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非脊椎动物Kv2基因A至I RNA编辑位点的鉴定和形成机制的初步研究
Editing Sites Identification and Formation Mechanism Study of the A to I Editing of Invertebrates Kv2 Gene
【作者】 尹姮；
【导师】 金勇丰；
【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2010， 硕士
【摘要】 RNA编辑的分子机制和生理功能的研究已经非常广泛,但其在生物进化过程中的意义仍然是一个谜团。本研究从约占动物界总种数85%的节肢动物门出发,通过基因组序列与RT-PCR序列的比对,鉴定Kv2基因第二外显子A至I RNA编辑靶位点。结果发现,尽管各物种所在纲和目不尽相同,进化年代上也相差甚远,但是却有三个编辑位点Y/C,S/G,I/V是非常保守的,表明这种修饰对蛋白质的功能是有重要作用的。不仅如此,同一个目或同一个纲亲缘关系相近的物种,往往会共享一些编辑位点,同时保留一些自己特有的编辑位点。A至I RNA编辑酶ADAR作用的底物一般认为是由外显子与其下游内含子的一小段序列回折形成的双链RNA,而根据本研究对节肢动物门中模式生物家蚕(Bombyx mori)、果蝇(Drosophila melanogaster)、意大利蜜蜂(Apis mellifera)二级结构的预测,发现这些物种Kv2基因的exon2这段序列自身就可以形成完美的RNA编辑结构。由此运用细胞转染的方法建立了一套体内编辑系统来验证这一预测。结果显示果蝇的Kv2基因片段发生编辑,但家蚕和意大利蜜蜂的不能被编辑。继而我们推断果蝇的Kv2基因编辑是由exon2自身形成二级结构介导的,而家蚕和蜜蜂的Kv2基因编辑需要除exon2以外的片段辅助形成二级结构介导。同时,利用生物信息学手段重新预测出了家蚕和蜜蜂ECS序列(编辑互补序列)。
【Abstract】 The molecular mechanism and physiological function of recoding by A-to-I RNA editing is well known, but its evolutionary significance remains a mystery. The RNA editing of the Kv2 K~+channel from different classes of Arthropoda are extensively analyzed. By comparing the RNA RT-PCR sequence with the genomic sequence, we verify several editing sites that change codon usage within exon 2. It is notable that three of these editing sites were conserved in most species, suggesting that a crucial role of function during hundreds of millions of years’ evolution. We also found that in the same classis, different species share identical editing sites, meantime own their specific editing sites. We all know that the occurrence of RNA editing is catalyzed by the ADAR enzyme family. Their editing substrates are RNA stem-loop structures that are occasionally formed by an exon sequence and an inverted repeat located in the downstream intron. Surprisingly, bioinformatical analysis of B.mori, D. melanogaster and A.mellifera kv2 RNA secondary structures show that the entire exon 2 sequence form a perfect double-stranded RNA structure that could be the editing substrate. Although it has been found in mammals Kv1.1 and Gabra-3 before, it is still rare that the editing sites and the editing complementary sequences are located in the same exon. Based on these results, experiments are undertaken to demonstrate our predictions. However, the results show that only D. melanogaster can be edited.