Ectopic expression of Drosophila ELAV and human HuD in Drosophila wing disc cells reveals functional distinctions and similarities.
Ectopic expression of Drosophila ELAV and human HuD in Drosophila wing disc cells reveals functional distinctions and similarities.
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发表时间：
2002-06
影响因子：
4
通讯作者：
G. Toba;J. Qui;S. Koushika;K. White
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作者：
G. Toba;J. Qui;S. Koushika;K. White
果蝇ELAV和人类HuD是两种具有显著序列同源性的神经元RNA结合蛋白，但它们各自在转录后调控中的作用不同。ELAV调节特定转录物的神经特异性选择性剪接，HuD稳定特异性mrna，否则这些mrna由于其3'非翻译区（UTR）中的富au元件（AREs）而不稳定。AREs是哺乳动物细胞转录物稳定性的主要决定因素。这些蛋白的作用被研究和比较，通过异位表达它们在果蝇的想象翅盘细胞，缺乏内源性表达的蛋白质。研究了两组绿色荧光蛋白报告基因（均由广表达启动子驱动）对ELAV和HuD异位表达的影响。每组由三个报告基因组成：(1)具有不间断开放阅读框（ORF）；(2)将组成剪接的内含子插入ORF；(3)将内含子nASI插入ORF，其剪接在神经元中受ELAV调节。两组的不同之处在于它们的3'UTR：具有are样特征的热休克蛋白70ab （Hsp70Ab）预告片或Actin 5C （Act5C）预告片。结果表明：(1)异位表达的ELAV和HuD均能促进Hsp70Ab 3'UTR基因的表达，而对Act5C 3'UTR基因的表达无促进作用；(2)这种增强伴随着mRNA水平的升高；(3)只有ELAV能诱导nASI的神经特异性剪接；(4)虽然HuD主要定位于细胞质，但ELAV既定位于细胞质，也定位于细胞核。
Drosophila ELAV and human HuD are two neuronal RNA binding proteins that show remarkable sequence homology, yet differ in their respective documented roles in post-transcriptional regulation. ELAV regulates neural-specific alternative splicing of specific transcripts, and HuD stabilizes specific mRNAs that are otherwise unstable due to AU-rich elements (AREs) in their 3' untranslated region (UTR). AREs are major determinants of transcript stability in mammalian cells. The role of each of these proteins was investigated and compared, by ectopically expressing them in Drosophila imaginal wing disc cells, which lack endogenous expression of either protein. The effect of the ectopic expression of ELAV and HuD was assessed on two sets of green fluorescent protein reporter transgenes, which were all driven with a broadly expressing promoter. Each set consisted of three reporter transgenes: (1) with an uninterrupted open reading frame (ORF); (2) with a constitutively spliced intron inserted into the ORF; and (3) with the intron nASI whose splicing is regulated in neurons by ELAV, inserted into the ORF. The two sets differed from each other only in their 3'UTR: Heat-shock-protein-70Ab (Hsp70Ab) trailer with ARE-like characteristics or Actin 5C (Act5C) trailer. Our results show that: (1) both ectopically expressed ELAV and HuD can enhance expression of transgenes with the Hsp70Ab 3'UTR, but not of transgenes with Act5C 3'UTR; (2) this enhancement is accompanied by an increase in mRNA level; (3) only ELAV can induce neural-specific splicing of nASI; and (4) although HuD is localized primarily to the cytoplasm, ELAV is localized to both the cytoplasm and the nucleus.