p100蛋白与U5 snRNP特异蛋白人Prp8和人Snu114相互结合作用的研究
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摘要
目的：p100蛋白首先作为EBNA2(Epstein-Barr virus nuclear antigen2, EB病毒细胞核抗原2)的转录调控激活因子被发现。HCA(hydrophobic cluster analysis,疏水簇分析)法发现p100蛋白是由4个重复的SN-like ((Staphylococcal nuclease-like,葡萄球菌核酸酶类似)功能片段和随后位于蛋白C末端的Tudor-SN(TD)功能片段组成。我们通过大量的研究发现p100蛋白作为一种共激活因子参与着基因转录调控,这种作用主要借助其SN样功能片段完成。但p100蛋白TD片段的功能目前并不清楚,但是我们通过比较不同蛋白的结构和功能发现：p100蛋白的TD功能片段与SMN (Survival motor neuron protein,运动神经元生存蛋白)的Turdor功能片段有较高的同源性,具有相似的结构和功能特点。而SMN是一种偶联基因转录调控和pre-mRNA (precusor messenger RNA,前体mRNA)剪接加工的双重功能蛋白,因此我们推测p100蛋白因为具有相似的TD功能片段而具有剪接加工的作用。
Pre-mRNA的剪接加工是在一个大的蛋白复合物一剪接体中进行的,而剪接体中重要的组分就是U5snRNP。 hPrp8蛋白和hSnu114蛋白因为能够直接与U5snRNA结合,因而组成U5snRNA的核心。我们研究发现p100蛋白TD功能片段能够与一组U5snRNP特异蛋白组分结合,这其中包括hPrp8蛋白和hSnu114蛋白。但是我们还需要鉴定：(1)它们是直接结合还是通过桥梁蛋白而间接结合?(2)如果是直接结合,它们通过哪一段功能片段结合?基于此,我们开展了相关研究工作。
方法：本课题分三部分进行研究,第一部分p100蛋白与hPrp8在细胞内的结合。首先利用GST (Glutathione S transferase,谷胱甘肽S转移酶)融合蛋白钓取法和Western Blot技术研究p100蛋白的TD片段与一组U5snRNP特异蛋白之间的作用。首先提取HeLa细胞的核裂解液,与p100蛋白TD功能片段的GST融合蛋白孵育一定时间后进行SDS-PAGE电泳,使用考马斯亮兰染色或银染分析结果。通过质谱分析p100蛋白TD片段所能结合的核蛋白。在此基础上,利用免疫共沉淀的方法和Western Blot技术研究p100蛋白与hPrp8蛋白在细胞内是否能够稳定结合,提取HeLa-p100稳定表达细胞株的核裂解液,一部分与抗Flag抗体(因为建立p100的稳定表达株时,p100后面带有一个Flag小肽)进行免疫沉淀(抗IgG抗体作为阴性对照),钓取核裂解液中的p100蛋白,然后用葡聚糖颗粒纯化与p100蛋白结合的复合物,之后进行SDS-PAGE电泳分离结合的蛋白组分,再用抗Flag抗体和抗hPrp8抗体检测p100蛋白与hPrp8蛋白能否在细胞内结合；另一部分HeLa-p100稳定表达细胞株的核裂解液与抗hPrp8抗体进行免疫沉淀,钓取核裂解液中的hPrp8蛋白,然后用葡聚糖颗粒纯化与hPrp8蛋白结合的复合物之后进行SDS-PAGE电泳分离结合的蛋白组分,用抗Flag抗体和抗hPrp8抗体再一次检测p100蛋白与hPrp8蛋白能否在细胞内结合。为了进一步确定p100蛋白与hPrp8蛋白结合的部位,再次使用GST融合蛋白钓取法和Western Blot技术,将p100蛋白SN片段和TD片段的GST融合蛋白(GST空载作为阴性对照)与HeLa-p100稳定表达株的核裂解液孵育一定时间后进行SDS-PAGE电泳,用抗hPrp8抗体检测是p100蛋白的SN样功能片段还是TD功能片段与hPrp8蛋白结合。
第二部分为了进一步确定p100蛋白与hPrp8蛋白结合的功能区域,需要构建以下重组质粒：(1)构建hPrp8和hSnu114全长序列真核表达质粒。应用逆转录聚合酶链式反应(reverse transcription polymerase chain reaction, RT-PCR)技术扩增hPrp8和hSnu114的全长序列,hPrp8分成4段彼此有一小段重复序列(约十几个碱基)的片段,hSnu114分成两个片段扩增。这些片段中都含有特异的酶切位点(在序列中是单一的酶切位点),目的是为将来能够连接成全长序列。由于hPrp8序列比较长(7.2kb),优先考虑使用重叠延伸PCR法将4个RT-PCR片段连成全长并亚克隆至真核表达质粒pcDNA3.1(+)中。hSnu114序列为2.9kb,可以利用自身含有的单一酶切位点将两段RT-PCR片段连成全长并亚克隆至真核表达质粒pcDNA3.1(-)中。这2种质粒可以作为体外翻译hPrp8和hSnu114蛋白时的DNA模板,用于研究p100蛋白与hPrp8和hSnu114蛋白在体外的相互作用；(2)hSnu114的pEGFP C I及带有6个组氨酸标签(设计反义引物时引入6个组氨酸密码子,后面为终止密码子)的pcDNA3.1(-)全长重组质粒。