利用酵母三杂交系统筛选人胚海马cDNA文库以获得能够与FMRP发生相互作用的mRNAs
摘要
一研究背景
脆性X综合征是最常见的遗传性智力低下疾病之一,其发病率男性约为1/4000,女性约为1/8000。临床上主要表现为中度到重度的智力低下,并常伴有巨睾、特殊面容、多动症以及癫痫发作等临床病理表现。目前已证实脆性X智力低下基因(FMR1)所编码的蛋白质FMRP的表达缺失是该病的致病原因,但具体的发病机制尚不清楚。
FMRP(脆性X智力低下蛋白)是一种RNA结合蛋白,含有两种RNA结合结构域:KH结构域(核酸蛋白K同源结构域)和RGG框(精氨酸甘氨酸簇);此外,还有一个核定位信号(Nuclear Localization Sequence,NLS)和一个核输出信号(Nuclear Export Sequence,NES),可以使FMRP穿梭于细胞核与细胞质之间。Damell等用FMRP与随机RNA序列选择性结合的方法(SELEX),发现FMRP的RGG盒特异性的结合于一种称为G四集体(G quartet)的RNA分子内茎环结构;而Denman等人发现除了G四聚体结构外,FMRP还可以识别U-rich的RNA序列。这些都说明FMRP有多个位点参与了与靶RNA的相互作用,并且其靶RNA可能具有一定的序列特异性。已有研究证实FMRP参与部分靶基因如FMR 1 mRNA、ribosome protein S5、eukaryotic translation elongation factorα等的表达调控;FMRP的结构特征也提示它的功能可能是参与其靶mRNAs从细胞核到细胞质,或从神经元细胞质到树突和突触后的蛋白质合成位点的转运;FMRP还可作为信使核糖核蛋白复合体(mRNPs)的组份,影响突触后及树突的具有翻译活性的多聚核糖体的功能。有观点认为细胞内FMRP的功能模式可能是:通过其核定位信号被转运至核内,入核后与数种蛋白质和特定亚群的mRNAs相互作用,形成mRNP复合体;然后此复合体通过核输出信号输出至胞浆并定位于突触后位点。在此过程中,mRNP复合体中的mRNAs一直处于无活性状态,直到突触的信号输入改变FMRP的活性,从而起始靶mRNAs的翻译。因此,脆性X综合征患者的FMRP缺失,可能引起某些靶mRNAs的翻译异常,从而导致一系列的临床病理表现。筛查并研究FMRP的靶mRNAs将有助于揭示该病的致病机制,寻找FMRP的靶mRNAs也一直都是研究的热点。
Brown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确定了数百种FMRP的靶mRNAs;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发现了一系列的靶基因。这两组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很大的影响。而目前所进行的筛查实验几乎都是利用体外(in vitro)筛查的方法,且研究对象大多是实验动物或哺乳动物细胞,与人体内的实际情况有较大的差异,难免会遗漏一些可能很重要的相互作用信息。
二研究目的
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的,可以快速检测RNA-蛋白质相互作用的体内(in vivo)筛查体系。它可以更加准确、更加生动地动态反映真核细胞内的相互作用情况。已有人利用该系统对FMRP与靶RNA AF040097和AF040099的相互作用进行过研究,但是将该系统应用于大规模的筛查工作尚未见报道。
我们希望利用酵母三杂交系统对人胎脑海马cDNA文库进行大规模的筛查,确定在该体内(in vivo)环境与FMRP发生了相互作用的靶mRNAs。这对于现有的体外筛查结果将是一种有益的补充,并有可能筛选出新的有意义的相关致病基因。
三实验方法与实验结果
本课题以19周胎龄的人胎脑海马总RNA为材料,分离得到1μg mRNA,逆转录合成平末端双链cDNA,琼脂糖凝胶电泳显示cDNA片段大小介于50 bp～3 Kb之间,绝大部分集中于200 bp～1 Kb。采用滤膜截留的方法回收100 bp以上的cDNA片段,用于构建cDNA质粒文库。限制性内切酶SmaⅠ消化RNA表达质粒pRH5',琼脂糖凝胶电泳回收线性化载体DNA后再进行碱性磷酸酶去磷酸化处理,纯化回收处理后的线性化载体。经验证,该载体自连转化效率＜5×10~3/μg,与ΦX174/λHindⅢ片段的连接转化效率＞2×10~5/μg。利用上述逆转录合成的cDNA片段和处理好的pRH5'线性化质粒载体构建酵母三杂交的cDNA质粒文库。每200 ng线性化pRH5'质粒载体与大约200 ng cDNA片段进行一次连接反应并转化大肠杆菌感受态细胞,在10个150 mm LB/Amp固体培养基平板上得到大约10～15万个克隆,载体与cDNA片段的连接转化效率＞2×10~5/μg。随机挑取培养平板上的克隆进行菌落PCR,有效克隆比例＞95%,插入片段长度介于约100 bp～2 Kb之间。该连接转化反应共进行了10次,从100个LB/Amp平板上共收集了大约1.5×10~6个大肠杆菌克隆。分离这些克隆的质粒即制得酵母三杂交的cDNA质粒文库。
