Alternative Splicing Of The FMR1 Gene And Its Implications

Fragile X syndrome is the most common inherited mental retardation disorder. The disease-determining gene, fragile X mental retardation 1 gene(FMR1), lies on chromosome Xq27.3. This gene spans 38 kb in the genome and contains 17 exons and 16 introns. It encodes a selectively RNA-binding protein called fragile X mental retardation protein(FMRP). It had been found that the gene has multiple alternative spliced forms and alternative splicing(AS) plays a major role in the functioning and regulation of the FMR1 gene. But the physiological function of alternatively spliced products remains unknown.To study the AS of FMR1 gene in different species, comparative genomics approaches were used. Conserved coding sequences, intron sequences and splicing expression profiles were compared, and evolutionary selection pressure analysis of exons were performed. The results showed that complex alternative splicing of FMR1 gene exists in different species and there is species-specific AS of the gene. The results also showed that AS is closely related to the evolution of FMR1 gene, especially the splicing of exon 12.To further study the AS profiles of FMR1 gene in human, T cloning-sequencing approach was employed to analyze the alternatively spliced transcripts in human tissues. Two novel splicing products were found. In addition to the novel products found in our previous research, it implies that the AS of the FMR1 gene is far more complex than what have been understood. The results also showed that ISO1 did not dominate in these tissues, while ISO7 and ISO17, both lacking exon 12, were the dominant splicing products, whose relative abundance was different in these tissues.To evaluate the effect of novel exon on FMR1 gene expression, functional studies on the transcript containing novel exon were carried out. The recombinant eukaryotic expression vectors with full-length coding sequence and the novel transcript were constructed, which was named p EGFP-N2-FMR1 and p EGFP-N2-△FMR1 respectively. Immunofluorescence analysis showed that the inclusion of the novel exon led to alter the localization of FMRP in HEK293 T and He La cells.To further study the effect of the novel transcript on FMRP function, recombinant lentiviral vector with the novel transcript was constructed, named p LEX-MCS-△FMR1, and RNA microarray analysis was performed in HEK293 T cells with the over expression of the novel transcript. Compared with the control, a total of 117 altered specific m RNAs were identified. The SLC7A11 gene may be involved in the regulation of FMRP signaling pathway.This study may lay the foundation for the impact of alternative splicing on the functioning of FMRP, and provides a new direction for the pathogenic mechanism of FXS.