Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom.
Proc Natl Acad Sci U S A. 2011 Jun 14;108(24):10010-5. doi: 10.1073/pnas.1017386108. Epub 2011 May 24.
Many protein-protein and protein-nucleic acid interactions have been experimentally characterized, whereas RNA-RNA interactions have generally only been predicted computationally. Here, we describe a high-throughput method to identify intramolecular and intermolecular RNA-RNA interactions experimentally by cross-linking, ligation, and sequencing of hybrids (CLASH). As validation, we identified 39 known target sites for box C/D modification-guide small nucleolar RNAs (snoRNAs) on the yeast pre-rRNA. Novel snoRNA-rRNA hybrids were recovered between snR4-5S and U14-25S. These are supported by native electrophoresis and consistent with previously unexplained data. The U3 snoRNA was found to be associated with sequences close to the 3' side of the central pseudoknot in 18S rRNA, supporting a role in formation of this structure. Applying CLASH to the yeast U2 spliceosomal snRNA led to a revised predicted secondary structure, featuring alternative folding of the 3' domain and long-range contacts between the 3' and 5' domains. CLASH should allow transcriptome-wide analyses of RNA-RNA interactions in many organisms.
许多蛋白质-蛋白质和蛋白质-核酸相互作用已经通过实验进行了表征，而 RNA-RNA 相互作用通常仅通过计算进行预测。在这里，我们描述了一种通过交联、连接和杂交体测序（CLASH）来实验鉴定分子内和分子间 RNA-RNA 相互作用的高通量方法。作为验证，我们在酵母前 rRNA 上鉴定了 39 个已知的框 C/D 修饰指南小核仁 RNA（snoRNA）的靶位点。在 snR4-5S 和 U14-25S 之间回收了新的 snoRNA-rRNA 杂交体。这些得到了天然电泳的支持，并且与以前未解释的数据一致。U3 snoRNA 被发现与靠近 18S rRNA 中央假结 3' 侧的序列相关，支持其在该结构形成中的作用。将 CLASH 应用于酵母 U2 剪接体 snRNA 导致了预测二级结构的修订，其特征是 3' 结构域的替代折叠和 3' 和 5' 结构域之间的长程接触。CLASH 应该允许在许多生物体中进行全转录组范围内的 RNA-RNA 相互作用分析。