利用钟南教授实验室提供的酵母菌株L40-ura3/DHyblex/MS2/pYESTrp3/FMR15'～3'(其中已转入DNA结合结构域杂合蛋白的表达载体pHybLex/MS2和诱饵杂合蛋白的表达载体pYESTrp3/FMR1 5'～3')对上述cDNA文库进行筛查。在第一轮筛查工作中共对120块SD/Trp-/His-/Ura-/Zeo/3-AT(30 mM)选择性固体培养基平板上的超过100万个酵母克隆进行了β-半乳糖苷酶活性检测(Colony Lift Assay),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PRH1和PRH2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。然后对这些质粒的外源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blastn(NCBI)比对分析,排除重复的序列,共得到50种不同的靶基因信息。
由于酵母杂交系统难以避免的假阳性效应,并且在某些阳性酵母克隆中会同时存在2种甚至2种以上的目的RNA表达质粒,必须确定是哪一种或是哪几种目的RNA与FMRP发生了相互作用,所以我们再将上述50种可能的靶基因所对应的pRH5'/MS2/cDNA表达质粒分别重新转化酵母菌株(L40-ura3/pHyblex/MS2/pYESTrp3/FMR1 5'～3')——即回转验证实验,再次进行营养缺陷型选择性培养筛选和β-半乳糖苷酶活性检测。该回转实验在同等条件下进行了三次,其中31种仍显示阳性。
为了尽量避免假阳性的发生,我们验证了阳性酵母克隆的FMRP和目的RNA的依赖性,即用Western Blotting和RT-PCR的方法验证这些阳性酵母克隆中是否存在FMRP和目的RNA的表达。目前已完成13种阳性结果的验证,均呈阳性反应。
为了进一步加强结果的说服力,对这些可能阳性的目的RNAs分别与FMRP进行了体外结合实验以及EMSA验证,以确定这些在酵母水平显示与FMRP发生了相互作用的靶RNA是否可以在体外环境与FMRP结合。首先构建全长hFMRP的原核表达载体pET22b~+/FMR 1,在大肠杆菌BL21原核表达系统表中达6×His-taggedhFMRP,通过Ni-亲和层析柱纯化回收,共得到约100μg hFMRP,浓度15 ng/μl,纯度＞80%。利用带有T7 promoter的特异性引物从阳性的目的RNA表达质粒上PCR扩增阳性靶RNA的体外转录模板,并在体外体系利用T7 RNA polymerase转录得到阳性的靶RNA片段,用Trizol Reagent纯化回收目的RNA片段并定量为50 ng/μl。在体外结合缓冲体系内进行FMRP与目的RNA的结合反应,随后进行EMSA(凝胶电泳阻滞实验)验证。目前已经进行了13种(即上述已完成酵母克隆FMRP和目的RNA依赖性验证的)靶RNA与FMRP的体外结合验证,其中8种为阳性,它们是:roundaboutaxon guidance receptor(Robo),thioredoxin reductase 1 mRNA(TXNRD 1),ribosomeprotein L13a mRNA(RPL13a),eukaryotic translational elongation factor 1 gammamRNA,fatty acid binding protein 5 mRNA,hypothetical protein SB145 mRNA,hypothetical protein MGC 4707 mRNA,cDNA DKFZp6668189;另外还有4种疑似阳性,1种显示阴性。
四结论和讨论
我们筛选到的31种靶RNA中没有与已报道的靶RNAs完全相同的序列,只有4种与其他实验室的筛查结果相似:文献报道eukaryotic translation elongation factor 1alpha和ribosomal protein S5能够与FMRP结合并受其调控,而我们筛查到的结果中包含eukaryotic translation elongation factor 1 gamma和ribosomal protein S9,L23,L13a,虽不能完全对应,但在功能上存在密切的联系;其余27种都未见报道。我们认为这种差异可能是因为实验材料和筛查方法不同所造成的。
在我们的结果中有18种靶基因可能与神经系统的发育分化相关,另外还涉及一些结构蛋白、代谢相关酶以及转录翻译调控因子等。在经过体外结合实验验证的8种阳性靶mRNA中,膜受体蛋白Robo已被大量的研究证明与果蝇中枢神经系统的突触的发育分化密切相关;TXNRD1(硫氧化还原蛋白还原酶1)则通过氧化还原反应调控细胞的生长、分化、凋亡以及信号传导等多种生理过程。
本课题证实了酵母三杂交系统是一种简单高效的、可以快速大规模筛查RNA-蛋白质相互作用的体内筛查体系。我们得到的筛查结果是对现有FMRP靶RNA库的有力补充,可为揭示脆性X综合症的发病机制提供新的线索。
