The precise function of FMRP, the protein deficient in the Fragile X syndrome, is poorly understood. Identification of its RNA binding properties, the presence of a Nuclear Localization Signal and of a Nuclear Export Signal, and its association to polysomes, have suggested that it may be involved in mRNA transport, stability, translatability and/or in modulating protein synthesis at synapses. Our major goals are: a) The identification and functional analysis of protein interactors of FMRP. b)the identification of mRNA binding specifically to FMRP, and of the target RNA sequences/structures that are responsible for the specificity of binding. c) The analysis of the role of these protein and mRNA interactions in model systems, either cell in culture, drosophila mutants, and transgenic mice. Four proteins interacting with FMRP have been recently identified by us: a nuclear RNA binding protein, NUFIP1 (initial characterization by Bardoni et al.(1999), a family of two highly conserved cytoplasmic proteins (CYFIP1 and 2), and NUCIP1. These proteins have no known function (but carry some recognizable functional domains), and will be characterized by analyzing their precise intracellular localization in human and mouse cells (normal or deficient in FMRP), their own RNA binding properties (for NUFIP1), and their effect on specific binding of FMRP on its own mRNA. As the FMR/FXR family, NUFIP1 and CYFIP family have each a single homologue in drosophila, their function and interaction will be studied in drosophila, using mutants with deficient or altered expression of these genes. We have initiated a detailed characterization between full-length FMRP (produced in baculovirus) and its own mRNA. A very specific binding site was identified, that corresponds to a peculiar RNA structure. Structural studies will be performed at nucleotide resolution, by chemical and enzymatic probing and computer modeling. This interaction will be studied in vivo using a reporter system involving the TAT protein of HIV. Mutations affecting the binding site on the mRNA or the KH domains of FMRP will be constructed, and their effect on the interaction and on FMR1 mRNA export, stability or translatability will be studied in neuronal PC12 cells stably expressing the constructs. Search for similar RNA structures in other mRNAs should identify other possible targets of FMRP. This will be complemented by in vitro and in vivo selection experiments (SELEX, and adaptions to the TAT interaction system), to identify RNA sequence motifs binding selectively to FMRP. The FMR1 mRNA mutants that show the most interesting features will be expressed in transgenic mice, that will be crossed to FMR1 KO mice.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD040612-02
Application #
6536365
Study Section
Special Emphasis Panel (ZHD1-MRG-C (15))
Program Officer
Oster-Granite, Mary Lou
Project Start
2001-06-15
Project End
2004-05-31
Budget Start
2002-06-01
Budget End
2003-05-31
Support Year
2
Fiscal Year
2002
Total Cost
$125,000
Indirect Cost
Name
Institute of Molecular Biology/Cellular
Department
Type
DUNS #
City
Strasbourg
State
Country
France
Zip Code
Didiot, Marie-Cecile; Subramanian, Murugan; Flatter, Eric et al. (2009) Cells lacking the fragile X mental retardation protein (FMRP) have normal RISC activity but exhibit altered stress granule assembly. Mol Biol Cell 20:428-37
Didiot, Marie-Cecile; Tian, Zhaoxia; Schaeffer, Celine et al. (2008) The G-quartet containing FMRP binding site in FMR1 mRNA is a potent exonic splicing enhancer. Nucleic Acids Res 36:4902-12
Schenck, Annette; Qurashi, Abrar; Carrera, Pilar et al. (2004) WAVE/SCAR, a multifunctional complex coordinating different aspects of neuronal connectivity. Dev Biol 274:260-70
Schenck, Annette; Bardoni, Barbara; Langmann, Caillin et al. (2003) CYFIP/Sra-1 controls neuronal connectivity in Drosophila and links the Rac1 GTPase pathway to the fragile X protein. Neuron 38:887-98
Schenck, Annette; Van de Bor, Veronique; Bardoni, Barbara et al. (2002) Novel features of dFMR1, the Drosophila orthologue of the fragile X mental retardation protein. Neurobiol Dis 11:53-63