分别选择Sal I/BamH I和Xba I/BamH I限制性内切酶将hSnu114全长序列定向克隆至pEGFP C I和pcDNA3.1(-)载体中。由于hPrp8序列较长,在构建这2种质粒时存在一定的困难,但现有研究表明hPrp8和hSnu114这2种蛋白在细胞内是以复合物的形式存在的,即通过hSnu114蛋白可以预见hPrp8蛋白与p100蛋白之间的作用。利用免疫荧光技术研究hSnu114和hPrp8蛋白与p100蛋白在细胞内结合的情况。将hSnu114的pEGFP C I重组质粒瞬时转染HeLa/COS7细胞,观察胞内绿色荧光,凡是有绿色荧光的地方就表示hSnu114蛋白的存在,待细胞贴璧后(转染后6-8个小时)先后加入第一抗体(与胞内p100蛋白结合)和红色荧光标记的第二抗体(与一抗结合),于荧光倒置显微镜下观察同一视野下的荧光定位。此外,还可以应用hSnu114的带有6个组氨酸标签的pcDNA3.1(-)全长重组质粒,利用免疫共沉淀的方法研究hPrp8蛋白与p100蛋白之间的作用机制。(3)构建hPrp8和hSnu114功能片段的pGEX-4T-1重组质粒,利用GST基因融合系统在体外表达相应功能片段的GST融合蛋白。hPrp8和hSnu114都存在着介导不同生物功能的功能片段。根据文献报道我们设计了相关的功能片段,(4)构建hPrp8和hSnu114功能片段的pBluescript SK重组质粒。这些重组质粒可作为体外翻译的DNA模板,在体外翻译出相应功能片段用同位素标记的蛋白。构建hPrp8和hSnu114功能片段的pGEX-4T-1重组质粒和pBluescript SK里组质粒的目的是进一步研究hPrp8、 hSnu114和p100蛋白三者之间的作用区域。
第三部分利用TNT⑧T7体外翻译系统在体外合成带有35S标记的p100蛋白和hPrp8功能片段,用GST pull down (GST融合蛋白钓取1法研究hPrp8的哪一段功能片段能够与p100蛋白的Tudor片段结合：(1)在体外应用GST gene fusion system表达p100蛋白TD功能片段的GST融合蛋白(GST-pl00-TD)(GST空载表达的蛋白作为阴性对照),与体外翻译35S标记的hPrp8蛋白的功能片段孵育后,经SDS-PAGE电泳和放射性自显影观察感光底片,GST阴性对照未出现hPrp8蛋白功能片段的体外翻译产物而GST-p100-TD出现了其相应体外翻译产物的情况,我们认为是阳性结果；(2)hPrp8各功能片段的GST融合蛋白与体外翻译35S标记的p100蛋白一起孵育,SDS-PAGE电泳和放射性自显影观察感光底片p100蛋白体外翻译产物出现的情况。
结果：第一部分p100蛋白与hPrp8在细胞内结合的研究结果显示：(1)p100蛋白能够与一组U5snRNP特异蛋白结合,经过质谱分析p100蛋白TD片段能够结合的U5snRNP特异蛋白为hPrp8蛋白、hBrr2蛋白、hSnu114蛋白,分子量分别为220kDa、200kDa和116kDa；(2)p100蛋白可与hPrp8蛋白在细胞内结合,但不能够确定它们是直接结合还是间接结合；(3)p100蛋白的TD片段能够与hPrp8蛋白在体外直接结合而SN片段不能与其结合。
为了鉴定p100蛋白的TD功能片段与hPrp8的哪一段功能片段结合,第二部分成功构建了以下重组质粒：(DhPrp8和hSnu114全长序列pcDNA3.1(+/-)真核表达质粒；②hSnu114的pEGFP C I和带有6个组氨酸标签的pcDNA3.1(-)全长重组质粒；③hPrp8和hSnu114功能片段的pGEX-4T-1重组质粒；④hPrp8和hSnu114功能片段的pBluescript SK重组质粒,并应用这些功能性重组质粒在体外研究hPrp8和hSnu114蛋白与转录共激活因子p100蛋白之间的作用机制。
在成功获得这些重组质粒的基础上,第三部分p100蛋白与hPrp8蛋白在体外结合的研究结果显示p100蛋白的TD片段与hPrp8蛋白的第二个功能片段(Domain2)稳定结合。
结‘论：本课题研究结果表明(1)p100蛋白参与组成U5snRNP复合物；(2)p100蛋白的TD片段能够与hPrp8的功能片段2稳定结合；(3)p100蛋白可能通过结合hPrp8功能片段2,进而参与前体mRNA的剪接加工作用。
Objective:A novel coactivator, p100, was recently found to bind to the Epstein-Barr virus nuclear antigen2(EBNA-2) and coactivate gene expression mediated by the EBNA-2acidic domain. HCA method found that p100consisted of four repeat SN-like fragment and C-terminal Tudor-SN (TD) fragment. We found that p100participated in the gene transcriptional