binding protein/sex hormone-binding globulin (ABP/SHBG) gene is regulated by promoters P1 and PA. P1 regulates the mRNA encoding secreted ABP/SHBG, whereas PA regulates an alternate mRNA which encodes a modified protein that is targeted to the nucleus. Promoter PA is GC rich, consisting of 70-80% GC residues. During routine BLAST sequence analysis it was discovered that this GC-rich region is highly related to the human fragile X-related protein 2 (FXR-2) 5'-untranslated RNA sequence. Furthermore, the nucleotide coding sequence of the initial 14 FXR-2 amino acid residues was identical in the ABP/SHBG gene. The 5'-untranslated FXR-2 sequence contains triplet (CGG) repeats, which are also present in the rat ABP/SHBG gene. The meiotic instability of CGG repeats in the human fragile X (FMR1) gene causes the fragile X mental retardation syndrome. The data presented here suggest that the ABP/SHBG and FXR-2 genes overlap with each gene transcribed in the opposite direction. In support of this structure, the human ABP/SHBG and the FXR-2 genes map to the same site on chromosome 17. Thus, the ABP/SHBG gene contains triplet repeats in the alternate promoter PA. It will be of particular interest to determine if triplet instability affects ABP/SHBG gene expression. A triplet instability in the X-linked androgen receptor gene causes spinal and bulbar muscular atrophy.

• Khandjian EW et al.
• Novel isoforms of the fragile X related protein FXR1P are expressed during myogenesis.
• Hum Mol Genet. 1998; 7: 2121-8
• Display abstract

• The fragile X syndrome results from transcriptional silencing of the FMR1 gene and the absence of its encoded FMRP protein. Two autosomal homologues of the FMR1 gene, FXR1 and FXR2, have been identified and the overall structures of the corresponding proteins are very similar to that of FMRP. Using antibodies raised against FXR1P, we observed that two major protein isoforms of relative MW of 78 and 70 kDa are expressed in different mammalian cell lines and in the majority of mouse tissues. In mammalian cells grown in culture as well as in brain extracts, both P78and P70isoforms are associated with mRNPs within translating polyribosomes, similarly to their closely related FMRP homologues. In muscle tissues as well as in murine myoblastic cell lines induced to differentiate into myotubes, FXR1P78and P70isoforms are replaced by novel unpredicted isoforms of 81-84 kDa and a novel FXR1 exon splice variant was detected in muscle RNA. While P81-84isoforms expressed after fusion into myotubes in murine myoblast cell lines grown in culture are associated with polyribosomes, this is not the case when isolated from muscle tissues since they sediment with lower S values. Immunohistochemical studies showed coexpression of FMRP and FXR1P70and P78in the cytoplasm of brain neurons, while in muscle no FMRP was detected and FXR1P81-84were mainly localized to structures within the muscle contractile bands. The complex expression pattern of FXR1P suggests tissue-specific expression for the various isoforms of FXR1 and the differential expression of FMRP and FXR1Ps suggests that in certain types of cells and tissues, complementary functions may be fulfilled by the various FMRP family members.

• Nanba E
• [Mental retardation and fragile X syndrome]
• No To Shinkei. 1998; 50: 317-23

• Zafatayeff S, Zahed L, Souraty N, Megarbane A
• [Retrospective study of nine Lebanese families with fragile X syndrome and review of the literature]
• J Med Liban. 1998; 46: 317-20
• Display abstract

• The Fragile X syndrome is the most common inherited form of mental retardation. Despite its incidence, which is estimated at 1/4000 boys, only 9 families have been documented so far in Lebanon, of which 3 have been partially investigated. This syndrome therefore seems to be largely ignored by physicians. Although no treatment is yet available for the Fragile X syndrome, the diagnosis of the disorder in a child is essential in order to provide the family with genetic counselling. The most critical point is still to convince the family of the need for such an evaluation and relieve the parents of any feeling of guilt.

• Donnenfeld AE
• Fragile X syndrome.
• Indian J Pediatr. 1998; 65: 513-8
• Display abstract

• Fragile X syndrome is the most common familial form of mental retardation. This X-linked disorder affects one in every 1000 males and one in every 2000 females. The female carrier rate in the general population is estimated to be 1/600. A fragile site at the distal long arm of the X chromosome (Xq 27.3) is the hallmark cytogenetic feature of the syndrome. Clinical features include physical as well as cognitive and neuropsychological deficits. Although fragile X syndrome follows an X-linked pattern of inheritance (which explains the predominance of affected males), females can also be affected. Many inconsistencies exist between the genetic inheritance pattern of fragile X and traditional Mendelian inheritance tenets of most X-linked diseases. Due to recent molecular advances, our understanding of the perplexing genetic issues surrounding fragile X syndrome has grown and diagnostic techniques have become both reliable and readily available.

• de Vries BB, Halley DJ, Oostra BA, Niermeijer MF
• The fragile X syndrome.
• J Med Genet. 1998; 35: 579-89
• Display abstract

• The fragile X syndrome is characterised by mental retardation, behavioural features, and physical features, such as a long face with large protruding ears and macro-orchidism. In 1991, after identification of the fragile X mental retardation (FMR1) gene, the cytogenetic marker (a fragile site at Xq27.3) became replaced by molecular diagnosis. The fragile X syndrome was one of the first examples of a "novel" class of disorders caused by a trinucleotide repeat expansion. In the normal population, the CGG repeat varies from six to 54 units. Affected subjects have expanded CGG repeats (>200) in the first exon of the FMR1 gene (the full mutation). Phenotypically normal carriers of the fragile X syndrome have a repeat in the 43 to 200 range (the premutation). The cloning of the FMR1 gene led to the characterisation of its protein product FMRP, encouraged further clinical studies, and opened up the possibility of more accurate family studies and fragile X screening programmes.

• Brown L
• The carrier rate of fragile X.
• Aust Fam Physician. 1998; 27: 577-8

• Hmadcha A, De Diego Y, Pintado E
• Assessment of FMR1 expression by reverse transcriptase-polymerase chain reaction of KH domains.
• J Lab Clin Med. 1998; 131: 170-3
• Display abstract

• The fragile X syndrome is the most frequent form of inherited mental retardation. This is caused by the transcriptional inactivation of the FMR1 gene. The KH domain is an evolutionarily conserved sequence motif present in many RNA-binding proteins including the fragile X mental retardation gene product. We have studied the expression of the gene in fresh leukocytes derived from patients and normal controls by using a reverse transcriptase-polymerase chain reaction (RT-PCR) protocol that amplifies the region of the FMR1 that contains the KH1 and KH2 domains and that has not been used in previous studies. As expected, normal expression was observed in control subjects and carriers, but FMR1 mRNA was absent in male patients with fragile X syndrome. This method was also proved to be useful for testing the expression of FMR1 in samples from several species and tissues. In all cases we obtained a similar and unique transcript. We suggest that RT-PCR from the KH domains could be the method of choice for studying FMR1 expression.

