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Identification of RNA-binding protein targets with HyperTRIBE

Nature Protocolsvolume 13pages18291849 (2018) | Download Citation


RNA-binding proteins (RBPs) accompany RNA from birth to death, affecting RNA biogenesis and functions. Identifying RBP–RNA interactions is essential to understanding 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 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 of the RNA-editing enzyme ADAR (adenosine deaminase acting on RNA) (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. Therefore, we developed HyperTRIBE by incorporating a previously characterized hyperactive mutation, E488Q, into the ADARcd. This strategy increases the editing efficiency and reduces sequence bias, which markedly increases the sensitivity of this technique without sacrificing specificity. HyperTRIBE provides a more powerful strategy for identifying RNA targets of RBPs with an easy experimental and computational protocol at low cost, that can be performed not only in flies, but also in mammals. The HyperTRIBE experimental protocol described below can be carried out in cultured Drosophila S2 cells in 1 week, using tools available in a common molecular biology laboratory; the computational analysis requires 3 more days.

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1. TRIBE: hijacking an RNA-editing enzyme to identify cell-specific targets of RNA-binding proteins:

2. Mechanistic implications of enhanced editing by a HyperTRIBE RNA-binding protein:


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We thank K. Abruzzi and J. Sherk for their helpful comments on improving the manuscript. We thank J. Rodriguez for his contribution in developing some of the scripts that are now part of the HyperTRIBE software. This work was supported by the Howard Hughes Medical Institute, NIH EUREKA grant DA037721 and NIH grant 5R01AG052465.

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  1. Department of Biology, Howard Hughes Medical Institute and National Center for Behavioral Genomics, Brandeis University, Waltham, MA, USA

    • Reazur Rahman
    • , Weijin Xu
    • , Hua Jin
    •  & Michael Rosbash


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R.R., W.X., H.J. and M.R. wrote the manuscript. W.X. and H.J. developed the experimental protocol and wrote the relevant sections of the protocol. R.R. developed the software and wrote the bioinformatics sections of the protocol.

Competing interests

The authors declare that a PCT patent application (PCT patent application no. PCT/US2016/05425) has been filed based on the HyperTRIBE method described in this paper.

Corresponding author

Correspondence to Michael Rosbash.

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