Identification of RNA-binding protein targets with HyperTRIBE.
Identification of RNA-binding protein targets with HyperTRIBE.
复制标题
使用 HyperTRIBE 鉴定 RNA 结合蛋白靶点。
DOI：
10.1038/s41596-018-0020-y
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发表时间：
2018-08
期刊：
影响因子：
14.8
通讯作者：
Rosbash M
中科院分区：
文献类型：
--
作者：
Rahman R;Xu W;Jin H;Rosbash M
RNA binding proteins (RBPs) accompany RNA from birth to death, affecting RNA biogenesis and functions. Identifying RBP-RNA interactions is essential to understand their complex roles in different cellular processes. However, detecting in vivo RNA targets of RBPs, especially in a small number of discrete cells, has been a technically challenging task. We have previously developed a novel technique called TRIBE (Targets of RNA-binding proteins Identified By Editing) to overcome this problem. TRIBE expresses a fusion protein consisting of a queried RBP and the catalytic domain from RNA editing enzyme ADAR (ADARcd), which marks target RNA transcripts by converting adenosine to inosine near the RBP binding sites. These marks can be subsequently identified via high-throughput sequencing. In spite of its usefulness, TRIBE is constrained by a low editing efficiency and editing-sequence bias from the ADARcd. So, we developed HyperTRIBE by incorporating a previously characterized hyperactive mutation, E488Q, into the ADARcd. This strategy increases the editing efficiency and reduce sequence bias, which dramatically increased sensitivity of this technique without sacrificing specificity. HyperTRIBE provides a more powerful strategy to identify RNA targets of RBPs with an easy experimental and computational protocol at low cost not only in flies but also in mammals. The HyperTRIBE experimental protocol described below can be carried out in cultured Drosophila S2 cells in one week using tools available in a common molecular biology laboratory, and the computational analysis requires 3 more days. HyperTRIBE uses a hyperactive RNA editing enzyme fused to an RNA-binding protein (RBP), to mark the target RNA transcripts of the RBP by converting adenosine to inosine near the binding sites with increased efficiency and reduced sequence bias.
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影响因子：
16
作者：
Rodriguez, Joseph;Menet, Jerome S.;Rosbash, Michael
通讯作者：
Rosbash, Michael
影响因子：
25
作者：
Hrvatin S;Hochbaum DR;Nagy MA;Cicconet M;Robertson K;Cheadle L;Zilionis R;Ratner A;Borges-Monroy R;Klein AM;Sabatini BL;Greenberg ME
通讯作者：
Greenberg ME
DOI：
10.3791/2034
发表时间：
2010-07-02
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Hafner, Markus;Landthaler, Markus;Tuschl, Thomas
通讯作者：
Tuschl, Thomas
影响因子：
8.8
作者：
Artegiani, Benedetta;Lyubimova, Anna;Clevers, Hans
通讯作者：
Clevers, Hans
影响因子：
48
作者：
Lapointe CP;Wilinski D;Saunders HA;Wickens M
通讯作者：
Wickens M