Double-stranded RNA induces mRNA degradation in Trypanosoma brucei
Double-stranded RNA induces mRNA degradation in Trypanosoma brucei
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DOI：
10.1073/pnas.95.25.14687
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发表时间：
1998-12-08
影响因子：
11.1
通讯作者：
Ullu, E
中科院分区：
文献类型：
--
作者：
Ngô, H;Tschudi, C;Ullu, E
双链RNA（dsRNA）在秀丽隐杆线虫中具有遗传干扰作用，在某些情况下也可能是植物表型共抑制的基础。在构建用于转染锥虫细胞的质粒载体时，我们偶然发现，在体内表达cy-tubulin mRNA 5'非翻译区（5' UTR）的dsRNA导致具有显著形态学改变和胞质分裂特异性阻断的多核细胞。合成的α-微管蛋白5' UTR dsRNA的转染，而不是单独的任一链的转染，引起相同的表型。在dsRNA转染时，微管蛋白mRNA，而不是相应的前体mRNA，被快速且特异性地降解，导致α-微管蛋白合成的缺陷。转染的细胞不再能够进行胞质分裂并最终死亡。从这些锥虫的细胞骨架结构的分析显示缺陷的鞭毛轴丝和鞭毛附着区，一个复杂的皮质结构，我们建议是必不可少的建立在胞质分裂的分裂沟的路径的微管。最后，双链RNA介导的mRNA降解并不局限于α-微管蛋白mRNA，还可以应用于其他细胞mRNA，从而建立了一个强大的工具来遗传操纵这些重要的原生动物寄生虫。
Double-stranded RNA (dsRNA) recently has been shown to give rise to genetic interference in Caenorhabditis elegans and also is likely to be the basis for phenotypic cosuppression in plants in certain instances. While constructing a plasmid vector for transfection of trypanosome cells, we serendipitously discovered that in vivo expression of dsRNA of the cy-tubulin mRNA 5' untranslated region (5' UTR) led to multinucleated cells with striking morphological alterations and a specific block of cytokinesis. Transfection of synthetic alpha-tubulin 5' UTR dsRNA, but not of either strand individually, caused the same phenotype, On dsRNA transfection, tubulin mRNA, but not the corresponding pre-mRNA, was rapidly and specifically degraded, leading to a deficit of alpha-tubulin synthesis. The transfected cells were no longer capable of carrying out cytokinesis and eventually died. Analysis of cytoskeletal structures from these trypanosomes revealed defects in the microtubules of the flagellar axoneme and of the flagellar attachment zone, a complex cortical structure that we propose is essential for establishing the path of the cleavage furrow at cytokinesis. Last, dsRNA-mediated mRNA degradation is not restricted to alpha-tubulin mRNA but can be applied to other cellular mRNAs, thus establishing a powerful tool to genetically manipulate these important protozoan parasites.