Fragile X Related Protein1Target Identification And Effect The Translation Of IQCE

Fragile X syndrome (fragile X syndrome, FXS) is one of the most commoninherited mental retardation disease worldwide.99%of the patients due to thedecrease expression or loss of function of fragile X mental retardation protein（FMRP）.Scientists found that a very similar gene of FMR1in autosomal,has beennamed for FXR1.FXR1encodes a protein called FXR1P.FXR1P is one of fragile XRNA binding protein(RBP) family members,confirmed it has been involved in manyaspects of RNA regulation,including mRNA transport, stability andtranslation.Discovery and identification of FXR1P new mRNA targets for research thefunction of FXR1P as well as the pathogenesis of Syndrome X has a very importantsignificance.Objective: Use reporter gene system to identifying the new mRNA targets ofFXR1P.Reporter gene system preliminary study the interaction relationship betweenFXR1P and its mRNA targets,and also can study the effect to it targets translation.Method:1Bioinformatic analysis FMRP protein family, PAR-CLIP andRIP-chip experiment data,obtain the interaction sites of the FMRP protein family andits target,and analysis FXR1P interaction sites.2Using genetic engineering techniquesto build recombinant reporter gene vectors： pSEAP-B2M ORF,pSEAP-TNF3’UTR,pSEAP-FXR13’UTR and pSEAP-IQCE3’UTR.restriction endonuclease andsequence analysis recombinant reporter gene vectors.3Lipofectamine2000transfection expression vector pcDNA3.1(-)/FXR1to Hela cells and rat myocardialcells and expression vector transfection efficiency detected by western blot.4Inaccordance with certain requirements of packet, the over-expression vector pcDNA3.1(-)/FXR1,control vector pSV-β-Gal and report the gene vector pSEAP-insertedwere transfected into Hela cells and rat cardiomyocytes.β-galactosidase enzymeassay kit detect the activity of β-galactosidase,chemiluminescence kit detect theactivity of secreted alkaline phosphatase. Results:1) Bioinformatics analysis showed that FXR1P and FMRP proteinfamily RNAs target interaction sites are mainly located in the genome exon regionsand mRNA3’UTR and CDS.2) Build the pSEAP-B2M ORF the pSEAP-TNF3’UTR,pSEAP-FXR13’UTR pSEAP-IQCE3’UTR recombinant reporter gene vectors, byrestriction endonuclease and sequencing analysis shows vector was constructedsuccessfully.3) Western blot results show pcDNA3.1(-)/FXR1recombinant vectorwas successful transfected to hela cells and rat cardiomyocytes and expression FXR1Pprotein.4) In HeLa cells the influence of overexpression of FXR1P have nostatistically significant on recombinant reporter gene expression which contains theassumed target IQCE3’UTR and FXR13’UTR.However,in rat cardiomyocytesoverexpression of FXR1P can significantly reduced expression of the recombinantreporter gene which contains IQCE3’UTR but not FXR13’UTR.Conclusion:1. Bioinformatics analysis showed that FMRP protein family (including FXR1P)genome interaction sites are mainly located in exon region, and mRNA binding sitesare mainly located in the the CDS and3’UTR.2. FXR1P have different binding effect to IQCE in HeLa cells and ratcardiomyocytes，indicate FXR1P interact with targets have organized specific.3. In rat cardiomyocytes IQCE3’UTR is a mRNA target of FXR1P，Prompt thatFXR1P have negative regulation on IQCE expression.