The fragile X syndrome is an X linked mental retardation syndrome, characterised by behavioural and physical features such as a long face with large, protruding ears, macro-orchidism, and eye gaze avoidance. The prevalence of the fragile X syndrome was estimated as about 1/4000 for males and 1/6000 for females. Fragile X syndrome results from a mutation (a change in the typical DNA sequence) known as a trinucleotide repeat expansion. This means that a series of three particular nucleotides (CGG) in the DNA is greatly expanded beyond its normal size, disrupting the normal messages that need to be sent. This fact was discovered in 1991 by several teams of researchers studying the X chromosome. In the FMR-1 gene located on the X chromosome, most individuals have a CGG trinucleotide repeat that occurs between 5 and 50 times, the average being around 30. These individuals are normal with respect to fragile X syndrome, and usually carry no risk of transmitting it, although the 40-60 repeat range is sometimes considered a "gray zone" which may or may not be unstable (have a risk of expanding). Some individuals have CGG sequences that are repeated in the range of about 50 to about 200. These individuals are generally referred to as premutation carriers. This means that they carry the syndrome and can transmit it to their children. Premutation carriers, however, are not usually affected by fragile X syndrome. When the number of CGG repeats expands beyond 200, the individual usually has the full mutation. This means that they have fragile X syndrome and will experience the impairments and delays associated with the syndrome.
FMRP is an RNA binding protein associated with polyribosomes. A clue for the function of FMRP was given by the finding that FMRP contains motifs (two KH domains and one RGG box) characterizing other RNA binding proteins. Indeed, FMRP is able to bind RNA in vitro, with preference for poly (U) and poly (G) RNA homopolymers. FMRP was shown to colocalize with actively translating polysomes . The association of FMRP to polysomes suggested that it may be involved in the control of mRNA translation. Although some of the messenger RNA targets of this protein, including FMR 1, have been ascertained, many have yet to be identified .