• Gustavson KH
• [Fragile X chromosome--a male weakness]
• Lakartidningen. 1998; 95: 3547-8

• Drouin R, Angers M, Dallaire N, Rose TM, Khandjian W, Rousseau F
• Structural and functional characterization of the human FMR1 promoter reveals similarities with the hnRNP-A2 promoter region.
• Hum Mol Genet. 1997; 6: 2051-60
• Display abstract

• Fragile X mental retardation syndrome is associated with an expansion of a CGG repeat within the 5'UTR of the first exon of the FMR1 gene, abnormal methylation of the CpG island in the promoter region, and a transcriptional silencing of this gene. We studied transcriptional regulation of the FMR1 gene using protein footprint analysis of the active and inactive gene in vivo . We identified four footprints within the FMR1 promoter region which correspond to consensus binding sites of known transcription factors, alpha-PAL/NRF1, Sp1, H4TF1/Sp1-like and c-myc. These footprints were present in normal cells with a transcriptionally active FMR1 gene. The same footprints were present in different cell types: primary fibroblasts, lymphoblastoid cells and peripheral lymphocytes. However, for the 1.1 kb region analyzed, no footprints were detected in a variety of cell types derived from patients with fragile X syndrome which have a transcriptionally inactive FMR1 gene. A BLAST nucleotide search identified sequence similarities between the region of the FMR1 gene containing the footprints and an analogous region within the promoter region of the gene for the heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a member of a family of ribonucleoproteins implicated in mRNA processing and nuclear-cytoplasm transport. The nucleotide sequences identified in the hnRNP-A2 promoter region correspond to the same consensus binding sites showing DNA-protein interactions in the FMR1 gene. Our previous functional studies and the studies of others demonstrate that FMR proteins, like hnRNP-A2, are also ribonucleoproteins which appear to be involved in mRNA transport. The results from our footprint studies suggest that the expression of the FMR1 gene is regulated by the binding of specific transcription factors to sequence elements in the 5' region of the gene and that this expression may be regulated by elements in common with the hnRNP-A2 gene. Common regulation of these two genes might play an important role in the cooperative processing and transport of mRNA from the nucleus to the translation machinery.

• Oostra BA, Hoogeveen AT
• Animal model for fragile X syndrome.
• Ann Med. 1997; 29: 563-7
• Display abstract

• The fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by an expansion of a polymorphic CGG repeat upstream of the coding region in the FMR1 gene. The expansion blocks expression of the FMR1 gene due to methylation of the FMR1 promoter. Functional studies on the FMR1 protein have shown that the protein can bind RNA and might be involved in transport of RNAs from the nucleus to the cytoplasm. A role of FMR1 protein on translation of certain mRNAs has been suggested. An animal model for fragile X syndrome exists and these mice show some behavioural difficulties mimicking the human fragile X syndrome phenotype. This review presents what is known about the protein and what is learned from the animal model for fragile X syndrome.

• Arvio M, Peippo M, Simola KO
• Applicability of a checklist for clinical screening of the fragile X syndrome.
• Clin Genet. 1997; 52: 211-5
• Display abstract

• In a population of 340,000 in Southern Hame, Finland, there were 541 intellectually disabled adult males (> 16 years) known to the District Organisation for the Care of the Mentally Retarded in August 1993. Of these, 197 already had a confirmed etiological diagnosis, with 20 having the fragile X syndrome. The other 344 males were screened for the fragile X syndrome using a three-step method: a clinical checklist used by a specialist nurse, a clinical examination by a physician who was very familiar with the fragile X syndrome, and the FRAXA-locus gene test. Six new fragile X males were found. The minimum prevalence of the fragile X syndrome in the district was calculated to be 1:4400.

• Kar B
• Fragile X syndrome in India.
• J Indian Med Assoc. 1997; 95: 91-91

• Lin Q, Taylor SJ, Shalloway D
• Specificity and determinants of Sam68 RNA binding. Implications for the biological function of K homology domains.
• J Biol Chem. 1997; 272: 27274-80
• Display abstract

• Sam68, a specific target of the Src tyrosine kinase in mitosis, possesses features common to RNA-binding proteins, including a K homology (KH) domain. To elucidate its biological function, we first set out to identify RNA species that bound to Sam68 with high affinity using in vitro selection. From a degenerate 40-mer pool, 15 RNA sequences were selected that bound to Sam68 with Kd values of 12-140 nM. The highest affinity RNA sequences (Kd approximately 12-40 nM) contained a UAAA motif; mutation to UACA abolished binding to Sam68. Binding of the highest affinity ligand, G8-5, was assessed to explore the role of different regions of Sam68 in RNA binding. The KH domain alone did not bind G8-5, but a fragment containing the KH domain and a region of homology within the Sam68 subgroup of KH-containing proteins was sufficient for G8-5 binding. Deletion of the KH domain or mutation of KH domain residues analogous to loss-of-function mutations in the human Fragile X syndrome gene product and the Caenorhabditis elegans tumor suppressor protein Gld-1 abolished G8-5 binding. Our results establish that a KH domain-containing protein can bind RNA with specificity and high affinity and suggest that specific RNA binding is integral to the functions of some regulatory proteins in growth and development.

• Weiler IJ et al.
• Fragile X mental retardation protein is translated near synapses in response to neurotransmitter activation.
• Proc Natl Acad Sci U S A. 1997; 94: 5395-400
• Display abstract

• Local translation of proteins in distal dendrites is thought to support synaptic structural plasticity. We have previously shown that metabotropic glutamate receptor (mGluR1) stimulation initiates a phosphorylation cascade, triggering rapid association of some mRNAs with translation machinery near synapses, and leading to protein synthesis. To determine the identity of these mRNAs, a cDNA library produced from distal nerve processes was used to screen synaptic polyribosome-associated mRNA. We identified mRNA for the fragile X mental retardation protein (FMRP) in these processes by use of synaptic subcellular fractions, termed synaptoneurosomes. We found that this mRNA associates with translational complexes in synaptoneurosomes within 1-2 min after mGluR1 stimulation of this preparation, and we observed increased expression of FMRP after mGluR1 stimulation. In addition, we found that FMRP is associated with polyribosomal complexes in these fractions. In vivo, we observed FMRP immunoreactivity in spines, dendrites, and somata of the developing rat brain, but not in nuclei or axons. We suggest that rapid production of FMRP near synapses in response to activation may be important for normal maturation of synaptic connections.

