Global in situ profiling of RNA-RNA spatial interactions with RIC-seq
Global in situ profiling of RNA-RNA spatial interactions with RIC-seq
复制标题
使用 RIC-seq 对 RNA-RNA 空间相互作用进行全局原位分析
DOI：
10.1038/s41596-021-00524-2
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发表时间：
2021-05-21
期刊：
影响因子：
14.8
通讯作者：
Xue, Yuanchao
中科院分区：
文献类型：
--
作者：
Cao, Changchang;Cai, Zhaokui;Xue, Yuanchao
新的证据表明，RNA-RNA 相互作用对于控制多种生物过程至关重要，包括转录、RNA 剪接和蛋白质翻译。 RNA 原位构象测序 (RIC-seq) 是一种以单碱基分辨率捕获活细胞中蛋白质介导的 RNA-RNA 近端相互作用的技术。首先用甲醛处理细胞，以原位修复所有蛋白质介导的 RNA-RNA 相互作用。经过细胞透化和微球菌核酸酶消化后，近端相互作用的 RNA 在 3' 末端用 pCp-生物素标记，随后使用 T4 RNA 连接酶进行连接。然后，嵌合 RNA 被富集并转化为文库，用于双端测序。深度测序后，计算分析得出起始群体中近端相互作用 RNA 上每个碱基的相互作用强度分数。整个实验过程设计在 6 d 内完成，然后额外 8 d 进行计算分析。 RIC-seq技术可以公正地检测分子内和分子间的RNA-RNA相互作用，从而使其可用于重建RNA高阶结构和识别直接非编码RNA靶标。
Emerging evidence has demonstrated that RNA-RNA interactions are vital in controlling diverse biological processes, including transcription, RNA splicing and protein translation. RNA in situ conformation sequencing (RIC-seq) is a technique for capturing protein-mediated RNA-RNA proximal interactions globally in living cells at single-base resolution. Cells are first treated with formaldehyde to fix all the protein-mediated RNA-RNA interactions in situ. After cell permeabilization and micrococcal nuclease digestion, the proximally interacting RNAs are 3′ end-labeled with pCp-biotin and subsequently ligated using T4 RNA ligase. The chimeric RNAs are then enriched and converted into libraries for paired-end sequencing. After deep sequencing, computational analysis yields interaction strength scores for every base on proximally interacting RNAs in the starting populations. The whole experimental procedure is designed to be completed within 6 d, followed by an additional 8 d for computational analysis. RIC-seq technology can unbiasedly detect intra- and intermolecular RNA-RNA interactions, thereby rendering it useful for reconstructing RNA higher-order structures and identifying direct noncoding RNA targets.