Screening methods used to identify the mRNA-targets of FMRP were almost in vitro methods, it's far way from the real situation in living cells. Yeast three-hybrid system is a research method derived from that of yeast two-hybrid system. It provides a technic platform with a stable in vivo environment for the research of these interactions in eukaryotic cells. Although it had been used to study interactions between some potential RNA-targets with FMRP, there is no report about screening work in library scale. In our study, we are planning to screen the RNA-targets of FMRP in library scale using yeast three hybrid system. Finding some new RNA targets is expected.
A cDNA library of fetal hippocampus at the age of 19 weeks with the plasmid named pRH5' has been formed for yeast three-hybrid system. The purpose of the present work is to enlarge the size of cDNA library, and use the yeast three-hybrid system to screen the target mRNAs of FMRP in fetal hippocampus. Whole length cDNA of FMR1 were recombined into pYES plasmids, and then transferred into L40-ura yeast strains. The fusion-protein expressed by the pYES plasmids were used as the "bait" to screen the library to find the target mRNAs interacting with FMRP in the yeast cells.
About 1.5×10~6 clones have been screened on 120 plates by the X-gal filter paper screen method, and have extracted plasmid from 122 clones revealing positive result in theβ-galactosidase screen. Then the positive clone plasmids were transmitted into TOP10 E.coli., and re-extracted from them. 2,934 plasmids were obtained and sequenced analyzed with two primers respectively, and the sequences analyzed with blasten (NCBI), 50 kinds of pRH5'/cDNA-insert plasmids were identified. Re-transforming these potentially positive plasmids on by one back into the yeast cells followed byβ-galactosidase screening, 31 of 50 were proved to be positive. Up to now, 13 kinds of target-RNAs have been validated by in vitro binding with FMRP and EMSA, 8 of them were proved to be positive. Some of these positive target-mRNAs are associated with the development and differentiation of central nerve system, key-enzymes of some metabolic pathway and some structural proteins et al.
Through the above mentioned process, it has been identified that the yeast three-hybrid system is a simple and efficient method in validating the interaction between RNA and protein. As a RNA binding protein, FMRP can interact with many mRNAs acting as a key role in translation, transcription and cell differentiation process. These interactions are closely correlated with the molecular foundation of fragile X syndrome.
脆性X综合征是最常见的遗传性智力低下疾病之一,其发病率男性约为1/4000,女性约为1/8000。临床上主要表现为中度到重度的智力低下,并常伴有巨睾、特殊面容、多动症以及癫痫发作等临床病理表现。目前已证实脆性X智力低下基因(FMR1)所编码的蛋白质FMRP的表达缺失是该病的致病原因,但具体的发病机制尚不清楚。
