p100蛋白與U5-116蛋白的相互結(jié)合作用
發(fā)布時(shí)間：2018-09-17 20:39
【摘要】： 目的: 真核基因的表達(dá)過(guò)程,包括DNA轉(zhuǎn)錄、pre-mRNA剪接、翻譯等步驟。其中DNA轉(zhuǎn)錄和pre-mRNA剪接是兩個(gè)重要環(huán)節(jié),它們都要通過(guò)復(fù)雜的蛋白復(fù)合物來(lái)執(zhí)行功能,涉及到蛋白-DNA、蛋白-RNA和蛋白-蛋白之間的相互作用。大量研究表明基因轉(zhuǎn)錄和pre-mRNA剪接并非兩個(gè)獨(dú)立的過(guò)程,多種蛋白具有參與轉(zhuǎn)錄和剪接調(diào)控的雙重功能,從而將這兩個(gè)過(guò)程緊密地連接起來(lái),因此這些蛋白被稱(chēng)cross-talk蛋白。對(duì)于這些蛋白的了解有助于轉(zhuǎn)錄和剪接機(jī)制的深入探討。人類(lèi)p100蛋白是一個(gè)關(guān)鍵的轉(zhuǎn)錄調(diào)控子,能通過(guò)在啟動(dòng)子特異性激活因子和基本轉(zhuǎn)錄機(jī)器之間形成連接從而促進(jìn)基因轉(zhuǎn)錄。HCA(hydrophobic cluster analysis,疏水簇分析)法發(fā)現(xiàn)p100蛋白是由4個(gè)重復(fù)的SN-lik(Staphylococcal nuclease-like,葡萄球菌核酸酶類(lèi)似)功能片段和隨后位于蛋白C末端Tudor-SN(TSN)功能片段組成。大量的研究發(fā)現(xiàn)p100蛋白作為一種共激活因子參與基因轉(zhuǎn)錄調(diào)控,這種作用主要借助其重復(fù)的SN樣功能片段完成。前期我們課題組采用非變性膠電泳觀(guān)察剪接體復(fù)合物體外組裝的情況。觀(guān)察發(fā)現(xiàn)p100蛋白TSN片段參與并促進(jìn)剪接體的形成。但p100蛋白的TSN功能片段參與并促進(jìn)pre-mRNA剪接加工過(guò)程的具體分子機(jī)制還不明確。 Pre-mRNA的剪接加工是在一個(gè)大的蛋白復(fù)合物—剪接體中進(jìn)行的,而剪接體中重要的組分就是U5snRNP。U5-116蛋白因?yàn)槟軌蛑苯优cU5 snRNA結(jié)合,因而組成U5snRNP的核心,是U5的特異蛋白。本研究的主要目的在于研究p100蛋白,U5-116蛋白之間特異的結(jié)合方式和結(jié)合位點(diǎn),通過(guò)蛋白—蛋白相互作用研究探討p100蛋白的TSN功能片段參與并促進(jìn)Pre-mRNA剪接加工過(guò)程的分子機(jī)制,為隨后p100蛋白功能的研究奠定基礎(chǔ)。 方法: 本課題分三部分進(jìn)行研究,第一部分為確定能與p100蛋白,U5-116蛋白結(jié)合的hPrp8蛋白功能片段。首先根據(jù)hPrp8蛋白的結(jié)構(gòu)特點(diǎn)構(gòu)建hPrp8蛋白不同結(jié)構(gòu)域的GST融合蛋白表達(dá)質(zhì)粒。利用GST pull down方法用GST-hPrp8片段融合蛋白釣取體外翻譯帶有~(35)S標(biāo)記的p100蛋白,放射自顯影觀(guān)察結(jié)果,尋找hPrp8的功能部位。同樣方法研究U5-116蛋白與hPrp8蛋白結(jié)合的功能片段。 第二部分是p100蛋白與U5-116蛋白的結(jié)合。首先構(gòu)建pEGFP-CI-U5-116重組質(zhì)粒,利用CO-IP(Co-Immunoprecipitation,免疫共沉淀)技術(shù)研究p100蛋白與U5-116蛋白在細(xì)胞內(nèi)的結(jié)合情況。再構(gòu)建pERFP-CI-p100重組質(zhì)粒,利用激光共聚焦顯微鏡研究p100蛋白與U5-116蛋白細(xì)胞內(nèi)定位情況。 第三部分為了進(jìn)一步確定p100蛋白與U5-116蛋白結(jié)合的功能片段。使用GST融合蛋白釣取法和Western Blot技術(shù),用p100蛋白SN片段和TSN片段的GST融合蛋白(GST空載作為陰性對(duì)照)釣取細(xì)胞內(nèi)過(guò)表達(dá)的U5-116蛋白,Western Blot方法檢測(cè)。 結(jié)果: 第一部分hPrp8蛋白與p100蛋白,U5-116蛋白相互結(jié)合的功能片段研究結(jié)果顯示:p100蛋白不能與hPrp8蛋白2.1片段結(jié)合,能與2.2片段在體外直接結(jié)合。U5-116蛋白能與hPrp8蛋白的2.1,2.2片段在體外結(jié)合。 第二部分成功構(gòu)建了以下重組質(zhì)粒:①pEGFP-CI-U5-116②pERFP-CI-p100。過(guò)表達(dá)的p100蛋白與U5-116蛋白之間可以發(fā)生共沉淀,表明兩者細(xì)胞內(nèi)可以穩(wěn)定結(jié)合。利用激光共聚焦顯微鏡研究p100蛋白與U5-116蛋白定位情況。在轉(zhuǎn)染后的細(xì)胞內(nèi)觀(guān)察到兩者定位基本一致,主要分布于胞漿。 第三部分p100蛋白與U5-116蛋白在細(xì)胞外結(jié)合的研究結(jié)果顯示p100蛋白的TSN片段能夠與U5-116蛋白結(jié)合而SN片段不能與其結(jié)合。 結(jié)論: 本課題研究結(jié)果表明p100蛋白和U5-116蛋白都能與hPrp8蛋白體外直接結(jié)合,但結(jié)合部位不同。p100蛋白能夠與U5-116蛋白在細(xì)胞內(nèi),外均可結(jié)合。二者之間的結(jié)合是由p100-TSN片段介導(dǎo)的。
[Abstract]:Objective:
