Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen
Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen
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DOI：
10.3791/55255
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发表时间：
2017-05-01
影响因子：
1.2
通讯作者：
Wan, Yue
中科院分区：
文献类型：
--
作者：
Aw, Jong Ghut Ashley;Shen, Yang;Wan, Yue
了解 RNA 如何与自身以及与其他 RNA 相互作用是了解细胞中基于 RNA 的基因调控的关键。虽然 RNA-RNA 相互作用（例如 microRNA-mRNA 相互作用）的例子已被证明可以调节基因表达，但细胞中 RNA 相互作用发生的全部程度仍然未知。以前研究 RNA 相互作用的方法主要集中于与特定蛋白质或 RNA 种类相互作用的 RNA 子集。在这里，我们详细介绍了一种名为补骨脂素交联、连接和选定杂交体测序 (SPLASH) 的方法，该方法允许以公正的方式在体内全基因组范围内捕获 RNA 相互作用。 SPLASH 利用体内交联、邻近连接和高通量测序来识别全球分子内和分子间 RNA 碱基配对伙伴。 SPLASH 可应用于不同的生物体，包括细菌、酵母和人类细胞，以及不同的细胞条件，以促进了解不同细胞环境下 RNA 组织的动态。整个实验SPLASH协议大约需要5天才能完成，计算工作流程大约需要7天才能完成。
Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.