The Study On FTH1 And CMAS Interaction With FXR1P

Fragile X syndrome (FXS),which is one of the most common form of inherited cognitive impairment,are caused by the expression decrease or absence of FMRP(fragile X mental retardation protein), the coding products of FMR1(fragile X mental retardation 1). FXR1,a gene that shares great similarity with FMR1,is a housekeeping gene that plays an important role in the development process of nervous system and muscular system.FXR1P not only associate with mRNAs and proteins, The screening and identification of the series of interaction proteins of FXR1P will be helpful for the clarification of the function of FXR1,understand the regulatory mechanism of FXR1P,and then to comprehend the relationship between FXR1P and FXS.Objective:To analyze the interaction between FXR1P and FTH1 and CMAS by co-immunoprecipitation and observe the expression and intracellular co-localization of FXR1P and FTH1 and CMAS by laser scanning confocal technique,it helps to provide the experimental basis for studying FXR1P function and further exploring the relationship between FXR1P function and the pathogenesis of fragile X syndrome.Methods: In the Co-IP experiment, three eukaryotic fusion expression vectors, pCMV-HA-FXR1,pCMV-Myc-FTH1 and pCMV-Myc-CMAS were constructed.The interaction between FXR1P and FTH1,CMAS was respectively detected by coimm- unoprecipitation. GFP-tagged fusion protein(pEGFP-C1-FXR1)expression vector and RFP-tagged fusion protein (pDsRed1-Monomer-C1-FTH1 and pDsRed1-Mono- mer-C1-CMAS) expression vectors were respectively constructed,and cotransfected into 293T cells and Hela cells,the expression and intracellular co-localization of the products were detected by confocal laser scanning microscopy.Results:The six recombinant vectors,pCMV-HA-FXR1,pCMV-Myc-FTH1, pCMV-Myc-CMAS,pEGFP-C1-FXR1,pDsRed1-Monomer-C1-FTH1 and pDsRed1- Monomer-C1-CMAS,were constructed successfully.Western blotting confirmed the expression of fusion protein in the transfected 293T cells.Co-immunoprecipitation showed that when HA-FXR1 was immunoprecipitated by anti-HA polyclonal antibody,Myc-FTH1,Myc-CMAS were identified by western blotting with anti-Myc monoclonal antibody from immunoprecipitated complex,and vice versa. Fluorescence confocal microscopy showed that they had interactive material basis in spatial distribution, FXR1 and FTH1 were only localized in the cytoplasm,and FXR1 and CMAS were co-localized in the cytoplasm and in the nuclei.Conclusions: 1. Co-immunoprecipitation and cellular colocalization assay to confirm the interaction between FXR1P and FTH1,which shows they mutual locate in the cytoplasm.2. Co-immunoprecipitation and cellular colocalization assay to confirm the interaction between FXR1P and CMAS,which shows they mutual locate in the cytoplasm and in the nuclei,and CMAS expression migrates after it co-transfected into FXR1.