Identification And Functional Analysis Of Multiple Isoforms Of Fragile X Syndrome Related Protein FMRP

Fragile X syndrome (FXS) is one of the common causes of heritable mentalretardation, one important reason leading to FXS is the transcriptional silencing of thefragile X mental retardation protein (FMRP) expression caused by the instabilityamplification of the trinucleotide repeat sequence in FMR1gene. It is known that thefunction of FMRP is closely related to their own domain function and structure.Alternative splicing of mRNA transcript exists in FMR1of diverse species, producingdifferent transcripts and encoding various protein isoforms. Distinct FMRP isoformsmay lead to important differences in their expression and function. The main isoformof mFMRP (isoform1) is composed of17exons, while the other isoforms possessvarying degrees of exon sequences lacking. Twelve different transcript isoforms havebeen identified in the latest research, but approximate4-5of mFMRP isoforms havebeen identified so far. We still know little about the specific function of proteinisoform because of the nonexistence of the specific antibody for them.In this study, we identify the transcript isoforms in the brain tissue of FVB inbredmouse embryonic rat (E18) and adult rats (P28) respectively by RT-PCR combinedwith sequencing, and compare the frequency of utilization of each transcript isoformwith the others. The open reading frames (ORF) of these transcript isoforms arepredicted by using bioinformatics software for the analysis of differences among them.We construct the plasmids of fusion protein containing V5tag and isoform proteins, analyze the distribution of exogenous mFMRP isoforms in N1E-115, Neuro-2A andHEK-293cell lines, observe the affection of these isoforms structure for thedistribution, and explore the latent function and regulation mechanism of mFMRP.We find that:1. There are5patterns of sequence alternative splicing in the wholebrain tissues of E18and P28mice, and these patterns form7different mFmr1transcript isoforms. Choice of alternative splicing sites is made according to theGT-AG rule.2. The distribution of mFmr1transcript isoforms has peculiarity ofdevelopmental periods, shows different bias in different stages of neurodevelopment.The phenomenon may lead to the changes of the recognition and binding functionsbetween FMRP and RNA sequences, suiting the special features in individualdevelopmental periods.3. Individual mFMRP isoform has the diversity in expressionamong different types of cell lines whilst all mFMRP isoforms have diversity indistribution in one same cell line as well. It suggests that for the isoforms, thedifference of structure result in the difference of distribution and expression, andaffects functional expression at last.4. Varying location of V5-tag in the fusionprotein (either at N-terminal or at C-terminal) affects the expression and distributionof related protein isoforms.Our study identifies7mFmr1transcript isoforms isolated from mouse braintissue, of which the isoform6is detected for the first time. It is also confirmed thatthere are differences of isoform proteins’ distribution and expression in diverse celllines. Some key amino acid residue sites are speculated to be present in NLS domain,and may have affection in the FMRP transport into nucleus. This study providesfoundation for further exploration of distribution law and translational regulation ofmFMRP isoforms.