【作者】 刘剑；

【导师】 沈岩； 钟南；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 脆性X综合征,是最常见的遗传性智力低下疾病之一;其发病率在男性约为1/4 000,女性约为1/8 000。临床上主要表现为轻度到重度不等的智力低下,以及语言、行为功能障碍等。目前已经知道,它是由于X染色体长臂木端的脆性位点异常,导致FMR1基因所编码的脆性X智力低下蛋白(FMRP)缺失或低表达而引起的,但具体的发病机制仍不清楚。FMR1基因编码的蛋白FMRP是一种RNA结合蛋白,它含有三种RNA的结合结构域:核蛋白K同源框结构域、甘氨酸精氨酸簇(RGG box)和新发现的位于FMRP蛋白N末端的RNA结合结构域。研究表明,RGG box是FMRP与mRNA结合的主要部位。FMRP还含有一个核定位信号和一个核输出信号;通过这两个位点,FMRP蛋白参与靶RNA的转运,影响突触后及树突具有翻译活性的多聚核糖体的功能。脆性X综合征患者由于体内FMRP的缺失或低表达,可能使得相关mRNA的转运、定位或蛋白合成等出现异常,从而导致一系列复杂的临床病理表现。在揭示FMRP缺失与临床表现之间的关系时,寻找FMRP的靶mRNA成为了一个研究热点。筛查并研究FMRP的靶RNA将有助于揭示脆性X综合征致病的分子机制。已有研究者通过不同的方法确定了一系列可与FMRP相互作用的靶mRNA,例如Warren实验室利用免疫共沉淀和微阵列分析的方法,从鼠脑抽提物中找到了FMRP的数百种靶mRNA;Eberwine等人则利用抗体定位的RNA扩增技术(APRA),在哺乳动物细胞系内原位确定了一系列FMRP的靶基因。各组的筛查结果仅有部分相同,说明筛查方法和研究对象的差异会对筛查结果造成一定的影响。在本课题中,我们利用酵母三杂交系统对FMRP与RNA的相互作用进行研究。酵母三杂交系统是一种在酵母双杂交系统基础上发展而来、能够快速检测RNA-蛋白质相互作用的体内筛查体系,它更接近于活体细胞的实际情况,为研究蛋白质与RNA之间的相互作用提供了一个更加准确、有效的筛查平台。我们得到的靶mRNA,对于现有的筛查试验结果将是一种有益的补充,并且有可能筛选出新的有意义的相关致病基因。我们用钟南教授实验室提供的酵母菌株L40-ura3/pHyblex/MS2/pYESTrp3/FMR15′～3′对19周胎龄人胎脑海马cDNA文库进行筛查,共对120块SD/-Trp/-His/-Ura/Zeo/3-AT选择性固体培养板上的超过100万个酵母克隆进行了β-半乳糖苷酶活性检测,共得到122个阳性酵母克隆。分别提取阳性酵母克隆的质粒并转化感受态大肠杆菌细胞,利用针对目的RNA表达质粒pRH5′/MS2/cDNA的特异性引物对获得的12 000个大肠杆菌克隆进行菌落PCR,共得到2 934个pRH5′/MS2/cDNA-insert质粒。对这些质粒的外源cDNA插入片段进行双向序列测定,并对插入片段的核酸序列进行比对分析,共得到48种可能的靶基因信息。之后经过酵母回转验证实验,共得到22种三杂交筛选出的阳性结果。对于在酵母体内显示可与FMRP相结合的靶mRNA,为了进一步排除假阳性的可能,我们对阳性筛查结果进行了验证。首先检测阳性酵母克隆中是否表达了FMRP,以及是否含有相应的靶mRNA。上述验证为阳性的克隆,我们采用凝胶电泳迁移实验进行体外结合验证,22个待验证基因中,19个为阳性,2个为阴性,1个在体外转录过程中未得到有效的转录产物。我们筛查并验证得到的靶基因,它们的功能涉及神经系统的发育分化等多种生命过程,研究FMRP与这些靶RNA翻译的调控关系将有助于进一步理解脆性X综合征的发病机制。因此,我们利用RNA干扰技术,分析了FMRP对部分靶基因表达的影响。首先,根据FMRP的mRNA序列设计并化学合成siRNA转染细胞,提取细胞蛋白,通过western-blot的方法检测靶RNA编码蛋白质的表达情况,并根据候选靶基因表达产物的分布与性质,采用不同的方法对干扰后细胞的变化情况进行检测。实验结果表明:FMRP蛋白对靶基因TXNRD1和SEPTIN2的表达起负调控作用。这两个基因编码的蛋白,在人体各组织器官中广泛表达,并参与神经系统的发育、分化过程。TXNRD1编码的硫氧还蛋白还原酶是一种定位于细胞质的广泛表达的氧化还原反应的调控酶;有研究表明,硫氧还蛋白和硫氧还原蛋白还原酶在中枢神经系统中发挥着重要的作用,包括神经营养及神经元保护等。而基因SEPTIN2编码一种细胞骨架相关的GTPase,与细胞分裂过程中胞浆的运动有关,并在脑中含量丰富;在神经分化过程中,它可能通过加强胞吐复合物的功能而为极化的神经突生长所必需。我们的实验结果提示,它们可能参与了脆性X综合征的发病过程。通过我们的研究,证明了酵母三杂交系统是一种可以大规模筛查RNA-蛋白质相互作用的体内筛查体系。FMRP作为一种RNA结合蛋白,其靶mRNA在翻译、转录及细胞分化等多种生理过程中发挥着重要作用。通过筛选与实验验证,我们得到一系列可与FMRP相互作用的靶RNA,这是对现有FMRP靶RNA库的有益补充。此外,我们初步分析了FMRP对部分靶基因表达的调控,这些结果有可能为揭示脆性X综合征的发病机制提供新的线索。更多还原

【Abstract】 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.更多还原