regulation as a coactivator, this kind of interaction depend on the SN-like fragment. But the function of p100-TD is not clear, but we found the structure and function of p100-TD are the same as SMN-Tudor, they have similar properties. SMN is two-function protein because it participates in gene transcriptional regulation and pre-mRNA splicing. Thus we presume that p100protein may be has splicing function because it has similar TD.
Pre-messenger RNA (pre-mRNA) splicing takes place in the nucleus, catalyzed by a large RNA—protein complex called spliceosome. U5snRNP are important components of spliceosome. hPrp8(precursor mRNA processing) and hSnu114are core of U5snRNP because they bind to U5snRNA directly. We found that p100-TD interacted with a series of U5snRNP-specific proteins, include hPrp8and hSnu114. But we need to identify:(1) they bind directly or indirectly through bridge-protein.(2) which functional domain is to bind if they bind directly?
Methods:The experiment is divided into three parts:in the first part, p100protein interacts with hPrp8in vivo. Firstly, p100-TD interacts with a series of U5snRNP-specific proteins, which is researched by GST pull down and Western Blot. Then HeLa nuclear lysis solution is extracted, incubates with GST-p100-TD and SDS-PAGE by Coomacia Blue or silver staining. The nuclear proteins bound to p100-TD are analyzed by MALDI-TOF. And then, whether p100protein and hPrp8 interact with each other in vivo, we utilize CO-IP and western blot. Then the HeLa-p100stable cell nuclear lysis solutions are extracted and detect proteins which bind to pi00protein. And purifying the complexes which bind to p100protein by sepharose beads and analyzing the result by SDS-PAGE and western blot. Another lysis solution is precipitated by anti-hPrp8antibody, the result is analyzed by the same method as above. The result indicates that p100protein interacts with hPrp8in vivo. In order to confirm the binding site of p100and hPrp8by GST pull down and western blot, GST-p100-SN/GST-p100-TD incubate with HeLa-p100stable cell nuclear lysis solution, and are tested by SDS-PAGE. The interactions between hPrp8and p100-TD or p100-SN are detected by anti-hPrp8antibody.
