Targeted ribose methylation of RNA in vivo directed by tailored antisense RNA guides
Targeted ribose methylation of RNA in vivo directed by tailored antisense RNA guides
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DOI：
10.1038/383732a0
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发表时间：
1996-10-24
期刊：
影响因子：
64.8
通讯作者：
Bachellerie, JP
中科院分区：
文献类型：
--
作者：
Cavaille, J;Nicoloso, M;Bachellerie, JP
真核细胞的核糖体RNA通过特定核苷酸的核糖甲基化在转录后修饰(1)。这发生在核仁中，涉及一组具有与跨越甲基化位点(2-4)的rRNA序列互补的长区域(10-21个核苷酸)的小核仁RNA(SnoRNA)--特定位点的甲基化需要互补的snoRNA(5)。在这里，我们证明改变snoRNA的序列足以改变甲基化的特异性。哺乳动物细胞被基因工程改造为与任意rRNA序列互补的snoRNA，指导该序列中预测的核苷酸的甲基化。我们进一步鉴定了甲基化所需的引导RNA和底物RNA的结构特征，并利用这些结构特征设计了一个不含rRNA序列的外源转录本，当与适当的引导snoRNA共表达时，该转录本是位点特异性甲基化的。因此，内源性非核糖体RNA可以被靶向，可能为在转录后水平上改变基因表达提供一个高度选择性的工具。
EUKARYOTIC ribosomal RNAs are post-transcriptionally modified by methylation at the ribose sugar of specific nucleotides(1). This takes place in the nucleolus and involves a family of small nucleolar RNAs (snoRNAs) with long regions (10-21 nucleotides) complementary to rRNA sequences spanning the methylation site(2-4)-a complementary snoRNA is required for methylation at a specific site(5). Here we show that altering the sequence of the snoRNA is sufficient to change the specificity of methylation. Mammalian cells transfected with a snoRNA engineered to be complementary to an arbitrary rRNA sequence direct the methylation of the predicted nucleotide in that sequence. We have further identified structural features, both of the guide and substrate RNA, required for methylation and have used these to design an exogenous transcript, devoid of rRNA sequence, that is site-specifically methylated when co-expressed with an appropriate guide snoRNA. Endogenous non-ribosomal RNA can thus be targeted, possibly providing a highly selective tool for the alteration of gene expression at the post-transcriptional level.