【作者】 李晓冬；

【导师】 杨洁；

【作者基本信息】 天津医科大学 ， 免疫学， 2009， 硕士

【摘要】 目的:真核基因的表达过程,包括DNA转录、pre-mRNA剪接、翻译等步骤。其中DNA转录和pre-mRNA剪接是两个重要环节,它们都要通过复杂的蛋白复合物来执行功能,涉及到蛋白-DNA、蛋白-RNA和蛋白-蛋白之间的相互作用。大量研究表明基因转录和pre-mRNA剪接并非两个独立的过程,多种蛋白具有参与转录和剪接调控的双重功能,从而将这两个过程紧密地连接起来,因此这些蛋白被称cross-talk蛋白。对于这些蛋白的了解有助于转录和剪接机制的深入探讨。人类p100蛋白是一个关键的转录调控子,能通过在启动子特异性激活因子和基本转录机器之间形成连接从而促进基因转录。HCA（hydrophobic cluster analysis,疏水簇分析）法发现p100蛋白是由4个重复的SN-lik（Staphylococcal nuclease-like,葡萄球菌核酸酶类似）功能片段和随后位于蛋白C末端Tudor-SN（TSN）功能片段组成。大量的研究发现p100蛋白作为一种共激活因子参与基因转录调控,这种作用主要借助其重复的SN样功能片段完成。前期我们课题组采用非变性胶电泳观察剪接体复合物体外组装的情况。观察发现p100蛋白TSN片段参与并促进剪接体的形成。但p100蛋白的TSN功能片段参与并促进pre-mRNA剪接加工过程的具体分子机制还不明确。Pre-mRNA的剪接加工是在一个大的蛋白复合物—剪接体中进行的,而剪接体中重要的组分就是U5snRNP。U5-116蛋白因为能够直接与U5 snRNA结合,因而组成U5snRNP的核心,是U5的特异蛋白。本研究的主要目的在于研究p100蛋白,U5-116蛋白之间特异的结合方式和结合位点,通过蛋白—蛋白相互作用研究探讨p100蛋白的TSN功能片段参与并促进Pre-mRNA剪接加工过程的分子机制,为随后p100蛋白功能的研究奠定基础。方法:本课题分三部分进行研究,第一部分为确定能与p100蛋白,U5-116蛋白结合的hPrp8蛋白功能片段。首先根据hPrp8蛋白的结构特点构建hPrp8蛋白不同结构域的GST融合蛋白表达质粒。利用GST pull down方法用GST-hPrp8片段融合蛋白钓取体外翻译带有³⁵S标记的p100蛋白,放射自显影观察结果,寻找hPrp8的功能部位。同样方法研究U5-116蛋白与hPrp8蛋白结合的功能片段。第二部分是p100蛋白与U5-116蛋白的结合。首先构建pEGFP-CI-U5-116重组质粒,利用CO-IP（Co-Immunoprecipitation,免疫共沉淀）技术研究p100蛋白与U5-116蛋白在细胞内的结合情况。再构建pERFP-CI-p100重组质粒,利用激光共聚焦显微镜研究p100蛋白与U5-116蛋白细胞内定位情况。第三部分为了进一步确定p100蛋白与U5-116蛋白结合的功能片段。使用GST融合蛋白钓取法和Western Blot技术,用p100蛋白SN片段和TSN片段的GST融合蛋白（GST空载作为阴性对照）钓取细胞内过表达的U5-116蛋白,Western Blot方法检测。结果:第一部分hPrp8蛋白与p100蛋白,U5-116蛋白相互结合的功能片段研究结果显示:p100蛋白不能与hPrp8蛋白2.1片段结合,能与2.2片段在体外直接结合。U5-116蛋白能与hPrp8蛋白的2.1,2.2片段在体外结合。第二部分成功构建了以下重组质粒:①pEGFP-CI-U5-116②pERFP-CI-p100。过表达的p100蛋白与U5-116蛋白之间可以发生共沉淀,表明两者细胞内可以稳定结合。利用激光共聚焦显微镜研究p100蛋白与U5-116蛋白定位情况。在转染后的细胞内观察到两者定位基本一致,主要分布于胞浆。第三部分p100蛋白与U5-116蛋白在细胞外结合的研究结果显示p100蛋白的TSN片段能够与U5-116蛋白结合而SN片段不能与其结合。结论:本课题研究结果表明p100蛋白和U5-116蛋白都能与hPrp8蛋白体外直接结合,但结合部位不同。p100蛋白能够与U5-116蛋白在细胞内,外均可结合。二者之间的结合是由p100-TSN片段介导的。更多还原

【Abstract】 Objective:Eukaryotic gene expression process is consisting of DNA transcription, pre-mRNA splicing and translation.In this processes transcription and pre-mRNA splicing are the key processes and both of them require multi-component complex to function,concluding of protein-DNA,protein-RNA and protein-protein.Transcription and splicing are not independent events.They are functionally coordinated.Some proteins have transcription and splicing function,so they are called cross-talk protein. Studying these cross-talk proteins will help us known much about transcription and splicing.Human p100 protein is a novel transcriptional coactivator.HCA（hydrophobic cluster analysis）show that p100 consists of staphylococcal nuclease（SN）-like and Tudor（TSN）domains.The SN-like domains have been shown to function in transcription,but the function of TSN domain in the assembly of snRNP complexes and pre-mRNA splicing process.Pre-mRNA splicing is happened in a big protein complex -splicosome,in which there is U5 snRNP specific proteins,such as U5-116.The purpose of this study was to validate the hypothesis that p100 protein functions in pre- mRNA splicing,and to reveal Splicing processes molecule mechanism.Methods:In this report,there are three parts.First,GST gene fusion proteins with various fragments of hPrp8 were used to pull down in vitro translated full length p100 or U5-116 labeled with³⁵S.Western Blot experiments were also used to confirm the specific interaction domains of hPrp8.In the second part,interactions of p100 and U5-116 proteins in cells.We constructed the recombinant plasmids of pEGFP-CI-U5-116 and pERFP-CI-p100. The two recombinant plasmids are used to studies in cells.We coimmunoprecipitation of pl00 and U5-116 proteins to confirm the interaction between them.We also studied the colocalization of p100 and U5-116 proteins by immunofluorescence microscopy.In the last part,identify the domain of p100 protein interacts with U5-116.GST gene fusions with various fragments of p100 were used to pull down U5-116 protein in cells to identify the interaction domains of p100.Results:In the first part,we found that p100 protein can bind to hPrp8 domain 2.2,not hPrp8 domain 2.1;U5-116 can bind to hPrp8 domain 2.1,2.2. In the second part,we constructed the following recombinant plasmids successfully:①pEGFP-CI-U5-116②pERFP-CI-p100.Coimmunoprecipitation and Western Blot experiments also confirmed the interaction of over-express two proteins in cell.And we observed the colocalization of p100 protein and U5-116 protein by immunofluorescence microscopy.In the third part,p100-TSN fragment stably interacts with U5-116 protein.Conclusion:Our present study provides following views:p100 protein,U5-116 protein can binds to hPrp8 protein,but function domains of hPrp8 are different.p100 protein can interacts U5-116 protein.p100-TSN stably binds to U5-116 protein.更多还原