• Tamanini F et al.
• Differential expression of FMR1, FXR1 and FXR2 proteins in human brain and testis.
• Hum Mol Genet. 1997; 6: 1315-22
• Display abstract

• Lack of expression of the fragile X mental retardation protein (FMRP) results in mental retardation and macroorchidism, seen as the major pathological symptoms in fragile X patients. FMRP is a cytoplasmic RNA-binding protein which cosediments with the 60S ribosomal subunit. Recently, two proteins homologous to FMRP were discovered: FXR1 and FXR2. These novel proteins interact with FMRP and with each other and they are also associated with the 60S ribosomal subunit. Here, we studied the expression pattern of the three proteins in brain and testis by immunohistochemistry. In adult brain, FMR1, FXR1 and FXR2 proteins are coexpressed in the cytoplasm of specific differentiated neurons only. However, we observed a different expression pattern in fetal brain as well as in adult and fetal testis, suggesting independent functions for the three proteins in those tissues during embryonic development and adult life.

• Kurihara Y et al.
• Structural properties and RNA-binding activities of two RNA recognition motifs of a mouse neural RNA-binding protein, mouse-Musashi-1.
• Gene. 1997; 186: 21-7
• Display abstract

• mouse-Musashi-1 (m-Msi-1) is an RNA-binding protein, abundantly expressed in the developing mammalian central nervous system (CNS). m-Msi-1 contains two RNA recognition motifs (RRMs). In this study, we found that the N-terminal RRM of m-Msi-1 (MMA) binds strongly to poly(G) and weakly to poly(U) in a way similar to that of the full-length m-Msi-1 protein characterized previously. The C-terminal RRM of m-Msi-1 (MMB), however, does not bind to RNA. In addition, the circular dichroism (CD) spectra of the two RRMs showed that the alpha-helical content of MMA is significantly higher than that of MMB, indicating that some differences in the secondary structure may be responsible for the distinct RNA binding properties of MMA and MMB.

• Hammond LS, Macias MM, Tarleton JC, Shashidhar Pai G
• Fragile X syndrome and deletions in FMR1: new case and review of the literature.
• Am J Med Genet. 1997; 72: 430-4
• Display abstract

• The fragile X syndrome phenotype of mental retardation is almost always caused by abnormal CGG trinucleotide amplification within the FMR1 gene. Occasionally fragile X syndrome results from point mutations or deletions within or around the FMR1 locus. We have identified a mentally retarded African American male with typical fragile X phenotype and a 300-400 base pair intragenic deletion near the CGG repeat segment, present in his peripheral blood lymphocytes with no apparent mosaicism. His mother, who is not retarded, has a full FMR1 CGG expansion mutation with 700-900 repeats. A review of 23 published cases with FMR1 gene deletions shows full FMR1 mutation in the mother of only 1 other propositus, a male with FMR1 full mutation/premutation/deletion mosaicism of his cultured skin fibroblasts and peripheral blood lymphocytes. The various deletions within FMR1 and their clinical significance are reviewed.

• Beauvais P
• [Fragile X syndrome]
• Arch Pediatr. 1997; 4: 195-195

• Bardoni B, Sittler A, Shen Y, Mandel JL
• Analysis of domains affecting intracellular localization of the FMRP protein.
• Neurobiol Dis. 1997; 4: 329-36
• Display abstract

• Fragile X syndrome is the most frequent form of inherited mental retardation and it is caused by deficiency of FMRP, the protein encoded by the FMR1 gene. FMRP is a RNA binding protein of unknown function which is associated with ribosomes. FMRP is found in the cytoplasm, but it is endowed with a nuclear export signal (NES), encoded by exon 14, and a nuclear localization signal (NLS). Characterization of the FMRP NES and NLS domains is presented here. We show by site-directed mutagenesis that three leucine residues in exon 14 are functionally important for the cytoplasmic localization of FMRP. Changing these leucines to serine resulted in a nuclear localization, while another nonconservative change (leucine to tyrosine) did not show such an effect. We also show that the NLS activity is localized between residues 115 and 150, a region that lacks stretches of basic residues. Such stretches are typical of nuclear localization signals that act through the important alpha pathway. The region between residues 151 and 196 can reinforce the NLS activity. A truncated construct containing the N-terminal region of FMRP (residues 1-114) is strikingly concentrated in the nucleus. This suggests that it may contain a domain of strong affinity with a nuclear component.

• Das S et al.
• Methylation analysis of the fragile X syndrome by PCR.
• Genet Test. 1997; 1: 151-5
• Display abstract

• The fragile X syndrome is predominantly caused by a large expansion of a CGG trinucleotide repeat in the promoter region of the FMR1 gene, which is associated with methylation and downregulation of transcription. The molecular diagnosis of this disorder is based on repeat size and methylation analysis of the FMR1 gene usually by Southern blot analysis. We describe a PCR-based method for the analysis of methylation of the FMR1 gene, which involves bisulfite treatment of DNA prior to amplification. Fifty-two normal and 48 affected, premutation, or mosaic males were analyzed in a blinded study by this method. A prospective study of 30 males suspected of fragile X was also performed. Amplification specific for the methylated FMR1 sequence was readily observed in all individuals with a full mutation, whereas all normal and premutation individuals showed only amplification-specific for the unmethylated sequence, thus, allowing affected and unaffected males to be distinguished. A full mutation in the presence of mosaicism was also detectable by this method. Methylation-specific PCR appears to be a rapid and reliable tool for the diagnosis of fragile X males.

