【作者】 步天栩；

【导师】 姚智； 杨洁；

【作者基本信息】 天津医科大学 ， 免疫学， 2007， 博士

【摘要】 目的:真核基因准确表达是一个复杂的过程,其中DNA的转录和pre-mRNA的剪接均是基因表达中的重要环节,都需要蛋白复合物的精密调控。真核基因转录活性的调节依赖于转录调控复合物,它是由基本转录调控元件、特异性转录调控因子和共激活因子等组成的。pre-mRNA剪接加工则是在蛋白复合物—剪接体的调控下完成的。大量研究表明基因转录和pre-mRNA剪接并非两个绝对独立的过程,很多蛋白都可通过它们之间的偶联作用将基因转录和pre-mRNA剪接加工两个环节联系起来,使转录和剪接同时进行,于是出现了pre-mRNA的“共转录”（co-transcriptional）加工的新概念。人类p100蛋白是一种新发现的转录共激活因子,最初作为转录因子EBNA-2（Epstein-Barr virus nuclear antigen 2,EB病毒核抗原2）的共激活因子被发现。p100蛋白可作为桥梁参与多种转录因子和基本转录元件结合过程中,它可与基本转录元件的核心成员—RNA polⅡ（RNApolymeraseⅡ,RNA聚合酶Ⅱ）直接结合,从而参与转录前起始复合物的形成,并以此形式参与多条信号传导通路的基因转录调控。p100蛋白由4个重复的类似葡萄球菌核酸酶的片段（Staphylococcal nucleases,SN）以及随后的Tudor-SN（TD）片段组成。其SN片段可与多种转录调节因子结合,参与特异性基因转录活性调控;但TD片段的功能还不甚了解,研究发现它可与多种U5 snRNP特异性蛋白结合,例如U5-220K（hPrp8）、U5-200K（hBrr2）和U5-116K（hSnu114）等。此外,由于p100蛋白TD片段与SMN（survival of motor neuron,运动神经元生存蛋白）的TD片段具有高度保守性和结构上的相似性,而后者则参与pre-mRNA剪接加工,促进剪接体的形成,因此推测p100蛋白有可能通过其TD片段参与pre-mRNA剪接加工。本研究的主要目的在于探讨p100蛋白如何影响pre-mRNA剪接加工,以及它是否参与剪接体组装过程的调节,并且对其可能的机制进行分析和探讨。方法:本课题分为四部分进行研究。第一部分为非同位素标记的pre-mRNA体外剪接实验方法的建立。以AdML（adenovirus major late,腺病毒株晚期启动子）质粒为模板进行体外转录分别合成³²p标记的pre-mRNA和不标记的pre-mRNA,将它们作为底物分别加入到同位素标记剪接反应体系以及非标记的剪接反应体系中。采用放射性自显影技术观察传统的标记的剪接反应产物,而非标记的产物则通过溴化乙锭染色来探测,并对两种方法进行比较。第二部分是p100蛋白及其功能片段的纯化和鉴定。首先,采用以Flag为标签的融合蛋白纯化系统分别从HeLa-p100细胞株（稳定表达带有Flag标签的p100蛋白）以及p100蛋白过表达的COS-7细胞株的细胞裂解液中钓取和纯化p100蛋白。第二,采用GST（Glutathione S transferase,谷胱甘肽-S-转移酶）融合蛋白纯化系统分别从可表达p100-TD和p100-SN的GST融合蛋白的细菌裂解液中钓取并纯化p100-TD和p100-SN片段蛋白。第三部分是p100蛋白参与剪接加工研究。采用已建立的非同位素标记的体外剪接实验方法观察p100蛋白在pre-mRNA剪接加工过程中的潜在功能。在反应体系中不同剂量的纯化的p100蛋白观察p100蛋白剂量大小对于剪接反应的影响;随后观察加入p100蛋白后不同时间段反应进行的情况;采用RNAi（RNAinterference,RNA干扰）技术探讨p100蛋白是否是pre-mRNA剪接加工有效进行所需的重要物质,即将siRNA（small interference RNA,小干扰RNA）用脂质体转染到稳定表达p100蛋白的HeLa细胞中,使细胞内p100蛋白表达量明显降低,用Western blotting鉴定p100蛋白表达抑制情况后进行体外剪接实验,观察p100蛋白减少是否影响剪接反应。剪接反应产物均通过含7M尿素的8%变性PAGE凝胶电泳进行分离,随后用溴化乙锭染色观察。第四部分p100蛋白及其功能片段对剪接体组装的影响。体外剪接体组装实验是在同位素标记的剪接实验基础上,利用同位素标记的pre-mRNA和剪接中间产物示踪不同状态的剪接体复合物的存在以及变化情况,以便观察p100蛋白及其TD片段是否影响剪接体组装过程。结果:第一部分研究建立了采用溴化乙锭染色的非标记的pre-mRNA体外剪接实验方法,通过该方法可以清晰探测剪接反应的进行以及反应产物的变化。与传统的同位素标记剪接反应相比不足之处在于非特异性条带较多。第二部分结果表明采用Flag融合蛋白纯化系统以及GST融合蛋白纯化系统可以分别制备p100蛋白和p100功能片段—TD和SN。第三部分结果表明随着剪接反应体系中p100蛋白水平增加,剪接产物中成熟mRNA的量增加,而且剪接反应的第一步酯基转移反应的产物套索中间体量的积累明显较快,而对照蛋白BSA（bovine serum albumin,牛血清白蛋白）对于剪接活性并无影响;p100蛋白可随着时间延长加速剪接反应产物的积累;为了证实p100蛋白是否只在剪接反应的初期具有促进剪接的活性,在剪接反应开始20 min时才加入p100蛋白,其促进剪接效率的功能与反应前即将p100蛋白与HeLa细胞核提取物一起孵育的反应结果并无差别;运用RNAi技术,设计并合成的p100 siRNA可以有效地降低HeLa-p100细胞p100蛋白表达水平。在剪接反应中,当p100蛋白表达水平下降时pre-mRNA剪接加工明显受到抑制。当剪接反应中加入一定量的纯化的p100蛋白后,受抑制的剪接反应活性可部分恢复。第四部分,p100蛋白对于剪接体复合物组装的影响是通过同位素示踪剪接体聚集状态（spliceosome assembly）来分析的,结果表明p100蛋白可以促进所有3种剪接体复合物的形成,特别是形成复合物A生成以及复合物A向B转化的速度加快更为明显,p100蛋白的TD片段对剪接体组装的影响与全长p100蛋白一致。结论:通过溴化乙锭染色来鉴定反应产物的非标记pre-mRNA体外剪接反应的建立为在普通实验室中进行pre-mRNA剪接加工机制研究提供新途径,特别是在预试验方面,为剪接加工的相关研究奠定基础。本研究发现P100蛋白提高pre-mRNA的剪接速度和效率,它可在剪接反应初期以及中后期都具有作用,并可以促进进剪接体的组装。作为一个潜在的多功能蛋白,它可以通过不同结构片段与不同功能复合物相互作用,从而具有不同功能。因此p100蛋白不仅是基因转录共激活因子,它还可以调控pre-mRNA剪接加工。很有可能是通过与RNA polⅡ和U5 snRNP特异性蛋白（特别是Prp8）直接结合而参与转录和剪接过程。更多还原

