Characterization Of A Novel Alternatively Spliced Variant From Human FMR1 Gene And Function Of Its Encoded Product

Fragile X mental retardation 1 gene(FMR1)encodes a selective messenger RNA-binding protein,which is called fragile X mental retardation protein(FMRP).Its absence can cause to a common form of inherited intellectual disability,known as fragile X syndrome(FXS).The alternative splicing of FMR1 gene has a close relationship with FMRP expression.Different ways of splicing will produce different splice variants of FMR1,thereby altering FMRP’s structure and function and affecting the corresponding signaling pathways.However,how to detect various alternatively spliced isoforms and what are their biological functions are still poorly understood.In our previous work,we used clone-sequencing technology to analyze the expression of human FMR1 gene and a 140bp sequence splicing from the middle of intron 9 was detected.Bioinformatics showed that the novel cryptic exon insertion alters the open reading frame of FMR1 gene and creates a truncated novel polypeptide chain.To investigate the expression of the novel cryptic exon in peripheral blood cells of normal individuals,quantitative real-time PCR and Western blot were used to analyze the new alternatively spliced FMR1 transcript.The results of qPCR showed that the mRNA expression level of the novel cryptic exon normally distributed and WB showed that the new alternatively spliced FMR1 transcript all expressed in normal individuals.To explore the biological functions of the new alternatively spliced FMR1 transcript,we constructed the recombinant eukaryotic expression vectors with full-length coding sequence or the novel cryptic exon,which was named pEGFP-N2-wFMR1(wild type)and pEGFP-N2-tFMRl(truncated type),respectively.After transfecting into HEK293T cells,western blot showed that wild type and truncated type proteinwere expressed successfully in HEK293T cells,and Immunofluorescence revealed that wild type protein mainly expressed in the cytoplasm,while truncated type protein mainly expressed in cell nucleus.To further evaluate whether the novel cryptic exon has an effect on FMR1 gene expression and to understand its underlying molecular mechanisms,we constructed the lentiviral vector with the novel transcript of human FMR1 gene and established stably transfected HEK293T cells.RNA microarray analysis was employed to detect the differentially expressed genes,and 545 altered specific mRNAs were identified,among which,BEX1 gene may be involved in FMRP related signaling pathways.Our study will refine our understanding of FMRP’s signaling networks and provide the theoretical basis for the establishment of candidate schemes for the FXS therapy.