FMRP(脆性X智力低下蛋白)是一种RNA结合蛋白,含有两种RNA结合结构域:KH结构域(核酸蛋白K同源结构域)和RGG框(精氨酸甘氨酸簇);此外,还有一个核定位信号(Nuclear Localization Sequence,NLS)和一个核输出信号(Nuclear Export Sequence,NES),可以使FMRP穿梭于细胞核与细胞质之间。Damell等用FMRP与随机RNA序列选择性结合的方法(SELEX),发现FMRP的RGG盒特异性的结合于一种称为G四集体(G quartet)的RNA分子内茎环结构;而Denman等人发现除了G四聚体结构外,FMRP还可以识别U-rich的RNA序列。这些都说明FMRP有多个位点参与了与靶RNA的相互作用,并且其靶RNA可能具有一定的序列特异性。已有研究证实FMRP参与部分靶基因如FMR 1 mRNA、ribosome protein S5、eukaryotic translation elongation factorα等的表达调控;FMRP的结构特征也提示它的功能可能是参与其靶mRNAs从细胞核到细胞质,或从神经元细胞质到树突和突触后的蛋白质合成位点的转运;FMRP还可作为信使核糖核蛋白复合体(mRNPs)的组份,影响突触后及树突的具有翻译活性的多聚核糖体的功能。有观点认为细胞内FMRP的功能模式可能是:通过其核定位信号被转运至核内,入核后与数种蛋白质和特定亚群的mRNAs相互作用,形成mRNP复合体;然后此复合体通过核输出信号输出至胞浆并定位于突触后位点。在此过程中,mRNP复合体中的mRNAs一直处于无活性状态,直到突触的信号输入改变FMRP的活性,从而起始靶mRNAs的翻译。因此,脆性X综合征患者的FMRP缺失,可能引起某些靶mRNAs的翻译异常,从而导致一系列的临床病理表现。筛查并研究FMRP的靶mRNAs将有助于揭示该病的致病机制,寻找FMRP的靶mRNAs也一直都是研究的热点。
Brown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确定了数百种FMRP的靶mRNAs;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发现了一系列的靶基因。这两组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很大的影响。而目前所进行的筛查实验几乎都是利用体外(in vitro)筛查的方法,且研究对象大多是实验动物或哺乳动物细胞,与人体内的实际情况有较大的差异,难免会遗漏一些可能很重要的相互作用信息。
二研究目的
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的,可以快速检测RNA-蛋白质相互作用的体内(in vivo)筛查体系。它可以更加准确、更加生动地动态反映真核细胞内的相互作用情况。已有人利用该系统对FMRP与靶RNA AF040097和AF040099的相互作用进行过研究,但是将该系统应用于大规模的筛查工作尚未见报道。
我们希望利用酵母三杂交系统对人胎脑海马cDNA文库进行大规模的筛查,确定在该体内(in vivo)环境与FMRP发生了相互作用的靶mRNAs。这对于现有的体外筛查结果将是一种有益的补充,并有可能筛选出新的有意义的相关致病基因。
三实验方法与实验结果
本课题以19周胎龄的人胎脑海马总RNA为材料,分离得到1μg mRNA,逆转录合成平末端双链cDNA,琼脂糖凝胶电泳显示cDNA片段大小介于50 bp～3 Kb之间,绝大部分集中于200 bp～1 Kb。采用滤膜截留的方法回收100 bp以上的cDNA片段,用于构建cDNA质粒文库。限制性内切酶SmaⅠ消化RNA表达质粒pRH5',琼脂糖凝胶电泳回收线性化载体DNA后再进行碱性磷酸酶去磷酸化处理,纯化回收处理后的线性化载体。经验证,该载体自连转化效率＜5×10~3/μg,与ΦX174/λHindⅢ片段的连接转化效率＞2×10~5/μg。利用上述逆转录合成的cDNA片段和处理好的pRH5'线性化质粒载体构建酵母三杂交的cDNA质粒文库。每200 ng线性化pRH5'质粒载体与大约200 ng cDNA片段进行一次连接反应并转化大肠杆菌感受态细胞,在10个150 mm LB/Amp固体培养基平板上得到大约10～15万个克隆,载体与cDNA片段的连接转化效率＞2×10~5/μg。随机挑取培养平板上的克隆进行菌落PCR,有效克隆比例＞95%,插入片段长度介于约100 bp～2 Kb之间。该连接转化反应共进行了10次,从100个LB/Amp平板上共收集了大约1.5×10~6个大肠杆菌克隆。分离这些克隆的质粒即制得酵母三杂交的cDNA质粒文库。
利用钟南教授实验室提供的酵母菌株L40-ura3/DHyblex/MS2/pYESTrp3/FMR15'～3'(其中已转入DNA结合结构域杂合蛋白的表达载体pHybLex/MS2和诱饵杂合蛋白的表达载体pYESTrp3/FMR1 5'～3')对上述cDNA文库进行筛查。在第一轮筛查工作中共对120块SD/Trp-/His-/Ura-/Zeo/3-AT(30 mM)选择性固体培养基平板上的超过100万个酵母克隆进行了β-半乳糖苷酶活性检测(Colony Lift Assay),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PRH1和PRH2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。然后对这些质粒的外源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blastn(NCBI)比对分析,排除重复的序列,共得到50种不同的靶基因信息。
由于酵母杂交系统难以避免的假阳性效应,并且在某些阳性酵母克隆中会同时存在2种甚至2种以上的目的RNA表达质粒,必须确定是哪一种或是哪几种目的RNA与FMRP发生了相互作用,所以我们再将上述50种可能的靶基因所对应的pRH5'/MS2/cDNA表达质粒分别重新转化酵母菌株(L40-ura3/pHyblex/MS2/pYESTrp3/FMR1 5'～3')——即回转验证实验,再次进行营养缺陷型选择性培养筛选和β-半乳糖苷酶活性检测。该回转实验在同等条件下进行了三次,其中31种仍显示阳性。
为了尽量避免假阳性的发生,我们验证了阳性酵母克隆的FMRP和目的RNA的依赖性,即用Western Blotting和RT-PCR的方法验证这些阳性酵母克隆中是否存在FMRP和目的RNA的表达。目前已完成13种阳性结果的验证,均呈阳性反应。
