Xu Y X, Liu L, Michaeli S
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
Isr Med Assoc J. 2000 Jul;2 Suppl:58-62.
Trypanosomatids are the causative agent of several major parasitic diseases including African trypanosomiasis, American trypanosomiasis, and leishmaniasis. These parasites possess unique RNA-processing mechanisms including trans-splicing of pre-mRNA and RNA editing of mitochondrial transcripts. In this study, we identified a trypanosomatid novel group of small nucleolar RNAs that belong to the box C/D snoRNA, which were shown to guide ribose methylation on rRNA. Three snoRNA genes were identified; snoRNA-2 carrying a single snoRNA and g2 and b2 coding for a single or multiple snoRNAs, respectively. Mapping of the methylation sites guided by snoRNA-2 using two different approaches suggest that snoRNA-2 has the potential to guide methylation on both 5.8S and 18S rRNAs. The trypanosomes follow the same guide-methylation rule established for yeast and for mammals. As a first attempt to change the methylation pattern of target RNAs, we generated transgenic parasites carrying the B2 and snoRNA-2, which were engineered to shift the methylation site on rRNA. Despite efficient expression of these tagged snoRNAs, the novel methylation site was not generated. However, efficient expression of tagged snoRNAs in transgenic parasites opens the possibility of engineering novel methylation sites on different target RNAs in vivo.
锥虫是包括非洲锥虫病、美洲锥虫病和利什曼病在内的几种主要寄生虫病的病原体。这些寄生虫具有独特的RNA加工机制，包括前体mRNA的反式剪接和线粒体转录本的RNA编辑。在本研究中，我们鉴定出一组属于盒C/D小核仁RNA（snoRNA）的新型锥虫小核仁RNA，它们被证明可指导核糖体RNA（rRNA）上的核糖甲基化。鉴定出了三个snoRNA基因；snoRNA-2携带单个snoRNA，而g2和b2分别编码单个或多个snoRNA。使用两种不同方法对由snoRNA-2指导的甲基化位点进行定位表明，snoRNA-2有可能指导5.8S和18S rRNA上的甲基化。锥虫遵循为酵母和哺乳动物确立的相同指导甲基化规则。作为改变靶RNA甲基化模式尝试的第一步，我们构建了携带B2和snoRNA-2的转基因寄生虫，对其进行改造以改变rRNA上的甲基化位点。尽管这些标记的snoRNA高效表达，但并未产生新的甲基化位点。然而，转基因寄生虫中标记的snoRNA的高效表达为在体内对不同靶RNA进行新型甲基化位点工程改造开辟了可能性。