In the second part, we constructed the pcDNA3.1(+/-)-hPrp8/hSnu114and pEGFP C I-hSnull4and pcDNA3.1(-)-hSnu114(His)6tail full-length. The full-length of hPrp8and hSnu114are amplified by RT-PCR. hPrp8was amplified through four overlapping fragments, hSnul14was amplified through two overlapping fragments. These fragments contain specific and sole enzyme-digestion sites in order to construct the full-length of hPrp8and hSnu114. Because hPrp8sequence is very long (7.2kb), overlap extension PCR method is used to ligate the four overlapping fragments into full-length, and the two large fragments which produced by overlap extension PCR were sub-cloned into pcDNA3.1(+). hSnull4sequence is2.9kb in size, the two RT-PCR fragments were ligated into full-length by the sole enzyme-digestion sites which it contains itself and were sub-cloned into pcDNA3.1(-). The two recombinant plasmids are used to studies in vitro. Furthermore, we constructed the pEGFP C I-hSnull4and pcDNA3.1(-)-hSnu114(His)6tail full-length, now it has been shown that hPrp8and hSnu114proteins are a complex in cell. So the two recombinant plasmids are used to study the interaction between their and p100protein. In addition, we constructed the pGEX-4T-1/pBluescript SK -hPrp8/hSnu114functional domains recombinant plasmids. hPrp8and hSnull4include different functional domains which play different roles in organism. So we designed the corresponding functional domains in order to further study the interaction domains between hPrp8and hSnu114and p100protein in vitro.
In the third part, firstly hPrp8and hSnu114functional domains were expressed by GST gene fusion system in vitro. The synthesized protein were labeled35S.And then study the interaction domains between hPrp8and hSnu114and p100-TD by TNT T7Coupled Reticulocyte Lysate Systems,.
Results:In the first part,(1)p100protein interacts with U5snRNP-specific proteins, these proteins include hPrp8, hBrr2and hSnull4, their molecular weighs are respectively220kDa、200kDa and116kDa;(2)p100protein interacts with hPrp8in vivo, but we can not confirm that they interact directly or not;(3)p100-TD interacts with hPrp8, p100-SN can not.
In the second part, we constructed the following recombinant plasmids successfully in order to identify p100-TD interacts with which domain of hPrp8:①pcDNA3.1(+/-)-hPrp8/hSnu114;②pEGFP C I-hSnull4and pcDNA3.1(-)-hSnu114(His)6tail full-length;③PGEX-4T-1-hPrp8/hSnu114functional domains recombinant plasmids;④pBluescript SK-hPrp8/hSnu114functional domains recombinant plasmids. These functional plasmids are used to study the interaction mechanism between hPrp8and hSnu114a transcriptional coactivator p100protein.
