Functional Analysis Of746T>C Mutation In The3′Untranslated Region Of Human FMR1Gene

Introduction(3’ untranslate region,3’UTR) are known to play crucial roles in thepost-transcrip-tional regulation of gene expression including modulation of mRNAstabitily, subcelluar location and translation efficiency, but also to be the target formicroRNA (miRNA) to silence the gene. Fragile X syndrome（FXS）is one of thecommon heritable form of mental retardation, which is a X-linked incomplete dominantgenetic disease. The progressive expansion of polymorphic (CGG)n trinucleotiderepeats in promoter region of FMR1gene at Xq27.3and methylation of CpG islandcause inactivation of FMR1. MiRNAs are a family of about19~23nucleotide,non-coding single-stranded small molecule RNA. Mature miRNAs form antisensebases pairs with the3′UTR of mRNAs to inhibit translation or destruct the targetmRNAs. We infer that the mutation of FMR13’UTR may lead to functional changingand relate to miRNAs Previous studies show that miRNAs are closely related withnervous system diseases. As a typical representative of single gene inheritance nervoussystem disease, it has not been detailed report whether the FMR1gene is associatedwith the miRNA. ObjectiveAnalysis the structure of FMR13’UTR and combined bioinformatics softwarewith in vitro experiments to confirm the regulation of miRNA on the FMR1geneexpression which would play an exploratory role in the pathogenesis of FXS.MethodsThe conservation of hFMR13′UTR were analysis by the computer program andthe position of the mutations reported in previous were predicted. Then a chimericconstruct expressing with firefly luciferase with the FMR13′UTR and its mutationswere developed. The neurogenic SH-SY5Y cells and the non-neurogenic HEK-293cells were transfected. And assay the relative activities of firefly luciferase by using theDual-luciferase Reporter. Quantity of luciferase mRNA of two cells is analysis by thequantitative real-time PCR. Electrophoretic mobility shift assay is used to analysis thespecific combination with cytoplasmic protein and RNA fragments include wild typeand mutant. Then the construct is co-transfected with miRNA chimeric construct, herelative activities of firefly luciferase is assyed.Results1.746T>C were found to located in the conversed region on the hFMR13′UTRIn the conservative analysis, three conversed regions: CS1, CS2, CS3were foundon the hFMR13′UTR. With the computer programs, the mutant reported previouslywere found to located in the three conversed regions and potentially target of miRNA.2. The746T>C mutant on the hFMR13′UTR down-regulate the expression of reportergene on the post-transcriptional level.The HEK293cells and SH-SY5Y cells were transfeted with the two constructs.The results showed that the activities of the746T>C mutant on the hFMR13′UTRluciferase construct was lower to50%than the wild type (P<0.01), whereas the othermutant constructs showed no significantly difference (P>0.05). The real-time PCRshowed the746T>C mutant produced more luciferase mRNA than the wild type(P<0.01) 3. The down-regulation to the reporter gene of746T>C mutant on hFMR13′UTR mayrelated to the3′UTR binding protein and miRNAs.The result of electrophoretic mobility shift assay showed that both wild type andmutant RNA fragment can specifically combine with cytoplasmic protein. But differentbands are observed between wild type and mutant. Then the hFMR13′UTR expressionconstructs or mutation constructs were co-transfected with miRNA expression vector.Our result showed that co-transfecting with mir-19b, the mutation construct resulted ina significantly difference in luciferase activity comparing to the wild type in both twocell, the effect of mir-19b have Species-specific.ConclusionThe hFMR13′UTR T>C mutation leaded down-regulation to the reporter gene onthe post-transcriptional level, the mutation located in the conversed region, and itseffect related to the cytoplasmic3′UTR binding protein and miRNAs...