• Corbin F, Bouillon M, Fortin A, Morin S, Rousseau F, Khandjian EW
• The fragile X mental retardation protein is associated with poly(A)+ mRNA in actively translating polyribosomes.
• Hum Mol Genet. 1997; 6: 1465-72
• Display abstract

• The fragile X syndrome results from a transcriptional silencing of the FMR1 gene and the absence of its encoded protein. FMRP is a cytoplasmic RNA-binding protein, whose specific cellular function is still unknown. We present evidence that virtually all detectable cytoplasmic FMRP in mouse NIH 3T3 and human HeLa cells is found strictly in association with mRNA in actively translating polyribosomes. Furthermore, FMRP released from polyribosomes is associated with ribonucleoprotein complexes with sedimentation coefficients of 60-70S and selection on oligo(dT)-cellulose reveals that this association is specific to poly(A)-containing mRNPs. This association with actively translating polyribosomes is not affected by alteration of translational processes induced by serum stimulation and starvation in NIH 3T3 cells, suggesting that FMR1 expression is not cell cycle regulated and that FMRP might have a house-keeping function. FXR2 protein, which is closely related to FMRP, is also detected associated with mRNPs in translating polyribosomes. The results strongly suggest that FMRP might be a mRNA chaperone interacting with mRNP complexes.

• Willemsen R et al.
• Rapid antibody test for diagnosing fragile X syndrome: a validation of the technique.
• Hum Genet. 1997; 99: 308-11
• Display abstract

• To date, the identification of patients and carriers of the fragile X syndrome has been carried out by DNA analysis by means of the polymerase chain reaction and Southern blot analysis. This direct DNA analysis allows both the size of the CGG repeat and methylation status of the FMR1 gene to be determined. We have recently presented a rapid antibody test on blood smears based on the presence of FMRP, the protein product of the FMR1 gene, in lymphocytes from normal individuals and the absence of FMRP in lymphocytes from patients. Here, we have tested the diagnostic value of this new technique by studying FMRP expression in 173 blood smears from normal individuals and fragile X patients. The diagnostic power of the antibody test is "perfect" for males, whereas the results are less specific for females.

• Feng Y, Absher D, Eberhart DE, Brown V, Malter HE, Warren ST
• FMRP associates with polyribosomes as an mRNP, and the I304N mutation of severe fragile X syndrome abolishes this association.
• Mol Cell. 1997; 1: 109-18
• Display abstract

• Fragile X mental retardation is caused by the lack of FMRP, a selective RNA-binding protein associated with ribosomes. A missense mutation, I304N, has been found to result in an unusually severe phenotype. We show here that normal FMRP associates with elongating polyribosomes via large mRNP particles. Despite normal expression and cytoplasmic mRNA association, the I304N FMRP is incorporated into abnormal mRNP particles that are not associated with polyribosomes. These data indicate that association of FMRP with polyribosomes must be functionally important and imply that the mechanism of the severe phenotype in the I304N patient lies in the sequestration of bound mRNAs in nontranslatable mRNP particles. In the absence of FMRP, these same mRNAs may be partially translated via alternative mRNPs, although perhaps abnormally localized or regulated, resulting in typical fragile X syndrome.

• Barnicoat A
• Screening for fragile X syndrome: a model for genetic disorders?
• BMJ. 1997; 315: 1174-5

• van Rijn MA, de Vries BB, Tibben A, van den Ouweland AM, Halley DJ, Niermeijer MF
• DNA testing for fragile X syndrome: implications for parents and family.
• J Med Genet. 1997; 34: 907-11
• Display abstract

• The fragile X syndrome is an X linked, semidominant mental retardation disorder caused by the amplification of a CGG repeat in the 5' UTR of the FMR1 gene. Nineteen fragile X families in which the mutated FMR1 gene segregated were evaluated. The implications of the diagnosis for the parents and family were studied through pedigree information, interviews, and questionnaires. Information about the heredity of fragile X syndrome was only disseminated by family members to a third (124/366) of the relatives with an a priori risk of being a carrier of the fragile X syndrome. Twenty-six percent (94/366) of the relatives were tested. Transmission of information among first degree relatives seemed satisfactory but dropped off sharply with increasing distance of the genetic relationship, leaving 66% uninformed. This is particularly disadvantageous in an X linked disease. Of those subjects tested, 42% (39/94) had a premutation and 18% (17/94) had a full mutation. On average, in each family one new fragile X patient and two new carriers were found. When people have the task of transmitting genetic information to their relatives, they usually feel responsible and capable; however, reduced acquaintance and contact with more distant relative severely reduces the effectiveness of such transfer of information in fragile X families.

• Loesch DZ
• FMR1 fully expanded mutation with minimal methylation in a high functioning fragile X male.
• J Med Genet. 1997; 34: 350-350

• Fridell RA, Benson RE, Hua J, Bogerd HP, Cullen BR
• A nuclear role for the Fragile X mental retardation protein.
• EMBO J. 1996; 15: 5408-14
• Display abstract

• 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).

• Eberhart DE, Malter HE, Feng Y, Warren ST
• The fragile X mental retardation protein is a ribonucleoprotein containing both nuclear localization and nuclear export signals.
• Hum Mol Genet. 1996; 5: 1083-91
• Display abstract

• Fragile X syndrome is a frequent cause of mental retardation resulting from the absence of FMRP, the protein encoded by the FMR1 gene. FMRP is an RNA-binding protein of unknown function which is associated with ribosomes. To gain insight into FMRP function, we performed immunolocalization analysis of FMRP truncation and fusion constructs which revealed a nuclear localization signal (NLS) in the amino terminus of FMRP as well as a nuclear export signal (NES) encoded by exon 14. A 17 amino acid peptide containing the FMRP NES, which closely resembles the NES motifs recently described for HIV-1 Rev and PKI, is sufficient to direct nuclear export of a microinjected protein conjugate. Sucrose gradient analysis shows that FMRP ribosome association is RNA-dependent and FMRP is found in ribonucleoprotein (RNP) particles following EDTA treatment. These data are consistent with nascent FMRP entering the nucleus to assemble into mRNP particles prior to export back into the cytoplasm and suggests that fragile X syndrome may result from altered translation of transcripts which normally bind to FMRP.

• Reyniers E et al.
• Mean corpuscular hemoglobin is not increased in Fmr1 knockout mice.
• Hum Genet. 1996; 97: 49-50
• Display abstract

• A slight increase in mean corpuscular hemoglobin (MCH) has been reported in erythrocytes from human fragile X patients. As it is difficult to perform case-controlled studies in patients with fragile X syndrome, we studied MCH in erythrocytes from transgenic mice with an Fmr1 knockout. None of the knockout mice showed increased MCH levels when compared with normal littermates. We conclude that it is unlikely that the FMR1 gene product has an effect on MCH.

