RNA-RNA interactions enable specific targeting of noncoding RNAs to nascent pre-mRNAs and chromatin sites

We developed a general method based on RNA Antisense Purification (RAP) to identify the intermolecular RNA-RNA interactions of a target RNA (RAP-RNA). RAP-RNA identifies endogenous RNA-RNA complexes through in vivo crosslinking, RNA capture with antisense oligonucleotides, and high-throughput RNA sequencing. This approach provides a systematic view of other RNAs that interact with a target RNA, and furthermore can distinguish between direct and indirect RNA-RNA interactions through the use of crosslinking reagents with different reactivities with proteins and nucleic acids. We applied this method to numerous small and large noncoding RNAs, including U1 snRNA, Malat1 lncRNA, Xist lncRNA, U3 snoRNA, U17/Snora73a snoRNA and U12 snRNA. Overall design: We examined the RNA and chromatin interactions of ncRNAs in mouse embryonic stem cells. We developed and applied three related protocols: RAP-RNA[AMT], RAP-RNA[FA], and RAP-RNA[FA-DSG]. In the RAP-RNA[AMT] protocol, we fixed direct RNA-RNA hybrids in mouse embryonic stem (ES) cells with 4''-aminomethyltrioxalen (AMT), a psoralen-derivative crosslinker; AMT generates inter-strand crosslinks between uridine bases in RNA but does not react with proteins. In the RAP-RNA[FA] protocol, we used a different crosslinking strategy to capture both direct and indirect RNA-RNA interactions: we fixed ES cells using formaldehyde (FA), which crosslinks protein-RNA and protein-protein interactions and thus can capture both indirect interactions as well as direct interactions that are caged or flanked by proteins. In the RAP-RNA[FA-DSG] protocol, we fixed with both FA and disuccinimidyl glutarate (DSG), a strong protein-protein crosslinker, to more efficiently capture RNAs linked indirectly through multiple protein intermediates.

本研究开发了一种基于RNA反义纯化（RNA Antisense Purification, RAP）的通用方法，用于鉴定靶RNA的分子间RNA-RNA相互作用，该方法即RAP-RNA。RAP-RNA通过体内交联、反义寡核苷酸介导的RNA捕获以及高通量RNA测序技术，识别内源性RNA-RNA复合物。该方法可系统性解析与靶RNA相互作用的其他RNA，此外还可通过使用对蛋白质与核酸具有不同反应活性的交联试剂，区分直接与间接RNA-RNA相互作用。
我们将该方法应用于多种大小各异的非编码RNA，包括U1小核RNA（U1 snRNA）、Malat1长链非编码RNA（Malat1 lncRNA）、Xist长链非编码RNA（Xist lncRNA）、U3小核仁RNA（U3 snoRNA）、U17/Snora73a小核仁RNA以及U12小核RNA（U12 snRNA）。
整体实验设计：我们检测了小鼠胚胎干细胞中非编码RNA（ncRNA）与RNA及染色质的相互作用。本研究开发并应用了三种相关实验方案：RAP-RNA[AMT]、RAP-RNA[FA]以及RAP-RNA[FA-DSG]。在RAP-RNA[AMT]方案中，我们使用4''-氨甲基三氧补骨脂素（4''-aminomethyltrioxalen, AMT）——一种补骨脂素类交联剂——对小鼠胚胎干细胞（ES细胞）内的直接RNA-RNA杂交体进行交联固定；AMT可在RNA的尿嘧啶碱基之间形成链间交联，但不与蛋白质发生反应。在RAP-RNA[FA]方案中，我们采用了另一种交联策略，以同时捕获直接与间接RNA-RNA相互作用：使用甲醛（FA）固定ES细胞，甲醛可交联蛋白质-RNA与蛋白质-蛋白质相互作用，因此既能捕获间接相互作用，也能捕获被蛋白质包裹或侧翼结合有蛋白质的直接相互作用。在RAP-RNA[FA-DSG]方案中，我们同时使用甲醛与二琥珀酰亚胺戊二酸酯（disuccinimidyl glutarate, DSG）——一种强效蛋白质-蛋白质交联剂——进行固定，以更高效地捕获通过多种蛋白质中介间接相连的RNA。