In the third part, p100-TD fragment stably interacts with hPrp8-Domain2in vitro. Conclusion:Our present study provides following views:①p100is a novel component of U5snRNP;②p100-TD stably interacts with hPrp8Domin2;③p100 may be involves in pre-mRNA splicing by interacting hPrp8Domain2.
Pre-mRNA的剪接加工是在一个大的蛋白复合物一剪接体中进行的,而剪接体中重要的组分就是U5snRNP。 hPrp8蛋白和hSnu114蛋白因为能够直接与U5snRNA结合,因而组成U5snRNA的核心。我们研究发现p100蛋白TD功能片段能够与一组U5snRNP特异蛋白组分结合,这其中包括hPrp8蛋白和hSnu114蛋白。但是我们还需要鉴定：(1)它们是直接结合还是通过桥梁蛋白而间接结合?(2)如果是直接结合,它们通过哪一段功能片段结合?基于此,我们开展了相关研究工作。
方法：本课题分三部分进行研究,第一部分p100蛋白与hPrp8在细胞内的结合。首先利用GST (Glutathione S transferase,谷胱甘肽S转移酶)融合蛋白钓取法和Western Blot技术研究p100蛋白的TD片段与一组U5snRNP特异蛋白之间的作用。首先提取HeLa细胞的核裂解液,与p100蛋白TD功能片段的GST融合蛋白孵育一定时间后进行SDS-PAGE电泳,使用考马斯亮兰染色或银染分析结果。通过质谱分析p100蛋白TD片段所能结合的核蛋白。在此基础上,利用免疫共沉淀的方法和Western Blot技术研究p100蛋白与hPrp8蛋白在细胞内是否能够稳定结合,提取HeLa-p100稳定表达细胞株的核裂解液,一部分与抗Flag抗体(因为建立p100的稳定表达株时,p100后面带有一个Flag小肽)进行免疫沉淀(抗IgG抗体作为阴性对照),钓取核裂解液中的p100蛋白,然后用葡聚糖颗粒纯化与p100蛋白结合的复合物,之后进行SDS-PAGE电泳分离结合的蛋白组分,再用抗Flag抗体和抗hPrp8抗体检测p100蛋白与hPrp8蛋白能否在细胞内结合；另一部分HeLa-p100稳定表达细胞株的核裂解液与抗hPrp8抗体进行免疫沉淀,钓取核裂解液中的hPrp8蛋白,然后用葡聚糖颗粒纯化与hPrp8蛋白结合的复合物之后进行SDS-PAGE电泳分离结合的蛋白组分,用抗Flag抗体和抗hPrp8抗体再一次检测p100蛋白与hPrp8蛋白能否在细胞内结合。为了进一步确定p100蛋白与hPrp8蛋白结合的部位,再次使用GST融合蛋白钓取法和Western Blot技术,将p100蛋白SN片段和TD片段的GST融合蛋白(GST空载作为阴性对照)与HeLa-p100稳定表达株的核裂解液孵育一定时间后进行SDS-PAGE电泳,用抗hPrp8抗体检测是p100蛋白的SN样功能片段还是TD功能片段与hPrp8蛋白结合。
第二部分为了进一步确定p100蛋白与hPrp8蛋白结合的功能区域,需要构建以下重组质粒：(1)构建hPrp8和hSnu114全长序列真核表达质粒。应用逆转录聚合酶链式反应(reverse transcription polymerase chain reaction, RT-PCR)技术扩增hPrp8和hSnu114的全长序列,hPrp8分成4段彼此有一小段重复序列(约十几个碱基)的片段,hSnu114分成两个片段扩增。这些片段中都含有特异的酶切位点(在序列中是单一的酶切位点),目的是为将来能够连接成全长序列。由于hPrp8序列比较长(7.2kb),优先考虑使用重叠延伸PCR法将4个RT-PCR片段连成全长并亚克隆至真核表达质粒pcDNA3.1(+)中。hSnu114序列为2.9kb,可以利用自身含有的单一酶切位点将两段RT-PCR片段连成全长并亚克隆至真核表达质粒pcDNA3.1(-)中。这2种质粒可以作为体外翻译hPrp8和hSnu114蛋白时的DNA模板,用于研究p100蛋白与hPrp8和hSnu114蛋白在体外的相互作用；(2)hSnu114的pEGFP C I及带有6个组氨酸标签(设计反义引物时引入6个组氨酸密码子,后面为终止密码子)的pcDNA3.1(-)全长重组质粒。分别选择Sal I/BamH I和Xba I/BamH I限制性内切酶将hSnu114全长序列定向克隆至pEGFP C I和pcDNA3.1(-)载体中。由于hPrp8序列较长,在构建这2种质粒时存在一定的困难,但现有研究表明hPrp8和hSnu114这2种蛋白在细胞内是以复合物的形式存在的,即通过hSnu114蛋白可以预见hPrp8蛋白与p100蛋白之间的作用。利用免疫荧光技术研究hSnu114和hPrp8蛋白与p100蛋白在细胞内结合的情况。将hSnu114的pEGFP C I重组质粒瞬时转染HeLa/COS7细胞,观察胞内绿色荧光,凡是有绿色荧光的地方就表示hSnu114蛋白的存在,待细胞贴璧后(转染后6-8个小时)先后加入第一抗体(与胞内p100蛋白结合)和红色荧光标记的第二抗体(与一抗结合),于荧光倒置显微镜下观察同一视野下的荧光定位。此外,还可以应用hSnu114的带有6个组氨酸标签的pcDNA3.1(-)全长重组质粒,利用免疫共沉淀的方法研究hPrp8蛋白与p100蛋白之间的作用机制。(3)构建hPrp8和hSnu114功能片段的pGEX-4T-1重组质粒,利用GST基因融合系统在体外表达相应功能片段的GST融合蛋白。hPrp8和hSnu114都存在着介导不同生物功能的功能片段。根据文献报道我们设计了相关的功能片段,(4)构建hPrp8和hSnu114功能片段的pBluescript SK重组质粒。这些重组质粒可作为体外翻译的DNA模板,在体外翻译出相应功能片段用同位素标记的蛋白。构建hPrp8和hSnu114功能片段的pGEX-4T-1重组质粒和pBluescript SK里组质粒的目的是进一步研究hPrp8、 hSnu114和p100蛋白三者之间的作用区域。
