探讨调控人SND1蛋白表达的分子机制

发布时间：2018-06-18 19:09

本文选题：SND1蛋白 + 生物信息学　；参考：《天津医科大学》2012年硕士论文

【摘要】：研究目的：人SND1蛋白被发现是一种潜在的多功能蛋白,目前已证实其在基因转录、RNA干扰、细胞应激、脂肪代谢等多个生物活动中发挥重要作用。因此,SND1蛋白表达异常势必对其生物功能的发挥产生不同的影响,而目前人们对调控SND1蛋白表达的机制却知之甚少。任何一种蛋白质的表达都需要经过转录起始、pre-mRNA的转录、转录后修饰、蛋白翻译、翻译后修饰等众多精细复杂而协调有序的调控过程。本文旨在从基因转录起始水平探讨调控人SND1蛋白表达的分子机制,并从mRNA和蛋白水平初步探讨调控其表达的分子机制,进而深入了解其表达如何影响生理或病理过程的分子机制。方法：本课题分三部分进行,第一部分：利用生物信息学数据库和软件对SND1基因特点进行预测与分析,推测SND1基因启动子的核心区域及参与调控SND1基因转录起始的关键转录因子。第二部分：结合生物信息学分析结果,构建包含SND1基因启动子的虫荧光素酶质粒,通过虫荧光素酶活性检测法和染色质免疫沉淀技术对其启动子活性及转录因子的作用进行检测和验证。第三部分：通过RNA核酸印迹实验明确SND1的mRNA水平上是否存在多种转录本,其可能翻译后形成SND1蛋白的亚型。通过GST融合蛋白钓取法检测SND1蛋白在体外能否与自身结合形成二聚体或者多聚体,该结构可能参与蛋白本身转运、储存、降解等生物过程。结果：①分析出人SND1基因的启动子特点：其启动子序列中缺乏典型的TATA盒结构,含有3个CAAT序列和多个GC盒。CpG岛位于-650～-+295区域内(以转录起始位点为+1,上游为正,下游为负)。②成功构建人SND1核心启动子及其截短片段的虫荧光素酶质粒,即,SND1(-493/+14)-luc和SND1(-863/-493)-luc.③转录因子Sp1和stat6都能增加SND1(-863/+14)-luc质粒的虫荧光素酶活性,其中Sp1对其表达的调控作用很强,而stat6对其表达的调控作用较弱,都存在剂量依赖性。④转录因子Spl.stat6和NF-Y在体内均能与人SND1启动子结合。⑤TGF-β信号传导通路可能通过Spl参与调控人SND1的表达,IL-4/stat6信号传导通路不参与调控人SND1的表达。⑥正常和应激状态下,HeLa细胞和MD-MB-231细胞中人SND1的mRNA都只检测到一个转录本。⑦人SND1蛋白通过SN结构域在体外与其自身蛋白结合,而TD和TSN结构域在体外不能与其自身蛋白结合。⑧人SND1蛋白的SNl,SN2,SN3和SN4各个单独的结构域均能在体外与其自身蛋白结合,SN3的结合能力最强,SN1的结合能力最弱。结论：①人SND1基因的启动子序列中缺乏典型的TATA盒结构,含有3个CAAT序列和多个GC盒。CpG岛位于-650～+295区域内(以转录起始位点为+1,上游为正,下游为负)。②转录因子Sp1、stat6和NF-Y参与调控人SND1基因的转录,Sp1可能通过TGF-β信号传导通路在调控过程中发挥重要作用。③正常和应激状态下细胞SND1的mRNA仅有一个转录本,不存在多个剪切状态。④人SND1蛋白在体外通过SN结构域与自身蛋白结合形成二聚体或者多聚体。
[Abstract]:Objective: human SND1 protein has been found to be a potential multifunctional protein. It has been proved to play an important role in many biological activities such as gene transcription, RNA interference, cell stress, fat metabolism and so on. Therefore, the abnormal expression of SND1 protein is bound to have different effects on its biological function, and people are currently regulating the SND1 eggs. The mechanism of white expression is little known. Any protein expression needs to be transcriptional starting, pre-mRNA transcription, posttranscriptional modification, protein translation, post-translational modification and so on and many sophisticated and orderly regulatory processes. This paper aims to explore the molecular mechanism of regulating the expression of SND1 protein from the gene transcriptional starting level and from M RNA and protein levels initially explore the molecular mechanisms regulating its expression, and further understand how its expression affects the molecular mechanism of physiological or pathological processes.
Methods: the subject is divided into three parts. The first part: using the bioinformatics database and software to predict and analyze the characteristics of the SND1 gene, the core region of the SND1 gene promoter and the key transcription factors involved in the regulation of the transcription initiation of the SND1 gene. The second part: the results of bioinformatics analysis and the construction of the SND1 base The promoter activity and transcription factors were detected and verified by the insect luciferase activity assay and chromatin immunoprecipitation technique. The third part: the existence of multiple transcripts on the mRNA level of SND1 by RNA nucleic acid blotting, and it may be translated into SND1 eggs after the translation. White subtype. By GST fusion protein fishing, SND1 protein can be combined with itself to form two polymer or polymer in vitro. This structure may be involved in the biological processes such as protein itself transport, storage, degradation and other biological processes.
Results: (1) the promoter characteristic of human SND1 gene was analyzed: the promoter sequence lacks typical TATA box structure, contains 3 CAAT sequences and multiple GC box.CpG islands are located in -650 ~ -+295 region (the transcription initiation site is +1, upstream is positive, and the downstream is negative). Enzyme plasmids, that is, both SND1 (-493/+14) -luc and SND1 (-863/-493) -luc. (-863/-493) -luc. 3 transcription factors Sp1 and STAT6 can increase the activity of the luciferase activity of the SND1 (-863/+14) -luc plasmid, in which the regulation of Sp1 on its expression is strong, and the regulation of the expression is weak, all stored in a dose dependent manner. Combined with human SND1 promoter. 5 TGF- beta signaling pathway may participate in the regulation of human SND1 expression through Spl, and IL-4/stat6 signaling pathway does not participate in the regulation of human SND1 expression. 6. In normal and stressful States, only one transcript is detected in HeLa cells and MD-MB-231 cells in SND1 mRNA. The TD and TSN domains can not be combined with their own proteins in vitro. The individual domains of SNl, SN2, SN3 and SN4 of SND1 protein can be combined with their own proteins in vitro. The binding ability of SN3 is the strongest, and the ability of SN1 to bind is the weakest.
Conclusion: (1) there is a lack of typical TATA box structure in the promoter sequence of human SND1 gene, which contains 3 CAAT sequences and multiple GC boxes in the region of -650 to +295 (the transcriptional starting site is +1, the upstream is positive, and the downstream is negative). The pathway plays an important role in the process of regulation. (3) there is only one transcript in the mRNA of cell SND1 in normal and stressful States, and there are no multiple shear states. (4) human SND1 protein forms a two polymer or polymer through the combination of the SN domain with its own protein in vitro.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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