Identification of RNA-binding protein targets with HyperTRIBE

• Published in: Nature protocols Vol. 13; no. 8; pp. 1829 - 1849
• Main Authors: Rahman, Reazur, Xu, Weijin, Jin, Hua, Rosbash, Michael
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.08.2018
• Subjects: Adenosine; Adenosine deaminase; Adenosine Deaminase - genetics; Adenosine Deaminase - metabolism; Animals; Bias; Binding proteins; Binding Sites; Biosynthesis; Catalysis; Cell Line; Computation; Computer applications; Death; Drosophila; Editing; Enzymes; Evolution; Fruit flies; Fusion protein; Gene targeting; Identification and classification; Methods; Molecular biology; Mutant Proteins - genetics; Mutant Proteins - metabolism; Next-generation sequencing; Novels; Prejudice; Protein Binding; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Ribonucleic acid; RNA; RNA - metabolism; RNA editing; RNA sequencing; RNA-binding protein; RNA-Binding Proteins - metabolism; Software; Target detection; Target recognition; United States
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/s41596-018-0020-y

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.