天然无内含子mRNA转运出核顺式作用元件的预测与功能鉴定

发布时间：2018-05-10 08:38

本文选题：天然无内含子mRNA + 转运出核　；参考：《大连医科大学》2017年硕士论文

【摘要】：背景:mRNA转运出核一直是RNA领域的研究热点与难点,近些年的研究中发现mRNA中存在调控其转运出核的顺式作用元件[1]。在高等生物中,出核转运与剪接过程是关联十分密切的。在mRNA发生剪接过程时,剪接复合体会直接招募出核转运复合体并共同完成剪接与出核两种生命活动[2-5]。删除基因的内含子后,mRNA会因为剪接事件的终止而被阻滞在细胞核内[6]。所以在剪接与转运出核相偶联的mRNA中,内含子就成为转运出核的顺式作用元件[7]。天然无内含子mRNA占全部mRNA的5%,其中包含着像干扰素、组蛋白等对生物体的生命过程非常重要的基因[8]。天然无内含子mRNA其本身因不具有内含子而不会发生剪接过程,那么这些天然无内含子的mRNA是否同样存在转运出核的顺式作用元件呢?本实验室在之前对4个天然无内含子mRNA(HSPB3、IFNα1、IFNβ1,、c-Jun)的研究中,发现4条mRNA中的保守序列CAR-E(促细胞质区域聚集元件)能够促进已阻滞在细胞核内的β-球蛋白c DNA转运出核[9]。那么在更大范围的天然无内含子mRNA中是否同样存在这样功能的顺式作用元件?这些顺式作用元件的结合蛋白是什么?目前的研究依然没有解决这些问题。本文通过对天然无内含子mRNA转运出核机制的研究完善mRNA转运出核机制。方法:1.通过生物信息学比对软件MEME对天然无内含子的mRNA数据库(Intronless Gene Database)进行比对,检索其中存在的共有片段。2.将获取的共有片段与已验证阻滞在细胞核内的β-球蛋白的c DNA进行重组连接,通过原位荧光杂交技术(FISH)寻找可以促进c DNA发生出核转运的片段。3.通过生物信息学软件FIMO对天然无内含子mRNA数据库进行反检索,找到含有促进c DNA出核序列的天然无内含子mRNA,并对其中预测的顺式作用元件进行碱基删除、突变,检测其在天然无内含子mRNA中是否具有促进出核的功能。4.构建预测元件、β-球蛋白c DNA与p CDNA5载体的重组质粒。利用Flp-In系统在FRT-Hela细胞中构建稳转细胞株。并通过MS2-MBP蛋白纯化体系与质谱分析方法确定顺式作用元件的结合蛋白[10]。结果:通过生物信息学软件MEME将679个天然无内含子mRNA数据进行比对,发现其中含有多条共有序列。FISH实验结果显示在预测的顺式作用元件中有4条能够促进β-球蛋白c DNA的mRNA出核转运,我们分别命名为motif1-AS、motif 1V-AS、motif 4-AS、motif 5。天然无内含子mRNA KRN1基因中含有9个预测元件motif1V-AS,其中6个元件位置相近我们将这6个元件整体命名为CAR-C。通过分子克隆的方法将CAR-C进行删除与突变,FISH实验结果显示删除与突变后的KRN1 mRNA会被阻滞在细胞核内。完成稳转细胞构建后我们通过MS2-MBP蛋白结合体系,得到预测元件的结合蛋白,并通过聚丙烯酰胺凝胶电泳(PAGE)后的银染实验发现促出核元件结合蛋白与非促出核元件结合蛋白存在特异性条带。结论:实验数据表明预测中具有促进β-球蛋白c DNA转运出核功能的元件在天然无内含子mRNA中同样起到促进其转运出核的功能。
[Abstract]:Background: mRNA transshipment is always a hot and difficult problem in the field of RNA. In recent years, it is found that there is a cis acting element in mRNA that regulates its transshipment, [1]. is closely related to the process of transshipment and splicing in higher organisms. In the splicing process of mRNA, the splicing compound directly recruits nuclear transfer complex. After combining and co completing the introns of the two life activities [2-5]. deletion genes, mRNA will be blocked in the nucleus [6]. because of the termination of the splicing event, so the intron is the cis acting element of the transshipment nucleus, [7]. natural intron mRNA, which accounts for 5% of all mRNA, because of the termination of the splicing event. It contains the [8]. natural intron, such as interferon, histone, and so on, which is very important for the life process of the organism, mRNA itself because it does not have introns and does not have the splicing process. Then, do these natural introns mRNA also have a cis component that transshipped the nucleus? The laboratory was before the 4 natural In the study of the intron mRNA (HSPB3, IFN alpha 1, IFN beta 1, c-Jun), it was found that the conserved sequence CAR-E in 4 mRNA (cytoplasmic regional aggregation elements) could promote the nucleation of the beta globulin C DNA transshipment out of the nucleus in the nucleus, then whether there is a cis acting element in the larger natural intron mRNA. What are the binding proteins of these cis acting elements? The current research still does not solve these problems. This paper improves the mechanism of mRNA transshipment through the study of the mechanism of natural intron mRNA transshipment. Method: 1. the mRNA database (Intronless Gene Database) of natural intron, by bioinformatics comparison software MEME In comparison, the common fragment of the existing fragment.2. was retrieved to recombine the obtained common fragments with the C DNA that had been verified to be blocked in the nucleus. Through in situ fluorescence hybridization (FISH), a fragment of.3. that could promote the nuclear transport of C DNA was found through the bioinformatics software FIMO to the natural intron mRNA data. The library carries out reverse retrieval and finds the natural intron mRNA containing the promoter of the C DNA nucleation sequence, and deletes the bases for the cis acting elements predicted in it, and detects whether it has the function.4. construction prediction element in the natural intron mRNA, and the recombinant plasmid of the beta globulin C DNA and P CDNA5 carrier. Use Flp-In. The system constructs a stable cell line in FRT-Hela cells. Through the MS2-MBP protein purification system and mass spectrometry analysis, the binding protein [10]. results of cis acting elements are determined. By comparing the 679 natural intron mRNA data by the bioinformatics software MEME, it is found that the results of the.FISH experiment containing multiple common sequences are shown in the experimental data. 4 of the predicted cis elements can promote the mRNA nuclear transfer of beta globulin C DNA. We named motif1-AS, motif 1V-AS, motif 4-AS, motif 5. naturally without intron mRNA KRN1 gene containing 9 predictive elements motif1V-AS, of which 6 components are similar to the 6 components. CAR-C was deleted and mutated by the method of subcloning. The results of FISH experiment showed that the deletion and mutation of KRN1 mRNA would be blocked in the nucleus. After the completion of the cell construction, we obtained the binding protein of the predictive element through the MS2-MBP protein binding system, and the silver staining experiment after the polyacrylic acid gel electrophoresis (PAGE) was found to be promoted. There is a specific band between the binding protein of nuclear element binding protein and non stimulating nuclear element. Conclusion: the experimental data show that the element that can promote the function of nucleation of beta globulin C DNA transport in the natural intron mRNA also plays a role in promoting the transfer of nucleus in the natural intron mRNA.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

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