腺病毒主要晚期转录单位上URE调控元件结合蛋白的筛选及其初步功能研究
发布时间：2018-11-11 09:45
【摘要】：腺病毒在宿主细胞中的生活周期以病毒DNA复制的起始为界限被分为早期感染阶段和晚期感染阶段。早期基因编码病毒的调控蛋白，晚期基因主要编码结构蛋白，早晚期阶段基因的表达是一个受到各个水平严密调控的过程。腺病毒的主要晚期转录单位(Maior late transcription unit，MLTU)上有5个poly A信号而将其分为L1到L5 5个转录区。腺病毒生活周期中，MLTU上5个转录区的使用存在一个显著的时间变化：早期阶段MLTU转录水平比较低，并且主要只利用了MLTU上L1一个转录区，L1编码和病毒包装有关的非结构蛋白；而在晚期阶段MLTU转录水平升高，并且腺病毒利用了MLTU上所有5个转录区，并且腺病毒大部分晚期蛋白尤其是病毒结构蛋白都从这5个转录区转录表达出。所以，在腺病毒早晚期感染阶段转换的调控中，有一个重要问题就是：腺病毒如何在不同的感染阶段使用MLTU上不同的转录区?即MLTU上不同poly A位点使用转换的机制。 研究发现L1 poly A位点上游存在一个上游调控元件(upstream regulatory element，URE)，在早期感染阶段，URE能够使转录出的包含URE的L2、L3转录本在核内不稳定，不能有效进入胞浆成熟mRNA库，从而抑制L2、L3的基因表达，使L1成为早期阶段主导使用的转录本。本实验室利用HeLa细胞核液与URE RNA的紫外交联的实验发现，，URE可同至少两个30kd左右的蛋白结合。为进一步研究URE在异源poly A信号上游时抑制基因表达的机制，本课题采用酵母三杂交的方法筛选HeLa cDNA文库，寻找URE结合蛋白，并对候选的RNA结合蛋白后进行功能研究，发现该蛋白参与URE抑制基因表达的机制，为研究腺病毒早晚期转换调控的机制和细胞核内转录后水平的调控提供新的依据。 酵母三杂交系统由三部分组成：DNA结合结构域融合蛋白、杂交RNA分子、DNA激活结构域融合蛋白。在利用cDNA文库筛选URE RNA结合蛋白时，URE分子是“诱饵”，融合有DNA激活结构域的cDNA文库质粒是“诱饵”要寻找的“猎物”。 我们首先测定了预制HeLa cDNA文库的滴度，克隆扩增了文库的3×10~7个克隆，为文库原始容量的10倍，有效保证扩增后的文库的完整性。然后，通过对URE序列的碱基分析，发现URE上有RNA聚合酶Ⅲ的终止子，所以选择下游的MD功能片段作为三杂交中的“诱饵”序列。我们还使用了RNA structure软件对MD序列的两种“诱饵”RNA分子进行了二级结构的分析。预测的两种“诱饵”RNA分子在最低自由能下的二级结构显示，leader-MD-MS2序列没有稳定的异源序列间的配对，各部分能够保持
[Abstract]:The life cycle of adenovirus in host cells is divided into early infection stage and late infection stage according to the threshold of viral DNA replication. The early gene encodes the regulating protein of virus, the late gene mainly encodes structural protein, and the expression of gene in the morning and evening stage is closely regulated at all levels. There are 5 poly A signals on the main advanced transcriptional unit (Maior late transcription unit,MLTU) of adenovirus, which are divided into L1 to L55 transcriptional regions. During the life cycle of adenovirus, there was a significant time change in the use of five transcriptional regions on MLTU: at the early stage, the transcription level of MLTU was relatively low, and only one transcriptional region of L1 on MLTU was mainly utilized. L1 encodes nonstructural proteins related to viral packaging; At the late stage, MLTU transcription level increased, and adenovirus utilized all five transcriptional regions of MLTU, and most of the adenovirus advanced proteins, especially viral structural proteins, were transcribed from these five transcriptional regions. Therefore, one of the important questions in the regulation of the transition of adenovirus infection stage is: how does adenovirus use different transcriptional regions on MLTU at different stages of infection? That is, different poly A sites on MLTU use conversion mechanism. It was found that there was an upstream regulatory element (upstream regulatory element,URE in the upstream of L1 poly A locus. In the early stage of infection, URE could make the L2L3 transcripts containing URE unstable in the nucleus and could not effectively enter the cytoplasmic mature mRNA library. Thus, the gene expression of L2 and L3 was inhibited, and L1 became the dominant transcripts in the early stage. In our laboratory, we found that URE can bind to at least two 30kd proteins by ultraviolet crosslinking of HeLa nuclear fluid with URE RNA. In order to further study the mechanism of URE inhibiting gene expression in the upstream of heterologous poly A signal, HeLa cDNA library was screened by yeast three-hybrid method to search for URE binding protein, and the function of candidate RNA binding protein was studied. It is found that this protein is involved in the mechanism of URE inhibiting gene expression, which provides a new basis for the study of the regulation mechanism of adenovirus in the morning and evening and the regulation of posttranscriptional level in the nucleus. Yeast three-hybrid system consists of three parts: DNA binding domain fusion protein, hybrid RNA molecule, and DNA activated domain fusion protein. When cDNA library was used to screen URE RNA binding protein, URE molecule was "bait", and cDNA library plasmid with DNA activation domain was the "prey" to be looked for. We first measured the titer of the prefabricated HeLa cDNA library, and cloned and amplified 3 脳 10 ~ 7 clones of the library, 10 times the original capacity of the library, which effectively ensured the integrity of the expanded library. Then, based on the base analysis of URE sequence, the Terminator of RNA polymerase 鈪
