Cavaillé J, Nicoloso M, Bachellerie J P
Laboratoire de Biologie Moléculaire Eucaryote du CNRS, Université Paul-Sabatier, Toulouse, France.
Nature. 1996 Oct 24;383(6602):732-5. doi: 10.1038/383732a0.
Eukaryotic ribosomal RNAs are post-transcriptionally modified by methylation at the ribose sugar of specific nucleotides. 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--a complementary snoRNA is required for methylation at a specific site. 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 coexpressed 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.
真核生物核糖体RNA在转录后通过特定核苷酸核糖糖基的甲基化进行修饰。这种修饰发生在核仁中，涉及一类小核仁RNA（snoRNA），其具有与跨越甲基化位点的rRNA序列互补的长区域（10 - 21个核苷酸）——特定位点的甲基化需要一个互补的snoRNA。我们在此表明，改变snoRNA的序列足以改变甲基化的特异性。用设计为与任意rRNA序列互补的snoRNA转染的哺乳动物细胞会指导该序列中预测核苷酸的甲基化。我们进一步确定了甲基化所需的引导RNA和底物RNA的结构特征，并利用这些特征设计了一种不含rRNA序列的外源转录本，当与适当的引导snoRNA共表达时，该转录本会进行位点特异性甲基化。因此，内源性非核糖体RNA可以成为靶点，这可能为转录后水平上改变基因表达提供一种高度选择性的工具。
