【作者】 刘珊珊；

【导师】 金英花；

【作者基本信息】 吉林大学 ， 细胞生物学， 2015， 博士

【摘要】 p53在细胞中的主要功能是作为转录因子与靶基因启动子区域特定DNA序列结合,调控基因转录。大量研究表明p53蛋白在维持细胞正常生长及抑制肿瘤细胞增殖过程中起到重要的监控作用。p53能够对细胞内外各种应激信号产生反应,在细胞中发挥类似信号分子的作用,因此自身也受到各种调控机制的调控。随着对RNA研究的深入,发现p53蛋白与RNA密切相关。研究显示p53蛋白本身具有RNA结合能力,并且与p53相互作用的多种蛋白质都是RNA结合蛋白。进一步研究发现一些非编码RNA或m RNA能够通过结合p53相互作用蛋白质而调控p53的功能,如p53 m RNA和lnc RNA Apela等。这提示RNA分子可能在p53蛋白与其他蛋白的相互作用中广泛存在并发挥重要调控作用。因此,本文针对RNA相关的p53相互作用蛋白展开研究,目的在于揭示RNA相关的p53相互作用蛋白以及RNA分子在p53蛋白调控网络中的作用。为了筛选鉴定RNA相关的p53相互作用蛋白,首先改进了免疫共沉淀方法的条件,在进行免疫共沉淀之前,分别用RNase A以及RNase抑制剂处理细胞裂解液,从而分别消化或保护细胞中的RNA分子,对细胞转染过表达的Flag-p53进行免疫共沉淀,分别获得通过RNA介导与p53相互作用的蛋白,以及本身与p53相互作用却受到RNA分子阻碍的p53结合蛋白。通过质谱分析及实验验证,发现了由RNA介导的p53相互作用蛋白NCL、ILF3以及HSPD1和RNA阻碍结合的p53相互作用蛋白hn RNPC。通过细胞内外相互作用实验验证,确定这些蛋白分子、RNA和p53三者的相互作用关系,同时分析鉴定蛋白相互作用的具体结构域。随后通过RNA干扰及过表达实验,分析了这些蛋白在p53调控网络中的作用,以及它们依赖于p53的生物学功能。结果发现这些蛋白分子与p53的相互作用受到RNA分子的调控,而且这些蛋白分子在p53调控网络中具有重要作用。为了分析介导p53与其他蛋白相互作用的RNA,运用HITS-CLIP技术对p53结合的RNA进行了大规模筛选,通过生物信息学分析及免疫共沉淀等实验验证,获得了一批可能与p53直接相互作用的RNA分子,并就p53对这些RNA分子的调控方式进行了分析。此外,本研究还对长非编码RNA MEG3的相互作用蛋白进行了探索性研究。为了揭示MEG3在肿瘤细胞中的功能及分子机制,建立MTRAP-MS(MS2-tagged RNA affinitypurification and Mass spectrometry)方法并筛选获得一系列MEG3结合的蛋白质,并利用GO(Gene Onology)分析对这些MEG3结合蛋白进行细胞组成、分子功能与生物学过程三方面的分类分析,为建立MEG3-蛋白质功能网络奠定了基础。取得的主要进展:1.对免疫共沉淀技术进行改进,建立了RNA相关p53相互作用蛋白的筛选鉴定方法。制备过表达Flag-p53融合蛋白的细胞裂解液,分别用RNase A及RNase抑制剂进行处理,达到消化或保护细胞裂解液中RNA分子的目的。然后利用Flag标签抗体进行免疫共沉淀实验,运用SDS-PAGE胶分离富集到的Flag-p53蛋白及其复合物,考马斯亮蓝染色找到差异性蛋白条带并进行质谱分析,获得hn RNPC、NCL、ILF3和HSPD1等4个RNA相关的p53相互作用蛋白。2.发现RNA作为连接分子介导p53与NCL、ILF3及HSPD1相互作用,并发现NCL与HSPD1蛋白可调控p53蛋白转录活性。构建NCL、ILF3和HSPD1真核表达载体,利用免疫共沉淀技术确定NCL、ILF3和HSPD1通过RNA与p53蛋白结合;抑制NCL、ILF3以及HSPD1表达水平,利用双荧光素酶报告基因技术检测发现NCL和HSPD1蛋白能够调控p53蛋白的转录活性。3.发现hn RNPC可以与p53直接相互作用,并且这种相互作用可以被RNA分子阻抑。GST pulldown以及免疫共沉淀等多个实验证明hn RNPC通过自身的RNA结合结构域(RRM domain)与p53直接相互作用,同时证明RNA分子能够显著降低hn RNPC与p53的结合能力。4.抑制hn RNPC表达可导致p53蛋白稳定性增加及转录活性增强,并促进p53依赖的阿霉素诱导的细胞凋亡效应,抑制细胞增殖。说明hn RNPC具有抑制p53蛋白稳定性以及转录活性的功能。5.运用HITS-CLIP和RIP技术,发现p53蛋白能够结合PLAGC1、TERC及mi R143HG等多种RNA分子。抑制p53表达可降低PLAGC1及TERC的表达水平,提示p53可能通过与PLAGC1及TERC结合调控其表达。6.对MTRAP-MS方法获得的长链非编码RNA MEG3候选结合蛋白进行生物信息学分析,发现MEG3结合蛋白主要分布于细胞核和线粒体中,大多数蛋白具有核苷酸结合蛋白的特性,参与生物大分子复合物的组装过程。综上,本研究证实RNA分子参与p53蛋白-蛋白相互作用,发现RNA分子可以介导p53与NCL、ILF3及HSPD1相互作用,同时证明hn RNPC能够与p53蛋白直接相互作用,并且RNA分子可以阻碍这一相互作用。揭示了hn RNPC调控p53的具体分子机制和生物学功能。获得了约1100条与p53相互作用的RNA分子。上述发现证实RNA分子在p53-蛋白相互作用中发挥着重要的作用,并与p53相互作用蛋白共同对p53的功能进行精密调控。更多还原