The process of eukaryotic gene expression, including DNA transcription, pre-mRNA splicing, translation and so on. DNA transcription and pre-mRNA splicing are two important links. They all perform their functions through complex protein complexes, involving protein-DNA, protein-RNA and protein-protein interactions. These proteins are known as cross-talk proteins. Understanding of these proteins is helpful in understanding the transcriptional and splicing mechanisms. Human P100 protein is a key transcriptional regulator. HCA (hydrophobic cluster analysis) showed that P100 protein was a functional fragment of four repeated SN-liks (Staphylococcal nuclease-like) and subsequently located at the end of protein C. Tudor-SN (TSN) functional fragment composition. A large number of studies have found that P100 protein as a co-activator involved in gene transcription regulation, this role mainly through its repeated SN-like functional fragments. Previously, our team used non-denaturing gel electrophoresis to observe the assembly of splice complexes in vitro. However, the specific molecular mechanism of TSN functional fragment of P100 protein participating in and promoting pre-mRNA splicing is not clear.
Pre-mRNA splicing is performed in a large protein complex-splicing body, and the important component of splicing body is the protein U5snRNP.U5-116, which can bind directly to U5 snRNA. Therefore, the core of U5snRNP is the specific protein of U5. The main purpose of this study is to study the specificity of P100 protein and U5-116 protein. The binding mode and binding site were used to investigate the molecular mechanism of TSN functional fragment of P100 protein involved in the splicing process of Pre-mRNA through protein-protein interaction.
Method:
The first part is to determine the functional fragment of hPrp8 protein which can bind to P100 and U5-116 proteins. Firstly, the GST fusion protein expression plasmids of different domains of hPrp8 protein were constructed according to the structural characteristics of hPrp8 protein. 35) S-labeled P100 protein, autoradiography results, to find the functional site of hPrp8. The same method was used to study the functional fragments of U5-116 protein binding to hPrp8 protein.
The second part is the binding of P100 protein to U5-116 protein. First, the pEGFP-CI-U5-116 recombinant plasmid was constructed. The binding of P100 protein to U5-116 protein was studied by CO-IP (Co-Immuno precipitation) technique. Then the pERFP-CI-p100 recombinant plasmid was constructed and the P100 protein to U5-116 egg was studied by laser confocal microscopy. Localization of leukocytes.
In the third part, in order to further determine the functional fragment of P100 protein binding to U5-116 protein, the over-expressed U5-116 protein was detected by GST fusion protein fishing method and Western Blot technique, using the GST fusion protein of P100 protein SN fragment and TSN fragment as negative control.
Result:
The results showed that P100 protein could not bind to fragment 2.1 of hPrp8 protein and could bind directly to fragment 2.2 in vitro. U5-116 protein could bind to fragment 2.1 and 2.2 of hPrp8 protein in vitro.