【Abstract】 Objective:Eukaryotic gene expression is a complex stepwise process,in which transcription and pre-mRNA splicing are the key nuclear processes and both of them require multi-component complex to function.Transcription is regulated by protein complex composed of transcription factors,basal transcriptional machinery and a group of coactivators,while pre-mRNA splicing is carried out by spliceosome.Several studies have shown that pre-mRNA splicing occurred coordinately with the synthesization of nascent pre-mRNA.Therefore, transcription and splicing are not independent events,they are functionally coordinated.Co-transcriptional splicing appears as a new concept.Human p100 protein is a novel transcriptional coactivator,which was first identified as a transcriptional coactivator of Epstein-Barr virus nuclear antigen 2（EBNA-2）. Since it can directly interact with RNA polymeraseⅡ（RNA polⅡ）,p100 protein may facilitate the access of other transcription factor to the basal transcription machinery and form the pre-initiation complex of transcription.Thus,it could regulate the transcription in several signaling pathway,p100 consists of Staphylococcal nuclease（SN）-like and Tudor（TD）domains of which the SN-like domains have been shown to function in transcription,but the function of TD domain has remained elusive.However,our previous study showed that it could interact with U5 snRNP specific proteins,such as U5-220K（hPrp8）, U5-200K（hBrr2）and U5-116K（hSnu114）.p100 TD is homologous to SMN （survival of motor neuron protein）TD domain,which functions in the assembly of snRNP complexes and pre-mRNA splicing process.The purpose of this study was to validate the hypothesis that p100 protein functions in pre-mRNA splicing,and to reveal whether it could participate in the regulation of splicosome assembly.Methods:In this report,we demonstrate a novel function for p100 protein as a protein involves in pre-mRNA splicing and spliceosome assembly.Firstly,in the study of methodology of pre-mRNA splicing in vitro assay,two different splicing methods in vitro,³²P labeled and unlabeled pre-mRNA as the substrates in the reaction were investigated.The radiolabeled splicing reaction products were visualized by autoradiography,while the unlabeled products were observed by Ethidium Bromide（EB）staining.Secondly,the purified p100 protein and its functional domains were prepared by Flag-tag fusion protein purification system and GST（glutathione S transferase）fusion protein system respectively.The lysates of HeLa cells stable expressing Flag-tagged p100 and p100 over-expressed COS-7 cells were incubated with anti-Flag M2 agarose respectively.GST fusion proteins of p100-TD and p100-SN were bound on glutathione-Sepharose 4B beads when incubated with bacterial lysates.After the whole steps of purification,the purified p100 protein and its functional domains were detected by SDS-PAGE （polyacrylamide gel electrophoresis）with silver staining.Thirdly,to investigate the potential function of p100 protein in pre-mRNA splicing,the unlabeled in vitro splicing reaction was carried out.Different amount of purified p100 protein was added in the reaction system;and the time course of splicing reaction with addition of p100 was also examined.RNA interference was performed to examine whether the p100 protein is essential in the effective splicing reaction.The effect of p100 siRNA was examined by western blotting.All the results