• Loesch DZ
• Fragile X: clinical associations.
• Am J Med Genet. 1996; 64: 413-4

• Buckanovich RJ, Yang YY, Darnell RB
• The onconeural antigen Nova-1 is a neuron-specific RNA-binding protein, the activity of which is inhibited by paraneoplastic antibodies.
• J Neurosci. 1996; 16: 1114-22
• Display abstract

• Nova-1, a protein expressed in tumors and neurons, is a target antigen in a human paraneoplastic motor disorder [paraneoplastic opsoclonus-myoclonus ataxia (POMA)]. We evaluated the relationship between the function of Nova-1 and its role as a disease antigen. We show that Nova-1 is a neuron-specific RNA-binding protein with sequence and functional similarities to FMR-1. Nova-1 mRNA is restricted to the subcortical nervous system, and the protein binds to RNA with high affinity. Nova-1 KH domains mediate this RNA binding, and point mutations within them abrogate binding. POMA disease antisera (6/6) recognize the third KH domain but not an inactive point mutant, and affinity-purified antibody blocks Nova-1 RNA binding. Thus, a cardinal feature of POMA is the production of antibodies that inhibit Nova-1-RNA interactions, suggesting such inhibition may cause the neurological disease.

• Mariappan SV et al.
• Solution structures of the individual single strands of the fragile X DNA triplets (GCC)n.(GGC)n.
• Nucleic Acids Res. 1996; 24: 784-92
• Display abstract

• Three-dimensional structures of the fragile X triplet repeats (GCC)n and (GGC)n are derived by using one- dimensional/two-dimensional NMR. Under a wide range of solution conditions (10-150 mM NaCl,pH6-7)(GCC)5-7 strands form exclusively slipped hairpins with a 3' overhanging C. The slipped hairpins of (GCC)n strands show the following structural characteristics: (i) maximization of Watson-Crick G.C pairs; (ii) formation of C.C mispairs at the CpG steps in the stem; (iii) C2'-endo, anti conformations for all the nucleotides. The ability of (GCC)n strands to form hairpin structures more readily than complementary (GGC)n strands suggests preferential slippage during replication and subsequent expansion of the (GCC)n strands. In addition, the C.C. mispairs at the CpG site of (GCC)n hairpins account for their exceptional substrate efficiencies for human methyltransferase. Gel electrophoresis data show that (GGC)n strands form both hairpin and mismatched duplex structures in 10-150 mM NaCl (ph 6-7) for n < 10, but for n > or + 11 hairpin structures are exclusively present. However, (GGC)n strands remain predominantly in the duplex state for n=4-11 under NMR solution conditions, which require DNA concentrations 100- to 1000-fold higher than in gel electrophoresis. NMR analyses of [(GGC)n]2 duplexes for n=4-6 show the presence of Watson-Crick G.C and mismatched G anti G syn pairs. The mismatches adjacent to the CpG step introduce local structural flexibility in these duplexes. Similar structural properties are also expected in the stem of the hairpins formed by (GGC)n strands.

• Turner G, Webb T, Wake S, Robinson H
• Prevalence of fragile X syndrome.
• Am J Med Genet. 1996; 64: 196-7
• Display abstract

• The much-quoted prevalence figure of 1:1,000 males for fragile X syndrome is an overestimate in a mixed ethnic population. A reexamination of the individuals from whom those data were derived using molecular diagnostic techniques demonstrates a more realistic figure of 1:4,000 males.

• Tamanini F et al.
• FMRP is associated to the ribosomes via RNA.
• Hum Mol Genet. 1996; 5: 809-13
• Display abstract

• The FMR1 transcript is alternatively spliced and generates different splice variants coding for FMR1 proteins (FMRP) with a predicted molecular mass of 70-80 kDa. FMRP is widely expressed and localized in the cytoplasm. To study a possible interaction with other cellular components, FMRP was isolated and characterized under non-denaturing conditions. Under physiological salt conditions FMRP appears to have a molecular mass of > 600 kDa, indicating a binding to other cellular components. This interaction is disrupted in the presence of high salt concentrations. The dissociation conditions to free FMRP from the complex are similar to the dissociation of FMRP from RNA as shown before. The binding of FMRP from the complex is also disrupted by RNAse treatment. That the association of FMRP to a high molecular weight complex possibly occurs via RNA, is further supported by the observation that the binding of FMRP, containing an lle304Asn substitution, to the high molecular weight complex is reduced. An equal reduced binding of mutated FMRP to RNA in vitro was observed before under the same conditions. The reduced binding of FMRP with the lle304Asn substitution further indicates that the interaction to the complex indeed occurs via FMRP and not via other RNA binding proteins. In a reconstitution experiment where the low molecular mass FMRP (70-80 kDa) is mixed with a reticulocyte lysate (enriched in ribosomes) it was shown that FMRP can associate to ribosomes and that this binding most likely occurs via RNA.

• Dejgaard K, Leffers H
• Characterisation of the nucleic-acid-binding activity of KH domains. Different properties of different domains.
• Eur J Biochem. 1996; 241: 425-31
• Display abstract

• The KH module is a sequence motif recently identified in a number of diversified RNA-binding proteins and suggested to be the functional element responsible for RNA binding. So far, however, this hypothesis has not received direct experimental support. We have expressed the three KH-domains from heterogeneous nuclear ribonucleoprotein K (hnRNP-K), the poly(C)-binding proteins PCBP-1 and PCBP-2, the first three to four domains from the high-density binding protein HBP, the one and a half domain from the archaeon Halobacterium halobium ORF139 and one and a half domain of the fragile-X protein FMR1 in Escherichia coli and analysed their nucleic-acid-binding properties in vitro. The results showed that the in vitro poly(rC)-binding activity of hnRNP-K can be assigned to KH-domain 3, whereas both domains 1 and 3 in the PCBPs bind poly(rC). In addition, all these domains exhibit binding activity towards other nucleic acids, albeit at a significantly lower level. The first KH domain from the FMR1 protein binds poly(rG) and single-stranded and double-stranded DNA. The N-terminal three or four domains from HBP bind poly(rG) and, at a much lower level, single-stranded and double-stranded DNA. Thus, single KH domains are discrete and independent nucleic-acid-binding units. Moreover, different KH domains bind different nucleic acids, suggesting that KH domains are composed of a conserved, weakly nucleic-acid-binding, structure that is fine tuned, by sequence variation, resulting in sequence-specific nucleic-acid-binding entities.