In the second part, the following recombinant plasmids were successfully constructed: (1) pEGFP-CI-U5-116; (2) pERFP-CI-p100. The co-precipitation between the overexpressed P100 protein and U5-116 protein indicated that the two proteins could bind stably in the cells. The localization of P100 protein and U5-116 protein was studied by confocal laser microscopy. The location is basically the same, mainly distributed in cytoplasm.
In the third part, the extracellular binding of P100 protein to U5-116 protein showed that the TSN fragment of P100 protein could bind to U5-116 protein and SN fragment could not bind to it.
Conclusion:
The results of this study showed that both P100 protein and U5-116 protein can bind directly to hPrp8 protein in vitro, but the binding sites are different. P100 protein can bind to U5-116 protein both in vitro and in vivo.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392
本文編號(hào)：2247028
[Abstract]:Objective:
The process of eukaryotic gene expression, including DNA transcription, pre-mRNA splicing, translation and so on. DNA transcription and pre-mRNA splicing are two important links. They all perform their functions through complex protein complexes, involving protein-DNA, protein-RNA and protein-protein interactions. These proteins are known as cross-talk proteins. Understanding of these proteins is helpful in understanding the transcriptional and splicing mechanisms. Human P100 protein is a key transcriptional regulator. HCA (hydrophobic cluster analysis) showed that P100 protein was a functional fragment of four repeated SN-liks (Staphylococcal nuclease-like) and subsequently located at the end of protein C. Tudor-SN (TSN) functional fragment composition. A large number of studies have found that P100 protein as a co-activator involved in gene transcription regulation, this role mainly through its repeated SN-like functional fragments. Previously, our team used non-denaturing gel electrophoresis to observe the assembly of splice complexes in vitro. However, the specific molecular mechanism of TSN functional fragment of P100 protein participating in and promoting pre-mRNA splicing is not clear.
Pre-mRNA splicing is performed in a large protein complex-splicing body, and the important component of splicing body is the protein U5snRNP.U5-116, which can bind directly to U5 snRNA. Therefore, the core of U5snRNP is the specific protein of U5. The main purpose of this study is to study the specificity of P100 protein and U5-116 protein. The binding mode and binding site were used to investigate the molecular mechanism of TSN functional fragment of P100 protein involved in the splicing process of Pre-mRNA through protein-protein interaction.
Method:
The first part is to determine the functional fragment of hPrp8 protein which can bind to P100 and U5-116 proteins. Firstly, the GST fusion protein expression plasmids of different domains of hPrp8 protein were constructed according to the structural characteristics of hPrp8 protein. 35) S-labeled P100 protein, autoradiography results, to find the functional site of hPrp8. The same method was used to study the functional fragments of U5-116 protein binding to hPrp8 protein.
The second part is the binding of P100 protein to U5-116 protein. First, the pEGFP-CI-U5-116 recombinant plasmid was constructed. The binding of P100 protein to U5-116 protein was studied by CO-IP (Co-Immuno precipitation) technique. Then the pERFP-CI-p100 recombinant plasmid was constructed and the P100 protein to U5-116 egg was studied by laser confocal microscopy. Localization of leukocytes.
In the third part, in order to further determine the functional fragment of P100 protein binding to U5-116 protein, the over-expressed U5-116 protein was detected by GST fusion protein fishing method and Western Blot technique, using the GST fusion protein of P100 protein SN fragment and TSN fragment as negative control.
Result:
The results showed that P100 protein could not bind to fragment 2.1 of hPrp8 protein and could bind directly to fragment 2.2 in vitro. U5-116 protein could bind to fragment 2.1 and 2.2 of hPrp8 protein in vitro.
In the second part, the following recombinant plasmids were successfully constructed: (1) pEGFP-CI-U5-116; (2) pERFP-CI-p100. The co-precipitation between the overexpressed P100 protein and U5-116 protein indicated that the two proteins could bind stably in the cells. The localization of P100 protein and U5-116 protein was studied by confocal laser microscopy. The location is basically the same, mainly distributed in cytoplasm.
In the third part, the extracellular binding of P100 protein to U5-116 protein showed that the TSN fragment of P100 protein could bind to U5-116 protein and SN fragment could not bind to it.
Conclusion:
The results of this study showed that both P100 protein and U5-116 protein can bind directly to hPrp8 protein in vitro, but the binding sites are different. P100 protein can bind to U5-116 protein both in vitro and in vivo.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392
【相似文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 李曉冬;p100蛋白與U5-116蛋白的相互結(jié)合作用[D];天津醫(yī)科大學(xué);2009年
，本文編號(hào)：2247028
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2247028.html
最近更新
教材專(zhuān)著
