title: Transport kinetics of FMRP containing the I304N mutation of severe fragile X syndrome in neurites of living rat PC12 cells.
abstract: Lack of fragile X mental retardation protein (FMRP) causes the fragile X syndrome, a common form of inherited mental retardation. The syndrome usually results from the expansion of a CGG repeat in the FMR1 gene with consequent transcriptional silencing of FMR1. However, one missense mutation (Ile304Asn) was reported in the second KH domain of the protein involved in RNA binding. The protein containing this mutation showed an impaired function, leading to an extremely severe phenotype. In the present report, we have studied the role of FMRP I304N in living PC12 cells to better understand the (dys) function of this mutant FMRP. We have generated an FMR1 I304N-EGFP stably transfected PC12 cell line with an inducible expression system (Tet-On) for regulated expression of the FMRP I304N-EGFP fusion protein. After Dox-induction, FMRP I304N-EGFP was localized in the neurites of PC12 cells; however, no granules were formed as has been recently demonstrated for the normal FMRP. Time-lapse microscopy in combination with bleaching technology illustrated that although FMRP I304N-EGFP does not form visible granules, the transport into the neurites is microtubule dependent. Immunoprecipitation with antibodies against GFP demonstrates that FMRP I304N-EGFP coprecipitate with both the 60S ribosomal protein P0 and FXR1P, suggesting that the mutant FMRP is still able to form complexes, however, with different characteristics compared to normal FMRP.
authors.affiliation: Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Mariëtte
authors.initials: M
authors.lastname: Schrier
authors.forename: Lies-Anne
authors.initials: LA
authors.lastname: Severijnen
authors.forename: Surya
authors.initials: S
authors.lastname: Reis
authors.forename: Maria
authors.initials: M
authors.lastname: Rife
authors.forename: Sandra
authors.initials: S
authors.lastname: van't Padje
authors.forename: Gert
authors.initials: G
authors.lastname: van Cappellen
authors.forename: Ben A
authors.initials: BA
authors.lastname: Oostra
authors.forename: Rob
authors.initials: R
authors.lastname: Willemsen
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001370:Axonal Transport / Q000235:genetics; D003594:Cytoplasmic Granules / Q000235:genetics / Q000378:metabolism; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008870:Microtubules / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D018993:Myelin P0 Protein / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism* / Q000648:ultrastructure; D016716:PC12 Cells; D021381:Protein Transport / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D018993:Myelin P0 Protein; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C096916:FXR1 protein, human; C497685:Fmr1 protein, rat