第三部分利用TNT⑧T7体外翻译系统在体外合成带有35S标记的p100蛋白和hPrp8功能片段,用GST pull down (GST融合蛋白钓取1法研究hPrp8的哪一段功能片段能够与p100蛋白的Tudor片段结合：(1)在体外应用GST gene fusion system表达p100蛋白TD功能片段的GST融合蛋白(GST-pl00-TD)(GST空载表达的蛋白作为阴性对照),与体外翻译35S标记的hPrp8蛋白的功能片段孵育后,经SDS-PAGE电泳和放射性自显影观察感光底片,GST阴性对照未出现hPrp8蛋白功能片段的体外翻译产物而GST-p100-TD出现了其相应体外翻译产物的情况,我们认为是阳性结果；(2)hPrp8各功能片段的GST融合蛋白与体外翻译35S标记的p100蛋白一起孵育,SDS-PAGE电泳和放射性自显影观察感光底片p100蛋白体外翻译产物出现的情况。
结果：第一部分p100蛋白与hPrp8在细胞内结合的研究结果显示：(1)p100蛋白能够与一组U5snRNP特异蛋白结合,经过质谱分析p100蛋白TD片段能够结合的U5snRNP特异蛋白为hPrp8蛋白、hBrr2蛋白、hSnu114蛋白,分子量分别为220kDa、200kDa和116kDa；(2)p100蛋白可与hPrp8蛋白在细胞内结合,但不能够确定它们是直接结合还是间接结合；(3)p100蛋白的TD片段能够与hPrp8蛋白在体外直接结合而SN片段不能与其结合。
为了鉴定p100蛋白的TD功能片段与hPrp8的哪一段功能片段结合,第二部分成功构建了以下重组质粒：(DhPrp8和hSnu114全长序列pcDNA3.1(+/-)真核表达质粒；②hSnu114的pEGFP C I和带有6个组氨酸标签的pcDNA3.1(-)全长重组质粒；③hPrp8和hSnu114功能片段的pGEX-4T-1重组质粒；④hPrp8和hSnu114功能片段的pBluescript SK重组质粒,并应用这些功能性重组质粒在体外研究hPrp8和hSnu114蛋白与转录共激活因子p100蛋白之间的作用机制。
在成功获得这些重组质粒的基础上,第三部分p100蛋白与hPrp8蛋白在体外结合的研究结果显示p100蛋白的TD片段与hPrp8蛋白的第二个功能片段(Domain2)稳定结合。
结‘论：本课题研究结果表明(1)p100蛋白参与组成U5snRNP复合物；(2)p100蛋白的TD片段能够与hPrp8的功能片段2稳定结合；(3)p100蛋白可能通过结合hPrp8功能片段2,进而参与前体mRNA的剪接加工作用。
Objective:A novel coactivator, p100, was recently found to bind to the Epstein-Barr virus nuclear antigen2(EBNA-2) and coactivate gene expression mediated by the EBNA-2acidic domain. HCA method found that p100consisted of four repeat SN-like fragment and C-terminal Tudor-SN (TD) fragment. We found that p100participated in the gene transcriptional regulation as a coactivator, this kind of interaction depend on the SN-like fragment. But the function of p100-TD is not clear, but we found the structure and function of p100-TD are the same as SMN-Tudor, they have similar properties. SMN is two-function protein because it participates in gene transcriptional regulation and pre-mRNA splicing. Thus we presume that p100protein may be has splicing function because it has similar TD.
Pre-messenger RNA (pre-mRNA) splicing takes place in the nucleus, catalyzed by a large RNA—protein complex called spliceosome. U5snRNP are important components of spliceosome. hPrp8(precursor mRNA processing) and hSnu114are core of U5snRNP because they bind to U5snRNA directly. We found that p100-TD interacted with a series of U5snRNP-specific proteins, include hPrp8and hSnu114. But we need to identify:(1) they bind directly or indirectly through bridge-protein.(2) which functional domain is to bind if they bind directly?
