干扰素调节因子3第二内含子内剪接异构体1的转录调控

发布时间：2018-05-17 03:23

本文选题：干扰素调节因子3 + 启动子　；参考：《南京医科大学》2011年博士论文

【摘要】：干扰素调节因子3 (interferon regulatory factor 3, IRF-3)是调节Ⅰ型干扰素(IFN-α/β)基因表达的关键转录因子,在固有免疫反应和获得性免疫反应中的起重要性作用。目前,国内外关于IRF-3的研究几乎全部集中在IRF-3参与的抗病毒自身免疫信号通路中的作用,而关于IRF-3的自身转录调控机制尚不十分清楚。生物信息学分析结果表明IRF-3第一内含子和第二内含子内有新的转录起始位点(transcription start site, TSS),表明该基因有多个启动子和多个剪接异构体,但现有文献未见该分子多个启动子鉴定和详细的剪接异构体分析的报道。本研究中通过5'末端快速cDNA末端扩增法(rapid amplification of cDNA ends, RACE),发现IRF-3第二内含子内存在两个新的转录起始位点,分别位于第三外显子前718bp和162bp,与之对应的新的剪接异构体命名为intron 2 variant 1(Int2V1)和intron 2 variant 2(Int2V2)。RT-PCR发现二者在多数组织细胞中表达,并存在表达差异。为探索新的剪接异构体Int2V1的转录调控机制,分别克隆不同长度的5'侧翼启动子区域进行荧光素酶功能分析,发现Int2V1的核心启动子位于转录起始位点上游159~100bp之间,结合点突变、过表达和RNA干扰实验以及凝胶迁移实验(electrophoretic mobility shift assay, EMSA)和染色质免疫沉淀法(chromatin immunoprecitation, ChIP)证实转录因子特异性蛋白1(specificity protein, Sp1)是调节其启动子活性及mRNA水平的关键转录因子。EB病毒(Epstein-Barr varius)感染患儿外周血中Int2V1 mRNA水平较健康患儿低。病毒双链RNA(double stranded RNA, dsRNA)类似物Poly(I:C)和双链DNA(double stranded DNA, dsDNA)类似物poly(dA:dT)转染293T细胞发现随着转染时间和剂量的增加Int2V1启动子活性随之增加。Poly(I:C)刺激组随时间延长Int2V1 mRNA表达量升高。poly(dA:dT)处理组,Int2V1在6-8小时表达量到达高峰,随后下降。本研究拓展了IRF-3启动子和剪接异构体的探索,完善了IRF-3转录调控的关键信息。
[Abstract]:Interferon regulatory factor 3 (IRF-3) is a key transcription factor in the regulation of IFN- 伪 / 尾 gene expression and plays an important role in the innate and acquired immune response. At present, almost all the studies on IRF-3 at home and abroad focus on the role of IRF-3 in the anti-virus autoimmune signaling pathway, but the mechanism of autotranscription regulation of IRF-3 is not very clear. Bioinformatics analysis showed that there were new transcription start sites in the first and second introns of IRF-3, indicating that the gene had multiple promoters and splicing isomers. However, the identification of multiple promoters and the detailed analysis of splicing isomers have not been reported in the literature. In this study, we found that there are two new transcriptional initiation sites in the second intron of IRF-3 by rapid cDNA terminal amplification method. The new splicing isomers were named intron 2 variant 1 (Int2V1) and intron 2 variant 2(Int2V2).RT-PCR, respectively, and they were found to be expressed differently in most tissue cells. In order to explore the transcriptional regulation mechanism of new splicing isomer Int2V1, luciferase function analysis was performed by cloning 5 'flanking promoter regions with different lengths. It was found that the core promoter of Int2V1 was located between the upstream 159~100bp of transcription initiation site, and the binding point was mutated. Overexpression and RNA interference assay, gel migration assay, electrophoretic mobility shift assay, EMSA) and chromatin immunoprecipitation (Chip-immunoprecipitation) confirmed that transcription factor-specific protein 1(specificity (Sp1) is the key transcription factor to regulate its promoter activity and mRNA level. EB disease The level of Int2V1 mRNA in peripheral blood of children with Epstein-Barr varius infection was lower than that of healthy children. 293T cells were transfected with virus double-stranded RNA(double stranded RNA, dsRNA) analogues PolyRNA(double stranded RNA, dsRNA): C) and double-stranded DNA(double stranded DNA, dsDNA) analogues polyDNA: DT). It was found that the activity of Int2V1 promoter increased with the increase of transfection time and dose, and the activity of Int2V1 promoter increased with the increase of transfection time and dose. The expression of Int2V1 reached its peak at 6-8 hours. Then it went down. This study expanded the exploration of IRF-3 promoter and splicing isomer and improved the key information of IRF-3 transcription regulation.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1

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