腺病毒主要晚期轉(zhuǎn)錄單位上URE調(diào)控元件結(jié)合蛋白的篩選及其初步功能研究
發(fā)布時(shí)間：2018-11-11 09:45
【摘要】：腺病毒在宿主細(xì)胞中的生活周期以病毒DNA復(fù)制的起始為界限被分為早期感染階段和晚期感染階段。早期基因編碼病毒的調(diào)控蛋白，晚期基因主要編碼結(jié)構(gòu)蛋白，早晚期階段基因的表達(dá)是一個(gè)受到各個(gè)水平嚴(yán)密調(diào)控的過(guò)程。腺病毒的主要晚期轉(zhuǎn)錄單位(Maior late transcription unit，MLTU)上有5個(gè)poly A信號(hào)而將其分為L(zhǎng)1到L5 5個(gè)轉(zhuǎn)錄區(qū)。腺病毒生活周期中，MLTU上5個(gè)轉(zhuǎn)錄區(qū)的使用存在一個(gè)顯著的時(shí)間變化：早期階段MLTU轉(zhuǎn)錄水平比較低，并且主要只利用了MLTU上L1一個(gè)轉(zhuǎn)錄區(qū)，L1編碼和病毒包裝有關(guān)的非結(jié)構(gòu)蛋白；而在晚期階段MLTU轉(zhuǎn)錄水平升高，并且腺病毒利用了MLTU上所有5個(gè)轉(zhuǎn)錄區(qū)，并且腺病毒大部分晚期蛋白尤其是病毒結(jié)構(gòu)蛋白都從這5個(gè)轉(zhuǎn)錄區(qū)轉(zhuǎn)錄表達(dá)出。所以，在腺病毒早晚期感染階段轉(zhuǎn)換的調(diào)控中，有一個(gè)重要問(wèn)題就是：腺病毒如何在不同的感染階段使用MLTU上不同的轉(zhuǎn)錄區(qū)?即MLTU上不同poly A位點(diǎn)使用轉(zhuǎn)換的機(jī)制。 研究發(fā)現(xiàn)L1 poly A位點(diǎn)上游存在一個(gè)上游調(diào)控元件(upstream regulatory element，URE)，在早期感染階段，URE能夠使轉(zhuǎn)錄出的包含URE的L2、L3轉(zhuǎn)錄本在核內(nèi)不穩(wěn)定，不能有效進(jìn)入胞漿成熟mRNA庫(kù)，從而抑制L2、L3的基因表達(dá)，使L1成為早期階段主導(dǎo)使用的轉(zhuǎn)錄本。本實(shí)驗(yàn)室利用HeLa細(xì)胞核液與URE RNA的紫外交聯(lián)的實(shí)驗(yàn)發(fā)現(xiàn)，，URE可同至少兩個(gè)30kd左右的蛋白結(jié)合。為進(jìn)一步研究URE在異源poly A信號(hào)上游時(shí)抑制基因表達(dá)的機(jī)制，本課題采用酵母三雜交的方法篩選HeLa cDNA文庫(kù)，尋找URE結(jié)合蛋白，并對(duì)候選的RNA結(jié)合蛋白后進(jìn)行功能研究，發(fā)現(xiàn)該蛋白參與URE抑制基因表達(dá)的機(jī)制，為研究腺病毒早晚期轉(zhuǎn)換調(diào)控的機(jī)制和細(xì)胞核內(nèi)轉(zhuǎn)錄后水平的調(diào)控提供新的依據(jù)。 酵母三雜交系統(tǒng)由三部分組成：DNA結(jié)合結(jié)構(gòu)域融合蛋白、雜交RNA分子、DNA激活結(jié)構(gòu)域融合蛋白。在利用cDNA文庫(kù)篩選URE RNA結(jié)合蛋白時(shí)，URE分子是“誘餌”，融合有DNA激活結(jié)構(gòu)域的cDNA文庫(kù)質(zhì)粒是“誘餌”要尋找的“獵物”。 我們首先測(cè)定了預(yù)制HeLa cDNA文庫(kù)的滴度，克隆擴(kuò)增了文庫(kù)的3×10~7個(gè)克隆，為文庫(kù)原始容量的10倍，有效保證擴(kuò)增后的文庫(kù)的完整性。然后，通過(guò)對(duì)URE序列的堿基分析，發(fā)現(xiàn)URE上有RNA聚合酶Ⅲ的終止子，所以選擇下游的MD功能片段作為三雜交中的“誘餌”序列。我們還使用了RNA structure軟件對(duì)MD序列的兩種“誘餌”RNA分子進(jìn)行了二級(jí)結(jié)構(gòu)的分析。預(yù)測(cè)的兩種“誘餌”RNA分子在最低自由能下的二級(jí)結(jié)構(gòu)顯示，leader-MD-MS2序列沒(méi)有穩(wěn)定的異源序列間的配對(duì)，各部分能夠保持
[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
本文編號(hào)：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
本文編號(hào)：2324468
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