为了进一步加强结果的说服力,对这些可能阳性的目的RNAs分别与FMRP进行了体外结合实验以及EMSA验证,以确定这些在酵母水平显示与FMRP发生了相互作用的靶RNA是否可以在体外环境与FMRP结合。首先构建全长hFMRP的原核表达载体pET22b~+/FMR 1,在大肠杆菌BL21原核表达系统表中达6×His-taggedhFMRP,通过Ni-亲和层析柱纯化回收,共得到约100μg hFMRP,浓度15 ng/μl,纯度＞80%。利用带有T7 promoter的特异性引物从阳性的目的RNA表达质粒上PCR扩增阳性靶RNA的体外转录模板,并在体外体系利用T7 RNA polymerase转录得到阳性的靶RNA片段,用Trizol Reagent纯化回收目的RNA片段并定量为50 ng/μl。在体外结合缓冲体系内进行FMRP与目的RNA的结合反应,随后进行EMSA(凝胶电泳阻滞实验)验证。目前已经进行了13种(即上述已完成酵母克隆FMRP和目的RNA依赖性验证的)靶RNA与FMRP的体外结合验证,其中8种为阳性,它们是:roundaboutaxon guidance receptor(Robo),thioredoxin reductase 1 mRNA(TXNRD 1),ribosomeprotein L13a mRNA(RPL13a),eukaryotic translational elongation factor 1 gammamRNA,fatty acid binding protein 5 mRNA,hypothetical protein SB145 mRNA,hypothetical protein MGC 4707 mRNA,cDNA DKFZp6668189;另外还有4种疑似阳性,1种显示阴性。
四结论和讨论
我们筛选到的31种靶RNA中没有与已报道的靶RNAs完全相同的序列,只有4种与其他实验室的筛查结果相似:文献报道eukaryotic translation elongation factor 1alpha和ribosomal protein S5能够与FMRP结合并受其调控,而我们筛查到的结果中包含eukaryotic translation elongation factor 1 gamma和ribosomal protein S9,L23,L13a,虽不能完全对应,但在功能上存在密切的联系;其余27种都未见报道。我们认为这种差异可能是因为实验材料和筛查方法不同所造成的。
在我们的结果中有18种靶基因可能与神经系统的发育分化相关,另外还涉及一些结构蛋白、代谢相关酶以及转录翻译调控因子等。在经过体外结合实验验证的8种阳性靶mRNA中,膜受体蛋白Robo已被大量的研究证明与果蝇中枢神经系统的突触的发育分化密切相关;TXNRD1(硫氧化还原蛋白还原酶1)则通过氧化还原反应调控细胞的生长、分化、凋亡以及信号传导等多种生理过程。
本课题证实了酵母三杂交系统是一种简单高效的、可以快速大规模筛查RNA-蛋白质相互作用的体内筛查体系。我们得到的筛查结果是对现有FMRP靶RNA库的有力补充,可为揭示脆性X综合症的发病机制提供新的线索。
The fragile X syndrome is an X linked mental retardation syndrome, characterised by behavioural and physical features such as a long face with large, protruding ears, macro-orchidism, and eye gaze avoidance. The prevalence of the fragile X syndrome was estimated as about 1/4000 for males and 1/6000 for females. Fragile X syndrome results from a mutation (a change in the typical DNA sequence) known as a trinucleotide repeat expansion. This means that a series of three particular nucleotides (CGG) in the DNA is greatly expanded beyond its normal size, disrupting the normal messages that need to be sent. This fact was discovered in 1991 by several teams of researchers studying the X chromosome. In the FMR-1 gene located on the X chromosome, most individuals have a CGG trinucleotide repeat that occurs between 5 and 50 times, the average being around 30. These individuals are normal with respect to fragile X syndrome, and usually carry no risk of transmitting it, although the 40-60 repeat range is sometimes considered a "gray zone" which may or may not be unstable (have a risk of expanding). Some individuals have CGG sequences that are repeated in the range of about 50 to about 200. These individuals are generally referred to as premutation carriers. This means that they carry the syndrome and can transmit it to their children. Premutation carriers, however, are not usually affected by fragile X syndrome. When the number of CGG repeats expands beyond 200, the individual usually has the full mutation. This means that they have fragile X syndrome and will experience the impairments and delays associated with the syndrome.