were analyzed by 8%denatured PAGE with 7M urea,and EB staining afterward.Fourthly,the in vitro splicing assay with radiolabel was carried out for the splicosome assembly in vitro reaction.Native PAGE analysis was performed and visualized by autoradiography and phosphorimager to investigate the roles of p100 protein and TD domain of it in the splicosome assembly.Results:Firstly,according to the comparison of the traditional radiolabeled pre-mRNA splicing assay with the new unlabeled one,the RNA products of in vitro splicing,such as mRNA,lariat-intermediate,etc,could be observed clearly, although there are more unspecific bands in the EB staining splicing assay than ³²P labeled one.Secondly,as a result of the purification of p110 protein,the only purified p100 protein could be obtained only from the p100 over-expressed COS-7 cells. The p100 protein prepared from the p100 stable expressing HeLa cells was found interacting with U5-220K（Prp8）和U5-200K（Brr2）stably.The p100 functional domains,p100-TD and p100-SN were purified by the GST fusion protein system successfully.Thirdly,to identify the effect of the p100 protein in the pre-mRNA splicing process,several splicing assays were carried out.The results indicated that the amount of mRNA was increased concomitantly with the increase in p100 protein level,while the BSA（bovine serum albumin）control did not affect the splicing activity;And the products of the first step of splicing was accumulated more quickly,p100 protein accelerated the mRNA accumulation in a time-dependent manner;To identify whether p100 is only functioning at the early stage of the splicing reaction,the addition of the purified p100 to the reaction 20 min after the initiation resulted in the same efficiency as pre-incubation of p100 with HeLa nuclear extract.In the study of RNAi（RNA interference）,p100 siRNA efficiently reduced the amount of p100 protein in the p100-HeLa nuclear lysates. And the splicing reaction was significantly inhibited with p100 absence. However,the impaired splicing activity was recovered by addition of p100 protein.Fourthly,the results of the effect of the p100 protein and its functional domains in splicosomal complex formation were confirmed with the native gel analysis.Consistent with the splicing result,the native gel analysis demonstrated that TD domain alone had a similar function in splicing as the full-length p100 protein,that is,it accelerated the kinetics of all the spliceosomal complex formation,including complex A,B and C.Conclusion.The unlabeled pre-mRNA splicing assay still needs to be optimized, but it could be used as the alternative option,especially for the preliminary study,p100 protein functions as not only a transcriptional coactivator,but also a potential factor that involving in the regulation of pre-mRNA splicing.It could directly interact with RNA polⅡand U5 snRNP（small nuclear ribonucleoprotein）specific proteins（such as Prp8）,and participate in the both events of transcription and pre-mRNA splicing.Thus our results suggest that p100 protein is a novel dual function regulator of gene expression that participates via distinct domains in both transcription and pre-mRNA splicing.更多还原