RNA-protein interaction mapping via MS2-or Cas13-based APEX targeting
RNA-protein interaction mapping via MS2-or Cas13-based APEX targeting
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DOI：
10.1073/pnas.2006617117
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发表时间：
2020-09-08
影响因子：
11.1
通讯作者：
Ting, Alice Y.
中科院分区：
文献类型：
--
作者：
Han, Shuo;Zhao, Boxuan Simen;Ting, Alice Y.
RNA-蛋白质相互作用是多种细胞过程的基础。需要改进的方法来以公正的方式系统地绘制活细胞中 RNA-蛋白质相互作用的图谱。我们使用两种方法将工程过氧化物酶 APEX2 靶向特定的细胞 RNA，以对蛋白质相互作用伙伴进行以 RNA 为中心的邻近生物素化。 MS2-MCP 系统和工程化 CRISPR-Cas13 系统均用于将 APEX2 高特异性地递送至人端粒酶 RNA hTR。一分钟的邻近生物素化捕获了 hTR 的候选结合伴侣，包括十多种以前未与 hTR 连接的蛋白质。我们验证了 hTR 与 N-6-甲基腺苷 (m(6)A) 去甲基酶 ALKBH5 之间的相互作用，并表明 ALKBH5 能够消除内源 hTR 上的 m(6)A 修饰。 ALKBH5 还调节端粒酶复合物的组装和活性。 MS2 和 Cas13 靶向 APEX2 可能有助于发现活细胞中新型 RNA-蛋白质相互作用。
RNA-protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA-protein interactions in living cells in an unbiased manner. We used two approaches to target the engineered peroxidase APEX2 to specific cellular RNAs for RNA-centered proximity biotinylation of protein interaction partners. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were used to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured candidate binding partners for hTR, including more than a dozen proteins not previously linked to hTR. We validated the interaction between hTR and the N-6-methyladenosine (m(6)A) demethylase ALKBH5 and showed that ALKBH5 is able to erase the m(6)A modification on endogenous hTR. ALKBH5 also modulates telomerase complex assembly and activity. MS2- and Cas13-targeted APEX2 may facilitate the discovery of novel RNA-protein interactions in living cells.