Methods:The experiment is divided into three parts:in the first part, p100protein interacts with hPrp8in vivo. Firstly, p100-TD interacts with a series of U5snRNP-specific proteins, which is researched by GST pull down and Western Blot. Then HeLa nuclear lysis solution is extracted, incubates with GST-p100-TD and SDS-PAGE by Coomacia Blue or silver staining. The nuclear proteins bound to p100-TD are analyzed by MALDI-TOF. And then, whether p100protein and hPrp8 interact with each other in vivo, we utilize CO-IP and western blot. Then the HeLa-p100stable cell nuclear lysis solutions are extracted and detect proteins which bind to pi00protein. And purifying the complexes which bind to p100protein by sepharose beads and analyzing the result by SDS-PAGE and western blot. Another lysis solution is precipitated by anti-hPrp8antibody, the result is analyzed by the same method as above. The result indicates that p100protein interacts with hPrp8in vivo. In order to confirm the binding site of p100and hPrp8by GST pull down and western blot, GST-p100-SN/GST-p100-TD incubate with HeLa-p100stable cell nuclear lysis solution, and are tested by SDS-PAGE. The interactions between hPrp8and p100-TD or p100-SN are detected by anti-hPrp8antibody.
In the second part, we constructed the pcDNA3.1(+/-)-hPrp8/hSnu114and pEGFP C I-hSnull4and pcDNA3.1(-)-hSnu114(His)6tail full-length. The full-length of hPrp8and hSnu114are amplified by RT-PCR. hPrp8was amplified through four overlapping fragments, hSnul14was amplified through two overlapping fragments. These fragments contain specific and sole enzyme-digestion sites in order to construct the full-length of hPrp8and hSnu114. Because hPrp8sequence is very long (7.2kb), overlap extension PCR method is used to ligate the four overlapping fragments into full-length, and the two large fragments which produced by overlap extension PCR were sub-cloned into pcDNA3.1(+). hSnull4sequence is2.9kb in size, the two RT-PCR fragments were ligated into full-length by the sole enzyme-digestion sites which it contains itself and were sub-cloned into pcDNA3.1(-). The two recombinant plasmids are used to studies in vitro. Furthermore, we constructed the pEGFP C I-hSnull4and pcDNA3.1(-)-hSnu114(His)6tail full-length, now it has been shown that hPrp8and hSnu114proteins are a complex in cell. So the two recombinant plasmids are used to study the interaction between their and p100protein. In addition, we constructed the pGEX-4T-1/pBluescript SK -hPrp8/hSnu114functional domains recombinant plasmids. hPrp8and hSnull4include different functional domains which play different roles in organism. So we designed the corresponding functional domains in order to further study the interaction domains between hPrp8and hSnu114and p100protein in vitro.
In the third part, firstly hPrp8and hSnu114functional domains were expressed by GST gene fusion system in vitro. The synthesized protein were labeled35S.And then study the interaction domains between hPrp8and hSnu114and p100-TD by TNT T7Coupled Reticulocyte Lysate Systems,.
Results:In the first part,(1)p100protein interacts with U5snRNP-specific proteins, these proteins include hPrp8, hBrr2and hSnull4, their molecular weighs are respectively220kDa、200kDa and116kDa;(2)p100protein interacts with hPrp8in vivo, but we can not confirm that they interact directly or not;(3)p100-TD interacts with hPrp8, p100-SN can not.
In the second part, we constructed the following recombinant plasmids successfully in order to identify p100-TD interacts with which domain of hPrp8:①pcDNA3.1(+/-)-hPrp8/hSnu114;②pEGFP C I-hSnull4and pcDNA3.1(-)-hSnu114(His)6tail full-length;③PGEX-4T-1-hPrp8/hSnu114functional domains recombinant plasmids;④pBluescript SK-hPrp8/hSnu114functional domains recombinant plasmids. These functional plasmids are used to study the interaction mechanism between hPrp8and hSnu114a transcriptional coactivator p100protein.
In the third part, p100-TD fragment stably interacts with hPrp8-Domain2in vitro. Conclusion:Our present study provides following views:①p100is a novel component of U5snRNP;②p100-TD stably interacts with hPrp8Domin2;③p100 may be involves in pre-mRNA splicing by interacting hPrp8Domain2.
引文
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