Characterization of dFMR1, a Drosophila melanogaster Homolog of the Fragile X Mental Retardation Protein

ABSTRACT 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.

[1]  I. Weiler,et al.  Abnormal dendritic spines in fragile X knockout mice: maturation and pruning deficits. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[2]  T. Gibson,et al.  Three-Dimensional Structure and Stability of the KH Domain: Molecular Insights into the Fragile X Syndrome , 1996, Cell.

[3]  M. Fortini,et al.  Neurogenic phenotypes and altered Notch processing in Drosophila Presenilin mutants , 1999, Nature.

[4]  J. Rine.  Gene overexpression in studies of Saccharomyces cerevisiae. , 1991, Methods in enzymology.

[5]  J. Sutcliffe,et al.  Variation of the CGG repeat at the fragile X site results in genetic instability: Resolution of the Sherman paradox , 1991, Cell.

[6]  J. Sutcliffe,et al.  Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome , 1991, Cell.

[7]  R I Richards,et al.  Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n , 1991, Science.

[8]  P. Rørth,et al.  A modular misexpression screen in Drosophila detecting tissue-specific phenotypes. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[9]  G. Rubin,et al.  seven in absentia, a gene required for specification of R7 cell fate in the Drosophila eye , 1990, Cell.

[10]  P. Lasko,et al.  Localized Bicaudal‐C RNA encodes a protein containing a KH domain, the RNA binding motif of FMR1 , 1995, The EMBO journal.

[11]  G. Rubin,et al.  Genetic transformation of Drosophila with transposable element vectors. , 1982, Science.

[12]  G. Dreyfuss,et al.  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 , 1996, Molecular and cellular biology.

[13]  G. Rubin,et al.  Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase , 1991, Cell.

[14]  S. Benzer,et al.  Molecular characterization and expression of sevenless, a gene involved in neuronal pattern formation in the Drosophila eye , 1987, Cell.

[15]  G. Rubin,et al.  Transposition of cloned P elements into Drosophila germ line chromosomes. , 1982, Science.

[16]  A T Hoogeveen,et al.  FMRP is associated to the ribosomes via RNA. , 1996, Human molecular genetics.

[17]  G. Rubin,et al.  The Role of the Genome Project in Determining Gene Function: Insights from Model Organisms , 1996, Cell.

[18]  G. Sutherland.  Fragile sites on human chromosomes , 1985 .

[19]  C. Petit,et al.  Abnormal pattern detected in fragile-X patients by pulsed-field gel electrophoresis , 1991, Nature.

[20]  H. Steller,et al.  Programmed cell death during Drosophila embryogenesis. , 1993, Development.

[21]  Edwin Reyniers,et al.  A point mutation in the FMR-1 gene associated with fragile X mental retardation , 1993, Nature Genetics.

[22]  G. Rubin,et al.  Localization of the sevenless protein, a putative receptor for positional information, in the eye imaginal disc of Drosophila , 1987, Cell.

[23]  D. Ledbetter,et al.  Fragile X syndrome: a unique mutation in man. , 1986, Annual review of genetics.

[24]  J. Mallet,et al.  Nucleus basalis magnocellularis and hippocampus are the major sites of FMR-1 expression in the human fetal brain , 1993, Nature Genetics.

[25]  T. Schedl,et al.  Mutations in gld-1, a female germ cell-specific tumor suppressor gene in Caenorhabditis elegans, affect a conserved domain also found in Src-associated protein Sam68. , 1995, Genes & development.

[26]  G. Dreyfuss,et al.  Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box. , 1992, The EMBO journal.

[27]  K. Davies,et al.  Physical mapping across the fragile X: Hypermethylation and clinical expression of the fragile X syndrome , 1991, Cell.

[28]  B. Cullen,et al.  A nuclear role for the Fragile X mental retardation protein. , 1996, The EMBO journal.

[29]  D. Schlessinger,et al.  Fragile X genotype characterized by an unstable region of DNA , 1991, Science.

[30]  G. Rubin,et al.  Regulation of the complex pattern of sevenless expression in the developing Drosophila eye. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[31]  R. Nussbaum,et al.  The fragile X mental retardation syndrome protein interacts with novel homologs FXR1 and FXR2. , 1995, The EMBO journal.

[32]  M. Fortini,et al.  Apoptotic Activities of Wild-Type and Alzheimer's Disease-Related Mutant Presenilins in Drosophila melanogaster , 1999, The Journal of cell biology.

[33]  R. Nussbaum,et al.  FXR1, an autosomal homolog of the fragile X mental retardation gene. , 1995, The EMBO journal.

[34]  G. Dreyfuss,et al.  Immunological methods for purification and characterization of heterogeneous nuclear ribonucleoprotein particles. , 1990, Methods in enzymology.

[35]  M. Gassmann,et al.  Tissue-specific expression of a FMR1/β-galactosidase fusion gene in transgenic mice , 1995 .

[36]  N. Perrimon,et al.  Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. , 1993, Development.

[37]  A. Poustka,et al.  Highly conserved 3' UTR and expression pattern of FXR1 points to a divergent gene regulation of FXR1 and FMR1. , 1995, Human molecular genetics.

