RNA互斥可变剪接相关的顺式元件及其二级结构的鉴定和进化保守性研究

发布时间：2018-11-04 16:12

【摘要】：可变剪接(Alternative splicing)能将一条Pre-mRNA剪接加工后产生不同的成熟mRNA,翻译产生不同的蛋白质,增加了蛋白质的多样性。互斥剪接是可变剪接的一种重要的方式,最典型的互斥可变剪接的例子是黑腹果蝇唐氏综合症细胞黏附因子(Dscam),果蝇的Dscam基因能够通过互斥剪接产生38,016种不同的异构体,但是由于该基因十分复杂,深入研究十分困难。目前对互斥剪接机制的研究尚不清楚。果蝇多效耐药性蛋白(multidrug-resistance protein,MRP)中也存在互斥剪接的现象,MRP基因能够通过互斥剪接产生16种不同的异构体,目前没有相关文献对MRP基因的互斥剪接机制的报道,因此对MRP基因及其它相关基因的剪接机制的研究有助于进一步对剪接机理的理解。首先,通过生物信息学的分析果蝇MRP基因的外显子8簇中存在两种情况,一种为7个可变外显子的情况,另一种为8个可变外显子的情况。在7个可变外显子的外显子8簇中发现一对RNA二级结构,在8个可变外显子的外显子8簇中发现两对RNA二级结构,实验验证结果表明：MRP基因7个可变外显子的外显子8簇中预测的RNA二级结构在互斥剪接中起调控作用,进一步实验还表明在8个可变外显子的外显子8簇中双向RNA二级结构能够调控互斥剪接。其次,本研究利用生物信息学手段比对分析了其它基因RNA二级结构调控互斥剪接现象。分析结果显示了3个基因存在RNA二级结构,这3个基因分别是Nc73EF基因、Srp基因和Ebl基因。其中Srp基因中发现了双向RNA二级结构,为进一步揭示互斥剪接机制提供了基础。对一部分节肢动物的代表性物种Dscam基因的外显子4簇、外显子6簇、外显子9簇进行了系统进化分析。分析结果显示：鳞翅目、膜翅目、鞘翅目Dscam基因及甲壳纲的外显子4簇、外显子6簇、外显子9簇均存在RNA二级结构,其中外显子6簇保守性最好,锚定序列在所有分析物种是高度保守,外显子4簇和外显子6簇具有物种序列特异性,但RNA二级结构均具有进化保守性,这些分析结果表明RNA二级结构介导互斥剪接的保守性。最后,为了进一步开展对RNA可变剪接在蛋白质水平上的研究,对模式生物果蝇的RNA剪接相关蛋白互作用网络进行了进化分析,并且对隆头蛛CStegodyphus mimosarum)剪接相关蛋白互作网络进行了系统的预测,分析结果显示：果蝇与人类和拟南芥的部分剪接相关蛋白互作网络存在进化保守性,并且在S. mimosarum中预测了19个剪接相关蛋白,构建了剪接相关蛋白互作网络。这些信息为进一步研究可变剪接相关蛋白提供了线索。
[Abstract]:Variable splicing (Alternative splicing) can produce different proteins after processing a single Pre-mRNA splicing and produce different proteins, which increases the diversity of proteins. Mutex splicing is an important way of variable splicing. The most typical example of mutex alternative splicing is the fact that the Dscam gene of (Dscam), the cell adhesion factor of Drosophila melanogaster Down syndrome, can produce 38016 different isomers through mutex splicing. However, due to the complexity of the gene, it is very difficult to further study. At present, the research of mutex splicing mechanism is not clear. The phenomenon of mutex splicing also exists in Drosophila multidrug resistance protein (multidrug-resistance protein,MRP). MRP gene can produce 16 different isomers through mutex splicing. There are no reports on the mechanism of MRP gene mutex splicing. Therefore, the study of splicing mechanism of MRP gene and other related genes is helpful to further understand the splicing mechanism. Firstly, there are two cases in the exon 8 cluster of Drosophila MRP gene by bioinformatics, one is for 7 variable exons and the other is for 8 variable exons. A pair of RNA secondary structures were found in exon 8 clusters of 7 variable exons, and two pairs of RNA secondary structures were found in exon 8 clusters with 8 variable exons. The experimental results show that the predicted secondary structure of RNA in exon 8 of 7 variable exons of MRP gene plays a regulatory role in mutex splicing. Further experiments show that the bidirectional RNA secondary structure can regulate the mutex splicing in the exon 8 clusters of 8 variable exons. Secondly, bioinformatics was used to compare and analyze the mutex splicing of other genes regulated by RNA secondary structure. The results showed that three genes had RNA secondary structure, and the three genes were Nc73EF gene, Srp gene and Ebl gene. The secondary structure of bidirectional RNA was found in Srp gene, which provides a basis for further revealing the mechanism of mutex splicing. The phylogenetic analysis of exon 4, exon 6 and exon 9 of Dscam gene was carried out. The results showed that Lepidoptera, Hymenoptera, Coleoptera Dscam gene and crustacean exon 4 cluster, exon 6 cluster, exon 9 cluster all had secondary structure of RNA, and exon 6 cluster was the most conserved. The anchoring sequence was highly conserved in all species, exon 4 and exon 6 were species specific, but the secondary structure of RNA was evolutionarily conserved. These results indicated that the secondary structure of RNA mediated the conservation of mutually exclusive splicing. Finally, in order to further study the protein level of variable splicing of RNA, the evolutionary analysis of the RNA splicing associated protein interaction network of Drosophila melanogaster was carried out. A systematic prediction of the interaction network of CStegodyphus mimosarum) splicing associated proteins was carried out. The results showed that the interaction networks between Drosophila and human beings and Arabidopsis thaliana were evolutionarily conserved, and the results showed that the interaction networks between Drosophila and human beings and Arabidopsis thaliana were evolutionarily conserved. Nineteen splicing associated proteins were predicted in S. mimosarum, and a splicing associated protein interaction network was constructed. This information provides clues for further study of variable splicing associated proteins.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78

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