Detection Of Novel Alternatively Spliced Variants From Human FMR1 Gene In Peripheral Blood And Preliminary Functional Study

Fragile X syndrome(FXS)is a common inherited mental retardation disorder.Its disease-determining gene is fragile X mental retardation 1 gene(FMR1),encoding fragile X mental retardation protein(FMRP).The FMR1 gene contains 17 exons and 16 introns,and complex alternative splicing phenomenon of FMR1 gene was observed in its initial transcript.Different alternatively spliced variants of FMR1 gene may encode FMRPs with different structures and functions.However,little is known about the biological function of various spliced variants.To investigate the alternative splicing of FMR1 gene in human peripheral blood,RT-PCR and clone-sequencing technology was employed to detect the types of alternatively spliced products of peripheral blood leukocytes in 10 normal individuals and to statistically analyze their expression abundance.The results showed that A total of 50 FMR1 gene alternatively spliced products were identified from 505 PCR product-T vector recombinant plasmids,including 27 novel alternatively spliced products that have not been reported.The highest abundance in the alternatively spliced products of FMR1 gene is ISO 17 and ISO 7,both of which involve exon 12 skipping;followed by ISO 1(containing the full-length sequence of the coding region)and ISO 13(using the second acceptor site of exon 17),while the frequency of other types of spliced products is not higher than 5.0%.The novel alternatively spliced variants identified in this study involved skipping of exons 3,4,and 11,insertion of 30bp and 87bp sequence spliced from the middle of intron 7,and 46 bp and 140bp sequence spliced from the middle of intron 9.It suggests that the alternatively spliced products of human FMR1 gene is far more complex than known.To explore the splicing mechanism of these novel spliced variants,bioinformatics analysis of the splice sites was performed in the Human Splicing Finder,a widely accepted splice site prediction server.The splicing signals of the 3’ spliced acceptor site and the 5’ spliced donor site of the 27 novel splice products were all predicted.To further confirm whether the protein encoded by the novel splicing product IS051-IS053 was present in normal human peripheral blood,this study used the specific antibody against its C-terminal amino acid sequence to identify the protein expression in human peripheral blood leukocytes through immunoblotting technology.The results showed that the novel transcript containing the 140 bp sequence insertion splicing from the middle of intron 9 was all detectably expressed in adult peripheral blood.To evaluate the effect of alternative splicing of FMR1 gene on the structure and function of FMRP,two structure models of the human FMRPs encode by full-length coding sequence and the transcript sequence of novel spliced isoform ISO51-IS053 were constructed by I-TASSER server,respectively.A section of functions were predicted and the results reveals that the intracellular location of FMRP truncated protein encoded by novel transcript has altered.To further evaluate whether the novel transcript has an effect on the biological function of the corresponding FMRP,a full-length eukaryotic expression vector containing the full-length coding region and a truncated version containing a novel alternatively spliced variant IS051 were constructed using molecular cloning techniques.The eukaryotic expression and overexpression studies is intended to perform in 293T cells and N2a cells.This study not only enriched the understanding of the alternative splicing complexity and diversity of FMR1 gene,but also laid the foundation for exploring the effects of alternative splicing on the function of FMRP and the pathogenesis of FXS.