• Oostra BA
• Fragile X syndrome in humans and mice.
• Acta Genet Med Gemellol (Roma). 1996; 45: 93-108

• Eberhart DE, Warren ST
• The molecular basis of fragile X syndrome.
• Cold Spring Harb Symp Quant Biol. 1996; 61: 679-87

• Liu Q et al.
• Molecular characterization of the protein products of the fragile X syndrome gene and the survival of motor neurons gene.
• Cold Spring Harb Symp Quant Biol. 1996; 61: 689-97

• Price DK, Zhang F, Ashley CT Jr, Warren ST
• The chicken FMR1 gene is highly conserved with a CCT 5'-untranslated repeat and encodes an RNA-binding protein.
• Genomics. 1996; 31: 3-12
• Display abstract

• The transcriptional silencing of the human gene, fragile X mental retardation 1 (FMR1), is due to abnormal methylation in response to an expanded 5'-untranslated CGG trinucleotide repeat and accounts for most cases of fragile X syndrome, a frequent inherited form of mental retardation. Although the encoded fragile X mental retardation protein (FMRP) is known to have properties of a RNA-binding protein, the precise function of FMRP remains to be elucidated. We report the cloning of the chicken homolog of FMR1 and show strong evolutionary conservation, with nucleotide and amino acid identities of 85 and 92%, respectively, between chicken and human. In place of the mammalian CGG trinucleotide repeat, a 99-nt tripartite repetitive element containing a CCT trinucleotide repeat flanked on both sides by dinucleotide repeats was identified. Blocks of highly conserved 3'-untranslated sequence were also found. Within the coding region, two copies each of the highly conserved K homology motif and the Arg-Gly-Gly (RGG) box motif, both ribonucleotide particle family domains implicated in RNA binding, were identified. Chicken FMRP was found to bind RNA in vitro, and this activity correlated with the presence of the carboxy-terminal portion of the protein that includes the RGG motifs.

• Willemsen R, Bontekoe C, Tamanini F, Galjaard H, Hoogeveen A, Oostra B
• Association of FMRP with ribosomal precursor particles in the nucleolus.
• Biochem Biophys Res Commun. 1996; 225: 27-33
• Display abstract

• The fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by an expansion of a polymorphic CGG repeat upstream the coding region of the FMR1 gene. These expansions are associated with hypermethylation of the FMR1 gene, which results in the absence of the gene product, the FMR1 protein (FMRP). The physiological function of FMRP remains to be determined. We studied the ultrastructural localization of FMRP at the electron microscopical level using the immunogold technique. FMRP is associated with ribosomes attached to the endoplasmic reticulum and with ribosomes free in the cytoplasm. In addition, FMRP is found in the nucleus where the protein is associated with the granular component of the nucleolus. The cellular function of FMRP is hypothesized in relation to its subcellular distribution.

• Sittler A, Devys D, Weber C, Mandel JL
• Alternative splicing of exon 14 determines nuclear or cytoplasmic localisation of fmr1 protein isoforms.
• Hum Mol Genet. 1996; 5: 95-102
• Display abstract

• Impaired expression of the FMR1 gene is responsible for the fragile X mental retardation syndrome. The FMR1 gene encodes a cytoplasmic protein with RNA-binding properties. Its complex alternative splicing leads to several isoforms, whose abundance and specific functions in the cell are not known. We have cloned in expression vectors, cDNAs corresponding to several isoforms. Western blot comparison of the pattern of endogenous FMR1 proteins with these transfected isoforms allowed the tentative identification of the major endogenous isoform as ISO 7 and of a minor band as an isoform lacking exon 14 sequences (ISO 6 or ISO 12), while some other isoforms (ISO 4, ISO 5) were not expressed at detectable levels. Surprisingly, in immunofluorescence studies, the transfected splice variants that exclude exon 14 sequences (and have alternate C-terminal regions) were shown to be nuclear. Such differential localisation was however not seen in subcellular fractionation studies. Analysis of various deletion mutants suggests the presence of a cytoplasmic retention domain encoded in exon 14 and of a nuclear association domain encoded within the first eight exons that appear however to lack a typical nuclear localisation signal.

• Mila M, Castellvi-Bel S, Sanchez A, Lazaro C, Villa M, Estivill X
• Mosaicism for the fragile X syndrome full mutation and deletions within the CGG repeat of the FMR1 gene.
• J Med Genet. 1996; 33: 338-40
• Display abstract

• The main mutation responsible for the fragile X syndrome is the expansion of an untranslated CGG repeat in the first exon of the FMR1 gene, associated with the hypermethylation of the proximal CpG island and the CGG repeat region, and repression of transcription of FMR1. Fragile X syndrome mosaicism has been described as the coexistence of the full mutation and the permutation. We present here two cases of mosaicism for the full mutation in the FMR1 gene and deletions involving the CGG repeat region. In one case the deletion removed 113 bp proximal to the CGG repeat and part of the repeat itself, leaving 30 pure repeats, and representing 17% of lymphocytes of the patient. The 5' breakpoint of this deletion falls outside the putative hotspot for deletions in the CGG region of FMR1. In the second case the deleted region only involved the CGG sequence (leaving 15 pure repeats), with normal sequences flanking the repeat; this deleted ("normal") FMR1 was estimated to be in about 31% of blood lymphocytes. This second case can be considered a true regression of the CGG FMR1 expansion to a normal sized allele, although in mosaic form.

• Mornet E, Simon-Bouy B
• [Molecular biology of fragile X syndrome: recent data and diagnostic applications]
• Arch Pediatr. 1996; 3: 814-21
• Display abstract

• Fragile X syndrome is the most common cause of inherited mental retardation. Since the identification of the mutation, a Cytosine-Guanine-Guanine repeat in the Fragile X Mental Retardation (FMR1) gene, the genetic counselling and the diagnosis of the disease have been dramatically improved. The nature of the mutation and its size must be integrated in the calculation of the risk of transmission of mental retardation and in the genetic counselling in the family. Out of 245 patients affected with mental retardation referred to our laboratory, we found 37 (15%) fragile X patients, allowing to screen for the mutation in the family and to propose prenatal diagnosis in carrier females.

• Siomi MC, Zhang Y, Siomi H, Dreyfuss G
• 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.
• Mol Cell Biol. 1996; 16: 3825-32
• Display abstract

• 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.