本文编号：2324468
[Abstract]:The life cycle of adenovirus in host cells is divided into early infection stage and late infection stage according to the threshold of viral DNA replication. The early gene encodes the regulating protein of virus, the late gene mainly encodes structural protein, and the expression of gene in the morning and evening stage is closely regulated at all levels. There are 5 poly A signals on the main advanced transcriptional unit (Maior late transcription unit,MLTU) of adenovirus, which are divided into L1 to L55 transcriptional regions. During the life cycle of adenovirus, there was a significant time change in the use of five transcriptional regions on MLTU: at the early stage, the transcription level of MLTU was relatively low, and only one transcriptional region of L1 on MLTU was mainly utilized. L1 encodes nonstructural proteins related to viral packaging; At the late stage, MLTU transcription level increased, and adenovirus utilized all five transcriptional regions of MLTU, and most of the adenovirus advanced proteins, especially viral structural proteins, were transcribed from these five transcriptional regions. Therefore, one of the important questions in the regulation of the transition of adenovirus infection stage is: how does adenovirus use different transcriptional regions on MLTU at different stages of infection? That is, different poly A sites on MLTU use conversion mechanism. It was found that there was an upstream regulatory element (upstream regulatory element,URE in the upstream of L1 poly A locus. In the early stage of infection, URE could make the L2L3 transcripts containing URE unstable in the nucleus and could not effectively enter the cytoplasmic mature mRNA library. Thus, the gene expression of L2 and L3 was inhibited, and L1 became the dominant transcripts in the early stage. In our laboratory, we found that URE can bind to at least two 30kd proteins by ultraviolet crosslinking of HeLa nuclear fluid with URE RNA. In order to further study the mechanism of URE inhibiting gene expression in the upstream of heterologous poly A signal, HeLa cDNA library was screened by yeast three-hybrid method to search for URE binding protein, and the function of candidate RNA binding protein was studied. It is found that this protein is involved in the mechanism of URE inhibiting gene expression, which provides a new basis for the study of the regulation mechanism of adenovirus in the morning and evening and the regulation of posttranscriptional level in the nucleus. Yeast three-hybrid system consists of three parts: DNA binding domain fusion protein, hybrid RNA molecule, and DNA activated domain fusion protein. When cDNA library was used to screen URE RNA binding protein, URE molecule was "bait", and cDNA library plasmid with DNA activation domain was the "prey" to be looked for. We first measured the titer of the prefabricated HeLa cDNA library, and cloned and amplified 3 脳 10 ~ 7 clones of the library, 10 times the original capacity of the library, which effectively ensured the integrity of the expanded library. Then, based on the base analysis of URE sequence, the Terminator of RNA polymerase 鈪
本文编号：2324468
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