【Abstract】 The main function of p53 is as a transcription factor bound to the target gene promoter region, which regulate m RNA transcription. Numerous studies show that p53 plays an important role in maintaining cell growth and inhibition of tumor cell proliferation. p53 can regulate gene expression according to various stress induced cell signals, similar to a signal molecule. So p53 is also regulated both at the levels of protein stability and biochemical activities by kinds of molecules. With the development of RNA study, p53 and RNA are closely related. Some studies show that p53 can bind RNA, and many p53 binding proteins are RNA binding proteins. Moreover, some RNAs are found to regulate p53 by binding to other proteins, such as p53 m RNA, lnc RNA Apela. These results suggest that RNA may participate or even play an important role in p53-protein interaction. Hense, we studied p53 interacting proteins to explore the function of these proteins and RNA in p53 interaction network.In order to identify RNA associated p53 interacting protein, we improved the condition of immunoprecipitation, adding RNase A or RNase Inhibitor in cell lysates to digest RNA or protect RNA. Then Flag-p53 immunoprecipitation was used to acquire some proteins that bound RNA to interact with p53 and some that were inhibited to interact with p53 by RNA. Using MS analysis of protein and experiments, we identified the interaction among RNA, protein and p53 and the specific domain of protein and p53 interaction. Then we used RNAi to knockdowned target gene expression and overexpressed target gene to explore p53 dependent function of these proteins in p53 interaction network. We found that RNA participated in protein-p53 interaction, and these proteins played an important role in p53 interaction network.In order to analyse RNA which participated in p53-proteins interaction, we chose HITS-CLIP method to screen and identify p53 binding RNA. We used bioinformatics to analyse a large number of p53 protein binding RNA obtained by HITS-CLIP, and verify the interaction between RNA and p53 by RIP.We also explore the function of lnc RNA MEG3 binding protein, in order to reveal biological functions of MEG3 in cell, we established a method called MTRAP-MS(MS2-tagged RNA affinity purification and Mass spectrometry), screened and obtained a series of MEG3 binding proteins. We used Gene Onology analysis to analyze the protein from three aspects, cell component, molecular function and biological process, for the establishment of functional networks of protein-MEG3 interactions.The main improvements:1. We improved the conditions of immunoprecipitation to identify whether RNA participate in p53-protein interactions. We overexpressed Flag-p53 in cells and used Flag-IP to enrich p53 complexes with RNase or RNase Inhibitor, in order to digest or protect cell RNA. Then we acquired hn RNPC, NCL, ILF3 and HSPD1 protein using Coomassie Blue Staining and MS analysis.2. We found the interaction of NCL, ILF3 and HSPD1 with p53 through RNA for the first time. We constructed eukaryotic expression vector of NCL, ILF3 and HSPD1 and identified the interaction of NCL, ILF3 and HSPD1 with p53 through RNA by Co-IP. We found NCL and HSPD1 regulate p53 transcriptional activity using dual luciferase reporter.3. We found that RNA interfered the interaction of hn RNPC and p53, and RNA for the first time. GST pulldown and other experiments were used to identify the direct interaction of RRM domain in hn RNPC and p53, and identify that RNA inhibited the interaction of hn RNPC and p53.4. Knockdown of hn RNPC increased p53 protein level and transcriptional acitivity, and also induced p53 dependent apoptosis. The results showed that hn RNPC inhibited p53 protein level and transcriptional activity.5. We used HITS-CLIP and RIP experiments to find that p53 could bind PLAGC1, TERC and mi R143 HG. Knockdown p53 expression could decrease PLAGC1 and TERC RNA level. The results suggested that p53 might regulate PLAGC1 and TERC expression through binding them.6. We used bioinformatics to analyze MEG3 binding proteins obtained by MTRAP-MS, and we found that MEG3 binding proteins mainly distributed in nucleus and mitochondria. Most proteins had the character of nucleotide binding and took part in macromolecular complexes assembly process.To sum up, we identified that RNA participated p53-protein interaction for the first time and successfully acquired RNA associated p53 interacting protein NCL, ILF3, HSPD1 and hn RNPC that was inhibited to bind p53 by RNA. The results explained the mechanism of the inhibition of hn RNPC to p53 and the function in cells. And we also obtained a serious of RNA associated p53 interacting proteins. All of these results showed that RNA participated in the interaction of p53 and other proteins to play an important role, and regulated p53 with p53 interacting proteins.更多还原