• Brown WT, Zhong N, Dobkin C
• Positive fragile X microsatellite associations point to a common mechanism of dynamic mutation evolution.
• Am J Hum Genet. 1996; 58: 641-3

• Kettani A, Kumar RA, Patel DJ
• Solution structure of a DNA quadruplex containing the fragile X syndrome triplet repeat.
• J Mol Biol. 1995; 254: 638-56
• Display abstract

• Both X-ray and NMR structural studies have defined the polymorphic nature of G-quadruplexes generated through mutual stacking of G.G.G.G tetrads by guanine rich telomeric sequences. Recently, the fragile X syndrome d(C-G-G)n triplet nucleotide repeat has been shown to form a stable quadruplex of undefined structure in monovalent cation solution. We have undertaken a structural characterization of the d(G-C-G-G-T3-G-C-G-G) undecanucleotide to elucidate the structural alignments associated with quadruplex formation by this oligomer which contains sequence elements associated with the fragile X syndrome triplet repeat. d(G-C-G-G-T3-G-C-G-G) in Na+ cation solution forms a quadruplex through dimerization of two symmetry related hairpins with the lateral connecting T3 loops positioned at opposite ends of the quadruplex. This novel NMR-molecular dynamics based solution structure contains internal G.C.G.C tetrads sandwiched between terminal G.G.G.G tetrads. Watson-Crick G.C base-pairs within individual hairpins dimerize through their major groove edges using bifurcated hydrogen bonds to form internal G(anti).C(anti).G(anti).C(anti) tetrads. Adjacent strands are anti-parallel to each other around the symmetric G-quadruplex which contains two distinct narrow and two symmetric wide grooves. By contrast, the terminal G-tetrads adopt G(syn).G(anti).G(syn).G(anti) alignments. The structure of the d(G-C-G-G-T3-G-C-G-G) quadruplex with its multi-layer arrangement of G.G.G.G and G.C.G.C tetrads greatly expands on our current knowledge of quadruplex folding topologies. Our results establish the pairing alignments that can be potentially utilized by the fragile X syndrome triplet repeat to form quadruplex structures through dimerization of hairpin stems. The formation of novel G.C.G.C tetrads through dimerization of Watson-Crick G.C base-pairs is directly relevant to the potential pairing alignments of helical stems in genetic recombination.

• Siomi MC, Siomi H, Sauer WH, Srinivasan S, Nussbaum RL, Dreyfuss G
• FXR1, an autosomal homolog of the fragile X mental retardation gene.
• EMBO J. 1995; 14: 2401-8
• Display abstract

• 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.

• Mahone M, Saffman EE, Lasko PF
• Localized Bicaudal-C RNA encodes a protein containing a KH domain, the RNA binding motif of FMR1.
• EMBO J. 1995; 14: 2043-55
• Display abstract

• The Bicaudal-C (Bic-C) gene of Drosophila melanogaster is required for correct targeting of the migrating anterior follicle cells and for specifying anterior position. Females lacking any wild type copies of Bic-C produce only eggshells open at the anterior end, because of the failure of the columnar follicle cells to migrate in the correct position at the nurse cell--oocyte boundary. Embryos which develop from eggs produced in females with only one wild type copy of Bic-C show defects in anterior patterning and an abnormal persistence of oskar RNA in anterior regions. We cloned Bic-C and found that, in ovaries, Bic-C RNA is expressed only in germline cells. Bic-C RNA is localized to the oocyte in early oogenesis, and later concentrates at its anterior cortex. The Bic-C protein includes five KH domains similar to those found in the human fragile-X protein FMR1. Alteration of a highly conserved KH domain codon by mutation abrogates in vivo Bic-C function. These results suggest roles for the Bic-C protein in localizing RNAs and in intercellular signaling.

• Morris A, Morton NE, Collins A, Macpherson J, Nelson D, Sherman S
• An n-allele model for progressive amplification in the FMR1 locus.
• Proc Natl Acad Sci U S A. 1995; 92: 4833-7
• Display abstract

• An n-allele model is developed for the FMR1 locus, which causes the fragile X syndrome, where n is the number of triplet repeats in the first exon. Frequencies in the general population and in index families are used to generate an n to n + delta transition matrix that predicts specific risks in satisfactory agreement with observation. However, until sequencing distinguishes between stable and unstable alleles with the same value of n, it is premature to infer whether allelic frequencies at the FMR1 locus are at equilibrium or, as some have suggested, are evolving toward higher frequencies of the pathogenic allele.

• Oostra BA, Halley DJ
• Complex behavior of simple repeats: the fragile X syndrome.
• Pediatr Res. 1995; 38: 629-37
• Display abstract

• The fragile X syndrome of mental retardation is one of the most common genetic diseases. The mutation causing this disease was the first of a new class of mutations involving repeat sequences disturbing gene function. Fragile X mutations consist of an expansion of a CGG trinucleotide repeat in the FMR1 gene, which is inactivated as a result of this expansion. The lack of FMR1 protein is believed to be responsible for the mental retardation. The mechanism and the timing of the repeat amplification are still not known. Characterization of the repeat has clarified the genetics of fragile X syndrome, and has given tools to establish the diagnosis and to determine carrier status.

• Verheij C et al.
• Characterization of FMR1 proteins isolated from different tissues.
• Hum Mol Genet. 1995; 4: 895-901
• Display abstract

• FMR1 protein expression was studied in different tissues. In human, monkey and murine tissues, high molecular mass FMR1 proteins (67-80 kDa) are found, as shown in lymphoblastoid cells lines. The identity of these proteins was confirmed by their absence in tissues from patients with the fragile X syndrome and a FMR1 knock-out mouse. An Ile367Asn substitution in the FMR1 protein did not alter the translation, processing and localization of FMR1 proteins in lymphoblastoid cells from a patient carrying this mutation. All the high molecular mass FMR1 proteins isolated from normal lymphoblastoid cells and cells from the patient with the Ile367Asn substitution were able to bind RNA. However, the FMR1 proteins of the patient had reduced affinity for RNA binding at high salt concentrations. In some human, monkey and murine tissues low molecular mass FMR1 proteins (39-41 kDa) were found, which had the same N terminus as the 67-90 kDa isoforms, but differ in their C terminus and are therefore most likely the result of carboxy-terminal proteolytic cleavage. These low molecular mass FMR1 proteins did not bind RNA, in contrast with the high molecular mass FMR1 proteins. The significance of these low molecular mass proteins remains to be studied.

