Identification And Analysis Of The FMRP-Associated MRNAs

Fragile X syndrome(FXS) is one of the most common forms of inherited mental retardation,which occurs in approximately 1 in 4 000 males and 1 in 8 000 females. Patients with FXS showed different clinical characteristics ranging from modest to severe mental retardation,as well as dysfunction of speech and behaviors.It is well known that FXS is caused by the mutation in FMR1 gene,which is located at the fragile site on q arm of X chromosome.Mutation in FMR1 gene leads to the absence of its gene product,the fragile X mental retardation protein(FMRP).However,the mechanism how the absence of FMRP causes fragile X syndrome is still not clear.FMRP is an RNA binding protein,which contains three kinds of RNA binding domain: hnRNP K-homology(KH) binding domains,an arginine / glycine-rich RNA-binding motif (RGG box) and a noval RNA-binding domain at the N-terminus of FMRP.Previous studies have shown that RGG box is the main RNA binding domain in FMRP by interacting with RNA via a G-quartet structure.FMRP also contains a nuclear localization signal(NLS) and a nuclear export signal(NES).It is reported to be involved in transporting and regulating the translation of target mRNAs via mRNE Due to loss of function of FMRP in FXS patients,mRNA transportation and translation may be abnormal,leading to a variety of complicated clinical characteristics.Many studies have been focusing on identifying target mRNAs to understand the pathogenesis of fragile X syndrome.Several strategies have been used to identify mRNA molecules associated with FMRP. Warren et al.identified 432 mRNAs associated with FMRP from mouse brain by co-immunoprecipitation and microarray assay.Eberwine and colleagues developed a method termed APRA(Antibody-Positioned RNA Amplification) to identify RNAs in the proximity of FMRP in vivo.However,there is only little overlapping among these results, indicating that the target mRNAs identified may be different based on the different screening methods and materials used.In this project,we used the yeast three-hybrid system to study the interaction between FMRP and candidate target mRNAs.The yeast three-hybrid system is derived from yeast two-hybrid system,and designed for in vivo detection and analysis of RNA-protein interactions in living cells.This system might better reflect the internal environment in living cells to enable the identification of naturally occurring RNA-protein partners and the dissection of higher-order RNA-protein complexes.Therefore,our results may be a useful supplement to current studies.Using this system,we may also identify some new targets.We screened a human fetal hippocampus cDNA library constructed in our lab before, using the yeast strain L40-ura3 / pHyblex / MS2 / pYESTrp3 / FMR1 5'～3' provided from Dr.Zhong.Totally,we performedβ-galactosidase activity assay on 120 SD / -Trp / -His / -Ura / Zeo / 3-AT plates for more than 1.0×10~6 clones and got 122 positive clones. Plasmids in positive clones were isolated and transformed into E.Coli competent cells. About 12 000 PCR was performed with primers specific to pRH5' to amplify the cDNA library plasmids,and 2 934 colonies containing pRH5' plasmids were obtained.The pRH5' plasmids in these transformants were extracted,followed by sequencing in dual directions of their cDNA-inserts and 48 kinds of candidate genes were identified.Then,22 out of 48 candidate target mRNAs were validated by retransformation.To eliminate false positives,we further validated our screening results.First,we confirmed whether positive reaction was induced by the interaction between FMRP and the mRNA target by western-blot and RT-PCR.For clones that were still positive by this analysis,we performed a nondenaturing agarose gel electrophoresis shift assay(AGESA) to verify that FMRP can bind the candidate mRNAs in vitro.Among 22 positive clones, except for 1 that we could not get transcript in vitro,19 showed positive and 2 negative in AGESA assay.The candidate mRNAs we obtained in our study encode proteins involved in various cellular processes,including neural development and differentiation.To better understand the pathogenesis of fragile X syndrome,we further investigated whether FMRP can regulate the expression of some candidates.For this purpose,we utilized RNA interference technology.First,we designed and chemically synthesized siRNA based on the mRNA sequence of FMR1.After transfecting cells,total proteins were extracted and subjected to western-blot analysis to determine the protein profile of target mRNAs.Based on the distribution and characteristics of proteins encoded by the targets,we also employed other methods to analyze the changes of cell properties after transfection.Our data showed that FMRP can negatively regulate the expression of target mRNAs,TXNRD1 and SEPTIN2. Proteins encoded by these two genes are widely expressed in most organs in human,and were reported to be involved in neural development and differentiation.TXNRD1 encodes a cytosolic enzyme,thioredoxin reductase 1(TrxR1),which plays a central role in controlling cellular redox homeostasis.Previous studies have shown that Trx and TrxR play important roles in the CNS,including neurotrophic and neuroprotective actions. SEPTIN2 encodes a cytoskeletal GTPase that have diverse roles in protein scaffolding, cytokinesis,and vesicle trafficking.Septin2,enriched in brain,may be required for polarized neurite outgrowth by facilitating the exocyst complex function during neuronal differentiation.Our results suggested that they may be involved in the pathogenesis of fragile X syndrome.Our study suggested that yeast three-hybrid system is a useful system for large-scale screening of RNA-protein interaction in vivo.The target mRNAs of FMRP,a RNA binding protein,play important role in various physiological processes,such as transcription, translation,as well as cell differentiation.From yeast three-hybrid screening and further validation,we identified a series of candidate mRNAs associated with FMRP,which provided a meaningful supplement to current targets.Furthermore,we analyzed the role of FMRP in regulating the expression of some target mRNAs.These data may provide some hints to the pathogenesis of fragile X syndrome.