TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of RNA-Binding Proteins.

TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of RNA-Binding Proteins.
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DOI:
10.1016/j.cell.2016.03.007
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发表时间:
2016-04-21
期刊:
影响因子:
64.5
通讯作者:
Rosbash, Michael
Rosbash, Michael
中科院分区:
生物学1区
文献类型:
--
作者:
McMahon, Aoife C.;Rahman, Reazur;Jin, Hua;Shen, James L.;Fieldsend, Allegra;Luo, Weifei;Rosbash, Michael

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RNA转录物由RNA结合蛋白(RBP)结合和调节。目前用于鉴定RBP的体内靶标的方法是不完善的,并且不适合于检查少量细胞。为了解决这些问题,我们开发了TRIBE(通过编辑识别的RNA结合蛋白的靶标),这是一种将RBP与果蝇RNA编辑酶阿达尔的催化结构域偶联并在体内表达融合蛋白的技术。RBP靶标标记有新的RNA编辑事件,并通过测序RNA来鉴定。我们已经使用TRIBE来鉴定三种RBP(Hrp48、dFMR 1和NonA)的靶标。TRIBE与包括CLIP在内的其他方法相比,我们已经从150个特定的果蝇神经元中确定了RBP靶点。TRIBE可以在没有抗体和少量特定细胞的情况下进行。
RNA transcripts are bound and regulated by RNA-binding proteins (RBPs). Current methods for identifying in vivo targets of a RBP are imperfect and not amenable to examining small numbers of cells. To address these issues, we developed TRIBE (Targets of RNA-binding proteins Identified By Editing), a technique that couples an RBP to the catalytic domain of the Drosophila RNA editing enzyme ADAR and expresses the fusion protein in vivo. RBP targets are marked with novel RNA editing events and identified by sequencing RNA. We have used TRIBE to identify the targets of three RBPs (Hrp48, dFMR1 and NonA). TRIBE compares favorably to other methods, including CLIP, and we have identified RBP targets from as little as 150 specific fly neurons. TRIBE can be performed without an antibody and in small numbers of specific cells.
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