• Quan F, Grompe M, Jakobs P, Popovich BW
• Spontaneous deletion in the FMR1 gene in a patient with fragile X syndrome and cherubism.
• Hum Mol Genet. 1995; 4: 1681-4

• Nagai K, Oubridge C, Ito N, Avis J, Evans P
• The RNP domain: a sequence-specific RNA-binding domain involved in processing and transport of RNA.
• Trends Biochem Sci. 1995; 20: 235-40
• Display abstract

• The RNP domain is found in a number of proteins involved in processing and transport of mRNA precursors. The crystal structure of a complex between the U1A spliceosomal protein and its cognate RNA hairpin at 1.92 A resolution reveals the molecular basis of sequence-specific RNA recognition by the RNP domain.

• Oostra BA, Willems PJ
• A fragile gene.
• Bioessays. 1995; 17: 941-7
• Display abstract

• 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.

• Sutherland GR et al.
• Sixth international workshop on the fragile X and X-linked mental retardation.
• Am J Med Genet. 1994; 51: 281-93

• Gasparini S et al.
• The low molecular weight protein which co-purifies with alpha-latrotoxin is structurally related to crustacean hyperglycemic hormones.
• J Biol Chem. 1994; 269: 19803-9
• Display abstract

• LMWP is the low molecular weight protein which copurifies with alpha-latrotoxin, the main neurotoxin from the black widow venom. It contains 70 residues and three disulfides. We found that its primary structure, including its 6 half-cystines, can be aligned with the amino acid sequences of crustacean hyperglycemic hormones (CHHs) which contain 72-73 residues and three disulfides. To further investigate this structural relationship, we produced a recombinant analog of LMWP in which the unique Met was changed in Leu (LMWPM35L). LMWPM35L was produced as a folded fusion protein in the periplasm of Escherichia coli and was generated in vitro by treating the fusion protein with cyanogen bromide. We showed that LMWPM35L and CHHs have an identical disulfide pairing pattern and possess some alpha-helical structure, as deduced from a comparison of their circular dichroism spectra. In addition, LMWPM35L and CHHs are consensually predicted to possess a helical structure within the region 13-17. Together, the data indicate that CHHs are structurally related to LMWPM35L and presumably also to LMWP. Finally, preliminary studies showed that LMWPM35L is not toxic to mice and does not form channels in lipid bilayers, two well-known properties of alpha-latrotoxin preparations.

• Verkerk AJ et al.
• Alternative splicing in the fragile X gene FMR1.
• Hum Mol Genet. 1993; 2: 1348-1348

• Ashley CT Jr, Wilkinson KD, Reines D, Warren ST
• FMR1 protein: conserved RNP family domains and selective RNA binding.
• Science. 1993; 262: 563-6
• Display abstract

• Fragile X syndrome is the result of transcriptional suppression of the gene FMR1 as a result of a trinucleotide repeat expansion mutation. The normal function of the FMR1 protein (FMRP) and the mechanism by which its absence leads to mental retardation are unknown. Ribonucleoprotein particle (RNP) domains were identified within FMRP, and RNA was shown to bind in stoichiometric ratios, which suggests that there are two RNA binding sites per FMRP molecule. FMRP was able to bind to its own message with high affinity (dissociation constant = 5.7 nM) and interacted with approximately 4 percent of human fetal brain messages. The absence of the normal interaction of FMRP with a subset of RNA molecules might result in the pleiotropic phenotype associated with fragile X syndrome.

• Siomi H, Siomi MC, Nussbaum RL, Dreyfuss G
• The protein product of the fragile X gene, FMR1, has characteristics of an RNA-binding protein.
• Cell. 1993; 74: 291-8
• Display abstract

• Fragile X syndrome is one of the most common human genetic diseases and the most common cause of hereditary mental retardation. The gene that causes fragile X syndrome, FMR1, was recently identified and sequenced and found to encode a putative protein of unknown function. Here we report that FMR1 contains two types of sequence motifs recently found in RNA-binding proteins: an RGG box and two heterogeneous nuclear RNP K homology domains. We also demonstrate that FMR1 binds RNA in vitro. Using antibodies to FMR1, we detect its expression in divergent organisms and in cells of unaffected humans, but fragile X-affected patients express little or no FMR1. These findings demonstrate that FMR1 expression is directly correlated with the fragile X syndrome and suggest that anti-FMR1 antibodies will be important for diagnosis of fragile X syndrome. Furthermore, the RNA binding activity of FMR1 opens the way to understanding the function of FMR1.

• Bachner D et al.
• Enhanced expression of the murine FMR1 gene during germ cell proliferation suggests a special function in both the male and the female gonad.
• Hum Mol Genet. 1993; 2: 2043-50
• Display abstract

• To elucidate the function of the FMR1 gene, we applied RNA in situ hybridization to cryosections of mice from different developmental stages. The murine Fmr-1 was found transcribed in a ubiquitous manner with an expression pattern similar to glyceraldehyd phosphate dehydrogenase, Gapdh, which was used as a control gene. A significant difference in the Fmr-1 expression pattern, however, was markedly enhanced expression specifically confined to the testis and the fetal ovary. In the immature and mature testis an elevated level of Fmr-1 expression is found in type A1 spermatogonia. Expression in the testis is observed in fetal life, reaches the highest level in the immature testis, and declines early in adult life. In the mature ovary no specific Fmr-1 expression signal was found but enhanced levels were seen in the fetal ovary. At this developmental stage proliferation of oogonia takes place. It is suggested that FMR1 serves a special function during germ cell proliferation in males and females. These findings are discussed in the light of the current observation that fragile X patients produce only sperm with a premutation sized allele. Two hypotheses are put forward: (1) In males lack of FMR1 function results in a premeiotic defect preventing spermatogonia with a full mutation to reach meiosis. A fragile X mutation can be passed on to offsprings only as a premutation (selection hypothesis). (2) Transition of a premutation allele to full mutation occurs in a postzygotic stage after separation of the germ line and is restricted to soma cells (restriction hypothesis). Expression of FMR1 in proliferating germ cells is in line with both hypothesis.

• Gibson TJ, Rice PM, Thompson JD, Heringa J
• KH domains within the FMR1 sequence suggest that fragile X syndrome stems from a defect in RNA metabolism.
• Trends Biochem Sci. 1993; 18: 331-3

• von Koskull H, Leisti J
• [Discovery of the gene defect in fragile X syndrome reveals a new mode of inheritance and improves diagnosis]
• Duodecim. 1992; 108: 1445-7

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