FMRP is an RNA binding protein associated with polyribosomes. A clue for the function of FMRP was given by the finding that FMRP contains motifs (two KH domains and one RGG box) characterizing other RNA binding proteins. Indeed, FMRP is able to bind RNA in vitro, with preference for poly (U) and poly (G) RNA homopolymers. FMRP was shown to colocalize with actively translating polysomes . The association of FMRP to polysomes suggested that it may be involved in the control of mRNA translation. Although some of the messenger RNA targets of this protein, including FMR 1, have been ascertained, many have yet to be identified .
Screening methods used to identify the mRNA-targets of FMRP were almost in vitro methods, it's far way from the real situation in living cells. Yeast three-hybrid system is a research method derived from that of yeast two-hybrid system. It provides a technic platform with a stable in vivo environment for the research of these interactions in eukaryotic cells. Although it had been used to study interactions between some potential RNA-targets with FMRP, there is no report about screening work in library scale. In our study, we are planning to screen the RNA-targets of FMRP in library scale using yeast three hybrid system. Finding some new RNA targets is expected.
A cDNA library of fetal hippocampus at the age of 19 weeks with the plasmid named pRH5' has been formed for yeast three-hybrid system. The purpose of the present work is to enlarge the size of cDNA library, and use the yeast three-hybrid system to screen the target mRNAs of FMRP in fetal hippocampus. Whole length cDNA of FMR1 were recombined into pYES plasmids, and then transferred into L40-ura yeast strains. The fusion-protein expressed by the pYES plasmids were used as the "bait" to screen the library to find the target mRNAs interacting with FMRP in the yeast cells.
About 1.5×10~6 clones have been screened on 120 plates by the X-gal filter paper screen method, and have extracted plasmid from 122 clones revealing positive result in theβ-galactosidase screen. Then the positive clone plasmids were transmitted into TOP10 E.coli., and re-extracted from them. 2,934 plasmids were obtained and sequenced analyzed with two primers respectively, and the sequences analyzed with blasten (NCBI), 50 kinds of pRH5'/cDNA-insert plasmids were identified. Re-transforming these potentially positive plasmids on by one back into the yeast cells followed byβ-galactosidase screening, 31 of 50 were proved to be positive. Up to now, 13 kinds of target-RNAs have been validated by in vitro binding with FMRP and EMSA, 8 of them were proved to be positive. Some of these positive target-mRNAs are associated with the development and differentiation of central nerve system, key-enzymes of some metabolic pathway and some structural proteins et al.
Through the above mentioned process, it has been identified that the yeast three-hybrid system is a simple and efficient method in validating the interaction between RNA and protein. As a RNA binding protein, FMRP can interact with many mRNAs acting as a key role in translation, transcription and cell differentiation process. These interactions are closely correlated with the molecular foundation of fragile X syndrome.
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