[38]  R. Nussbaum,et al.  Essential role for KH domains in RNA binding: Impaired RNA binding by a mutation in the KH domain of FMR1 that causes fragile X syndrome , 1994, Cell.

[39]  J C Reed,et al.  IAP family proteins--suppressors of apoptosis. , 1999, Genes & development.

[40]  Ben A. Oostra,et al.  Absence of expression of the FMR-1 gene in fragile X syndrome , 1991, Cell.

[41]  G M Rubin,et al.  A genetic screen for novel components of the Ras/Mitogen-activated protein kinase signaling pathway that interact with the yan gene of Drosophila identifies split ends, a new RNA recognition motif-containing protein. , 2000, Genetics.

[42]  M. Bate,et al.  The development of Drosophila melanogaster , 1993 .

[43]  G. Rubin,et al.  Systematic gain-of-function genetics in Drosophila. , 1998, Development.

[44]  A. Poustka,et al.  A microdeletion of less than 250 kb, including the proximal part of the FMR-I gene and the fragile-X site, in a male with the clinical phenotype of fragile-X syndrome. , 1992, American journal of human genetics.

[45]  G. Rubin,et al.  Kuzbanian Controls Proteolytic Processing of Notch and Mediates Lateral Inhibition during Drosophila and Vertebrate Neurogenesis , 1997, Cell.

[46]  I. Weiler,et al.  Fragile X mental retardation protein is translated near synapses in response to neurotransmitter activation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[47]  M. Klymkowsky,et al.  The appearance of acetylated alpha-tubulin during early development and cellular differentiation in Xenopus. , 1989, Developmental biology.

[48]  David E. Housman,et al.  Tissue specific expression of FMR–1 provides evidence for a functional role in fragile X syndrome , 1993, Nature Genetics.

[49]  J. Mandel,et al.  The FMR–1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation , 1993, Nature Genetics.

[50]  É. Khandjian,et al.  Novel isoforms of the fragile X related protein FXR1P are expressed during myogenesis. , 1998, Human molecular genetics.

[51]  S. Warren,et al.  FMR1 protein: conserved RNP family domains and selective RNA binding. , 1993, Science.

[52]  A. Poustka,et al.  Enhanced Fmr–1 expression in testis , 1993, Nature Genetics.

[53]  A. Poustka,et al.  Fragile X syndrome without CCG amplification has an FMR1 deletion , 1992, Nature Genetics.

[54]  M. Fortini,et al.  Modeling human neurodegenerative diseases in Drosophila: on a wing and a prayer. , 2000, Trends in genetics : TIG.

[55]  G. Dreyfuss,et al.  The pre-mRNA binding K protein contains a novel evolutionarily conserved motif. , 1993, Nucleic acids research.

[56]  A. Krainer,et al.  Distinct functions of the closely related tandem RNA-recognition motifs of hnRNP A1. , 1998, RNA.

[57]  D. Absher,et al.  FMRP associates with polyribosomes as an mRNP, and the I304N mutation of severe fragile X syndrome abolishes this association. , 1997, Molecular cell.

[58]  J. Mandel,et al.  Alternative splicing of exon 14 determines nuclear or cytoplasmic localisation of fmr1 protein isoforms. , 1996, Human molecular genetics.

[59]  D. Le Paslier,et al.  Isolation of sequences that span the fragile X and identification of a fragile X-related CpG island. , 1991, Science.

[60]  S. Warren,et al.  The fragile X mental retardation protein is a ribonucleoprotein containing both nuclear localization and nuclear export signals. , 1996, Human molecular genetics.

[61]  R. Nussbaum,et al.  The protein product of the fragile X gene, FMR1, has characteristics of an RNA-binding protein , 1993, Cell.

[62]  É. Khandjian,et al.  The fragile X mental retardation protein is associated with ribosomes , 1996, Nature Genetics.

[63]  Iva Greenwald,et al.  Presenilin is required for activity and nuclear access of Notch in Drosophila , 1999, Nature.

[64]  J. Mandel,et al.  Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome , 1991, Science.

[65]  G M Rubin,et al.  A screen for genes that function downstream of Ras1 during Drosophila eye development. , 1996, Genetics.

[66]  A. Malafosse,et al.  Expression of FMR1, FXR1, and FXR2 genes in human prenatal tissues. , 1999, Journal of neuropathology and experimental neurology.

[67]  R. Myers,et al.  A putative Drosophila homolog of the Huntington's disease gene. , 1999, Human molecular genetics.

[68]  A T Hoogeveen,et al.  Differential expression of FMR1, FXR1 and FXR2 proteins in human brain and testis. , 1997, Human molecular genetics.

[69]  S. Ceman,et al.  Isolation of an FMRP-Associated Messenger Ribonucleoprotein Particle and Identification of Nucleolin and the Fragile X-Related Proteins as Components of the Complex , 1999, Molecular and Cellular Biology.

[70]  Gerald M. Rubin,et al.  Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death , 1995, Cell.

[71]  C. Burd,et al.  Conserved structures and diversity of functions of RNA-binding proteins. , 1994, Science.